Jiuzhuan Huangjing Pills alleviate fatigue by preventing energy metabolism dysfunctions in mitochondria.

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Pan Shen, Wei-mei Yu, Bing Deng, Ting Ao, Yu-xuan Tao, Zhe-xin Ni, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3866681/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Aug, 2024 Read the published version in Journal of Functional Foods → Version 1 posted You are reading this latest preprint version Abstract Background Fatigue exerts a profound impact on the efficiency of work and learning, as well as overall health, in a significant portion of the global population. Unfortunately, current anti-fatigue medications have fallen short in delivering satisfactory outcomes, underscoring the imperative for extensive research into the development of therapeutic interventions to effectively manage fatigue and mitigate its associated adverse effects. Purpose The aim of this study was to investigate the efficacy of dietary supplement Jiuzhuan Huangjing Pills (JHP) in improving fatigue induced by exercise and to elucidate its underlying mechanisms. Methods The weight-loaded forced swimming test was employed to establish a fatigue model in mice. C2C12 cells stimulated with H 2 O 2 were employed to establish an in vitro oxidative stress model. Enzyme linked immunosorbent assays (ELISA) were conducted to measure oxidative stress, mitochondrial function, and energy metabolism-related markers in both in vivo and in vitro models. Immunofluorescence assays were performed to assess mitochondrial membrane potential and cell apoptosis. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was used to identify metabolites in tissues and the JHP-derived ingredients, respectively. Network pharmacology analysis and molecular docking were applied to reveal the potential key genes and pathways targeted by the main ingredients. Results JHP significantly increased the swimming time of mice and improved abnormal changes in fatigue indicators caused by intensity exercise. Mechanistically, JHP improved fatigue by protecting against structural damage and functional disorders of mitochondria through the reduction of oxidative stress, thereby preventing cell death and enhancing energy metabolism. Consistent with JHP, the ingredients derived from JHP also displayed similar protective effects against fatigue-induced oxidative stress-mediated mitochondrial damage and cellular apoptosis. Importantly, JHP alleviated oxidative stress mainly by modulating the abundances of organic acids and lipids. The main ingredients of JHP as bioactive components exert their effects by binding to key genes involved in pathways crucial in fatigue. Conclusions Taken together, our findings demonstrated that JHP can serve as a candidate dietary supplement to improve exercise-induced fatigue without causing adverse effects, acting through the modulation at both metabolite and gene levels to ensure cellular survival and energy metabolism, ultimately enhancing overall energy production in the body. Jiuzhuan Huangjing Pills Fatigue Dietary supplement Mitochondria Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Fatigue, a debilitating symptom that imposes limitations on physical and cognitive functions due to the interplay between performance and perceived fatigability, is characterized by an overwhelming sense of exhaustion that detrimentally impacts both physical and mental well-being [ 1 ]. The causes of fatigue can be diverse, including insufficient sleep, coexisting medical conditions, medication side effects, or intense exercise [ 2 , 3 ]. Furthermore, the pathogenesis of fatigue is multifaceted, involving factors such as inflammation, energy exhaustion, oxidative stress response, and dysregulation of nervous system [ 4 , 5 ]. Globally, fatigue serves as a fundamental element in a broad range of illnesses that affect a diverse patient demographic [ 5 , 6 ]. Efficiently managing fatigue is crucial, as it has a significant impact on a person's quality of life and functional abilities. Various medications, such as cerebral cortex stimulants [ 7 ], immunostimulants [ 8 ] and antidepressants [ 9 ] are currently employed to address fatigue by stimulating the brain and eliminating sleepiness for a relatively short period. However, the long-term effectiveness of these medications in managing fatigue is not promising [ 1 ]. Some traditional central stimulus drugs are mostly short-acting or even addictive, and even have underlying side effects [ 1 , 3 ]. Hence, there is a crucial need for extensive research and the development of innovative therapeutic interventions without causing any additional harm to address fatigue effectively and alleviate its resulting adverse effects. Jiuzhuan Huangjing Pills (JHP), consisting of a 1:1 ratio of Polygonati Rhizoma (PR) and Angelicae Sinensis Radix (ASR), has a medicinal history and dietary supplement usage spanning over a thousand years in China. This ancient prescription has been clinically proven to have therapeutic effects in anti-aging, blood nourishment, immune enhancement, and lipid regulation [ 10 ]. Interestingly, our previous studies had shown that JHP effectively alleviated mitochondrial dysfunction mainly through protecting the mitochondrial ultrastructure and promoting fatty acid β-oxidation to reduce oxidative stress damage [ 10 , 11 ]. Mitochondria, as a crucial organelle for supplying adequate levels of ATP for skeletal muscle high oxidative demands[ 12 ]. These effects of JHP result in improved energy production and an enhanced oxidative stress response, which are important factors in addressing fatigue. By targeting these key mechanisms, JHP may offer a promising approach to effectively manage fatigue and improve overall well-being in individuals experiencing this debilitating symptom. However, the extent to which JHP can enhance resistance to fatigue through improving mitochondrial functionality remains uncertain. Here, we identified the effects of JHP on muscle fatigue by mouse swimming experiment, and explored the underlying mechanism by metabolome detection, experimentation and bioinformatics analysis. Our findings in both in vivo and in vitro models demonstrated that the anti-fatigue capability of JHP by regulating metabolites associated with multifaceted metabolic pathways to effectively reduce oxidative stress levels, thereby preserving mitochondrial structures and functions. Network pharmacology and molecular docking further confirmed that main JHP-derived ingredients in skeletal muscle (SM) act as bioactive components to potentially regulate energy metabolism-related genes, thereby impacting the overall energy metabolism level of the body. These findings highlight that JHP serves as a useful nutrient supplement strategy for alleviating exercise-induced muscle fatigue and potentially mitigating fatigue related to various diseases. Material and methods Preparation and quality control of JHP PR and ASR were purchased from Wenshan Shengnong trueborn medicinal materials cultivation cooperation society (Yunnan, China). JZP was extracted by boiling in water at a ratio of 1:10 (w/v) for 1 h at atmospheric pressure according to our previous studies [ 10 , 11 ]. After filtration, the water extracts were concentrated. Afterward, JZP extract was lyophilized at − 70°C using a freeze dryer, resulting in a dried JZP extract with an extraction rate of 56.6%. All lyophilized extract powder was sealed and stored at − 80°C for further experiments. Quantification of ferulic acid and diosgenin in JHP extract was performed using ultra performance liquid chromatography (UPLC) (Agilent, CA, USA) (refer to supplementary material and methods for details). Animals and experimental design Kunming mice weighing 18–22 g were obtained from SPF Biotech (Beijing, China). All experimental animal procedures in this work were conducted in accordance with national guidelines and were approved by the Ethics Committee of the Experimental Animal Center of Yunnan University of Traditional Medicine (Approval No. R-062021109). The mice were randomly divided into 5 different groups, each consisting of 6 mice: (1) normal control group (CON) receiving normal saline, (2) model group (MOD) undergoing swimming exercise and receiving saline, (3) PQR group administered with PQR at a dose of 0.6 g/kg for a duration of 4 weeks, (4) low-dose JHP-treated group (JHP-L) given JHP at a dose of 1.8 g/kg for a duration of 4 weeks, (5) high-dose JHP-treated group (JHP-H) administered JHP at a dose of 3.6 g/kg for a duration of 4 weeks. Weight-loaded forced swimming test The forced swimming test was carried out in a swimming pool measuring 36×36×48 cm, filled with warm water 40 cm deep and maintained at a temperature of 25 ± 1°C. The tail root of each mouse was loaded with lead blocks, equivalent to 5% of their body weight [ 13 ]. Following the administration of the respective drugs, each mouse underwent an exhaustive swimming exercise. In instances where mice were found to be floating during the final minute, they were encouraged to swim by stirring the water with a glass rod. Swimming times were recorded until the mice remained submerged at the bottom of the swimming pool for a continuous duration of 8 seconds. After the swimming test, the mice were taken out of the water and dried with a paper towel. Cell culture, differentiation and treatment