Cortex Lycii attenuates night sweat in Yin-deficient rat by downregulating β 2 AR/cAMP signaling: A proteomic approach

Cortex Lycii attenuates night sweat in Yin-deficient rat by downregulating β 2 AR/cAMP signaling: A proteomic approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cortex Lycii attenuates night sweat in Yin-deficient rat by downregulating β 2 AR/cAMP signaling: A proteomic approach Meixia Huang, Zhiyuan Hong, Jie Wang, Xiangxiang Liu, Yingzheng Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4023446/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: To investigate the therapeutic effect of Cortex Lycii on the sweat secretion of YDH rats and its underlying mechanism. Methods : Male SD rats (N=36) were randomly assigned into Control, YDH, and DI groups (N=12 each group). YDH model rats were constructed by oral administration of thyroxin and reserpine. After two-weeks treatment of Cortex Lycii , the pharmacodynamics was assessed then the protein profile was obtained using a tandem mass tag proteomic approach. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis was used for bioinformatics analysis of differential expressed proteins (DEPs), critical DEPs were validated by Western blot. The receptor involved in the Cortex Lycii were confirmed by using β 2 AR receptor agonist (isoproterenol)- and M 3 R receptor agonist (pilocarpine)-induced sweat secretion in YDH rats. Results : Cortex Lycii attenuates sweat secretion in YDH rats, and down-regulated the expression of AQP 5 and β 2 -receptor on paw pads of YDH rats. Proteomics analysis revealed 189 differential proteins between the model and control groups, of which 124 differential proteins were remarkably reversed by Cortex Lycii . The overlapping DEPs in the model and Cortex Lycii groups were mainly enriched in the β 2 AR/cAMP pathway. Moreover, Cortex Lycii considerably attenuated the agonistic effect of isoprenaline induced sweat secretion. Conclusion : This study demonstrated the effect of Cortex Lycii on sweat secretion in Yin-deficient rats. The mechanism on the ground may refer to the inhibition of β 2 AR/cAMP, followed by the suppression of the downstream PKA/ezrin/CFTR signaling pathway to reduce ion exchange and the inhibition of IP 3 /Ca 2+ /AQP 5 . The finding may provide new research on the mechanism of night sweats in TCM and their amelioration by Cortex Lycii . Biological sciences/Plant sciences Health sciences/Pathogenesis Yin deficiency heat sweat proteomic aquaporin-5 β2AR/cAMP Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. INTRODUCTION Night sweats are a frequently encountered symptom in patients, and it is characterized by increased sweating during sleep, regardless of the ambient temperature 1 . This condition is a complication of many diseases, and it frequently occurs in menopause 2 , hyperthyroidism 3 , gastroesophageal reflux disease 4 , pulmonary tuberculosis 5 , and some malignant tumors 4,6,7 . Night sweats seriously affect patients’ sleep and can even lead to insomnia and anxiety, which seriously influence their quality of life. However, modern medicine offers no desirable clinical treatment option. Night sweat, a common occurrence in individual’s daily routines, is a characteristic of Yin deficiency in traditional Chinese medicine (TCM). According to TCM theory, pathological and physiological states are often analyzed comprehensively and systematically, and a series of interrelated symptoms occurring at a particular stage of diseases is called syndromes. Although modern medicine recognizes that different diseases have distinct pathological mechanisms, they may be classified as the same syndrome according to the overall concept of TCM. For example, conditions such as type 2 diabetes, hyperthyroidism, tuberculosis, and menopausal syndrome are categorized as Yin deficiency syndrome in TCM according to their common symptoms of hot flashes, night sweats, red tongue, reduced fur, and rapid pulse. TCM treats Yin deficiency heat (YDH) by focusing on two main approaches: nourishing Yin and suppressing internal heat 8 . Cortex Lycii is a popular medicine for treating night sweats induced by YDH, and it is the root bark of Lycium Chinense Mill 9 . The first traditional Chinese medical book was published in 221 B.C., in which Shennong Ben Cao Jing described the effect of Cortex Lycii on suppressing internal heat. Since then, Cortex Lycii has been used in the treatment of Yin deficiency and night sweats 10,11 . According to the literature reviews, Cortex Lycii is still widely used in modern TCM for the clinical treatment of night sweats caused by YDH. YDH syndrome, a well-known illness in TCM, has received significant attention in recent decades. Clinical and experimental findings revealed that Yin deficiency syndrome has similar pathological features to systemic endocrine disorders, including abnormal energy metabolism and immune depression. Systems biology provides a possible method for understanding syndromes in Chinese medicine because they share the same holistic view. Proteomics, the study of large-scale protein expression profiles in blood and body fluids, is a widely used method in systems biology; it offers several advantages in studying TCM syndromes and understanding the mechanisms of herbal medicines in TCM 7,12 . Proteomics is currently used to investigate the hypothalamic mechanisms that contribute to lipid and glucose metabolism disorders in Yin deficiency syndrome 13,14 . It is also used to identify the serum protein markers associated with pathological features, such as immune depression, coagulation cascade, and glucose metabolism disorders 15–17 . However, the precise molecular mechanisms of night sweats in YDH and the therapeutic mechanisms by which Cortex Lycii alleviates this symptom are poorly understood. In our previous study, we evaluated the Pharmacodynamics of Cortex Lycii on YDH rats by using different doses of Cortex Lycii . The results show that a clinically equivalent doses of Cortex Lycii can significantly lower the body temperature and reduced sweat secretion of YDH rats. In this study, we utilized TMT-labeled proteomics to investigate the changes in serum protein levels in YDH rats following Cortex Lycii treatment. We also applied a series of bioinformatics methods to reveal the mechanism of night sweats in YDH and the therapeutic mechanism of Cortex Lycii against this symptom.The workflow of our study is shown in Fig.1 . 2. METHODS AND MATERIALS 2.1 Reagents , animals, and assay kits Specific pathogen-free (SPF) male Sprague–Dawley (SD) rats weighing 300±20 g were provided by the Animal Center of Fujian University of Traditional Chinese Medicine (FJTCM) [Animal license number: SCXK (Zhe) 2019–0002]. They were housed in an SPF laboratory with an ambient temperature of 25 °C and a 12 h light–dark cycle, and the animals had free access to standard rat diet and water. All experimental protocols were approved by the FJTCM Laboratory Animal Welfare Ethics Committee. The rats were handled in strict compliance with ARRIVE guidelines. Cortex Lycii was purchased from the Third Affiliated Hospital of FJTCM and authenticated by Professor Che Su-rong from FJTCM. Voucher specimens were stored in the Key Laboratory of TCM in Fujian Higher Education Institutions. In this study, the following primary antibodies were used: anti-AQP 5 (ab78486, Abcam), anti-M 3 R (ab87199, Abcam), anti-β 2 AR (ab182136, Abcam), anti-PRKAR1A+PRKAR1B (ab139695, Abcam), anti-calmodulin 1/2/3 (ab45689, Abcam), anti-NHERF-1 (ab3452, Abcam), anti-Raf1 (ab173539, Abcam), and anti-MEK1+MEK2 (ab178876, Abcam). 2.2 Preparation of Cortex Lycii extract Cortex Lycii extracts were prepared through the following steps: crude Cortex Lycii powder was soaked in 75% ethanol (1:10, w/v) for 1 h, extracted at reflux for 1 h, and filtered. The residue was extracted following the same method twice, and the filtrate of the three extracts was collected and concentrated by rotary evaporation. Subsequently, 0.5% CMC-Na solution was used to dissolve the extract, and the final concentration was adjusted to 6 g/mL (clinically equivalent dose). The extract was stored at 4 °C. 2.3 UPLC/MS analysis for the quality control of Cortex Lycii extract Cortex Lycii extract quality was monitored using an ultrahigh-performance liquid chromatography (UPLC) system (Vanquish, Thermo Fisher Scientific). The analytical conditions were as follows: Waters ACQUITY UPLC H-Class instrument and Waters UPLC BEH C18 column (1.7 m, 2.1×100 mm). The mobile phase comprised 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multistep linear elution gradient program was as follows: 0–3.5 min, 95%–85% A; 3.5–6 min, 85%–70% A; 6–6.5 min, 70%–70% A; 6.5–12 min, 70%–30% A; 12.5–18 min, 30%–0% A; 25–26 min, 0%–95% A; 26–30 min, 95%–95% A; and 26–30 min, 95%–95% A. The column temperature was set at 40 °C, the flow rate was set at 0.4 mL/min, and the sample injection volume was set at 5 L. Cortex Lycii extract was redissolved in 80% methanol 18 . The MS and MS/MS data were obtained using an Orbitrap Exploris 120 mass spectrometer and Xcalibur software in the IDA acquisition mode. The mass range was 100–1500 during each acquisition cycle. From each cycle, the top four were selected and screened, resulting in the retrieval of corresponding MS/MS data. The other conditions were as follows: sheath gas flow rate: 30 Arb; auxiliary gas flow rate: 10 Arb; ion transfer tube temperature: 350 °C; vaporizer temperature: 350 °C, full MS resolution: 60,000; MS/MS resolution: 15,000. In the NCE mode, the collision energy was 16/32/48 at 5.5 kV (positive) or −4 kV (negative) spray voltage. 2.4 Experiments in animals and sample collection After 1 week of accommodation, 36 SD rats were randomly divided into these three groups: control group (saline group, N=12), model group (YDH group, N=12), and Cortex Lycii group (DI group, N=12). The rats from the model and DI groups were administered with thyroxine (92 mg/mL/day) and reserpine (0.61 mg/mL/day) via gavage once a day for 1 week 8 . Those in the control group were given an equal volume of sterile saline solution (1 mL/100 g) by gavage. On day 8, the rats from the DI group were treated with Cortex Lycii (6 g/mg) for 14 days. Equal amounts of saline were given to the model and control groups. Body weight, body temperature, and sweat spots were measured once a day throughout the experiment. The blood sample was collected by the abdominal aortic method 1 h after the last medication, and animals were then subject to euthanasia. The blood sample was centrifuged at 3000×g for 10 min to separate the serum. The serum was aliquoted and stored at −80 °C for subsequent ELISA and proteomic experiments. The paw pads were collected from each group of rats, and the protein expression in these paw pads was determined by Western blot. 2. 5 Measurement of sweat spots Sweat coloring experiments were performed on each group of rats by using Hotan Takagaki reagent A solution (2% iodine dissolved in absolute ethanol) and B solution (soluble starch mixed with castor oil). After the sweat spots reacted with the reagent and turned blue-black, photographs were obtained. The proportion of the blue-black part of the foot pad was calculated with software image J. 2. 6 ELISA Serum samples were collected as mentioned above. Serum levels of cAMP and cGMP were measured using a commercially available ELISA kit (Commercially available) following the manufacturer’s instructions 19 . 2. 7 TMT-LC-MS/MS The serum sample from the animal experiment was used in proteomics analysis. Pierce TM Top 14 Abundant Protein Depletion Spin Columns (ThermoFisher Scientific) were used to remove the top 14 high-abundance proteins. Finally, the protein concentration was measured according to the manufacturer’s instructions using a BCA kit. The protein solution was reduced for 30 min at 56 °C with 5 mM dithiothreitol and alkylated for 15 min at room temperature in darkness with 11 mM iodoacetamide. Thereafter, the protein sample was diluted with 100 mM TEAB in less than 2 M urea. Trypsin was added at a trypsin-to-protein mass ratio of 1:50 for the first overnight digestion and a trypsin-to-protein mass ratio of 1:100 for the second digestion for 4 h. Finally, the C18 SPE column desalted the peptides 20 . Subsequently, 0.5 M TEAB was used to dissolve the tryptic peptides. Each peptide channel was labeled with its own TMT reagent (according to the manufacturer’s procedure, ThermoFisher Scientific) and incubated at room temperature for 2 h. About 5 L of each sample was pooled, desalted, and analyzed by MS to assess the labeling efficiency. The pooled samples were quenched with 5% hydroxylamine, desalted using a Strata X C18 SPE column (Phenomenex), and vacuum centrifuged for drying. The peptides were exposed to sodium/iodide symporter sources. Tandem mass spectrometry (MS/MS) was carried out with the use of Q Exactive TM Plus (Thermos) equipment that was linked online to a UPLC system 21 . The electrospray voltage was set at 2.0 kV, the entire MS scan resolution was set to 60,000, and the scan range was 350–1600 m/z. The top 20 most abundant precursors were selected for further MS/MS analyses under a 30 s dynamic exclusion period. Normalized collision energy (NCE) of 28% was used for HCD fragmentation. The Orbitrap detected the fragments with a resolution of 30,000. The initial fixed mass was fixed at 100 m/z. The AGC target was set to 1E5, with an intensity threshold of 3.3E4 and a maximum injection time of 60 ms. The generated MS/MS data were searched against the human SwissProt database (20422 items) concatenated with the reverse decoy database using the MaxQuant search engine (v.1.6.15.0). Trypsin/P was identified as a cleavage enzyme with the capability to catalyze up to two missed cleavages. The initial search utilized a mass tolerance of 20 ppm for precursor ions, whereas the main search used a mass tolerance of 5 ppm. By contrast, the mass tolerance of 0.02 Da was used for fragment ions. Carbamidomethylation of Cys was designated as a fixed modification, whereas acetylation of the protein’s N-terminus and oxidation of Met were designated as variable modifications. FDR decreased to 1%. 2. 8 Bioinformatics a nalysis Molecular functions, cellular components, and biological processes of the identified proteins were analyzed using the GO database (http://geneontology). Protein signaling pathways were annotated by using the KEGG pathway database (http://www.genome.jp/kegg/ map-per.ht). Protein interaction networks were analyzed by STRING software (http://string-db.org/). Functional annotation allowed for the inference of the biological functions associated with the output protein in the context of sweating 15 . 2.9 Western blot Protein was extracted from rats’ foot pads and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analyses. In brief, an appropriate amount of protein was added to each lane of 10% SDS-PAGE gel. The membranes were blocked for 15 min by using QuickBlock™ Blocking Buffer for Western blot. The blots were then incubated overnight at 4 °C with the following primary antibodies: GAPDH, β-actin, M 3 R, β 2 AR, AQP 5 , PKA, CaM, raf 1 , and NHE-RF1. At room temperature, the membranes were incubated with HRP-conjugated secondary antibody for 1 h. The indicator proteins on membranes were observed using the BeyoECL Star chemiluminescence kit (Beyotime Biotechnology, Shanghai, China). 2.10 Effects of Cortex Lycii on isoproterenol- or pilocarpine-induced sweat secretion in YDH rats Isoproterenol (β 2 -receptor agonist) or pilocarpine (M 3 -receptor agonist) was used to induce sweats in YDH rats to examine the effects of Cortex Lycii . A total of 36 YDH rats were randomized into six groups (each group comprised six rats): control (Group A), model (Group B), model + pilocarpine (Group C), model + pilocarpine +DI (Group D), model + isoprenaline (Group E), and model + isoprenaline + DI (Group F). The rats in groups B–F were administered with thyroxine (92 mg/mL/day) and reserpine (0.61 mg/mL/day) via gavage once a day for 7 days, whereas the control group was given an equal volume of sterile saline solution (1 mL/100 g) by gavage. On day 8, the rats in groups D and F were treated with Cortex Lycii (6 g/mg) for 14 days. Equal amounts of saline were given to the model and control groups. On day 20, the rats in groups C and D were administered with pilocarpine (0.035 g/kg) via subcutaneous injection. Meanwhile, the rats in groups E and F were administered with isoprenaline (0.255 mg/kg) via subcutaneous injection. Sweat spots were measured daily throughout the experiment. 2.11 Statistical analysis All data were presented as the mean ± standard deviation (SD). The three groups were analyzed by ANOVA and post-hoc Tukey test in SPSS 21.0 software (SPSS, Armonk, NY, USA). Graphs were generated with origin 2019b software. 3. RESULTS 3.1 UPLC-MS/MS analysis of Cortex Lycii extracts Qualitative analysis was conducted on the chemical components of Cortex Lycii by UPLC-MS/MS, and the total ion flow diagrams of negative and positive ion modes were obtained. The results are shown in Fig. 2 . Given the information of fragment ions in MS and reported literature, a total of 20 characteristic compounds were identified and labeled from Cortex Lycii. Information on these compounds is provided in Tables 1 and 2 . Table 1 Characteristic compounds identified and labeled from Cortex Lycii in the negative ion mode No Compounds t R （ min ） Formula m/z Ion mode MS fragment 1 Methyl hexadecanoate 11.9854 C 17 H 34 O 2 315.2534 [M+HCOO] - 315.25357,297.246019,313.241226,113.097391,316.256824 2 Caffeic acid 2.1406 C 9 H 8 O 4 179.0349 [M-H] - 135.045466,179.035719,134.037798,59.014108,107.050197 3 FA 18:2+1O 14.0061 C 18 H 32 O 3 295.2278 [M-H] - 59.013963,295.226337,277.214661,171.103228,195.138771 4 9-HODE 17.0497 C 18 H 32 O 3 295.2277 [M-H] - 295.226597,277.215156,171.103544,195.138428,59.014004 5 9-HOTrE 12.1908 C 18 H 30 O 3 293.2123 [M-H] - 275.203876,293.209425,171.103411,121.102168,183.13874 6 9-Hydroxy-10,12,15-octadecatrienoic acid 12.6889 C 18 H 30 O 3 293.2123 [M-H] - 293.215469,275.20352,171.103141,121.10197,183.138649 7 Cinnamic acid 5.0228 C 9 H 8 O 2 147.0449 [M-H] - 147.044513,59.013855,75.008341,87.008677,129.020466 8 Citric acid 0.8170 C 6 H 8 O 7 191.0194 [M-H] - 111.009041,113.013595,87.008725,193.022596,85.029638 Table 2 Characteristic compounds identified and labeled from Cortex Lycii in the positive ion mode. No Compounds t R （ min ） Formula m/z Ion mode MS fragment 1 Apigenin 8.2545 C 15 H 10 O 5 271.0608 [M+H] + 271.059817,272.064126,153.017964,211.075674,119.048474 2 BETAINE 26.7755 C 5 H 11 NO 2 140.0682 [M+Na] + 140.068983,141.113364,74.096389,112.087003,85.076399 3 Cinnamyl alcohol 10.9963 C 9 H 10 O 135.0806 [M+H] + 135.080771,136.083288,91.054483,107.084969,79.053978 4 Kukoamine B 3.9129 C 28 H 42 N 4 O 6 531.3189 [M+H] + 222.112786,532.323417,223.117422,294.189458,123.04396 5 Scopoletin 3.2805 C 10 H 8 O 4 193.0498 [M+H] + 193.050097,133.028093,178.026077,137.060352,165.053629 6 Moupinamide 7.2157 C 18 H 19 NO 4 314.1392 [M+H] + 177.05483,314.136449,121.064657,145.027994,93.069921 7 Paeonol 8.8080 C 9 H 10 O 3 167.0706 [M+H] + 167.069711,43.017894,149.059696,121.06481,125.058985 8 Coumaroyl tyramine 6.9857 C 17 H 17 NO 3 284.128 [M+H] + 147.044578,284.129045,121.0648,119.048613,93.069955 9 N-cis-Caffeoyltyramine 6.3647 C 17 H 17 NO 4 300.123 [M+H] + 163.038218,300.121594,121.064597,145.027891,135.045038 10 1,2-Dehydro-alpha-cyperone 10.9963 C 15 H 20 O 217.1589 [M+H] + 217.159194,161.096842,189.162798,175.111477,133.101639 11 Benzenepropanamide, N-[2-(acetyloxy)-1-(phenylmethyl)ethyl]-alpha-(benzoylamino) 11.1657 C 27 H 28 N 2 O 4 445.2122 [M+H] + 105.033494,194.117093,224.108788,117.070165,134.097014 12 Lyciumin A 6.8196 C 42 H 51 N9O 12 874.3709 [M+H] + 486.199942,136.07546,468.186945,874.374862,856.351538 3.2 Cortex Lycii attenuated Y in deficiency syndrome and sweat secretion in YDH rats Cortex Lycii has been reported to significantly improve the biological indicators of Yin deficiency model animals 22,23 . In this study, a YDH model was established according to references through the oral administration of thyroxine combined with reserpine 24 . Compared with control rats, YDH rats showed YDH symptoms, including irritability, easy fright, excessive sweats, tachypnea, and dry hair. After the animal experiment was completed, the body weight of rats in the YDH group significantly decreased ( P < 0.05) compared with that of the control group, whereas that of rats in the Cortex Lycii （DI） group increased significantly ( Fig. 3C ). YHD rats had elevated body temperatures compared with the controls, and Cortex Lycii was effective in reducing body temperatures to levels similar to those of the controls ( Fig. 3D ). As illustrated in Figs. 3E–3G , we observed an increase in the serum cAMP level and cAMP/cGMP ratio in the YDH model group, but the serum cGMP level decreased. Meanwhile, a decrease in serum cAMP level and cAMP/cGMP ratio was observed in DI rats, but minimal changes were recorded in the cGMP level of this group. As illustrated in Figs. 3I and 3J , in the model group, β 2 receptors were dramatically upregulated and M 3 receptors were downregulated. β 2 AR was substantially downregulated in the DI group, but no statistical difference was observed for M 3 receptors. The above results were consistent with the findings of previous reports 13 , indicating that we successfully established a model of YDH and Cortex Lycii successfully reversed the symptom of Yin deficiency syndrome. Hotan Takagaki reagent solutions are a well-established diagnostic tool to visualize and evaluate sweat spots. The sweating area on the paw pads of rats exhibited a blue-black discoloration. The blue-black pixels were then extracted from the footpad images, adjusted using the Auto Local Threshold function in ImageJ, and measured to the area fraction (sweating area %) as described in the reference 25 . Figs. 3 A and 3B shows that the amount of sweat spots in the YDH group rats was significantly higher than that in the control group ( P <0.01), and Cortex Lycii significantly reduced sweat secretion ( P <0.05). Moreover, the protein expression of AQP 5 was elevated in the YDH group, but this trend was reversed by Cortex Lycii treatment ( Fig. 3H ). 3.3 Quantitative analysis of differentially expressed proteins (DEPs) To investigate the mechanism of Cortex Lycii , we used a TMT-based proteomics approach to determine DEPs in the model, DI, and control groups. A total of 1473 proteins were identified; among them, 1219 were further quantified. The DEPs included proteins with expression change multiple > 1.2 and P < 0.05. Figs. 4A–4C show 189 DEPS in the YDH/control group (71 downregulated and 118 upregulated) and 124 DEPs in the DI/YDH group (112 downregulated and 12 upregulated). Among them, 54 overlapping differential proteins were upregulated in the YDH/control group but downregulated in the DI/YDH group ( Figs. 4D and 4E ). 3.4 Bioinformatics a nalysis The 54 overlapping differential proteins were selected for further bioinformatics analysis. Utilizing GO annotations including biological processes (BPs), cellular components (CCs), and molecular functions (MFs) to obtain functional insights into differential proteins. Fig. 5A shows the most closely associated enrichment. The first three terms of DEPs for BP were inositol biosynthesis process, cortical cytoskeleton organization, and actin cytoskeleton organization. The majority of the proteins were involved in protein kinase A catalytic subunit binding, actin binding, protein kinase activity for MF, actin cytoskeleton, cAMP-dependent protein kinase complex, and immunological synapse for cellular components. KEGG enrichment analysis has indicated identified several key pathways based on the DEPs. These pathways included Yersinia infection, regulation of actin cytoskeleton, parathyroid hormone synthesis and action, longevity regulating pathway-multiple species, insulin signaling pathway, Ras signaling pathway, inositol phosphate metabolism, and gastric acid secretion ( Fig. 5B ). Proteomics analysis showed that multiple differential proteins such as Impa1, Gmpr, Cnn2, Slc9a3r1, Tln1, Eif4b, Rab6a, Pfn1, CFL1, Alb,and Prkar1a were involved in the BPs of sweat secretion regulation such as inositol production, regulation of endoplasmic reticulum, calcium signaling regulation, and Na (+)/H (+) exchange. The expression of the differential proteins was elevated in the model group rats but decreased after Cortex Lycii treatment ( Table 3 ). Table 3 Overlapping DEPs involved in the regulation of sweat secretion Gene name M/S(up) DI/M(down) ratio P value ratio P value Impa1 inositol monophosphatase 1 1.689 0.0050 0.589 0.0047 Gmpr GMP reductase 1.313 0.0005 0.618 0.0011 Cnn2 calponin 2 1.682 0.0090 0.610 0.0081 Slc9a3r1 sodium/hydrogen exchange regulatory cofactor NHE-RF1 1.511 0.0008 0.637 0.0070 Tln 1 Tln 1 1.508 0.0007 0.623 0.0009 Eif4b Eukaryotic translation initiation factor 4B 1.667 0.0085 0.750 0.0334 Pfn1 Profilin-1 1.363 0.0286 0.748 0.0044 CFL1 Cofilin-1 1.423 0.0126 0.580 0.0054 Alb Serum albumin 1.601 0.0273 2.724 0.0449 Rab6a Ras-related protein Rab-6A 1.398 0.0017 0.699 0.0022 Prkar1a protein kinase cAMP-dependent type I regulatory subunit alpha 1.536 0.0296 0.5960 0.0389 3.5 Validation of DEPs by Western blot Western blot experiments were performed to validate the results of proteomics analysis. Prkar1a, Raf1, Slc9a3r1, and CaM were selected for this comparison. The results indicated that the ratios of the chosen protein were upregulated in the model group rats but downregulated after Cortex Lycii treatment ( Fig. 6 ). These results were consistent with the proteomics data. 3.6 Cortex Lycii attenuates sweat secretion induced by isoproterenol The results of proteomics and bioinformatics analyses indicated that the Ca 2+ signaling pathway was activated in YDH rats, which may be due to the activation of M 3 R and β 2 AR on sweat glands. Given that both the literature and our study showed a significant reduction in M 3 R receptor expression, we hypothesized that the activation of Ca 2+ signaling might be caused by β 2 AR excitation. To further confirm this hypothesis[Ed12191] , we conducted another experiment to explore the effect of Cortex Lycii on sweating after administering isoprenaline (β 2 AR agonist) or pilocarpine (M 3 R agonist) in YDH rats. As illustrated in Figs. 7A and 7B , local injection of isoprenaline and atropine significantly stimulated sweat secretion in model rats. In the DI group, the agonistic effect of isoprenaline on sweat secretion was considerably attenuated, but no statistical difference was found in the agonistic effect of atropine. The above results suggested that Cortex Lycii attenuated sweat secretion in YDH rats mainly via β2 receptors, not M3 receptors. 4 DISCUSSION YDH is a common condition in TCM. Oral administration of thyroxine and reserpine is a widely accepted method to establish the Yin deficiency model 24,26 . This model closely simulates the human YDH syndrome, making it highly appropriate for investigating the pathophysiology of YDH syndromes and the effects of herbal medicine to nourish Yin and suppress heat. In the clinical patient and animal models of YDH syndrome, the levels of β-receptors/cAMP increased and M-receptors/cGMP decreased significantly. Thus, the cAMP/cGMP ratio and the expression of β-receptors could be used to evaluate the models and therapeutic effect of medicines 27–29 . In our study, the rats of the YDH group were more irritable and aggressive than those in the control group, and their body weights decreased while their average temperature increased significantly. The number of sweat spots on the paw pad of the model rats increased, and the cAMP/cGMP ratio also increased in the serum of the model rats. These results were close to the clinical symptoms of Yin deficiency syndromes 30–33 , and they were reversed by administering Cortex Lycii for a 2-week treatment period. These results showed that a YDH model was successfully induced. Our experiments also demonstrated that the increased sweat secretion in YDH rats was effectively alleviated by Cortex Lycii treatment. Sweat secretion is primarily regulated by M 3 R and β 2 AR, as indicated by the elucidated molecular mechanism 34 . M 3 R is stimulated by cholinergics and produces inositol 1,4,5-trisphosphatase (IP 3 ) in the cytosol, which can trigger Ca 2+ release from the endoplasmic reticulum (ER) so that the intracellular Ca 2+ concentration increases. The increase in the intracellular Ca 2+ concentration can promote H 2 O efflux, ion efflux, and ion influx 35–37 . β 2 -Adrenoceptors, which are coupled to Gs proteins, are activated by catecholamines, ADR, and NA, thereby increasing cAMP production. cAMP, as one of the second messengers, can activate protein kinase A (PKA), which is cAMP-dependent, resulting in the phosphorylation and opening of the L-type VDCC, which promotes Ca 2+ influx and regulates sweat secretion 34,38,39 . Impa1 is critical for the second messenger Ca 2+ signaling pathways because it can provide inositol, an essential precursor of membrane phospholipid phosphatidylinositol (PI) and its derivative inositol IP 3 40–42 . IP 3 mobilizes the ER to release Ca 2+ , increase intracellular calcium concentration, and regulate sweat secretion. Rab6a, which is situated within the Golgi apparatus, regulates membrane transport from the Golgi to the ER and plays a role in the localization and fusion of transport carriers 43 . Cnn2 is a filament-associated protein that binds calmodulin, participates in a variety of intracellular signaling pathways, and plays a key role in the Ca 2+ -dependent signaling pathway; it is a type of dynamic Ca 2+ sensor that responds to a wide range of [Ca 2+ ] and transmits signals downstream. The expression levels of proteins Impa1, Rab6a, and Cnn2 were upregulated in the model group, indicating their underlying mechanism by activating the Ca 2+ signaling pathway in YDH, which was reversed by Cortex Lycii . Prkar1a, the regulatory subunit of the cAMP-dependent protein kinases, is activated following cAMP binding to the regulatory subunits. Gmpr and Prkar1a are involved in the cAMP/PKA signaling pathway 44–46 . They were upregulated in the YDH group and significantly reduced after Cortex Lycii treatment, suggesting that cAMP/PKA signaling participated in YDH sweat secretion, which was also reversed by Cortex Lycii . Expressed in the parietal membrane of exocrine sweat glands, aquaporin-5 (AQP 5 ) maintains their secretory function 47,48 . The localization of AQP 5 in the plasma membrane is crucial for its role of water permeability, independent of gating regulation 49,50 . The trafficking of AQP 5 is mediated by ezrin through its interaction with the cytoskeleton, and ezrin is a cross-linker between the plasma membrane and the actin cytoskeletal network 51,52 . Slc9a3r1 can connect plasma membrane proteins to members of the ezrin/moesin/radixin family as a scaffold protein, thereby linking them to the actin cytoskeleton 53,54 . As demonstrated by the proteomic results, Slc9a3r1 expression was upregulated in rats of the model group but downregulated after Cortex Lycii intervention. This finding suggested that the therapeutic effect of Cortex Lycii on sweat in YDH rats was linked to the regulation of plasma membrane localization of AQP5 by ezrin, which was consistent with the subsequent protein validation results. Taken together, the findings of GO and KEGG analyses implicate an important role in the overactivation of Ca 2+ signaling and cAMP/PKA signaling in sweat secretion of YDH rats. Given that M 3 R expression was downregulated in the YDH model, we hypothesized that activation of Ca 2+ in YDH rats was the downstream effect of β-receptor activation but not the M 3 R mechanism. To confirm this hypothesis, we further verified the effects of Cortex Lycii on β 2 AR receptor agonist (isoproterenol)- and M 3 R receptor agonist (pilocarpine)-induced sweat secretion in YDH rats. The results showed that the effect of Cortex Lycii on YDH was related to the β 2 AR mechanism. We hypothesized that Cortex Lycii regulated YDH sweating by downregulating β 2 AR/cAMP and transmitting to the downstream PKA/ezrin/CFTR and IP 3 /Ca 2+ /AQP 5 pathway (Fig. 8 ). 