The C2C12 cells (mouse myoblast cells) were purchased from Procell (CL-0044, Hubei, China). The cells were maintained in a growth medium consisting of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serums and 1% penicillin-streptomycin for cell culture. The culture medium was refreshed every 48 h, and the cells were maintained in a 25 cm 2 culture flask and incubated at 37°C under a humidified atmosphere of 5% CO 2 . To perform differentiation experiments on C2C12 cells, follow the differentiation protocol described in the Ref. [ 3 ]. Briefly, when the cultures reached approximately 70–85% confluency, 70–85% of the cells were switched to DMEM containing 2% equine serum and 1% penicillin-streptomycin. The differentiation medium was maintained for 12 d until the formation of myotubes, with medium changes performed every 48 h. Subsequently, the differentiation medium was replaced with serum-free culture medium supplemented with different treatments as follows: a control group (CON) and a model group (MOD) received serum-free culture medium, a positive control group (POS) received 0.1 mg/mL of caffeine, a low-dose MM group (MM-L) received 5 µg/mL of MM, and a high-dose MM group (MM-H) received 10 µg/mL of MM. After 18 hours of intervention, all groups were stimulated with 480 µM H 2 O 2 for 6 hours [ 14 ]. Enzyme linked immunosorbent assays (ELISA) The concentrations of fatigue, oxidative stress, and mitochondrial-related indicators was detected using ELISA, including blood urea nitrogen (BUN), creatine kinase (CK), lactic acid (LA), lactate dehydrogenase (LDH), liver glycogen (LG), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), mitochondrial respiratory chain complex I and II (mitochondrial complex I and II), Ca 2+ -Mg 2+ and Na + -K + adenosine triphosphatase (ATPase), ATP, 2-aminoethanethiol dioxygenase (ADO), citrate synthase (CS), and succinate dehydrogenase (SDH). Briefly, to quantify these indicators, the absorbance in each well of the plate was measured using the SpectraMax Plus 384 Microplate Reader (Molecular Devices, CA, USA) after determining the protein concentrations by bicinchoninic acid protein (BCA) assay. Standard curves were generated using the standards provided in the assay kits specific to each indicator, and the concentrations of the respective indicators in each sample were calculated based on the standard curves. Immunofluorescence staining experiment Immunofluorescence staining was used to detect apoptosis by calcein-acetoxymethyl ester/propidium iodide (calcein-AM/PI), reactive oxygen species (ROS) by 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and mitochondrial membrane potential (ΔΨm) by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimi-dazolylcarbocyanine iodide (JC-1). After co-incubation, cells were washed twice with DMEM solution, then loaded with the respective kit corresponding to each indicator for 20–25 min at 37°C in the absence of light. Following that, the cells were washed twice with staining buffer, and the fluorescence was measured using an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany) for respective channels. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) for Identification metabolomics and of JHP-derived ingredients The samples from serum, SM and skeletal muscle mitochondria (SMM) were collected. Metabolomics profiling in serum, SM and SMM were performed using UPLC/MS system coupled with a ACQUITY UPLC® HSS T3 (2.1×150 mm, 1.8 µm) column (Waters, MA, USA). The raw data was processed using MS-DIAL (version 4.48). The filtering, normalization and orthogonal partial least squares-discriminant analysis (OPLS-DA) were performed by R package MetaboAnalystR (version 3.2.0) [ 15 ]. Metabolites with a significance level of P 1 were considered as metabolites with differential abundance. To identification of JHP-derived ingredients in SM, UPLC/MS system coupled with a shim-pack XR-ODS column (2.0 mm × 100 mm, 2.2 µm) (Shimadzu) was used. The MassHunter Qualitative Analysis B.06.00 was utilized to find out the JHP-derived ingredients in SM by comparing the MS data of SM from JHP-treated mice were compared with the MS data of SM from normally cultured mice. Network pharmacology analysis This was performed using the previously described method [ 16 ]. The target genes of JHP-derived ingredients in SM were obtained from traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) [ 17 ], integrative pharmacology-based research platform of traditional Chinese medicine (TCMIP) [ 18 ], and a bioinformatics analysis tool for molecular mechANism of traditional Chinese medicine (BATMAN-TCM) [ 19 ] databases. The target genes annotated in all databases were selected as potential target genes for JHP-derived ingredients in SM. For components not found in these databases, SwissTargetPrediction [ 20 ] and PharmMapper [ 21 ] were utilized for target gene prediction. The potential fatigue-related target genes of JHP-derived ingredients in SM were determined by considering the intersection between the potential target genes and genes in the top10 enriched Kyoto encyclopedia of genes and genomes (KEGG) pathways in SM (Fig. 5 C). The network was showed using Cytoscape (version 3.7.2) [ 22 ]. Molecular docking The 3D structures of 9 components, including13-hydroxyl-9,11-hexadecane dienoic acid (CID: 73194724), 3-butylidene-7-hydroxyphthalide (CID: 5281559), ligustilide (CID: 5319022), linoleic acid (CID: 5280450), linolenic acid (CID: 5280934), octadecenoic acid (CID: 5282750), stearic acid (CID: 5281), senkyunolide G (CID: 10013283) and silvaticol (CID: 10921396), were obtained from PubChem database ( https://pubchem.ncbi.nlm.nih.gov ). The 3D structure of 12 proteins, namely ALDH2 (P05091), AOC3 (Q16853), ATP12A (P54707), CREBBP (Q92793), EPHX1 (P07099), EP300 (Q09472), HMGCR (P04035), MAOA (P21397), MAOB (P27338), MIF (P14174), NOS2 (P35228) and PPARA (Q07869), were downloaded from RCSB protein data bank (RCSB PDB) ( http://www.rcsb.org/ ). Pymol (version 2.5.7) was used to manipulate the protein 3D structures, which involved removing solvent molecules and deleting the original ligands located at the active pocket to expose the active pocket in each protein. Subsequently, AutoDockTools (version 1.5.6) was used to add hydrogens for proteins. To perform docking of components with proteins and calculate the binding affinity for each docking, AutoDock Vina (version 1.5.6) and PLIP [ 23 ] was utilized. Statistical analysis Data were presented as mean ± standard deviation (SD) from the indicated number of independent experiments. Differences between two groups were assessed by the Wilcoxon rank sum test or unpaired Student’s t test. A significance level of P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) was used to indicated statistical significance between the groups. Results and discussion JHP significantly enhances the anti-fatigue capability of mice without exhibiting any significant toxicity To ensure the quality control of JHP, we used UPLC to quantify the content of ferulic acid and diosgenin in JHP (Fig. S1 ). Comparing with the standard reference compounds, we found that the content of ferulic acid in JHP was 1,159 µg/g, which meets the content determination requirement of Chinese Pharmacopoeia 2020 for ASR [ 24 ]. Additionally, the content of diosgenin was measured to be 416 µg/g, consistent with previous reports of PR [ 10 , 11 ]. These results indicate that the quality of JHP used in this study is satisfactory and can be utilized for subsequent experimental research. To determine the potential anti-fatigue effect of JHP, we developed a mouse swimming model and evaluated the efficacy of JHP at low and high doses over a four-week administration period. Moreover, PQR, a well-established anti-fatigue drug [ 25 ], was used as a positive control. Given that the primary objective of this study is to identify a non-toxic anti-fatigue medication, it is of utmost importance to prioritize the confirmation of the clinical safety of JHP. Consistent with our previous findings [ 10 , 11 ], the body weight and food intake of mice in the different dose groups of JHP were unaffected (Figs. 1 A and 1 B), indicating that JHP, as a botanical drug, did not exhibit significant toxicity. Subsequently, we evaluated the anti-fatigue effects of JHP from a pharmacological perspective. All doses of JHP significantly prolonged the swimming time of mice, even showing superior effects compared to PQR (Fig. 1 C). This suggests that JHP has potential anti-fatigue effects. After intense or prolonged exercise, levels of CK and LDH in muscle tissue may increase, which can be an indicator of muscle damage [ 26 ]. Additionally, exercise can lead to the production of LA, while increased energy expenditure in muscles during exercise can potentially lead to protein breakdown, further raising BUN levels. Thus, CK, LDH, LA and BUN are commonly used as biomarkers to assess fatigue induced by exercise [ 27 ]. Examination of these biomarkers in mouse blood demonstrated that all doses of JHP, compared to the MOD group, resulted in decreased expression levels of CK and LDH (Figs. 1 D and 1 E). Moreover, the upregulation of LA and BUN was alleviated in the JHP groups (Figs. 1 F and 1 G), similar to the effects observed in the PQR group. Together, these findings demonstrate that JHP possesses anti-fatigue effects with low