5 CONCLUSIONS This study elucidated the effect of Cortex Lycii on sweat secretion in Yin-deficient rats. The underlying mechanism may be associated with the inhibition of β 2 AR/cAMP, followed by the modulation of the downstream PKA/ezrin/CFTR signaling pathway, resulting in a decrease in ion exchange and IP 3 /Ca 2+ /AQP 5 to reduce sweat production. This work offers novel perspectives on the underlying mechanism of night sweats in TCM and the therapeutic efficacy of Cortex Lycii . Declarations Funding This work was supported by State Administration of Traditional Chinese Medicine Clinical Pharmacy High-level Key Discipline of Traditional Chinese Medicine Construction Project (Chinese Medicine Education Letter [2022] No. 226), Provincial Natural Science Foundation of Fujian（No. 2023J01857）, and the Basic Scientific Research Enhancement Program of Fujian University of Traditional Chinese Medicine (No. XJC2022013). CRediT authorship contribution statement Meixia Huang: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing-review & editing. Zhiyuan Hong: Investigation, Methodology, Data curation, Visualization, Writing-original draft. Jie Wang: Investigation, Methodology, Data curation. Xiangxiang Liu: Investigation, Methodology, Visualization. Yingzheng Wang: Investigation, Methodology, Writing-review & editing. Yinghao Wang: Conceptualization, Formal analysis, Funding acquisition, Writing-review & editing. All authors contributed to manuscript revision, read, and approved the submitted version. Declaration of competing interest The authors declare that this research was conducted in the absence of any commercialor financial relationships that could be construed as a potential conflict of interest. Data availability All data presented in this study are available from the corresponding author upon reasonable request. Acknowledgements We would like to thank Biorender for helping with the graphical abstract design. Ethics declarations All animal procedures were performed in accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Center of Fujian University of Traditional Chinese Medicine (Approve License: FJTCM PRE IACUC 2023103). Consent for publication Not applicable. Author details 1 College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, 350122, China. References National Cancer Institute. in PDQ Cancer Information Summaries (Bethesda (MD), 2002). Lee, E. et al. Vasomotor symptoms of menopause, autonomic dysfunction, and cardiovascular disease. Am. J. Physiol. Heart Circ. Physiol. 323 , H1270–H1280 (2022). Jayapaul, M., Williams, M. R., Davies, D. P. & Large, D. M. Recurrent painful unilateral gynaecomastia-interactions between hyperthyroidism and hypogonadism. Andrologia 38 , 31–33 (2006). Berdnikov, A., McPhee, S., LaBine, L. & Fatoye, T. Night sweats as a manifestation of gastroesophageal reflux disease. Can. Fam. Physician Med. Fam. Can. 66 , 901–903 (2020). Moyo, S. et al. Prevalence of bacteriologically confirmed pulmonary tuberculosis in South Africa, 2017-19: a multistage, cluster-based, cross-sectional survey. Lancet Infect. Dis. 22 , 1172–1180 (2022). Carr, C., Vertelney, H., Fronk, J. & Trieu, S. Dronabinol for the Treatment of Paraneoplastic Night Sweats in Cancer Patients: A Report of Five Cases. J. Palliat. Med. 22 , 1221–1223 (2019). Jiang, T. & Li, J. Review on the systems biology research of Yin-deficiency-heat syndrome in traditional Chinese medicine. Anat. Rec. Hoboken NJ 2007 306 , 2939–2944 (2023). Liu, C. et al. A review of the botany, ethnopharmacology, phytochemistry, pharmacology, toxicology and quality of Anemarrhena asphodeloides Bunge. J. Ethnopharmacol. 302 , 115857 (2023). Guo, Z. et al. Study on the mechanism of Cortex Lycii on lung cancer based on network pharmacology combined with experimental validation. J. Ethnopharmacol. 293 , 115280 (2022). Chen, J. et al. Research progress on chemical constituents and pharmacological studies on root bark of Lycium barbarum. Zhongguo Zhong Yao Za Zhi Zhongguo Zhongyao Zazhi China J. Chin. Mater. Medica 46 , 3066–3075 (2021). Gao, D. et al. Hypoglycemic effects and mechanisms of action of Cortex Lycii Radicis on alloxan-induced diabetic mice. Yakugaku Zasshi 127 , 1715–1721 (2007). Lian, F. et al. Effects of Liuwei Dihuang Granule on the outcomes of in vitro fertilization pre-embryo transfer in infertility women with Kidney-yin deficiency syndrome and the proteome expressions in the follicular fluid. Chin. J. Integr. Med. 20 , 503–509 (2014). Guan, W., Liu, Y., Li, X., Yang, B. & Kuang, H. iTRAQ-Based Proteomics to Reveal the Mechanism of Hypothalamus in Kidney- Yin Deficiency Rats Induced by Levothyroxine. Evid.-Based Complement. Altern. Med. ECAM 2019 , 3703596 (2019). Liu, C. et al. Serum Protein KNG1, APOC3, and PON1 as Potential Biomarkers for Yin- Deficiency-Heat Syndrome. Evid.-Based Complement. Altern. Med. ECAM 2016 , 5176731 (2016). Chen, J. et al. A group of serum proteins as potential diagnostic biomarkers for Yin- deficiency-heat syndrome. Anat. Rec. Hoboken NJ 2007 303 , 2086–2094 (2020). Liu, C.-M. et al. The Chinese herbal formula Zhibai Dihuang Granule treat Yin-deficiency- heat syndrome rats by regulating the immune responses. J. Ethnopharmacol. 225 , 271–278 (2018). Yi, W.-J. et al. Screening of potential biomarkers for Yin-deficiency-heat syndrome based on UHPLC-MS method and the mechanism of Zhibai Dihuang granule therapeutic effect. Anat. Rec. Hoboken NJ 2007 303 , 2095–2108 (2020). Chen, C. et al. Qinzhizhudan formula dampens inflammation in microglia polarization of vascular dementia rats by blocking MyD88/NF-κB signaling pathway: Through integrating network pharmacology and experimental validation. J. Ethnopharmacol. 318 , 116769 (2024). Ran, X., Dou, D., Chen, H. & Ren, G. The correlations of adverse effect and tonifying effect of ginseng medicines. J. Ethnopharmacol. 291 , 115113 (2022). Li, L. et al. SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability. Nat. Commun. 7 , 12235 (2016). Zhao, D. et al. Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer. Cancer Cell 23 , 464–476 (2013). Zhang, H. et al. Research on biological expression characteristics of mice models of deficiency heat syndrome and asthenia cold syndrome. China J. Tradit. Chin. Med. Pharm. 1516-1520[in Chinese] (2020). Luo, J. et al. Construction and Verification of Post-infectious Cough Model with Lung Heat and Yin Deficiency Syndrome in Juvenile Rats. J. Tradit. Chin. Med. 2366–2374 (2022) doi:10.13288/j.11-2166/r.2022.24.012[in Chinese]. Wang, P. et al. Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS. J. Pharm. Biomed. Anal. 53 , 631–645 (2010). Cui, C., Noh, J. H., Michel, M., Gorospe, M. & Schlessinger, D. STIM1, but not STIM2, Is the Calcium Sensor Critical for Sweat Secretion. J. Invest. Dermatol. 138 , 704–707 (2018). Zhai, Y. et al. Broad range metabolomics coupled with network analysis for explaining possible mechanisms of Er-Zhi-Wan in treating liver-kidney Yin deficiency syndrome of Traditional Chinese medicine. J. Ethnopharmacol. 234 , 57–66 (2019). Wang, X. et al. UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine. J. Pharm. Anal. 11 , 458–464 (2021). Weissmann, G., Goldstein, I., Hoffstein, S., Chauvet, G. & Robineaux, R. Yin/Yang modulation of lysosomal enzyme release from polymorphonuclear leukocytes by cyclic nucleotides. Ann. N. Y. Acad. Sci. 256 , 222–232 (1975). Zhao, J., Cai, C., Xie, M., Liu, J. & Wang, B. Investigation of the therapy targets of Yi-Qi-Yang-Yin-Hua-Tan-Qu-Yu recipe on type 2 diabetes by serum proteome labeled with iTRAQ. J. Ethnopharmacol. 224 , 1–14 (2018). Bryce, C. Persistent Night Sweats: Diagnostic Evaluation. Am. Fam. Physician 102 , 427–433 (2020). Grenacher-Horn, D. Night sweats. MMW Fortschr. Med. 161 , 40–44 (2019). Takahashi, T. A. & Johnson, K. M. Menopause. Med. Clin. North Am. 99 , 521–534 (2015). Kronenberg, F. Hot flashes: epidemiology and physiology. Ann. N. Y. Acad. Sci. 592 , 52–86; discussion 123-133 (1990). Hu, Y., Converse, C., Lyons, M. C. & Hsu, W. H. Neural control of sweat secretion: a review. Br. J. Dermatol. 178 , 1246–1256 (2018). Concepcion, A. R. et al. Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function. J. Clin. Invest. 126 , 4303–4318 (2016). Pabbidi, M. R. et al. Inhibition of cAMP-Dependent PKA Activates beta2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes. PloS One 11 , e0168505 (2016). Perez-Zoghbi, J. F., Mayora, A., Ruiz, M. C. & Michelangeli, F. Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i. Am. J. Physiol. Gastrointest. Liver Physiol. 295 , G671-681 (2008). Amano, T., Shitara, Y., Fujii, N., Inoue, Y. & Kondo, N. Evidence for beta-adrenergic modulation of sweating during incremental exercise in habitually trained males. J. Appl. Physiol. Bethesda Md 1985 123 , 182–189 (2017). Lindsay, S. L., Holmes, S., Corbett, A. D., Harker, M. & Bovell, D. L. Innervation and receptor profiles of the human apocrine (epitrichial) sweat gland: routes for intervention in bromhidrosis. Br. J. Dermatol. 159 , 653–660 (2008). Kawamura, K. & Ferris, J. P. Kinetic and mechanistic analysis of dinucleotide and oligonucleotide formation from the 5’-phosphorimidazolide of adenosine on Na(+)-montmorillonite. J. Am. Chem. Soc. 116 , 7564–7572 (1994). Luttrell, B. M. Cellular actions of inositol phosphates and other natural calcium and magnesium chelators. Cell. Signal. 6 , 355–362 (1994). Nourbakhsh, A., Collakova, E. & Gillaspy, G. E. Characterization of the inositol monophosphatase gene family in Arabidopsis. Front. Plant Sci. 5 , 725 (2014). Ma, R. et al. Involvement of Rab6a in organelle rearrangement and cytoskeletal organization during mouse oocyte maturation. Sci. Rep. 6 , 23560 (2016). Arige, V. et al. CREB regulates the expression of type 1 inositol 1,4,5-trisphosphate receptors. J. Cell Sci. 134 , jcs258875 (2021). Busca, R. & Ballotti, R. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Res. 13 , 60–69 (2000). De Jonge, C. J., Han, H. L., Mack, S. R. & Zaneveld, L. J. Effect of phorbol diesters, synthetic diacylglycerols, and a protein kinase C inhibitor on the human sperm acrosome reaction. J. Androl. 12 , 62–70 (1991). Asahina, M., Poudel, A. & Hirano, S. Sweating on the palm and sole: physiological and clinical relevance. Clin. Auton. Res. Off. J. Clin. Auton. Res. Soc. 25 , 153–159 (2015). Iizuka, T., Suzuki, T., Nakano, K. & Sueki, H. Immunolocalization of aquaporin-5 in normal human skin and hypohidrotic skin diseases. J. Dermatol. 39 , 344–349 (2012). Inoue, R. et al. Immunolocalization and translocation of aquaporin-5 water channel in sweat glands. J. Dermatol. Sci. 70 , 26–33 (2013). Zhang, M. et al. Localization of Na(+)-K(+)-ATPase alpha/beta, Na(+)-K(+)-2Cl- cotransporter 1 and aquaporin-5 in human eccrine sweat glands. Acta Histochem. 116 , 1374–1381 (2014). Benharouga, M. et al. The role of the C terminus and Na+/H+ exchanger regulatory factor in the functional expression of cystic fibrosis transmembrane conductance regulator in nonpolarized cells and epithelia. J. Biol. Chem. 278 , 22079–22089 (2003). Muroi, S.-I. & Isohama, Y. Ezrin Regulates Ca(2+) Ionophore-Induced Plasma Membrane Translocation of Aquaporin-5. Int. J. Mol. Sci. 22 , 13505 (2021). Barati, M. T. et al. Loss of NHERF-1 expression prevents dopamine-mediated Na-K-ATPase regulation in renal proximal tubule cells from rat models of hypertension: aged F344 rats and spontaneously hypertensive rats. Am. J. Physiol. Cell Physiol. 313 , C197–C206 (2017). Weinman, E. J. et al. Sodium-hydrogen exchanger regulatory factor 1 (NHERF-1) transduces signals that mediate dopamine inhibition of sodium-phosphate co-transport in mouse kidney. J. Biol. Chem. 285 , 13454–13460 (2010). Additional Declarations No competing interests reported. Supplementary Files Westernblotsupplementaryinformation.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4023446","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289144665,"identity":"2f53542e-25d5-49cb-a410-dcc969747005","order_by":0,"name":"Meixia Huang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Meixia","middleName":"","lastName":"Huang","suffix":""},{"id":289144667,"identity":"e5bb2436-5409-45da-a6d9-5ebd850a951d","order_by":1,"name":"Zhiyuan Hong","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zhiyuan","middleName":"","lastName":"Hong","suffix":""},{"id":289144668,"identity":"d02c0111-225f-485b-aeff-b86aa92e20f9","order_by":2,"name":"Jie Wang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Wang","suffix":""},{"id":289144670,"identity":"0b36a8d3-c1e2-42da-adf9-8ce57897c20a","order_by":3,"name":"Xiangxiang Liu","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiangxiang","middleName":"","lastName":"Liu","suffix":""},{"id":289144673,"identity":"c12a6492-6f06-490a-a45b-3c0fc4811117","order_by":4,"name":"Yingzheng Wang","email":"","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yingzheng","middleName":"","lastName":"Wang","suffix":""},{"id":289144674,"identity":"38a065e2-25dd-4244-8348-d542e4fb5874","order_by":5,"name":"Yinghao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYFACHhBhw8PP30C8Fkag2jQZyRkHSNNy2MagIYFIDQY3co8/+JlznseA4QDjh485RGnJS2zs3Xabx5y5gVly5jYitJjdyDFsZgRqsWw4wMbMS4KWczwGBxJI03KABC32Z94YzuzdlswjOeNgM3F+kWzPMfjwc5udPT9/88EPH4nRwiCQAGOB4ocowH+ASIWjYBSMglEwcgEAa9w46jBoBvcAAAAASUVORK5CYII=","orcid":"","institution":"Fujian University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Yinghao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-03-07 08:00:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4023446/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4023446/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54478334,"identity":"46530f46-0c5c-4beb-a53d-b9f7a88a8a53","added_by":"auto","created_at":"2024-04-11 07:30:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1421161,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003cstrong\u003e \u0026nbsp;\u003c/strong\u003eThe abstract graphic was created with BioRender.com (Agreement number: AA253WIO7E)\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/4a64b8efbaa00b67b0b556a5.jpg"},{"id":54478337,"identity":"30f2f0b5-3925-4797-a3a7-eff76d2a3de0","added_by":"auto","created_at":"2024-04-11 07:30:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":908367,"visible":true,"origin":"","legend":"\u003cp\u003eCompounds of \u003cem\u003eCortex Lycii \u003c/em\u003e(DI) scanned by LC-MS/MS.(A)\u003cstrong\u003e \u003c/strong\u003eTotal ion current of \u003cem\u003eCortex Lycii \u003c/em\u003e(DI) in the positive ion mode.\u003cstrong\u003e \u003c/strong\u003e(B) Total ion current of \u003cem\u003eCortex Lycii \u003c/em\u003e(DI) in the negative ion mode.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/7fa1a714daadc36d28283447.jpg"},{"id":54478332,"identity":"5264f589-1655-4c6c-a47f-0806f6c95e8c","added_by":"auto","created_at":"2024-04-11 07:30:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1147908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCortex Lycii \u003c/em\u003e(DI) recovered yin deficiency syndrome and attenuated the sweat secretion of YDH rats (A–B) Sweat spots were examined using Hotan Takagaki reagent, and the percentage of sweat ratio (%) was calculated by Image J (N = 6). (C–D) Mean weight and temperature of rats from each group at the animal experiment endpoint. (E–G) Serum content of cAMP and cGMP and cAMP/cGMP ratio were measured by ELISA (n = 6). (H–J) Protein levels of AQP\u003csub\u003e5\u003c/sub\u003e , β\u003csub\u003e2\u003c/sub\u003eAR, and M\u003csub\u003e3\u003c/sub\u003eR on the paw pads of the rats. Results are mean ± SD (N = 3). Data were presented as the mean ± SD. Significances were marked as \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, vs. the control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, vs. the YDH group. One-way ANOVA and post-hoc Tukey test were performed to analyze the data.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/62f60704bef93b1e98444af9.jpg"},{"id":54478335,"identity":"5091599c-848f-48a1-9535-00c63398529d","added_by":"auto","created_at":"2024-04-11 07:30:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":950275,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed proteins (DEPs). (A) Volcano plot of DEPs between the YDH rats and control rats. (B) Volcano plot of DEPs between the \u003cem\u003eCortex Lycii\u003c/em\u003e (DI) rats and YDH rats. Volcano plots based on collapsed changes in DEPs and P-values, with red and blue dots denoting upregulated and downregulated DEPs, respectively. (C) Number of DEPs between the YDH model group and the control group, as well as between the DI group and the YDH model group. (D-E) Venn diagram of DEPs. A total of 54 common DEPs were upregulated in YDH but downregulated in DI. Two common DEPs were downregulated in YDH but upregulated in DI.\u003c/p\u003e","description":"","filename":"figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/87e2205db002860f35256305.jpg"},{"id":54478338,"identity":"be0727a7-7db0-447b-8563-933876228a0c","added_by":"auto","created_at":"2024-04-11 07:30:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1508914,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional annotation and categories of the 54 DEPs related to sweat secretion. (A) Significantly enriched terms of BP, CC, and MF of proteins regulated by \u003cem\u003eCortex Lycii\u003c/em\u003e (DI) using the Gene Ontology (GO) database. (B) Pathway enrichment of the proteins regulated by DI. Red and blue indicate the number of targets and P values of DEPs, respectively.\u003c/p\u003e","description":"","filename":"figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/2a1c3f8eeeadeb2bd8b8b51e.jpg"},{"id":54478339,"identity":"f433b1bb-1087-4008-b364-9e6620e13e0a","added_by":"auto","created_at":"2024-04-11 07:30:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":624174,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blot analysis validated the DEPs identified in proteomics. (A–D) Protein expression of PRKAR1, Raf1, SLC9A3R1, and CaM. The protein levels of PRKAR1, SLC9A3R1, and CaM were standardized based on the respective level of β-actin. N = 3 independent experiments. The protein expression levels of Raf1 were standardized based on the respective level of GAPDH. N = 3 independent experiments. Note : YDH,Yin deficiency group; DI, Cortex Lycii group.\u003c/p\u003e","description":"","filename":"figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/72730aa28863cb2a8171b480.jpg"},{"id":54478340,"identity":"b0498535-c2fb-42c2-abae-37da6c94f1e2","added_by":"auto","created_at":"2024-04-11 07:30:18","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":992824,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCortex Lycii \u003c/em\u003e(DI) attenuated the sweat secretion of YDH rats activated by β2AR agonist (Isoproterenol) (A) The representative sweat spot images of paw pads. YDH rats were pretreated with DI for 14 days, and sweat secretion was induced via local injection of isoprenaline or trichothecene on the paw pads. (B) Sweat spots were examined using Hotan Takagaki reagent, and the percentage of sweat ratio (%) was calculated by Image J (N = 6).\u003c/p\u003e","description":"","filename":"figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/98e63f43f5e63c9451faa712.jpg"},{"id":54478342,"identity":"22d2c30c-381c-495d-b87e-29d377699bb2","added_by":"auto","created_at":"2024-04-11 07:30:18","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":670246,"visible":true,"origin":"","legend":"\u003cp\u003eM3R-related pathway and β2AR-related pathway involved in sweating\u003c/p\u003e","description":"","filename":"figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/7726399c9fc2edf3d7d104a1.jpg"},{"id":67474686,"identity":"285a6a48-2f8a-4dfa-a5fb-b0946b883198","added_by":"auto","created_at":"2024-10-25 12:16:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9258367,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/c74a4e9f-6509-4eae-add4-d410c4dbabec.pdf"},{"id":54478336,"identity":"92c50ba8-615a-46c9-b884-edba6ec7ecb0","added_by":"auto","created_at":"2024-04-11 07:30:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":130853,"visible":true,"origin":"","legend":"","description":"","filename":"Westernblotsupplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4023446/v1/a6edec0098199ac7487bdc54.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cortex Lycii attenuates night sweat in Yin-deficient rat by downregulating β 2 AR/cAMP signaling: A proteomic approach","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eNight sweats are a frequently encountered symptom in patients, and it is characterized by increased sweating during sleep, regardless of the ambient temperature\u003csup\u003e1\u003c/sup\u003e. This condition is a complication of many diseases, and it frequently occurs in\u0026nbsp;menopause\u003csup\u003e2\u003c/sup\u003e, hyperthyroidism\u003csup\u003e3\u003c/sup\u003e, gastroesophageal reflux disease\u003csup\u003e4\u003c/sup\u003e, pulmonary tuberculosis\u003csup\u003e5\u003c/sup\u003e, and some malignant tumors\u003csup\u003e4,6,7\u003c/sup\u003e. Night sweats seriously affect patients\u0026rsquo; sleep and can even lead to insomnia and anxiety, which seriously influence their quality of life. However, modern medicine offers no desirable clinical treatment option.\u003c/p\u003e\n\u003cp\u003eNight sweat, a common occurrence in individual\u0026rsquo;s daily routines,\u0026nbsp;is\u0026nbsp;a characteristic of Yin deficiency in traditional Chinese medicine (TCM). According to TCM theory, pathological and physiological states are often analyzed comprehensively and systematically, and a series of interrelated symptoms occurring at a particular stage of diseases is called syndromes. Although modern medicine recognizes that different diseases have distinct pathological mechanisms, they may be classified as the same syndrome according to the overall concept of TCM. For example, conditions such as type 2 diabetes, hyperthyroidism, tuberculosis, and menopausal syndrome are categorized as Yin deficiency syndrome in TCM according to their common symptoms of hot flashes, night sweats, red tongue, reduced fur, and rapid pulse.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTCM\u0026nbsp;treats Yin deficiency heat (YDH) by focusing on two main approaches: nourishing Yin and suppressing internal heat\u003csup\u003e8\u003c/sup\u003e. \u003cem\u003eCortex Lycii\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eis a popular medicine for treating night sweats induced by YDH, and it is\u0026nbsp;the root bark of \u003cem\u003eLycium Chinense\u0026nbsp;\u003c/em\u003eMill\u003csup\u003e9\u003c/sup\u003e. The first traditional Chinese medical book was published in 221 B.C., in which Shennong Ben Cao Jing described the effect of \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eon suppressing internal heat. Since then, \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003ehas been used\u0026nbsp;in the\u0026nbsp;treatment of\u0026nbsp;Yin deficiency and night sweats\u003csup\u003e10,11\u003c/sup\u003e.\u0026nbsp;According to the literature\u0026nbsp;reviews,\u0026nbsp;\u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eis still widely used in modern TCM for the clinical treatment of night sweats caused by\u0026nbsp;YDH.\u003c/p\u003e\n\u003cp\u003eYDH syndrome, a well-known illness in TCM, has received significant attention in recent decades. Clinical and experimental findings revealed that Yin deficiency syndrome has similar pathological features to systemic endocrine disorders, including abnormal energy metabolism and immune depression. Systems biology provides a possible method for understanding syndromes in Chinese medicine because they share the same holistic view. Proteomics, the study of large-scale protein expression profiles in blood and body fluids,\u0026nbsp;is a widely used method in systems biology; it offers several advantages in studying TCM syndromes and understanding the mechanisms of herbal medicines in TCM\u003csup\u003e7,12\u003c/sup\u003e.\u0026nbsp;Proteomics\u0026nbsp;is currently used to investigate the hypothalamic mechanisms that contribute to lipid and glucose metabolism disorders in Yin deficiency syndrome\u003csup\u003e13,14\u003c/sup\u003e. It is also used\u0026nbsp;to identify the serum protein markers associated with pathological features, such as immune\u0026nbsp;depression, coagulation cascade, and glucose metabolism disorders\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e. However, the precise molecular mechanisms of night sweats in YDH and the therapeutic mechanisms by which\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003ealleviates this symptom are poorly understood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our previous study, we evaluated the Pharmacodynamics of \u003cem\u003eCortex Lycii\u003c/em\u003e on YDH rats by using different doses of \u003cem\u003eCortex Lycii\u003c/em\u003e. The results show that a clinically equivalent doses of \u003cem\u003eCortex Lycii\u003c/em\u003e can significantly lower the body temperature and reduced sweat secretion of YDH rats.\u003c/p\u003e\n\u003cp\u003eIn this study, we utilized TMT-labeled proteomics to investigate the changes in serum protein levels in YDH rats following\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003etreatment. We also applied a series of bioinformatics methods to\u0026nbsp;reveal\u0026nbsp;the mechanism of night sweats in YDH and the therapeutic mechanism of \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eagainst this symptom.The workflow of our study is shown in \u003cstrong\u003eFig.1\u003c/strong\u003e.\u003c/p\u003e"},{"header":"2. METHODS AND MATERIALS","content":"\u003cp\u003e\u003cstrong\u003e2.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eReagents\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanimals,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and assay kits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific pathogen-free (SPF) male Sprague\u0026ndash;Dawley (SD) rats weighing\u0026nbsp;300\u0026plusmn;20 g\u0026nbsp;were provided by the Animal Center of Fujian University of Traditional Chinese Medicine\u0026nbsp;(FJTCM)\u0026nbsp;[Animal license number: SCXK (Zhe) 2019\u0026ndash;0002]. They were housed in an SPF laboratory with an ambient temperature of 25 \u0026deg;C and a 12 h light\u0026ndash;dark cycle, and the animals had free access to standard rat diet and water. All experimental protocols\u0026nbsp;were\u0026nbsp;approved by the\u0026nbsp;FJTCM\u0026nbsp;Laboratory Animal Welfare Ethics Committee. The\u0026nbsp;rats\u0026nbsp;were handled in strict compliance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003ewas purchased from the Third Affiliated Hospital of\u0026nbsp;FJTCM\u0026nbsp;and authenticated by Professor Che Su-rong from\u0026nbsp;FJTCM. Voucher specimens were\u0026nbsp;stored\u0026nbsp;in the Key Laboratory of\u0026nbsp;TCM\u0026nbsp;in Fujian Higher Education Institutions.\u0026nbsp;In this study, the following primary antibodies\u0026nbsp;were used: anti-AQP\u003csub\u003e5\u003c/sub\u003e (ab78486, Abcam), anti-M\u003csub\u003e3\u003c/sub\u003eR (ab87199,\u0026nbsp;Abcam), anti-\u0026beta;\u003csub\u003e2\u003c/sub\u003eAR (ab182136,\u0026nbsp;Abcam), anti-PRKAR1A+PRKAR1B (ab139695,\u0026nbsp;Abcam), anti-calmodulin 1/2/3 (ab45689,\u0026nbsp;Abcam), anti-NHERF-1 (ab3452,\u0026nbsp;Abcam), anti-Raf1 (ab173539,\u0026nbsp;Abcam), and anti-MEK1+MEK2 (ab178876,\u0026nbsp;Abcam).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePreparation of\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;Cortex Lycii\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;extract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eextracts\u0026nbsp;were prepared through the following steps: crude\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003epowder was soaked in 75% ethanol (1:10, w/v) for 1 h, extracted at reflux for 1 h, and filtered. The residue was extracted\u0026nbsp;following\u0026nbsp;the same method\u0026nbsp;twice, and the filtrate of the three extracts was collected and concentrated by rotary evaporation.\u0026nbsp;Subsequently,\u0026nbsp;0.5% CMC-Na solution\u0026nbsp;was used to dissolve the extract,\u0026nbsp;and the\u0026nbsp;final concentration\u0026nbsp;was adjusted to\u0026nbsp;6 g/mL (clinically equivalent dose). The extract was\u0026nbsp;stored at 4\u0026nbsp;\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3\u003c/strong\u003e \u003cstrong\u003eUPLC/MS analysis for the quality control of \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eextract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCortex Lycii\u003c/em\u003e extract quality was monitored using an ultrahigh-performance liquid chromatography (UPLC) system (Vanquish, Thermo Fisher Scientific). The analytical conditions were as follows: Waters ACQUITY UPLC H-Class instrument and Waters UPLC BEH C18 column (1.7 m, 2.1\u0026times;100 mm). The mobile phase comprised 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multistep linear elution gradient program was as follows: 0\u0026ndash;3.5 min, 95%\u0026ndash;85% A; 3.5\u0026ndash;6 min, 85%\u0026ndash;70% A; 6\u0026ndash;6.5 min, 70%\u0026ndash;70% A; 6.5\u0026ndash;12 min, 70%\u0026ndash;30% A; 12.5\u0026ndash;18 min, 30%\u0026ndash;0% A; 25\u0026ndash;26 min, 0%\u0026ndash;95% A; 26\u0026ndash;30 min, 95%\u0026ndash;95% A; and 26\u0026ndash;30 min, 95%\u0026ndash;95% A. The column temperature was set at 40 \u0026deg;C, the flow rate was set at 0.4 mL/min, and the sample injection volume was set at 5 L. \u003cem\u003eCortex Lycii\u003c/em\u003e extract was redissolved in 80% methanol\u003csup\u003e18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe MS and MS/MS data were obtained using an Orbitrap Exploris 120 mass spectrometer and Xcalibur software in the IDA acquisition mode. The mass range was 100\u0026ndash;1500 during each acquisition cycle. From each cycle, the top four were selected and screened, resulting in the retrieval of corresponding MS/MS data. The other conditions were as follows: sheath gas flow rate: 30 Arb; auxiliary gas flow rate: 10 Arb; ion transfer tube temperature: 350 \u0026deg;C; vaporizer temperature: 350 \u0026deg;C, full MS resolution: 60,000; MS/MS resolution: 15,000. In the NCE mode, the collision energy was 16/32/48 at 5.5 kV (positive) or \u0026minus;4 kV (negative) spray voltage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eExperiments\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in animals\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 1 week of accommodation, 36 SD rats were randomly divided into these three groups: control group (saline group, N=12), model group (YDH group, N=12), and \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003egroup (DI group, N=12).