toxicity, highlighting its potential as a functional adjunct in the category of botanical anti-fatigue agents. JHP safeguarded mitochondrial functions by reducing oxidative stress to enhance energy metabolism in fatigued mice Muscular contractions in active muscle fibers stimulate the production of ROS, with skeletal muscle serving as a primary source of ROS generation during exercise [ 28 ]. To elucidate the specific mechanism underlying the anti-fatigue effects of JHP, we first examined its regulatory effect on oxidative stress level in vivo . SOD and GSH-Px are recognized as first-line defense antioxidants that play a crucial role in protecting against free radicals and ROS, and the levels of SOD and GSH-Px can reflect ROS levels [ 29 ]. Remarkably, compared to the CON group, the levels of antioxidant enzymes SOD and GSH-Px were reduced in the liver of mice in the MOD group (Figs. 2 A and 2 B), leading to an elevation in the expression levels of lipid oxidation product MDA (Fig. 2 C). Different doses of JHP promoted the expression of SOD and GSH-Px while reducing the expression of MDA, which was consistent with the effects of PQR (Figs. 2 A-C). These results indicate that JHP can ameliorate the elevated oxidative stress levels induced by exercise-induced fatigue. Importantly, we observed a significant decrease in LG content in the MOD group (Fig. 2 D), indicating an impact on energy failure due to muscle fatigue [ 27 ], which was improved by JHP and PQR. Since mitochondria play a crucial role in energy production and oxidative stress [ 30 ], we hypothesized that this phenomenon is associated with the mitochondrial functional homeostasis. By assessing the expression levels of mitochondrial complex I and complex II in SM, we found that JHP and PQR restored the decreased expression levels induced by muscle fatigue (Fig. 2 E and 2 F). Moreover, the expression levels of Ca 2+ -Mg 2+ ATPase and Na + -K + ATPase were significantly decreased in the MOD group, while JHP and PQR significantly increased the expression levels of both ATPases (Figs. 2 G and 2 H). Similar trends were observed with ATP content (Fig. 2 I). Considering the indispensable role of the aforementioned protein complexes in energy production within mitochondria [ 30 ], these findings demonstrate that JHP can restore mitochondrial function by alleviating the elevated oxidative stress levels induced by exercise fatigue, ultimately enhancing energy metabolism and exerting its anti-fatigue effects. JHP alleviated the structural damage of mitochondria and cell death induced by H 2 O 2 in mouse myoblast cells To further validate our findings, we utilized an in vitro model of fatigue-induced damage in mouse myoblast cells (C2C12 cells) induced by H 2 O 2 . Caffeine, known for its ergogenic effect [ 31 ], was used as the positive control (POS) group. Consistent with the POS group, we found that JHP was able to alleviate the elevated levels of ROS caused by oxidative damage (Fig. S2 A) and promote the expression levels of mitochondrial complex I and complex II (Figs. S2B and S2C). The functionality of mitochondria largely depends on the integrity of their structure [ 32 ]. Crucially, the ΔΨm generated by the proton pump is a crucial component of the energy storage process during oxidative phosphorylation, and plays a key role in maintaining structural and functional homeostasis of mitochondria [ 33 ]. The unstable changes of ΔΨm induced by high ROS level can result in unnecessary loss of cellular vitality and contribute to various pathological conditions [ 33 ]. Therefore, we further investigated the using immunofluorescence assays. In the H 2 O 2 group, the significant increase in fluorescence intensity of J-monomers indicated a marked decrease in ΔΨm, suggesting in the disruption of mitochondrial structure and triggering the early events of cellular apoptosis (Figs. 3 A and 3 B). Effectively, JHP and caffeine mitigated the elevation in J-monomers fluorescence intensity, preserving the integrity of the mitochondrial structure (Figs. 3 A and 3 B). Moreover, we observed that the application of H 2 O 2 significantly increased oxidative stress and mitochondrial damage, leading to a considerable increase in cell apoptosis (Figs. 3 C and 3 D). Notably, the treatment with JHP and caffeine alleviated this increase in cell apoptosis (Figs. 3 C and 3 D). These results provide further evidence that JHP maintained energy metabolism levels and protected against cell death by lowering oxidative stress levels, mitigating mitochondrial structural damage, and restoring mitochondrial function. Effects of JHP on metabolites in fatigued mice Given the promising anti-fatigue effects observed in the JHP-L group according to the aforementioned results, we conducted serum metabolomics analysis in the MOD and JHP-L groups for investigation of the metabolic changes induced by JHP in mice. A total of 612 metabolites were identified (Fig. S3A and Table S1 ), and the OPLS-DA results revealed significant differences in overall metabolite abundance between the two groups (Fig. 4 A). Subsequent differential abundance analysis identified 142 metabolites with altered abundance (Fig. 4 B and Table S1 ), including 42 upregulated and 100 downregulated metabolites. Categorization of these differentially abundant metabolites demonstrated that a majority of lipids (24 out of 26) were downregulated, while organic acids (10 out of 13) were upregulated (Fig. 4 C). Organic acids are involved in energy metabolism by mediating the tricarboxylic acid (TCA) cycle, and contribute to providing redox equilibrium by participating in redox reactions and regulating the transcription of oxidase [ 34 ]. Additionally, lipid peroxidation is one of the important responses to oxidative stress, leading to membrane dysfunction in cells [ 35 ]. This indicates that JHP influences the abundance of metabolites closely related to oxidative stress from different class in distinct ways. Moreover, pathway enrichment analysis highlighted several pathways associated with fatigue that were enriched (Fig. 4 D), such as neuroactive light-receptor interaction, and metabolism of lipid, amino acid, and protein. Furthermore, in our in vitro model, we observed an increased expression of ADO in taurine and hypotaurine metabolism, which was consistent with the upregulation of downstream hypotaurine between the MOD and JHP groups (Fig. 4 E). Hypotaurine, a precursor of taurine, exhibits stronger antioxidant activity compared to taurine due to its faster reaction with superoxide radicals and hydroxyl radicals [ 36 ]. Moreover, it has been reported that hypotaurine plays a significant role in antioxidant activity, as it effectively scavenges oxidants released by human neutrophils, inhibits lipid peroxidation, and prevents the inactivation of superoxide dismutase caused by hydrogen peroxide [ 37 ]. These findings indicate that the fatigue-alleviating effect of JHP in mice may be attributed to the upregulation of hypotaurine, which is achieved by promoting the expression of ADO. To further explore the impact of exercise-induced fatigue on SM, we conducted a detailed analysis of the metabolic profiles in SM and SMM in the MOD and JHP groups. A total of 791 and 545 metabolites were identified in SM and SMM, respectively (Fig. S3B and S3C, Table S2 and S3). Consistent with the previous findings (Fig. 4 A), OPLS-DA revealed notable differences in the overall abundance of metabolites in both SM and SMM between the two groups (Figs. S4A and S4B). Differential abundance analysis detected 197 and 59 metabolites with differential abundance in SM and SMM, respectively (Fig. 5 A, Table S2 and S3). In both SM and SMM, the number of upregulated metabolites was higher than the number of downregulated metabolites (Fig. 5 A). In line with the findings from serum metabolites (Fig. 4 C), a significant proportion of lipids (25 out of 30 in SM and 12 out of 12 in SMM) exhibited downregulation, while organic acids (11 out of 16 in SM and 3 out of 3 in SMM) displayed an upregulated trend (Fig. 5 B). Pathway enrichment analysis of the differentially abundant metabolites in SM and SMM demonstrated their involvement in pathways associated with fatigue (Figs. 5 C and 5 D). Specifically, differentially abundant metabolites in SM were mainly participated in pathways related to energy and amino acid metabolism (Fig. 5 C), while those in SMM tended to involved in pathways associated with muscle contraction and metabolism of energy, lipid, amino acid and purine (Fig. 5 D). The TCA cycle serves as both the energy-producing engine in cells, fueling ATP synthesis through oxidative phosphorylation, and a critical regulator in mitigating cellular stress by regulating NADH/NADPH homeostasis and scavenging ROS to control cellular function and fate across various contexts [ 38 ]. Notably, we observed that the enzymes CS and SDH, which are involved in the TCA cycle and corresponding to the upregulated metabolites citrate and fumarate, exhibited increased expression levels after JHP administration compared to the H 2 O 2 group (Fig. 5 E). This finding shows the potential regulatory effects of JHP on the TCA cycle and its downstream metabolites. Collectively, the improvement of fatigue by JHP may occur through two key pathways: the upregulation of organic acids and their derivatives (such as hypotaurine) and the downregulation of lipids and lipid-related metabolites. These pathways worked together to maintain cellular redox homeostasis, thereby preserving mitochondrial structure and functional integrity. This preservation facilitated energy generation through the TCA cycle, as evidenced by the increased abundance of metabolites involved in the TCA cycle, such as citrate and fumarate. Altogether, these mechanisms contributed to the alleviation of fatigue observed following JHP administration. The ingredients derived from JHP in SM demonstrated the ability to protect against oxidative stress-induced mitochondrial homeostasis imbalance and apoptosis To investigate which bioactive ingredients of JHP enter the SM and exert their effects, we identified the ingredients derived from JHP in SM using UPLC-Q-TOF/MS (Fig. S5). By comparing with the ingredient information of PR and ASR previously established [ 39 , 40 ], we identified 4 compounds derived from PR (13-hydroxyl-9,11-hexadecane dienoic acid, disporopsin, linoleic acid and silvaticol) and 16 compounds derived from ASR (3-butylidene-7-hydroxyphthalide, dehydroligustilide-O-sulphate, dihydrophthalide-O-sulphate thiol, E-butylidenephthalide, ferulic acid, linolenic acid, linoleic acid, α-linolenic acid, ligustilide glucoside sulphate 1 (a derivative of ligustilide), phthalic acid, octadecenoic acid, stearic acid, senkyunolide D–O-sulphate, senkyunolide G and trans/cis-ferulica acid-4-sulphate), resulting in a total of 19 non-redundant JHP-derived ingredients in SM (Table 1). Using the H 2 O 2 -treated C2C12 cells model, we evaluated the anti-fatigue ability of the MM composed of main ingredients derived from JHP in SM. The proportions of each ingredient in MM were determined based on the chromatographic peak areas detected by UPLC-Q-TOF/MS (Table S4). We found that MM exhibited similar effects to caffeine in improving H 2 O 2 -induced cell toxicity (Fig. 6 ). Specifically, MM significantly alleviated the elevation of ROS levels induced by H 2 O 2 and restored the reduced levels of mitochondrial complex I and complex II induced by H 2 O 2 (Figs. 6 A-C). Moreover, immunofluorescence assays showed that MM effectively reduced the increased expression of J-monomers caused by H 2 O 2 (Figs. 6 D and 6 E) and mitigated cellular apoptosis induced by H 2 O 2 (Figs. 6 F and 6 G). These results indicated that MM possesses antioxidant abilities similar to JHP and can prevent necrosis by reducing the oxidative stress level to protect the integrity and stability of mitochondria, suggesting that these JHP-derived ingredients play a crucial role in the anti-fatigue effects of JHP. JHP-derived ingredients in SM potentially regulated key genes involved in pathways related to energy production To elucidate the pathways through which the JHP-derived ingredients in SM exert their anti-fatigue effects, we identified 12 potential fatigue-related target genes corresponding to the 9 ingredients according to the top10 KEGG pathways enriched by metabolites with differential abundance in SM after JHP administration (Figs. 5 C and 7 A, Table S5). These potential fatigue-related target genes were found to be involved in metabolism pathways related to metabolism of energy and amino acid (Fig. 7 A), which is consistent with the pathways associated with differentially abundant metabolites in SM (Fig. 5 C). Interestingly, some target genes were found to be associated with the upregulated metabolites observed in SM following JHP administration, including 6 organic acids and derivatives (citrate, fumaric acid, L-argininosuccinate, phenylacetylglycine, spermine and spermidine) and phosphoric acid belongs to homogeneous non-metal compounds (Fig. 7 A). These findings suggest that the ingredients derived from JHP may regulate the upregulation of metabolites, particularly organic acids and derivatives, by binding to and modulating the expression of genes involved in the respective pathways in SM. Furthermore, we employed molecular docking to further validate the results obtained from network pharmacology analysis. It is generally believed that a lower binding affinity between a receptor and a ligand is associated with a more stable receptor-ligand complex, indicating a stronger potential activity of the ligand [ 41 ]. In our results, most of the protein-ligand interactions exhibited low binding energies ( < − 5 kcal/mol), indicating a strong binding potential between these 9 JHP-derived ingredients and their potential target genes (Fig. 7 B). Importantly, 7 out of 9 ingredients derived from JHP, as candidate bioactive components, exhibited strong binding potentials with proteins involved in lipid oxidation, oxidative stress regulation and mitochondrial homeostasis. Specifically, ligustilide had a binding energy of − 7.9 kcal/mol with aldehyde dehydrogenase 2 (ALDH2) (Fig. 7 C), which is a non-cytochrome P450 mitochondrial aldehyde oxidizing enzyme and acts as a protector against oxidative stress by oxidizing toxic aldehydes derived from lipid peroxidation under oxidative stress [ 42 – 44 ]. Silvaticol, 13-hydroxyl-9,11-hexadecane dienoic acid and octadecenoic acid showed binding energies of − 7.6, − 5.8, − 5.8 kcal/mol, respectively, with 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (Figs. 7 D, S6A and S6B), which maintains cellular cholesterol homeostasis by serving as the rate-limiting step in the synthesis of cholesterol and other isoprenoids, and exerts regulatory functions over mitochondria metabolism [ 45 ]. Ligustilide and silvaticol potentially bound to monoamine oxidase A and B (MAOA and MAOB) with binding energies of − 8.4, − 6.9, − 8.1 kcal/mol, respectively (Figs. 7 E, S6C and 7 F). The activity of MAO is commonly utilized as an indicator to evaluate the impact of oxidative stress on mitochondrial functions [ 46 ]. And the downregulation of MAOA, which is involved in fat metabolism, has been associated with a reduction in in vivo fat oxidation [ 47 ]. Peroxisome proliferator activated receptor alpha (PPARA), acting as a crucial regulator of mitochondrial homeostasis by participating in lipid metabolism to modulate oxidative stress levels [ 48 – 50 ], was potentially bound by 3 ingredients including linolenic acid, stearic acid and linoleic acid with binding energies of − 7.9, − 6.8, − 5.5 kcal/mol, respectively (Figs. 7 G, S6D and S6E). Together, these findings suggest that JHP-derived ingredients in SM may potentially enhance overall energy metabolism by modulating key genes involved in lipid oxidation, oxidative stress regulation, and mitochondrial homeostasis, ultimately leading to improvements in fatigue. Conclusion In this study, we revealed that JHP, a traditional Chinese medicine formulation falling under the category of medicine food homology, can improve exercise-induced fatigue in mice without affecting body weight or food intake. Both in vivo and in vitro experiments have demonstrated that JHP exerts its anti-fatigue effects by reducing oxidative stress and preserving mitochondrial structural integrity, thus maintaining mitochondrial homeostasis to protect cell death and enhance energy production. Further metabolomic analysis has shown that JHP achieves its regulatory effects on oxidative stress by upregulating the abundance of organic acids and derivatives while downregulating the abundance of lipids and lipid-like molecules. Additionally, we identified the ingredients derived from JHP in SM and verified their similarity to JHP in terms of their ability to protect against fatigue-induced oxidative stress-mediated mitochondrial damage, subsequently preventing cellular apoptosis. Through network pharmacology analysis and molecular docking, we discovered potential interactions between candidate bioactive components in JHP and key genes involved in fatigue-related pathways, such as ligustilide and ALDH2, silvaticol and HMGCR, ligustilide and MAOA, silvaticol and MAOB, linolenic acid and PPARA. Taken together, our findings suggest that JHP can serve as a therapeutic agent to alleviate exercise-induced fatigue without causing adverse effects by regulating metabolite abundance and gene expression, leading to an enhancement of energy production via the mitigation of the dysfunction caused by increased oxidative stress-induced impairment of mitochondrial structural integrity (Fig. 8 ). Abbreviations JHP Jiuzhuan Huangjing Pills PR Polygonati Rhizoma ASR Angelicae Sinensis Radix PQR Panacis Quinquefolii Radix UPLC-MS ultra performance liquid chromatography-mass spectrometry BUN Blood urea nitrogen CK creatine kinase LA lactic acid LDH lactate dehydrogenase LG liver glycogen GSH-Px glutathione peroxidase SOD superoxide dismutase MDA malondialdehyde ATPase adenosine triphosphatase BCA bicinchoninic acid protein mitochondrial complex I mitochondrial respiratory chain complex I mitochondrial complex II mitochondrial respiratory chain complex II calcein-AM/PI calcein-acetoxymethyl ester/propidium iodide DCFH-DA 2′,7′-dichlorofluorescein diacetate ROS reactive oxygen species JC-1 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimi-dazolylcarbocyanine iodide ΔΨm mitochondrial membrane potential DMEM Dulbecco’s modified Eagle medium CON normal control group MOD model group JHP-L low-dose JHP-treated group JHP-H high-dose JHP-treated group C2C12 cells mouse myoblast cells POS positive control group MM-L low-dose MM group MM-H high-dose MM group