\u0026nbsp;The rats from the\u0026nbsp;model and DI groups were administered with thyroxine (92 mg/mL/day) and reserpine (0.61 mg/mL/day) via gavage once a day for\u0026nbsp;1 week\u003csup\u003e8\u003c/sup\u003e. Those in the control group were given an equal volume of sterile saline solution (1 mL/100 g) by gavage.\u003c/p\u003e\n\u003cp\u003eOn day 8, the\u0026nbsp;rats from the\u0026nbsp;DI group were treated with\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003e(6 g/mg) for 14 days. Equal amounts of saline were given to the model and control groups. Body weight, body temperature, and sweat spots were measured once a day throughout the experiment. The blood sample was collected by the abdominal aortic method 1 h after the last medication, and animals were then subject to euthanasia. The blood sample was centrifuged at 3000\u0026times;g for 10 min to separate the serum. The serum was aliquoted and stored at \u0026minus;80 \u0026deg;C for subsequent ELISA and proteomic experiments. The paw pads were collected from each group of rats, and the protein expression in these paw pads was determined by Western blot.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Measurement of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esweat spots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSweat coloring experiments were performed on each group of rats by using Hotan Takagaki reagent A solution (2%\u0026nbsp;iodine dissolved in absolute ethanol) and B solution (soluble starch mixed with castor oil). After the sweat spots reacted with the reagent and turned blue-black, photographs were obtained. The proportion of the blue-black\u0026nbsp;part\u0026nbsp;of\u0026nbsp;the foot pad was calculated with software image J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003cstrong\u003e6\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum\u0026nbsp;samples were collected\u0026nbsp;as mentioned above. Serum levels of cAMP and cGMP were measured using a commercially available ELISA kit (Commercially available) following the manufacturer\u0026rsquo;s instructions\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003cstrong\u003e7\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eTMT-LC-MS/MS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe serum sample from the animal experiment was used in proteomics analysis. Pierce\u003csup\u003eTM\u003c/sup\u003e Top 14 Abundant Protein Depletion Spin Columns (ThermoFisher Scientific) were used to remove the top 14 high-abundance proteins. Finally, the protein concentration was measured according to the manufacturer\u0026rsquo;s instructions using a BCA kit. The protein solution was reduced for 30 min at 56 \u0026deg;C with 5 mM dithiothreitol and alkylated for 15 min at room temperature in darkness with 11 mM iodoacetamide. Thereafter, the protein sample was diluted with 100 mM TEAB in less than 2 M urea. Trypsin was added at a trypsin-to-protein mass ratio of 1:50 for the first overnight digestion and a trypsin-to-protein mass ratio of 1:100 for the second digestion for 4 h. Finally, the C18 SPE column desalted the peptides\u003csup\u003e20\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSubsequently, 0.5 M TEAB was used to dissolve the tryptic peptides. Each peptide channel was labeled with its own TMT reagent (according to the manufacturer\u0026rsquo;s procedure, ThermoFisher Scientific) and incubated at room temperature for 2 h. About 5\u0026nbsp;L of each sample was pooled, desalted, and analyzed by MS to assess the labeling efficiency. The pooled samples were quenched with 5% hydroxylamine, desalted using a Strata X C18 SPE column (Phenomenex), and vacuum centrifuged for drying. The peptides were exposed to sodium/iodide symporter sources. Tandem mass spectrometry (MS/MS) was carried out with the use of Q Exactive\u003csup\u003eTM\u003c/sup\u003e Plus (Thermos) equipment that was linked online to a UPLC system\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe electrospray voltage was set at 2.0 kV, the entire MS scan resolution was set to 60,000, and the scan range was 350\u0026ndash;1600 m/z. The top 20 most abundant precursors were selected for further MS/MS analyses under a 30 s dynamic exclusion period. Normalized collision energy (NCE) of 28% was used for HCD fragmentation. The Orbitrap detected the fragments with a resolution of 30,000. The initial fixed mass was fixed at 100 m/z. The AGC target was set to 1E5, with an intensity threshold of 3.3E4 and a maximum injection time of 60 ms. The generated MS/MS data were searched against the human SwissProt database (20422 items) concatenated with the reverse decoy database using the MaxQuant search engine (v.1.6.15.0). Trypsin/P was identified as a cleavage enzyme with the capability to catalyze up to two missed cleavages. The initial search utilized a mass tolerance of 20 ppm for precursor ions, whereas the main search used a mass tolerance of 5 ppm. By contrast, the mass tolerance of 0.02 Da was used for fragment ions. Carbamidomethylation of Cys was designated as a fixed modification, whereas acetylation of the protein\u0026rsquo;s N-terminus and oxidation of Met were designated as variable modifications. FDR decreased to 1%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Bioinformatics\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003enalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular functions, cellular components, and biological processes of the identified proteins were analyzed using the GO database (http://geneontology). Protein signaling pathways were annotated\u0026nbsp;by\u0026nbsp;using the KEGG pathway database (http://www.genome.jp/kegg/ map-per.ht). Protein interaction networks were analyzed by STRING software (http://string-db.org/). Functional annotation allowed for the inference of the biological functions associated with the output protein in the context of sweating\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Western blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein was extracted from rats\u0026rsquo; foot pads and subjected to sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) and Western blot analyses. In brief, an appropriate amount of protein was added to each lane of 10% SDS-PAGE gel. The membranes were blocked for 15 min\u0026nbsp;by\u0026nbsp;using QuickBlock\u0026trade;\u0026nbsp;Blocking Buffer for Western blot. The blots were then incubated overnight at 4 \u0026deg;C with the following primary antibodies:\u0026nbsp;GAPDH,\u0026nbsp;\u0026beta;-actin, M\u003csub\u003e3\u003c/sub\u003eR, \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR, AQP\u003csub\u003e5\u003c/sub\u003e, PKA, CaM, raf\u003csub\u003e1\u003c/sub\u003e, and NHE-RF1. At room temperature,\u0026nbsp;the membranes were incubated with HRP-conjugated secondary antibody for 1 h. The indicator proteins on membranes were observed using the BeyoECL Star chemiluminescence kit (Beyotime Biotechnology, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10\u003c/strong\u003e \u003cstrong\u003eEffects of \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eon isoproterenol- or pilocarpine-induced sweat secretion in YDH rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsoproterenol (\u0026beta;\u003csub\u003e2\u003c/sub\u003e-receptor agonist) or pilocarpine (M\u003csub\u003e3\u003c/sub\u003e-receptor agonist) was used to induce sweats in YDH rats to examine the effects of \u003cem\u003eCortex Lycii\u003c/em\u003e.\u0026nbsp;A total of 36 YDH\u0026nbsp;rats were randomized into six groups\u0026nbsp;(each group comprised six rats): control (Group A), model (Group B), model\u0026nbsp;+ pilocarpine\u0026nbsp;(Group C), model\u0026nbsp;+ pilocarpine\u0026nbsp;+DI (Group D), model + isoprenaline (Group E), and model + isoprenaline + DI (Group F).\u0026nbsp;The rats in groups\u0026nbsp;B\u0026ndash;F\u0026nbsp;were administered with thyroxine (92 mg/mL/day) and reserpine (0.61 mg/mL/day) via gavage once a day for 7 days, whereas the control group was given an equal volume of sterile saline solution (1 mL/100 g) by gavage.\u003c/p\u003e\n\u003cp\u003eOn day 8, the rats in groups D and F were treated with \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003e(6 g/mg) for 14 days. Equal amounts of saline were given to the model and control groups.\u0026nbsp;On day\u0026nbsp;20,\u0026nbsp;the rats in groups C and D were\u0026nbsp;administered with\u0026nbsp;pilocarpine (0.035 g/kg)\u0026nbsp;via subcutaneous injection. Meanwhile, the\u0026nbsp;rats in groups E and F were\u0026nbsp;administered with\u0026nbsp;isoprenaline (0.255 mg/kg)\u0026nbsp;via subcutaneous injection. Sweat spots were measured daily throughout the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were presented as the mean \u0026plusmn; standard deviation (SD). The three groups were analyzed by ANOVA and post-hoc Tukey test in SPSS 21.0 software (SPSS, Armonk, NY, USA). Graphs were generated with origin 2019b software.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1 UPLC-MS/MS analysis of\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e\u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eextracts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQualitative analysis was conducted on the chemical components of \u003cem\u003eCortex Lycii\u003c/em\u003e by UPLC-MS/MS, and the total ion flow diagrams of negative and positive ion modes were obtained. The results are shown in \u003cstrong\u003eFig. 2\u003c/strong\u003e. Given the information of fragment ions in MS and reported literature, a total of 20 characteristic compounds were identified and labeled from \u003cem\u003eCortex Lycii.\u003c/em\u003e Information on these compounds is provided in \u003cstrong\u003eTables 1 and 2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Characteristic compounds identified and labeled from \u003cem\u003eCortex Lycii\u003c/em\u003e in the negative ion mode\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"792\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e\u003cstrong\u003et\u003csub\u003eR\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003emin\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormula\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e\u003cstrong\u003em/z\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIon mode\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS fragment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003eMethyl hexadecanoate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e11.9854\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e315.2534\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M+HCOO]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e315.25357,297.246019,313.241226,113.097391,316.256824\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003eCaffeic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e2.1406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e179.0349\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e135.045466,179.035719,134.037798,59.014108,107.050197\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003eFA 18:2+1O\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e14.0061\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e295.2278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e59.013963,295.226337,277.214661,171.103228,195.138771\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003e9-HODE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e17.0497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e295.2277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e295.226597,277.215156,171.103544,195.138428,59.014004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003e9-HOTrE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e12.1908\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e293.2123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e275.203876,293.209425,171.103411,121.102168,183.13874\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003e9-Hydroxy-10,12,15-octadecatrienoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e12.6889\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e293.2123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e293.215469,275.20352,171.103141,121.10197,183.138649\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003eCinnamic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e5.0228\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e147.0449\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e147.044513,59.013855,75.008341,87.008677,129.020466\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"4.292929292929293%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.22222222222222%\"\u003e\n \u003cp\u003eCitric acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.207070707070708%\"\u003e\n \u003cp\u003e0.8170\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.944444444444445%\"\u003e\n \u003cp\u003eC\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.1010101010101%\"\u003e\n \u003cp\u003e191.0194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.227272727272727%\"\u003e\n \u003cp\u003e[M-H]\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.005050505050505%\"\u003e\n \u003cp\u003e111.009041,113.013595,87.008725,193.022596,85.029638\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Characteristic compounds identified and labeled from \u003cem\u003eCortex Lycii\u003c/em\u003e in the positive ion mode.