SM skeletal muscle SMM skeletal muscle mitochondria OPLS-DA orthogonal partial least squares-discriminant analysis VIP variable importance in projection HMDB human metabolome database KEGG Kyoto encyclopedia of genes and genomes TCMSP traditional Chinese medicine systems pharmacology database and analysis platform TCMIP traditional Chinese medicine BATMAN-TCM bioinformatics analysis tool for molecular mechANism of traditional Chinese medicine SD standard deviation TCA tricarboxylic acid ADO 2-aminoethanethiol dioxygenase ALDH2 aldehyde dehydrogenase 2 CS citrate synthase HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase MAOA and MAOB monoamine oxidase A and B SDH succinate dehydrogenase PPARA peroxisome proliferator activated receptor alpha. Declarations CRediT authorship contribution statement Pan Shen : Investigation, Data curation, Writing – original draft. Wei-mei Yu : Investigation, Methodology, Resources, Writing – original draft. Bing Deng : Investigation, Resources, Writing – original draft. Ting Ao : Investigation, Data curation. Yu-xuan Tao : Data curation. Zhe-xin Ni : Investigation, Resources. Chao-ji Huang-fu : Investigation. Ning-ning Wang : Methodology. Yang-yi Hu : Methodology. De-zhi Sun : Resources. Zhi-jie Bai : Resources. Tian-tian Xia : Resources. Jie Yu: Funding acquisition. Yue Gao : Investigation, Funding acquisition. Xing-xin Yang : Conceptualization, Resources, Writing – review & editing, Funding acquisition. Cheng Wang : Supervision, Resources, Writing – review & editing. Wei Zhou : Supervision, Resources, Funding acquisition, Writing – review & editing. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82060707 and 82104381), the Innovation Team and Talents Cultivation Program of the National Administration of Traditional Chinese Medicine (Grant No. ZYYCXTD-D-202207), the Young Elite Scientists Sponsorship Program by CAST (Grant No. 2021-QNRC1-03), and the application and basis research project of Yunnan China (Grant No. 202205AF150019, 202105AG070012, 202201AW070016 and 202001AZ070001-006). Appendix A. Supplementary material The supplementary material for this paper includes supplementary material and methods, Figs. S1-6, and Table S1-5. Data available All the data supporting the findings of this study are available from the corresponding author upon reasonable request. References Rosenthal TC, Majeroni BA, Pretorius R, Malik K. Fatigue: an overview. Am Fam Physician. 2008;78:1173–9. Caldwell JA, Caldwell JL, Thompson LA, Lieberman HR. 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Supplementary Files Table1.xlsx Supplementarymaterial112.docx Supplementarytables.xlsx Graphicabstract.eps Cite Share Download PDF Status: Published Journal Publication published 01 Aug, 2024 Read the published version in Journal of Functional Foods → 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3866681","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267442850,"identity":"84abc57e-b821-4fe8-beb8-b9823af534fa","order_by":0,"name":"Pan Shen","email":"","orcid":"","institution":"Department of pharmaceutical sciences, Beijing institute of radiation medicine","correspondingAuthor":false,"prefix":"","firstName":"Pan","middleName":"","lastName":"Shen","suffix":""},{"id":267442851,"identity":"9e24754d-3595-4274-aa81-6ffe0a8742bc","order_by":1,"name":"Wei-mei Yu","email":"","orcid":"","institution":"Yunnan University of 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(A and B) \u003c/strong\u003eThe body weight \u003cstrong\u003e(A)\u003c/strong\u003e and food intake \u003cstrong\u003e(B)\u003c/strong\u003e of mice in the CON, MOD, PQR, JHP-L and JHP-H groups showed no significant differences over time.\u003cstrong\u003e (C) \u003c/strong\u003eJHP prolonged the exhaustive swimming time of mice under a weight-loaded condition.\u003cstrong\u003e(D-G) \u003c/strong\u003eJHP significantly alleviated the increased content of CK \u003cstrong\u003e(D)\u003c/strong\u003e, LDH \u003cstrong\u003e(E)\u003c/strong\u003e, LA \u003cstrong\u003e(F)\u003c/strong\u003e, and BUN \u003cstrong\u003e(G)\u003c/strong\u003e caused by exercise-induced fatigue. The significance of differences between each group and the MOD group was indicated above each bar. Ns, \u003cem\u003eP\u003c/em\u003e ≥ 0.05; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/1cd8ad94620481fd38a8913d.png"},{"id":49884631,"identity":"edf44781-74fa-4c95-b6a0-07972461266d","added_by":"auto","created_at":"2024-01-19 17:07:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":363552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJHP improved oxidative stress-mediated mitochondrial dysfunction induced by exercise fatigue.\u003c/strong\u003e \u003cstrong\u003e(A-C)\u003c/strong\u003e JHP enhanced the expression of key enzymes involved in oxidative stress including SOD \u003cstrong\u003e(A)\u003c/strong\u003e and GSH-Px \u003cstrong\u003e(B)\u003c/strong\u003e, resulting in a reduction in MDA levels \u003cstrong\u003e(C)\u003c/strong\u003e in the MOD group. \u003cstrong\u003e(D)\u003c/strong\u003e JHP significantly increased the decreased LG levels induced by exercise fatigue. \u003cstrong\u003e(E-I)\u003c/strong\u003e JHP enhanced mitochondrial complex I \u003cstrong\u003e(E)\u003c/strong\u003e, complex II \u003cstrong\u003e(F)\u003c/strong\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e ATPase \u003cstrong\u003e(G)\u003c/strong\u003e, and Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e ATPase \u003cstrong\u003e(H)\u003c/strong\u003e activities, thereby improving the decreased ATP levels \u003cstrong\u003e(I)\u003c/strong\u003e associated with exercise fatigue. The significance of differences between each group and the MOD group was shown above each bar. Ns, \u003cem\u003eP\u003c/em\u003e ≥ 0.05; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/b4f26a1851b97ccdcaada7c0.png"},{"id":49884177,"identity":"42ea0117-a71c-4154-8683-8729e1adcec7","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1108697,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJHP preserved mitochondrial structural integrity and promotes cell survival. (A and B)\u003c/strong\u003e Immunofluorescence staining results \u003cstrong\u003e(A)\u003c/strong\u003e and relative fluorescence intensity quantification \u003cstrong\u003e(B)\u003c/strong\u003e of J-aggregates and J-monomers in the CON, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, POS, JHP-L, and JHP-H groups. Red fluorescence represented J-aggregates (higher mitochondrial membrane potential), while green fluorescence represented J-monomers (lower mitochondrial membrane potential, indicative of an early event in apoptosis). \u003cstrong\u003e(C and D)\u003c/strong\u003e Immunofluorescence staining results \u003cstrong\u003e(C)\u003c/strong\u003e and relative fluorescence intensity quantification \u003cstrong\u003e(D)\u003c/strong\u003e of calcein-AM and PI in the CON, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, POS, JHP-L and JHP-H groups. Green fluorescence represented calcein-AM (live cells), while red fluorescence represented PI (dead cells). ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/d376f34cfed5e293567005f4.png"},{"id":49884178,"identity":"8b8268bf-d5ce-420b-b321-dd3761e76f5b","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":541946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJHP regulated the abundance of serum metabolites involved in fatigue-related pathways in mice.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e The OPLS-DA showed overall differences in serum metabolite profiles between the MOD and JHP groups. The parameters of the OPLS-DA model were: R\u003csup\u003e2\u003c/sup\u003eY (the degree of variance explained by the model for the dependent variable (Y)) = 0.952, Q\u003csup\u003e2\u003c/sup\u003e (model prediction accuracy) = 0.785. \u003cstrong\u003e(B)\u003c/strong\u003e S-plot displayed the changes in metabolite levels between the JHP and MOD groups. Red and blue dots represented upregulated and downregulated metabolites, respectively. FC, fold change between JHP and MOD. Larger dots indicated a higher difference magnitude.\u003cstrong\u003e (C) \u003c/strong\u003eChord diagram represented the categories of differentially abundant metabolites. The red and blue numbers in parentheses indicated the numbers of upregulated and downregulated metabolites, respectively. \u003cstrong\u003e(D)\u003c/strong\u003e Bubble plot displayed the top 10 KEGG pathways enriched by metabolites with differential abundance. Larger bubbles indicated a higher number of metabolites in that pathway, and darker color indicated a higher enrichment level of that pathway. \u003cstrong\u003e(E)\u003c/strong\u003e Expression levels of the enzyme ADO upstream of upregulated hypotaurine in the CON, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, POS, JHP-L and JHP-H groups. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/688dee9c956ee2094bd6b2e3.png"},{"id":49884181,"identity":"e076ae8e-3359-4cb9-97d8-eb51d42d83de","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":590209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJHP modulates the abundance of metabolites in SM and SMM, regulating energy metabolism. (A)\u003c/strong\u003e Scatter plot showing the fold change of differentially abundant metabolites in SM and SMM between the JHP and MOD groups. Red and blue represented upregulated and downregulated metabolites, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e The Sankey diagram depicted the categories of metabolites with differential abundance in SM and SMM. \u003cstrong\u003e(C and D)\u003c/strong\u003e Top 10 KEGG pathways enriched by metabolites with differential abundance in SM \u003cstrong\u003e(C)\u003c/strong\u003e and SMM \u003cstrong\u003e(D)\u003c/strong\u003e. Larger squares indicated a higher number of metabolites in that pathway, and a redder color indicated a higher enrichment level of that pathway. \u003cstrong\u003e(E)\u003c/strong\u003e Expression changes of two key enzymes, CS and SDH, in the TCA cycle in the CON, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, POS, JHP-L and JHP-H groups. Ns, \u003cem\u003eP\u003c/em\u003e ≥ 0.05; *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/53e3478ae1073f8b83e917a4.png"},{"id":49884630,"identity":"cda1aa25-0a71-4696-bb33-b0bc74a684d4","added_by":"auto","created_at":"2024-01-19 17:07:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":826341,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe main ingredients derived from JHP in SM improved mitochondrial damage-mediated cell apoptosis caused by oxidative stress.\u003c/strong\u003e \u003cstrong\u003e(A-C)\u003c/strong\u003e The main ingredients derived from JHP in SM reduced ROS levels \u003cstrong\u003e(A)\u003c/strong\u003e, increased the expression of mitochondrial complex I \u003cstrong\u003e(B)\u003c/strong\u003e and complex II \u003cstrong\u003e(C)\u003c/strong\u003e in the CON, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, POS, MM-L, and MM-H groups. \u003cstrong\u003e(D and E)\u003c/strong\u003e Immunofluorescence staining results \u003cstrong\u003e(D)\u003c/strong\u003e and relative fluorescence intensity quantification \u003cstrong\u003e(E)\u003c/strong\u003e of J-aggregates and J-monomers in the CON, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, POS, MM-L, and MM-H groups. Red fluorescence represented J-aggregates, while green fluorescence represented J-monomers. \u003cstrong\u003e(F and G)\u003c/strong\u003e Immunofluorescence staining results \u003cstrong\u003e(F)\u003c/strong\u003e and relative fluorescence intensity quantification \u003cstrong\u003e(G)\u003c/strong\u003e of calcein-AM and PI in the CON, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, POS, JHP-L, and JHP-H groups. Green fluorescence represented calcein-AM, while red fluorescence represented PI. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/c3fe6165454e0607b0066111.png"},{"id":49884182,"identity":"e8c5bd55-8283-4cf2-b247-d371505edde0","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1576882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe candidate bioactive compounds derived from JHP in SM potentially interacted with key proteins involved in lipid metabolism, oxidative stress, and mitochondrial homeostasis.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Network pharmacology analysis revealed the association network among ingredients, target genes, KEGG pathways and metabolites. Green, red, yellow, and purple represented different categories. Red and blue arrows indicated upregulated and downregulated metabolites, respectively. \u003cstrong\u003e(B)\u003c/strong\u003eHeatmap displayed the binding energy between each candidate bioactive compound and target protein. Darker color indicates lower binding affinity. The gray color represented that the compound cannot bind to the target protein based on the network pharmacology analysis. \u003cstrong\u003e(C-G)\u003c/strong\u003e Possible structures generated by the molecular docking of ligustilide and ALDH2 \u003cstrong\u003e(C)\u003c/strong\u003e, silvaticol and HMGCR \u003cstrong\u003e(D)\u003c/strong\u003e, ligustilide and MAOA \u003cstrong\u003e(E)\u003c/strong\u003e, silvaticol and MAOB\u003cstrong\u003e (F)\u003c/strong\u003e, linolenic acid and PPARA \u003cstrong\u003e(G)\u003c/strong\u003e. The numbers in parentheses represent the binding energy between the components and the proteins.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/c1cca44818ad47033731db20.png"},{"id":49884632,"identity":"6142ea1d-d890-4cbe-bf35-0c859fb9a010","added_by":"auto","created_at":"2024-01-19 17:07:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":346803,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of the mechanisms underlying the improvement of exercise fatigue by JHP. \u003c/strong\u003eJHP could serve as a dual-purpose therapeutic agent for alleviating exercise-induced fatigue. Mechanistically, JHP primarily exerted its anti-fatigue efficacy by modulating the metabolite abundance and gene expression, resulting in a reduction of fatigue-induced oxidative stress. Building upon this foundation, JHP mitigated mitochondrial structural and functional damage to ensure cell survival, ultimately resulting in enhanced energy production.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/e1e88f7cf11ce573823e75dd.png"},{"id":58340639,"identity":"41414736-7267-40e7-aef1-bdd180e879f5","added_by":"auto","created_at":"2024-06-14 06:42:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6329261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/27518880-bb86-4274-bc2d-70fb6098d45b.pdf"},{"id":49884176,"identity":"548bcbd1-ae5e-4dea-9779-f697d98ab613","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13647,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/4d529b6aff5bf1f4d23cfc3b.xlsx"},{"id":49884183,"identity":"ee238d5b-d653-4418-8c99-8f88c0435f2a","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1167322,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial112.docx","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/baf6035720edca1702e889e9.docx"},{"id":49884186,"identity":"1f298776-5b77-43c9-97bf-9ca0d18d7595","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":467488,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/a43aee5c7c1c99f97655f537.xlsx"},{"id":49884187,"identity":"b5f362f7-1c92-4a13-a6c6-d9abc66c853e","added_by":"auto","created_at":"2024-01-19 16:59:04","extension":"eps","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":5944518,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.eps","url":"https://assets-eu.researchsquare.com/files/rs-3866681/v1/745061bca9721695b9004656.eps"}],"financialInterests":"No competing interests reported.","formattedTitle":"Jiuzhuan Huangjing Pills alleviate fatigue by preventing energy metabolism dysfunctions in mitochondria.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFatigue, a debilitating symptom that imposes limitations on physical and cognitive functions due to the interplay between performance and perceived fatigability, is characterized by an overwhelming sense of exhaustion that detrimentally impacts both physical and mental well-being [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The causes of fatigue can be diverse, including insufficient sleep, coexisting medical conditions, medication side effects, or intense exercise [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Furthermore, the pathogenesis of fatigue is multifaceted, involving factors such as inflammation, energy exhaustion, oxidative stress response, and dysregulation of nervous system [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Globally, fatigue serves as a fundamental element in a broad range of illnesses that affect a diverse patient demographic [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Efficiently managing fatigue is crucial, as it has a significant impact on a person's quality of life and functional abilities. Various medications, such as cerebral cortex stimulants [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], immunostimulants [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and antidepressants [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] are currently employed to address fatigue by stimulating the brain and eliminating sleepiness for a relatively short period. However, the long-term effectiveness of these medications in managing fatigue is not promising [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Some traditional central stimulus drugs are mostly short-acting or even addictive, and even have underlying side effects [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hence, there is a crucial need for extensive research and the development of innovative therapeutic interventions without causing any additional harm to address fatigue effectively and alleviate its resulting adverse effects.\u003c/p\u003e \u003cp\u003eJiuzhuan Huangjing Pills (JHP), consisting of a 1:1 ratio of Polygonati Rhizoma (PR) and Angelicae Sinensis Radix (ASR), has a medicinal history and dietary supplement usage spanning over a thousand years in China. This ancient prescription has been clinically proven to have therapeutic effects in anti-aging, blood nourishment, immune enhancement, and lipid regulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Interestingly, our previous studies had shown that JHP effectively alleviated mitochondrial dysfunction mainly through protecting the mitochondrial ultrastructure and promoting fatty acid β-oxidation to reduce oxidative stress damage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Mitochondria, as a crucial organelle for supplying adequate levels of ATP for skeletal muscle high oxidative demands[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These effects of JHP result in improved energy production and an enhanced oxidative stress response, which are important factors in addressing fatigue. By targeting these key mechanisms, JHP may offer a promising approach to effectively manage fatigue and improve overall well-being in individuals experiencing this debilitating symptom. However, the extent to which JHP can enhance resistance to fatigue through improving mitochondrial functionality remains uncertain.\u003c/p\u003e \u003cp\u003eHere, we identified the effects of JHP on muscle fatigue by mouse swimming experiment, and explored the underlying mechanism by metabolome detection, experimentation and bioinformatics analysis. Our findings in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models demonstrated that the anti-fatigue capability of JHP by regulating metabolites associated with multifaceted metabolic pathways to effectively reduce oxidative stress levels, thereby preserving mitochondrial structures and functions. Network pharmacology and molecular docking further confirmed that main JHP-derived ingredients in skeletal muscle (SM) act as bioactive components to potentially regulate energy metabolism-related genes, thereby impacting the overall energy metabolism level of the body. These findings highlight that JHP serves as a useful nutrient supplement strategy for alleviating exercise-induced muscle fatigue and potentially mitigating fatigue related to various diseases.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003ePreparation and quality control of JHP\u003c/p\u003e \u003cp\u003ePR and ASR were purchased from Wenshan Shengnong trueborn medicinal materials cultivation cooperation society (Yunnan, China). JZP was extracted by boiling in water at a ratio of 1:10 (w/v) for 1 h at atmospheric pressure according to our previous studies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. After filtration, the water extracts were concentrated. Afterward, JZP extract was lyophilized at \u0026minus;\u0026thinsp;70\u0026deg;C using a freeze dryer, resulting in a dried JZP extract with an extraction rate of 56.6%. All lyophilized extract powder was sealed and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further experiments. Quantification of ferulic acid and diosgenin in JHP extract was performed using ultra performance liquid chromatography (UPLC) (Agilent, CA, USA) (refer to supplementary material and methods for details).\u003c/p\u003e \u003cp\u003eAnimals and experimental design\u003c/p\u003e \u003cp\u003eKunming mice weighing 18\u0026ndash;22 g were obtained from SPF Biotech (Beijing, China). All experimental animal procedures in this work were conducted in accordance with national guidelines and were approved by the Ethics Committee of the Experimental Animal Center of Yunnan University of Traditional Medicine (Approval No. R-062021109). The mice were randomly divided into 5 different groups, each consisting of 6 mice: (1) normal control group (CON) receiving normal saline, (2) model group (MOD) undergoing swimming exercise and receiving saline, (3) PQR group administered with PQR at a dose of 0.6 g/kg for a duration of 4 weeks, (4) low-dose JHP-treated group (JHP-L) given JHP at a dose of 1.8 g/kg for a duration of 4 weeks, (5) high-dose JHP-treated group (JHP-H) administered JHP at a dose of 3.6 g/kg for a duration of 4 weeks.\u003c/p\u003e \u003cp\u003eWeight-loaded forced swimming test\u003c/p\u003e \u003cp\u003eThe forced swimming test was carried out in a swimming pool measuring 36\u0026times;36\u0026times;48 cm, filled with warm water 40 cm deep and maintained at a temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. The tail root of each mouse was loaded with lead blocks, equivalent to 5% of their body weight [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Following the administration of the respective drugs, each mouse underwent an exhaustive swimming exercise. In instances where mice were found to be floating during the final minute, they were encouraged to swim by stirring the water with a glass rod. Swimming times were recorded until the mice remained submerged at the bottom of the swimming pool for a continuous duration of 8 seconds. After the swimming test, the mice were taken out of the water and dried with a paper towel.\u003c/p\u003e \u003cp\u003eCell culture, differentiation and treatment\u003c/p\u003e \u003cp\u003eThe C2C12 cells (mouse myoblast cells) were purchased from Procell (CL-0044, Hubei, China). The cells were maintained in a growth medium consisting of Dulbecco\u0026rsquo;s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serums and 1% penicillin-streptomycin for cell culture. The culture medium was refreshed every 48 h, and the cells were maintained in a 25 cm\u003csup\u003e2\u003c/sup\u003e culture flask and incubated at 37\u0026deg;C under a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. To perform differentiation experiments on C2C12 cells, follow the differentiation protocol described in the Ref. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Briefly, when the cultures reached approximately 70\u0026ndash;85% confluency, 70\u0026ndash;85% of the cells were switched to DMEM containing 2% equine serum and 1% penicillin-streptomycin. The differentiation medium was maintained for 12 d until the formation of myotubes, with medium changes performed every 48 h. Subsequently, the differentiation medium was replaced with serum-free culture medium supplemented with different treatments as follows: a control group (CON) and a model group (MOD) received serum-free culture medium, a positive control group (POS) received 0.1 mg/mL of caffeine, a low-dose MM group (MM-L) received 5 \u0026micro;g/mL of MM, and a high-dose MM group (MM-H) received 10 \u0026micro;g/mL of MM. After 18 hours of intervention, all groups were stimulated with 480 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 6 hours [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEnzyme linked immunosorbent assays (ELISA)\u003c/p\u003e \u003cp\u003eThe concentrations of fatigue, oxidative stress, and mitochondrial-related indicators was detected using ELISA, including blood urea nitrogen (BUN), creatine kinase (CK), lactic acid (LA), lactate dehydrogenase (LDH), liver glycogen (LG), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), mitochondrial respiratory chain complex I and II (mitochondrial complex I and II), Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e and Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e adenosine triphosphatase (ATPase), ATP, 2-aminoethanethiol dioxygenase (ADO), citrate synthase (CS), and succinate dehydrogenase (SDH). Briefly, to quantify these indicators, the absorbance in each well of the plate was measured using the SpectraMax Plus 384 Microplate Reader (Molecular Devices, CA, USA) after determining the protein concentrations by bicinchoninic acid protein (BCA) assay. Standard curves were generated using the standards provided in the assay kits specific to each indicator, and the concentrations of the respective indicators in each sample were calculated based on the standard curves.\u003c/p\u003e \u003cp\u003eImmunofluorescence staining experiment\u003c/p\u003e \u003cp\u003eImmunofluorescence staining was used to detect apoptosis by calcein-acetoxymethyl ester/propidium iodide (calcein-AM/PI), reactive oxygen species (ROS) by 2\u0026prime;,7\u0026prime;-dichlorofluorescein diacetate (DCFH-DA), and mitochondrial membrane potential (ΔΨm) by 5,5\u0026prime;,6,6\u0026prime;-tetrachloro-1,1\u0026prime;,3,3\u0026prime;-tetraethyl-benzimi-dazolylcarbocyanine iodide (JC-1). After co-incubation, cells were washed twice with DMEM solution, then loaded with the respective kit corresponding to each indicator for 20\u0026ndash;25 min at 37\u0026deg;C in the absence of light. Following that, the cells were washed twice with staining buffer, and the fluorescence was measured using an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany) for respective channels.\u003c/p\u003e \u003cp\u003eUltra-performance liquid chromatography-mass spectrometry (UPLC-MS) for Identification metabolomics and of JHP-derived ingredients\u003c/p\u003e \u003cp\u003eThe samples from serum, SM and skeletal muscle mitochondria (SMM) were collected. Metabolomics profiling in serum, SM and SMM were performed using UPLC/MS system coupled with a ACQUITY UPLC\u0026reg; HSS T3 (2.1\u0026times;150 mm, 1.8 \u0026micro;m) column (Waters, MA, USA). The raw data was processed using MS-DIAL (version 4.48). The filtering, normalization and orthogonal partial least squares-discriminant analysis (OPLS-DA) were performed by R package MetaboAnalystR (version 3.2.0) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Metabolites with a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and a variable importance in projection (VIP) value\u0026thinsp;\u0026gt;\u0026thinsp;1 were considered as metabolites with differential abundance.\u003c/p\u003e \u003cp\u003eTo identification of JHP-derived ingredients in SM, UPLC/MS system coupled with a shim-pack XR-ODS column (2.0 mm \u0026times; 100 mm, 2.2 \u0026micro;m) (Shimadzu) was used. The MassHunter Qualitative Analysis B.06.00 was utilized to find out the JHP-derived ingredients in SM by comparing the MS data of SM from JHP-treated mice were compared with the MS data of SM from normally cultured mice.