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"794\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e\u003cstrong\u003et\u003csub\u003eR\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003emin\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFormula\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e\u003cstrong\u003em/z\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e\u003cstrong\u003eIon mode\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMS fragment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eApigenin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e8.2545\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e271.0608\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e271.059817,272.064126,153.017964,211.075674,119.048474\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eBETAINE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e26.7755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e140.0682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+Na] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e140.068983,141.113364,74.096389,112.087003,85.076399\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eCinnamyl alcohol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e10.9963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e135.0806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e135.080771,136.083288,91.054483,107.084969,79.053978\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eKukoamine B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e3.9129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e42\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e531.3189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e222.112786,532.323417,223.117422,294.189458,123.04396\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eScopoletin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e3.2805\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e193.0498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e193.050097,133.028093,178.026077,137.060352,165.053629\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eMoupinamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e7.2157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eNO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e314.1392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e177.05483,314.136449,121.064657,145.027994,93.069921\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003ePaeonol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e8.8080\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e167.0706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e167.069711,43.017894,149.059696,121.06481,125.058985\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eCoumaroyl tyramine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e6.9857\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e284.128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e147.044578,284.129045,121.0648,119.048613,93.069955\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eN-cis-Caffeoyltyramine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e6.3647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eNO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e300.123\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e163.038218,300.121594,121.064597,145.027891,135.045038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003e1,2-Dehydro-alpha-cyperone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e10.9963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e217.1589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e217.159194,161.096842,189.162798,175.111477,133.101639\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eBenzenepropanamide, N-[2-(acetyloxy)-1-(phenylmethyl)ethyl]-alpha-(benzoylamino)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e11.1657\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e445.2122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e105.033494,194.117093,224.108788,117.070165,134.097014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"3.5220125786163523%\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.42138364779874%\"\u003e\n \u003cp\u003eLyciumin A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.80503144654088%\"\u003e\n \u003cp\u003e6.8196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.30817610062893%\"\u003e\n \u003cp\u003eC\u003csub\u003e42\u003c/sub\u003eH\u003csub\u003e51\u003c/sub\u003eN9O\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.29559748427673%\"\u003e\n \u003cp\u003e874.3709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9245283018867925%\"\u003e\n \u003cp\u003e[M+H] \u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.723270440251575%\"\u003e\n \u003cp\u003e486.199942,136.07546,468.186945,874.374862,856.351538\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eattenuated Y\u003c/strong\u003e\u003cstrong\u003ein\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;deficiency syndrome and sweat secretion in YDH rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCortex Lycii\u003c/em\u003e has been reported to significantly improve the biological indicators of Yin deficiency model animals\u003csup\u003e22,23\u003c/sup\u003e. In this study, a YDH model was established according to references through the oral administration of thyroxine combined with reserpine\u003csup\u003e24\u003c/sup\u003e. Compared with control rats, YDH rats showed YDH symptoms, including irritability, easy fright, excessive sweats, tachypnea, and dry hair.\u003cu\u003e\u0026nbsp;\u003c/u\u003eAfter\u0026nbsp;the animal experiment\u0026nbsp;was completed, the body weight of rats in the YDH group\u0026nbsp;significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) compared with that of the control group, whereas that of rats in the \u003cem\u003eCortex Lycii\u003c/em\u003e（DI）\u0026nbsp;group increased\u0026nbsp;significantly\u0026nbsp;(\u003cstrong\u003eFig. 3C\u003c/strong\u003e). YHD rats had elevated body temperatures compared with the controls, and \u003cem\u003eCortex Lycii\u003c/em\u003e was effective in reducing body temperatures to levels similar to those of the controls (\u003cstrong\u003eFig. 3D\u003c/strong\u003e). As illustrated in \u003cstrong\u003eFigs. 3E\u0026ndash;3G\u003c/strong\u003e, we observed an increase in the serum cAMP level and cAMP/cGMP ratio in the YDH model group, but the serum cGMP level decreased. Meanwhile, a decrease in serum cAMP level and cAMP/cGMP ratio was observed in DI rats, but minimal changes were recorded in the cGMP level of this group. As illustrated in \u003cstrong\u003eFigs. 3I and 3J\u003c/strong\u003e, in the model group, \u0026beta;\u003csub\u003e2\u003c/sub\u003e receptors were dramatically upregulated and M\u003csub\u003e3\u003c/sub\u003e receptors were downregulated. \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR was substantially downregulated in the DI group, but no statistical difference was observed for M\u003csub\u003e3\u003c/sub\u003e receptors. The above results were consistent with the findings of previous reports\u003csup\u003e13\u003c/sup\u003e, indicating that we successfully established a model of YDH and \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003esuccessfully reversed the symptom of Yin deficiency syndrome.\u003c/p\u003e\n\u003cp\u003eHotan Takagaki reagent solutions are a well-established diagnostic tool to visualize and evaluate sweat spots. The sweating area on the paw pads of rats exhibited a blue-black discoloration. The blue-black pixels were then extracted from the footpad images, adjusted using the Auto Local Threshold function in ImageJ, and measured to the area fraction (sweating area %) as described in the reference\u003csup\u003e25\u003c/sup\u003e. Figs.\u003cstrong\u003e\u0026nbsp;3\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e and\u003cstrong\u003e\u0026nbsp;3B\u003c/strong\u003e shows that the amount of sweat spots in the YDH group rats was significantly higher than that in the control group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), and \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003esignificantly reduced sweat secretion (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u0026nbsp;Moreover, the protein expression of\u0026nbsp;AQP\u003csub\u003e5\u003c/sub\u003e was elevated in the YDH group, but this trend was reversed by \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003etreatment (\u003cstrong\u003eFig. 3H\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Quantitative analysis of differentially expressed proteins (DEPs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mechanism of \u003cem\u003eCortex Lycii\u003c/em\u003e, we used a TMT-based proteomics approach to\u0026nbsp;determine\u0026nbsp;DEPs in\u0026nbsp;the\u0026nbsp;model,\u0026nbsp;DI,\u0026nbsp;and\u003cem\u003e\u0026nbsp;\u003c/em\u003econtrol groups. A total of 1473 proteins were identified; among them, 1219 were further quantified. The DEPs included proteins with expression change multiple \u0026gt; 1.2 and P \u0026lt; 0.05. \u003cstrong\u003eFigs.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4A\u0026ndash;4C\u003c/strong\u003e show 189 DEPS in the YDH/control group (71 downregulated and 118 upregulated) and 124 DEPs in the DI/YDH group (112 downregulated and 12 upregulated). Among them, 54 overlapping differential proteins were upregulated in the YDH/control group but downregulated in the DI/YDH group (\u003cstrong\u003eFigs.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e4D and 4E\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Bioinformatics\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003enalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 54 overlapping differential proteins were selected for further bioinformatics analysis. Utilizing GO annotations including biological processes (BPs), cellular components (CCs), and molecular functions (MFs) to obtain functional insights into differential proteins. \u003cstrong\u003eFig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5A\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eshows the most closely associated enrichment.\u0026nbsp;The\u0026nbsp;first\u0026nbsp;three\u0026nbsp;terms of DEPs for BP were inositol biosynthesis process, cortical cytoskeleton organization, and actin cytoskeleton organization. The majority of the proteins were involved in protein kinase A catalytic subunit binding,\u0026nbsp;actin binding, protein kinase activity for MF, actin cytoskeleton, cAMP-dependent protein kinase complex, and immunological synapse for cellular components.\u003c/p\u003e\n\u003cp\u003eKEGG\u0026nbsp;enrichment analysis\u0026nbsp;has\u0026nbsp;indicated\u0026nbsp;identified several key pathways based on the DEPs. These pathways included\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eYersinia\u003c/em\u003e infection, regulation of actin cytoskeleton, parathyroid hormone synthesis and action, longevity regulating pathway-multiple species, insulin signaling pathway, Ras signaling pathway, inositol phosphate metabolism, and gastric acid secretion (\u003cstrong\u003eFig.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5B\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProteomics analysis showed that multiple differential proteins such as Impa1, Gmpr, Cnn2, Slc9a3r1, Tln1, Eif4b, Rab6a, Pfn1, CFL1, Alb,and Prkar1a were involved in the BPs of sweat secretion regulation such as inositol production, regulation of endoplasmic reticulum, calcium signaling regulation, and Na (+)/H (+) exchange. The expression of the differential proteins was elevated in the model group rats but decreased after \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003etreatment (\u003cstrong\u003eTable 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eOverlapping DEPs involved in the regulation of sweat secretion\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"786\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.193384223918574%\" colspan=\"2\"\u003e\n \u003cp\u003eM/S(up)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.3206106870229%\" colspan=\"2\"\u003e\n \u003cp\u003eDI/M(down)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003eratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003eratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eImpa1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003einositol monophosphatase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.689\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0050\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0047\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eGmpr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eGMP reductase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.313\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eCnn2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003ecalponin 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.682\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0090\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.610\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0081\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eSlc9a3r1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003esodium/hydrogen exchange regulatory cofactor NHE-RF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.637\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0070\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eTln 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eTln 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.508\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.623\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0009\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eEif4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eEukaryotic translation initiation factor 4B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0334\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003ePfn1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eProfilin-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.363\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.748\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0044\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eCFL1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eCofilin-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.423\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0054\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eAlb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eSerum albumin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.601\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e2.724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0449\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003eRab6a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eRas-related protein Rab-6A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.398\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0017\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.699\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.414758269720101%\"\u003e\n \u003cp\u003ePrkar1a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"54.07124681933842%\"\u003e\n \u003cp\u003eprotein kinase cAMP-dependent type I regulatory subunit alpha\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.9058524173028%\"\u003e\n \u003cp\u003e1.536\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.287531806615776%\"\u003e\n \u003cp\u003e0.0296\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.5960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.16030534351145%\"\u003e\n \u003cp\u003e0.0389\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Validation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDEPs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;by Western blot\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot experiments were performed to validate the results of\u0026nbsp;proteomics\u0026nbsp;analysis. Prkar1a, Raf1, Slc9a3r1,\u0026nbsp;and CaM\u0026nbsp;were selected for this comparison.\u0026nbsp;The results indicated that the ratios of the chosen protein were\u0026nbsp;upregulated\u0026nbsp;in the model group\u0026nbsp;rats\u0026nbsp;but\u0026nbsp;downregulated\u0026nbsp;after\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003etreatment\u0026nbsp;(\u003cstrong\u003eFig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e6\u003c/strong\u003e). These results were consistent with the proteomics data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eattenuates sweat\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;secretion induced by isoproterenol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of proteomics and bioinformatics analyses indicated that the Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathway was activated in YDH rats, which may be due to the activation of M\u003csub\u003e3\u003c/sub\u003eR and \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR on sweat glands. Given that both the literature and our study showed a significant reduction in M\u003csub\u003e3\u003c/sub\u003eR receptor expression, we hypothesized that the activation of Ca\u003csup\u003e2+\u003c/sup\u003e signaling might be caused by \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR excitation. To further confirm this hypothesis[Ed12191] , we conducted another experiment to explore the effect of \u003cem\u003eCortex Lycii\u003c/em\u003e on sweating after administering isoprenaline (\u0026beta;\u003csub\u003e2\u003c/sub\u003eAR agonist) or pilocarpine (M\u003csub\u003e3\u003c/sub\u003eR agonist) in YDH rats. As illustrated in \u003cstrong\u003eFigs. 7A and 7B\u003c/strong\u003e, local\u0026nbsp;injection of isoprenaline and atropine significantly stimulated sweat secretion in model rats.\u0026nbsp;In the DI group, the\u0026nbsp;agonistic effect of isoprenaline on sweat secretion\u0026nbsp;was considerably attenuated, but no statistical difference was found in the agonistic effect of atropine. The above results suggested that\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003eattenuated sweat secretion in YDH rats mainly via \u0026beta;2 receptors, not M3 receptors.\u003c/p\u003e\n\u003cdiv id=\"_com_1\" language=\"JavaScript\"\u003e\u003cbr\u003e\u003c/div\u003e"},{"header":"4 DISCUSSION","content":"\u003cp\u003eYDH is a common condition in TCM. Oral administration of thyroxine and reserpine\u003csup\u003e\u0026nbsp;\u003c/sup\u003eis a widely accepted method to establish the Yin deficiency model\u003csup\u003e24,26\u003c/sup\u003e. This model closely simulates the human YDH syndrome, making it highly appropriate for investigating the pathophysiology of YDH syndromes and the effects of herbal medicine to nourish Yin and suppress heat. In the clinical patient and animal models of YDH syndrome, the levels of\u0026nbsp;\u0026beta;-receptors/cAMP increased and M-receptors/cGMP decreased significantly. Thus, the\u0026nbsp;cAMP/cGMP\u0026nbsp;ratio\u0026nbsp;and the expression of\u0026nbsp;\u0026beta;-receptors\u0026nbsp;could be used to evaluate the models and therapeutic effect of medicines\u003csup\u003e27\u0026ndash;29\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;our\u0026nbsp;study, the rats of the YDH group were more irritable and aggressive than those in the control group, and their body weights decreased while their average temperature increased significantly.\u0026nbsp;The number of sweat spots on the\u0026nbsp;paw\u0026nbsp;pad of the model rats increased, and the\u0026nbsp;cAMP/cGMP\u0026nbsp;ratio\u0026nbsp;also increased in the serum of the model rats. These results were close to the clinical symptoms of Yin deficiency syndromes\u003csup\u003e30\u0026ndash;33\u003c/sup\u003e, and they were reversed by administering \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003efor a 2-week treatment period. These results showed that a YDH model was successfully induced.\u0026nbsp;Our experiments also demonstrated that the increased sweat secretion in YDH rats was effectively alleviated by\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003etreatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSweat secretion is primarily regulated by M\u003csub\u003e3\u003c/sub\u003eR\u0026nbsp;and \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR, as indicated by the elucidated molecular mechanism\u003csup\u003e34\u003c/sup\u003e. M\u003csub\u003e3\u003c/sub\u003eR\u0026nbsp;is stimulated by cholinergics and produces inositol 1,4,5-trisphosphatase (IP\u003csub\u003e3\u003c/sub\u003e) in the cytosol,\u0026nbsp;which\u0026nbsp;can trigger\u0026nbsp;Ca\u003csup\u003e2+\u003c/sup\u003e release from the endoplasmic reticulum (ER)\u0026nbsp;so that\u0026nbsp;the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration\u0026nbsp;increases. The increase in the intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentration\u0026nbsp;can\u0026nbsp;promote H\u003csub\u003e2\u003c/sub\u003eO efflux, ion efflux, and ion influx\u003csup\u003e35\u0026ndash;37\u003c/sup\u003e. \u0026beta;\u003csub\u003e2\u003c/sub\u003e-Adrenoceptors, which are coupled to Gs proteins, are activated by catecholamines, ADR, and NA, thereby increasing cAMP production. cAMP,\u0026nbsp;as\u0026nbsp;one of\u0026nbsp;the second messengers,\u0026nbsp;can\u0026nbsp;activate protein kinase A (PKA),\u0026nbsp;which is\u0026nbsp;cAMP-dependent,\u0026nbsp;resulting in the phosphorylation and opening of the L-type VDCC, which promotes Ca\u003csup\u003e2+\u003c/sup\u003e influx and regulates sweat secretion\u003csup\u003e34,38,39\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImpa1 is critical for the second messenger Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathways because it\u0026nbsp;can\u0026nbsp;provide\u0026nbsp;inositol, an essential precursor of membrane phospholipid phosphatidylinositol (PI) and its derivative inositol IP\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e40\u0026ndash;42\u003c/sup\u003e. IP\u003csub\u003e3\u003c/sub\u003e mobilizes the ER to release Ca\u003csup\u003e2+\u003c/sup\u003e, increase intracellular calcium concentration, and regulate sweat secretion.\u0026nbsp;Rab6a, which is situated within the Golgi apparatus, regulates membrane transport from the Golgi to the ER and plays a role in the localization and fusion of transport carriers\u003csup\u003e43\u003c/sup\u003e.\u0026nbsp;Cnn2 is a filament-associated protein that binds calmodulin, participates in a variety of intracellular signaling pathways, and plays a key role in the Ca\u003csup\u003e2+\u003c/sup\u003e-dependent signaling pathway; it is a type of dynamic Ca\u003csup\u003e2+\u003c/sup\u003e sensor that responds to a wide range of\u0026nbsp;[Ca\u003csup\u003e2+\u003c/sup\u003e]\u0026nbsp;and transmits signals downstream. The expression levels of proteins Impa1, Rab6a, and Cnn2 were upregulated in the model group, indicating their underlying mechanism by activating the Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathway in YDH, which was reversed by\u003cem\u003e\u0026nbsp;Cortex Lycii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003ePrkar1a, the regulatory subunit of the cAMP-dependent protein kinases, is activated following cAMP binding to the regulatory subunits.\u0026nbsp;Gmpr and Prkar1a are involved in the cAMP/PKA signaling pathway\u003csup\u003e44\u0026ndash;46\u003c/sup\u003e.\u0026nbsp;They were upregulated in the YDH group and significantly reduced after\u003cem\u003e\u0026nbsp;Cortex Lycii\u003c/em\u003e treatment, suggesting that\u003cem\u003e\u0026nbsp;\u003c/em\u003ecAMP/PKA signaling participated in YDH\u0026nbsp;sweat secretion,\u0026nbsp;which was also\u0026nbsp;reversed by\u003cem\u003e\u0026nbsp;Cortex Lycii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eExpressed in the parietal membrane of exocrine sweat glands, aquaporin-5 (AQP\u003csub\u003e5\u003c/sub\u003e) maintains their secretory function\u003csup\u003e47,48\u003c/sup\u003e.\u0026nbsp;The localization of AQP\u003csub\u003e5\u003c/sub\u003e in the plasma membrane is crucial for its role of water permeability, independent of gating regulation\u003csup\u003e49,50\u003c/sup\u003e. The trafficking of AQP\u003csub\u003e5\u003c/sub\u003e is mediated by ezrin through its interaction with the cytoskeleton, and ezrin is a cross-linker between the plasma membrane and the actin cytoskeletal network\u003csup\u003e51,52\u003c/sup\u003e.\u0026nbsp;Slc9a3r1\u0026nbsp;can\u0026nbsp;connect plasma membrane proteins to members of the ezrin/moesin/radixin family as a scaffold protein, thereby linking them to the actin cytoskeleton\u003csup\u003e53,54\u003c/sup\u003e. As demonstrated by the proteomic results, Slc9a3r1 expression was upregulated in rats of the model group but downregulated after \u003cem\u003eCortex Lycii\u003c/em\u003e intervention.\u0026nbsp;This finding suggested that the therapeutic effect of \u003cem\u003eCortex Lycii\u003c/em\u003e on sweat in YDH rats was linked to the regulation of plasma membrane localization of AQP5 by ezrin, which was consistent with the subsequent protein validation results.\u003c/p\u003e\n\u003cp\u003eTaken together, the findings of GO and KEGG analyses implicate an important role in\u0026nbsp;the overactivation of Ca\u003csup\u003e2+\u003c/sup\u003e signaling and cAMP/PKA signaling in sweat secretion of YDH rats.\u0026nbsp;Given that M\u003csub\u003e3\u003c/sub\u003eR expression was downregulated in the YDH model, we hypothesized that\u0026nbsp;activation of Ca\u003csup\u003e2+\u003c/sup\u003e in YDH rats was the downstream effect of \u0026beta;-receptor activation but not the M\u003csub\u003e3\u003c/sub\u003eR mechanism. To confirm this hypothesis,\u0026nbsp;we further verified the effects of \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eon \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR receptor agonist (isoproterenol)- and M\u003csub\u003e3\u003c/sub\u003eR receptor agonist (pilocarpine)-induced sweat secretion in YDH rats.\u0026nbsp;The results showed that the effect of \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eon YDH was related to the \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR mechanism.\u0026nbsp;We hypothesized that\u003cem\u003e\u0026nbsp;Cortex Lycii\u0026nbsp;\u003c/em\u003eregulated YDH sweating by downregulating \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR/cAMP and transmitting to the downstream PKA/ezrin/CFTR and IP\u003csub\u003e3\u003c/sub\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e/AQP\u003csub\u003e5\u0026nbsp;\u003c/sub\u003epathway\u0026nbsp;\u003cstrong\u003e(Fig.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e8\u003c/strong\u003e).\u003c/p\u003e"},{"header":"5 CONCLUSIONS","content":"\u003cp\u003eThis study elucidated the effect of \u003cem\u003eCortex Lycii\u0026nbsp;\u003c/em\u003eon sweat secretion in Yin-deficient rats. The underlying mechanism may be associated with the inhibition of \u0026beta;\u003csub\u003e2\u003c/sub\u003eAR/cAMP, followed by the modulation of the downstream PKA/ezrin/CFTR signaling pathway, resulting in a decrease in ion exchange and IP\u003csub\u003e3\u003c/sub\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e/AQP\u003csub\u003e5\u003c/sub\u003e to reduce sweat production. This work offers novel perspectives on the underlying mechanism of night sweats in TCM and the therapeutic efficacy of\u003cem\u003e\u0026nbsp;Cortex Lycii\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by State Administration of Traditional Chinese Medicine Clinical Pharmacy High-level Key Discipline of Traditional Chinese Medicine Construction Project (Chinese Medicine Education Letter [2022] No. 226), Provincial Natural Science Foundation of Fujian（No.\u0026nbsp;2023J01857）, and the Basic Scientific Research Enhancement Program of Fujian University of Traditional Chinese Medicine (No. XJC2022013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeixia Huang:\u003c/strong\u003e Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing-review \u0026amp; editing. \u003cstrong\u003eZhiyuan Hong:\u003c/strong\u003e Investigation, Methodology, Data curation, Visualization, Writing-original draft. \u003cstrong\u003eJie Wang:\u003c/strong\u003e Investigation, Methodology, Data curation. \u003cstrong\u003eXiangxiang Liu:\u003c/strong\u003e Investigation, Methodology, Visualization. \u0026nbsp;\u003cstrong\u003eYingzheng Wang:\u003c/strong\u003e Investigation, Methodology, Writing-review \u0026amp; editing. \u003cstrong\u003eYinghao Wang:\u003c/strong\u003e Conceptualization, Formal analysis, Funding acquisition, Writing-review \u0026amp; editing. All authors contributed to manuscript revision, read, and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this research was conducted in the absence of any commercialor financial relationships that could be construed as a potential conflict of\u0026nbsp;interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data presented in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Biorender for helping with the graphical abstract design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were performed in accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by\u0026nbsp;the Animal Center of Fujian University of Traditional Chinese Medicine\u0026nbsp;\u0026nbsp;(Approve License: FJTCM PRE IACUC 2023103).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eCollege of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, 350122, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNational Cancer Institute. in \u003cem\u003ePDQ Cancer Information Summaries\u003c/em\u003e (Bethesda (MD), 2002).\u003c/li\u003e\n\u003cli\u003eLee, E. \u003cem\u003eet al.\u003c/em\u003e Vasomotor symptoms of menopause, autonomic dysfunction, and cardiovascular disease. \u003cem\u003eAm. J. Physiol. Heart Circ. Physiol.\u003c/em\u003e \u003cstrong\u003e323\u003c/strong\u003e, H1270\u0026ndash;H1280 (2022).\u003c/li\u003e\n\u003cli\u003eJayapaul, M., Williams, M. R., Davies, D. P. \u0026amp; Large, D. M. Recurrent painful unilateral gynaecomastia-interactions between hyperthyroidism and hypogonadism. \u003cem\u003eAndrologia\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 31\u0026ndash;33 (2006).\u003c/li\u003e\n\u003cli\u003eBerdnikov, A., McPhee, S., LaBine, L. \u0026amp; Fatoye, T. Night sweats as a manifestation of gastroesophageal reflux disease. \u003cem\u003eCan. Fam. Physician Med. Fam. Can.\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 901\u0026ndash;903 (2020).\u003c/li\u003e\n\u003cli\u003eMoyo, S. \u003cem\u003eet al.\u003c/em\u003e Prevalence of bacteriologically confirmed pulmonary tuberculosis in South Africa, 2017-19: a multistage, cluster-based, cross-sectional survey. \u003cem\u003eLancet Infect. Dis.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1172\u0026ndash;1180 (2022).\u003c/li\u003e\n\u003cli\u003eCarr, C., Vertelney, H., Fronk, J. \u0026amp; Trieu, S. Dronabinol for the Treatment of Paraneoplastic Night Sweats in Cancer Patients: A Report of Five Cases. \u003cem\u003eJ. Palliat. Med.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 1221\u0026ndash;1223 (2019).\u003c/li\u003e\n\u003cli\u003eJiang, T. \u0026amp; Li, J. Review on the systems biology research of Yin-deficiency-heat syndrome in traditional Chinese medicine. \u003cem\u003eAnat. Rec. Hoboken NJ 2007\u003c/em\u003e \u003cstrong\u003e306\u003c/strong\u003e, 2939\u0026ndash;2944 (2023).\u003c/li\u003e\n\u003cli\u003eLiu, C. \u003cem\u003eet al.\u003c/em\u003e A review of the botany, ethnopharmacology, phytochemistry, pharmacology, toxicology and quality of Anemarrhena asphodeloides Bunge. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e302\u003c/strong\u003e, 115857 (2023).\u003c/li\u003e\n\u003cli\u003eGuo, Z. \u003cem\u003eet al.\u003c/em\u003e Study on the mechanism of Cortex Lycii on lung cancer based on network pharmacology combined with experimental validation. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e293\u003c/strong\u003e, 115280 (2022).\u003c/li\u003e\n\u003cli\u003eChen, J. \u003cem\u003eet al.\u003c/em\u003e Research progress on chemical constituents and pharmacological studies on root bark of Lycium barbarum. \u003cem\u003eZhongguo Zhong Yao Za Zhi Zhongguo Zhongyao Zazhi China J. Chin. Mater. Medica\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 3066\u0026ndash;3075 (2021).\u003c/li\u003e\n\u003cli\u003eGao, D. \u003cem\u003eet al.\u003c/em\u003e Hypoglycemic effects and mechanisms of action of Cortex Lycii Radicis on alloxan-induced diabetic mice. \u003cem\u003eYakugaku Zasshi\u003c/em\u003e \u003cstrong\u003e127\u003c/strong\u003e, 1715\u0026ndash;1721 (2007).\u003c/li\u003e\n\u003cli\u003eLian, F. \u003cem\u003eet al.\u003c/em\u003e Effects of Liuwei Dihuang Granule on the outcomes of in vitro fertilization pre-embryo transfer in infertility women with Kidney-yin deficiency syndrome and the proteome expressions in the follicular fluid. \u003cem\u003eChin. J. Integr. Med.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 503\u0026ndash;509 (2014).\u003c/li\u003e\n\u003cli\u003eGuan, W., Liu, Y., Li, X., Yang, B. \u0026amp; Kuang, H. iTRAQ-Based Proteomics to Reveal the Mechanism of Hypothalamus in Kidney- Yin Deficiency Rats Induced by Levothyroxine. \u003cem\u003eEvid.-Based Complement. Altern. Med. ECAM\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, 3703596 (2019).\u003c/li\u003e\n\u003cli\u003eLiu, C. \u003cem\u003eet al.\u003c/em\u003e Serum Protein KNG1, APOC3, and PON1 as Potential Biomarkers for Yin- Deficiency-Heat Syndrome. \u003cem\u003eEvid.-Based Complement. Altern. Med. ECAM\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 5176731 (2016).\u003c/li\u003e\n\u003cli\u003eChen, J. \u003cem\u003eet al.\u003c/em\u003e A group of serum proteins as potential diagnostic biomarkers for Yin- deficiency-heat syndrome. \u003cem\u003eAnat. Rec. Hoboken NJ 2007\u003c/em\u003e \u003cstrong\u003e303\u003c/strong\u003e, 2086\u0026ndash;2094 (2020).\u003c/li\u003e\n\u003cli\u003eLiu, C.-M. \u003cem\u003eet al.\u003c/em\u003e The Chinese herbal formula Zhibai Dihuang Granule treat Yin-deficiency- heat syndrome rats by regulating the immune responses. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e225\u003c/strong\u003e, 271\u0026ndash;278 (2018).\u003c/li\u003e\n\u003cli\u003eYi, W.-J. \u003cem\u003eet al.\u003c/em\u003e Screening of potential biomarkers for Yin-deficiency-heat syndrome based on UHPLC-MS method and the mechanism of Zhibai Dihuang granule therapeutic effect. \u003cem\u003eAnat. Rec. Hoboken NJ 2007\u003c/em\u003e \u003cstrong\u003e303\u003c/strong\u003e, 2095\u0026ndash;2108 (2020).\u003c/li\u003e\n\u003cli\u003eChen, C. \u003cem\u003eet al.\u003c/em\u003e Qinzhizhudan formula dampens inflammation in microglia polarization of vascular dementia rats by blocking MyD88/NF-\u0026kappa;B signaling pathway: Through integrating network pharmacology and experimental validation. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e318\u003c/strong\u003e, 116769 (2024).\u003c/li\u003e\n\u003cli\u003eRan, X., Dou, D., Chen, H. \u0026amp; Ren, G. The correlations of adverse effect and tonifying effect of ginseng medicines. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e291\u003c/strong\u003e, 115113 (2022).\u003c/li\u003e\n\u003cli\u003eLi, L. \u003cem\u003eet al.\u003c/em\u003e SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 12235 (2016).\u003c/li\u003e\n\u003cli\u003eZhao, D. \u003cem\u003eet al.\u003c/em\u003e Lysine-5 acetylation negatively regulates lactate dehydrogenase A and is decreased in pancreatic cancer. \u003cem\u003eCancer Cell\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 464\u0026ndash;476 (2013).\u003c/li\u003e\n\u003cli\u003eZhang, H. \u003cem\u003eet al.\u003c/em\u003e Research on biological expression characteristics of mice models of deficiency heat syndrome and asthenia cold syndrome. \u003cem\u003eChina J. Tradit. Chin. Med. Pharm.\u003c/em\u003e 1516-1520[in Chinese] (2020).\u003c/li\u003e\n\u003cli\u003eLuo, J. \u003cem\u003eet al.\u003c/em\u003e Construction and Verification of Post-infectious Cough Model with Lung Heat and Yin Deficiency Syndrome in Juvenile Rats. \u003cem\u003eJ. Tradit. Chin. Med.\u003c/em\u003e 2366\u0026ndash;2374 (2022) doi:10.13288/j.11-2166/r.2022.24.012[in Chinese].\u003c/li\u003e\n\u003cli\u003eWang, P. \u003cem\u003eet al.\u003c/em\u003e Thyroxine and reserpine-induced changes in metabolic profiles of rat urine and the therapeutic effect of Liu Wei Di Huang Wan detected by UPLC-HDMS. \u003cem\u003eJ. Pharm. Biomed. Anal.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 631\u0026ndash;645 (2010).\u003c/li\u003e\n\u003cli\u003eCui, C., Noh, J. H., Michel, M., Gorospe, M. \u0026amp; Schlessinger, D. STIM1, but not STIM2, Is the Calcium Sensor Critical for Sweat Secretion. \u003cem\u003eJ. Invest. Dermatol.\u003c/em\u003e \u003cstrong\u003e138\u003c/strong\u003e, 704\u0026ndash;707 (2018).\u003c/li\u003e\n\u003cli\u003eZhai, Y. \u003cem\u003eet al.\u003c/em\u003e Broad range metabolomics coupled with network analysis for explaining possible mechanisms of Er-Zhi-Wan in treating liver-kidney Yin deficiency syndrome of Traditional Chinese medicine. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e234\u003c/strong\u003e, 57\u0026ndash;66 (2019).\u003c/li\u003e\n\u003cli\u003eWang, X. \u003cem\u003eet al.\u003c/em\u003e UHPLC-MS/MS analysis of cAMP and cGMP in rat plasma as potential biomarkers of Yin-Yang disharmony in traditional Chinese medicine. \u003cem\u003eJ. Pharm. Anal.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 458\u0026ndash;464 (2021).\u003c/li\u003e\n\u003cli\u003eWeissmann, G., Goldstein, I., Hoffstein, S., Chauvet, G. \u0026amp; Robineaux, R. Yin/Yang modulation of lysosomal enzyme release from polymorphonuclear leukocytes by cyclic nucleotides. \u003cem\u003eAnn. N. Y. Acad. Sci.\u003c/em\u003e \u003cstrong\u003e256\u003c/strong\u003e, 222\u0026ndash;232 (1975).\u003c/li\u003e\n\u003cli\u003eZhao, J., Cai, C., Xie, M., Liu, J. \u0026amp; Wang, B. Investigation of the therapy targets of Yi-Qi-Yang-Yin-Hua-Tan-Qu-Yu recipe on type 2 diabetes by serum proteome labeled with iTRAQ. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e224\u003c/strong\u003e, 1\u0026ndash;14 (2018).\u003c/li\u003e\n\u003cli\u003eBryce, C. Persistent Night Sweats: Diagnostic Evaluation. \u003cem\u003eAm. Fam. Physician\u003c/em\u003e \u003cstrong\u003e102\u003c/strong\u003e, 427\u0026ndash;433 (2020).\u003c/li\u003e\n\u003cli\u003eGrenacher-Horn, D. Night sweats. \u003cem\u003eMMW Fortschr. Med.\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 40\u0026ndash;44 (2019).\u003c/li\u003e\n\u003cli\u003eTakahashi, T. A. \u0026amp; Johnson, K. M. Menopause. \u003cem\u003eMed. Clin. North Am.\u003c/em\u003e \u003cstrong\u003e99\u003c/strong\u003e, 521\u0026ndash;534 (2015).\u003c/li\u003e\n\u003cli\u003eKronenberg, F. Hot flashes: epidemiology and physiology. \u003cem\u003eAnn. N. Y. Acad. Sci.\u003c/em\u003e \u003cstrong\u003e592\u003c/strong\u003e, 52\u0026ndash;86; discussion 123-133 (1990).\u003c/li\u003e\n\u003cli\u003eHu, Y., Converse, C., Lyons, M. C. \u0026amp; Hsu, W. H. Neural control of sweat secretion: a review. \u003cem\u003eBr. J. Dermatol.\u003c/em\u003e \u003cstrong\u003e178\u003c/strong\u003e, 1246\u0026ndash;1256 (2018).\u003c/li\u003e\n\u003cli\u003eConcepcion, A. R. \u003cem\u003eet al.\u003c/em\u003e Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e, 4303\u0026ndash;4318 (2016).\u003c/li\u003e\n\u003cli\u003ePabbidi, M. R. \u003cem\u003eet al.\u003c/em\u003e Inhibition of cAMP-Dependent PKA Activates beta2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes. \u003cem\u003ePloS One\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e0168505 (2016).\u003c/li\u003e\n\u003cli\u003ePerez-Zoghbi, J. F., Mayora, A., Ruiz, M. C. \u0026amp; Michelangeli, F. Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i. \u003cem\u003eAm. J. Physiol. Gastrointest. Liver Physiol.\u003c/em\u003e \u003cstrong\u003e295\u003c/strong\u003e, G671-681 (2008).\u003c/li\u003e\n\u003cli\u003eAmano, T., Shitara, Y., Fujii, N., Inoue, Y. \u0026amp; Kondo, N. Evidence for beta-adrenergic modulation of sweating during incremental exercise in habitually trained males. \u003cem\u003eJ. Appl. Physiol. Bethesda Md 1985\u003c/em\u003e \u003cstrong\u003e123\u003c/strong\u003e, 182\u0026ndash;189 (2017).\u003c/li\u003e\n\u003cli\u003eLindsay, S. L., Holmes, S., Corbett, A. D., Harker, M. \u0026amp; Bovell, D. L. Innervation and receptor profiles of the human apocrine (epitrichial) sweat gland: routes for intervention in bromhidrosis. \u003cem\u003eBr. J. Dermatol.\u003c/em\u003e \u003cstrong\u003e159\u003c/strong\u003e, 653\u0026ndash;660 (2008).\u003c/li\u003e\n\u003cli\u003eKawamura, K. \u0026amp; Ferris, J. P. Kinetic and mechanistic analysis of dinucleotide and oligonucleotide formation from the 5\u0026rsquo;-phosphorimidazolide of adenosine on Na(+)-montmorillonite. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e116\u003c/strong\u003e, 7564\u0026ndash;7572 (1994).\u003c/li\u003e\n\u003cli\u003eLuttrell, B. M. Cellular actions of inositol phosphates and other natural calcium and magnesium chelators. \u003cem\u003eCell. Signal.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 355\u0026ndash;362 (1994).\u003c/li\u003e\n\u003cli\u003eNourbakhsh, A., Collakova, E. \u0026amp; Gillaspy, G. E. Characterization of the inositol monophosphatase gene family in Arabidopsis. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 725 (2014).\u003c/li\u003e\n\u003cli\u003eMa, R. \u003cem\u003eet al.\u003c/em\u003e Involvement of Rab6a in organelle rearrangement and cytoskeletal organization during mouse oocyte maturation. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 23560 (2016).\u003c/li\u003e\n\u003cli\u003eArige, V. \u003cem\u003eet al.\u003c/em\u003e CREB regulates the expression of type 1 inositol 1,4,5-trisphosphate receptors. \u003cem\u003eJ. Cell Sci.\u003c/em\u003e \u003cstrong\u003e134\u003c/strong\u003e, jcs258875 (2021).\u003c/li\u003e\n\u003cli\u003eBusca, R. \u0026amp; Ballotti, R. Cyclic AMP a key messenger in the regulation of skin pigmentation. \u003cem\u003ePigment Cell Res.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 60\u0026ndash;69 (2000).\u003c/li\u003e\n\u003cli\u003eDe Jonge, C. J., Han, H. L., Mack, S. R. \u0026amp; Zaneveld, L. J. Effect of phorbol diesters, synthetic diacylglycerols, and a protein kinase C inhibitor on the human sperm acrosome reaction. \u003cem\u003eJ. Androl.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 62\u0026ndash;70 (1991).\u003c/li\u003e\n\u003cli\u003eAsahina, M., Poudel, A. \u0026amp; Hirano, S. Sweating on the palm and sole: physiological and clinical relevance. \u003cem\u003eClin. Auton. Res. Off. J. Clin. Auton. Res. Soc.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 153\u0026ndash;159 (2015).\u003c/li\u003e\n\u003cli\u003eIizuka, T., Suzuki, T., Nakano, K. \u0026amp; Sueki, H. Immunolocalization of aquaporin-5 in normal human skin and hypohidrotic skin diseases. \u003cem\u003eJ. Dermatol.\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 344\u0026ndash;349 (2012).\u003c/li\u003e\n\u003cli\u003eInoue, R. \u003cem\u003eet al.\u003c/em\u003e Immunolocalization and translocation of aquaporin-5 water channel in sweat glands. \u003cem\u003eJ. Dermatol. Sci.\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 26\u0026ndash;33 (2013).\u003c/li\u003e\n\u003cli\u003eZhang, M. \u003cem\u003eet al.\u003c/em\u003e Localization of Na(+)-K(+)-ATPase alpha/beta, Na(+)-K(+)-2Cl- cotransporter 1 and aquaporin-5 in human eccrine sweat glands. \u003cem\u003eActa Histochem.\u003c/em\u003e \u003cstrong\u003e116\u003c/strong\u003e, 1374\u0026ndash;1381 (2014).\u003c/li\u003e\n\u003cli\u003eBenharouga, M. \u003cem\u003eet al.\u003c/em\u003e The role of the C terminus and Na+/H+ exchanger regulatory factor in the functional expression of cystic fibrosis transmembrane conductance regulator in nonpolarized cells and epithelia. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e278\u003c/strong\u003e, 22079\u0026ndash;22089 (2003).\u003c/li\u003e\n\u003cli\u003eMuroi, S.-I. \u0026amp; Isohama, Y. Ezrin Regulates Ca(2+) Ionophore-Induced Plasma Membrane Translocation of Aquaporin-5. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 13505 (2021).\u003c/li\u003e\n\u003cli\u003eBarati, M. T. \u003cem\u003eet al.\u003c/em\u003e Loss of NHERF-1 expression prevents dopamine-mediated Na-K-ATPase regulation in renal proximal tubule cells from rat models of hypertension: aged F344 rats and spontaneously hypertensive rats. \u003cem\u003eAm. J. Physiol. Cell Physiol.\u003c/em\u003e \u003cstrong\u003e313\u003c/strong\u003e, C197\u0026ndash;C206 (2017).\u003c/li\u003e\n\u003cli\u003eWeinman, E. J. \u003cem\u003eet al.\u003c/em\u003e Sodium-hydrogen exchanger regulatory factor 1 (NHERF-1) transduces signals that mediate dopamine inhibition of sodium-phosphate co-transport in mouse kidney. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e285\u003c/strong\u003e, 13454\u0026ndash;13460 (2010).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Yin deficiency heat, sweat, proteomic, aquaporin-5, β2AR/cAMP","lastPublishedDoi":"10.21203/rs.3.rs-4023446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4023446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose:\u003c/strong\u003e To investigate the therapeutic effect of \u003cem\u003eCortex Lycii \u003c/em\u003eon the sweat secretion of YDH rats and its underlying mechanism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Male SD rats (N=36) were randomly assigned into Control, YDH, and DI groups (N=12 each group). YDH model rats were constructed by oral administration of thyroxin and reserpine. After two-weeks treatment of \u003cem\u003eCortex Lycii\u003c/em\u003e, the pharmacodynamics was assessed then the protein profile was obtained using a tandem mass tag proteomic approach. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis was used for bioinformatics analysis of differential expressed proteins (DEPs), critical DEPs were validated by Western blot. The receptor involved in the \u003cem\u003eCortex Lycii \u003c/em\u003ewere confirmed by using β\u003csub\u003e2\u003c/sub\u003eAR receptor agonist (isoproterenol)- and M\u003csub\u003e3\u003c/sub\u003eR receptor agonist (pilocarpine)-induced sweat secretion in YDH rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: \u003cem\u003eCortex Lycii\u003c/em\u003e attenuates sweat secretion in YDH rats, and down-regulated the expression of AQP\u003csub\u003e5\u003c/sub\u003e and β\u003csub\u003e2\u003c/sub\u003e-receptor on paw pads of YDH rats. Proteomics analysis revealed 189 differential proteins between the model and control groups, of which 124 differential proteins were remarkably reversed by \u003cem\u003eCortex Lycii\u003c/em\u003e. The overlapping DEPs in the model and \u003cem\u003eCortex Lycii \u003c/em\u003egroups were mainly enriched in the β\u003csub\u003e2\u003c/sub\u003eAR/cAMP pathway. Moreover,\u003cem\u003e Cortex Lycii\u003c/em\u003e considerably attenuated the agonistic effect of isoprenaline induced sweat secretion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: This study demonstrated the effect of \u003cem\u003eCortex Lycii \u003c/em\u003eon sweat secretion in Yin-deficient rats. The mechanism on the ground may refer to the inhibition of β\u003csub\u003e2\u003c/sub\u003eAR/cAMP, followed by the suppression of the downstream PKA/ezrin/CFTR signaling pathway to reduce ion exchange and the inhibition of IP\u003csub\u003e3\u003c/sub\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e/AQP\u003csub\u003e5\u003c/sub\u003e. The finding may provide new research on the mechanism of night sweats in TCM and their amelioration by\u003cem\u003e Cortex Lycii\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Cortex Lycii attenuates night sweat in Yin-deficient rat by downregulating β 2 AR/cAMP signaling: A proteomic approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-11 07:30:13","doi":"10.21203/rs.3.rs-4023446/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f1f4ab49-ea84-4777-8a43-1d7c1d7d62b3","owner":[],"postedDate":"April 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30444266,"name":"Biological sciences/Plant sciences"},{"id":30444267,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2024-10-25T12:08:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-11 07:30:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4023446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4023446","identity":"rs-4023446","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}