\u003c/p\u003e \u003cp\u003eNetwork pharmacology analysis\u003c/p\u003e \u003cp\u003eThis was performed using the previously described method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The target genes of JHP-derived ingredients in SM were obtained from traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], integrative pharmacology-based research platform of traditional Chinese medicine (TCMIP) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and a bioinformatics analysis tool for molecular mechANism of traditional Chinese medicine (BATMAN-TCM) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] databases. The target genes annotated in all databases were selected as potential target genes for JHP-derived ingredients in SM. For components not found in these databases, SwissTargetPrediction [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and PharmMapper [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] were utilized for target gene prediction. The potential fatigue-related target genes of JHP-derived ingredients in SM were determined by considering the intersection between the potential target genes and genes in the top10 enriched Kyoto encyclopedia of genes and genomes (KEGG) pathways in SM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The network was showed using Cytoscape (version 3.7.2) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMolecular docking\u003c/p\u003e \u003cp\u003eThe 3D structures of 9 components, including13-hydroxyl-9,11-hexadecane dienoic acid (CID: 73194724), 3-butylidene-7-hydroxyphthalide (CID: 5281559), ligustilide (CID: 5319022), linoleic acid (CID: 5280450), linolenic acid (CID: 5280934), octadecenoic acid (CID: 5282750), stearic acid (CID: 5281), senkyunolide G (CID: 10013283) and silvaticol (CID: 10921396), were obtained from PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The 3D structure of 12 proteins, namely ALDH2 (P05091), AOC3 (Q16853), ATP12A (P54707), CREBBP (Q92793), EPHX1 (P07099), EP300 (Q09472), HMGCR (P04035), MAOA (P21397), MAOB (P27338), MIF (P14174), NOS2 (P35228) and PPARA (Q07869), were downloaded from RCSB protein data bank (RCSB PDB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Pymol (version 2.5.7) was used to manipulate the protein 3D structures, which involved removing solvent molecules and deleting the original ligands located at the active pocket to expose the active pocket in each protein. Subsequently, AutoDockTools (version 1.5.6) was used to add hydrogens for proteins. To perform docking of components with proteins and calculate the binding affinity for each docking, AutoDock Vina (version 1.5.6) and PLIP [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] was utilized.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from the indicated number of independent experiments. Differences between two groups were assessed by the Wilcoxon rank sum test or unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test. A significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**) and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***) was used to indicated statistical significance between the groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eJHP significantly enhances the anti-fatigue capability of mice without exhibiting any significant toxicity\u003c/p\u003e \u003cp\u003eTo ensure the quality control of JHP, we used UPLC to quantify the content of ferulic acid and diosgenin in JHP (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Comparing with the standard reference compounds, we found that the content of ferulic acid in JHP was 1,159 \u0026micro;g/g, which meets the content determination requirement of Chinese Pharmacopoeia 2020 for ASR [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, the content of diosgenin was measured to be 416 \u0026micro;g/g, consistent with previous reports of PR [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These results indicate that the quality of JHP used in this study is satisfactory and can be utilized for subsequent experimental research.\u003c/p\u003e \u003cp\u003eTo determine the potential anti-fatigue effect of JHP, we developed a mouse swimming model and evaluated the efficacy of JHP at low and high doses over a four-week administration period. Moreover, PQR, a well-established anti-fatigue drug [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], was used as a positive control. Given that the primary objective of this study is to identify a non-toxic anti-fatigue medication, it is of utmost importance to prioritize the confirmation of the clinical safety of JHP. Consistent with our previous findings [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], the body weight and food intake of mice in the different dose groups of JHP were unaffected (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating that JHP, as a botanical drug, did not exhibit significant toxicity. Subsequently, we evaluated the anti-fatigue effects of JHP from a pharmacological perspective. All doses of JHP significantly prolonged the swimming time of mice, even showing superior effects compared to PQR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This suggests that JHP has potential anti-fatigue effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter intense or prolonged exercise, levels of CK and LDH in muscle tissue may increase, which can be an indicator of muscle damage [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, exercise can lead to the production of LA, while increased energy expenditure in muscles during exercise can potentially lead to protein breakdown, further raising BUN levels. Thus, CK, LDH, LA and BUN are commonly used as biomarkers to assess fatigue induced by exercise [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Examination of these biomarkers in mouse blood demonstrated that all doses of JHP, compared to the MOD group, resulted in decreased expression levels of CK and LDH (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, the upregulation of LA and BUN was alleviated in the JHP groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), similar to the effects observed in the PQR group. Together, these findings demonstrate that JHP possesses anti-fatigue effects with low toxicity, highlighting its potential as a functional adjunct in the category of botanical anti-fatigue agents.\u003c/p\u003e \u003cp\u003eJHP safeguarded mitochondrial functions by reducing oxidative stress to enhance energy metabolism in fatigued mice\u003c/p\u003e \u003cp\u003eMuscular contractions in active muscle fibers stimulate the production of ROS, with skeletal muscle serving as a primary source of ROS generation during exercise [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To elucidate the specific mechanism underlying the anti-fatigue effects of JHP, we first examined its regulatory effect on oxidative stress level \u003cem\u003ein vivo\u003c/em\u003e. SOD and GSH-Px are recognized as first-line defense antioxidants that play a crucial role in protecting against free radicals and ROS, and the levels of SOD and GSH-Px can reflect ROS levels [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Remarkably, compared to the CON group, the levels of antioxidant enzymes SOD and GSH-Px were reduced in the liver of mice in the MOD group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), leading to an elevation in the expression levels of lipid oxidation product MDA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Different doses of JHP promoted the expression of SOD and GSH-Px while reducing the expression of MDA, which was consistent with the effects of PQR (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). These results indicate that JHP can ameliorate the elevated oxidative stress levels induced by exercise-induced fatigue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImportantly, we observed a significant decrease in LG content in the MOD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), indicating an impact on energy failure due to muscle fatigue [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which was improved by JHP and PQR. Since mitochondria play a crucial role in energy production and oxidative stress [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], we hypothesized that this phenomenon is associated with the mitochondrial functional homeostasis. By assessing the expression levels of mitochondrial complex I and complex II in SM, we found that JHP and PQR restored the decreased expression levels induced by muscle fatigue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Moreover, the expression levels of Ca\u003csup\u003e2+\u003c/sup\u003e-Mg\u003csup\u003e2+\u003c/sup\u003e ATPase and Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e ATPase were significantly decreased in the MOD group, while JHP and PQR significantly increased the expression levels of both ATPases (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Similar trends were observed with ATP content (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Considering the indispensable role of the aforementioned protein complexes in energy production within mitochondria [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], these findings demonstrate that JHP can restore mitochondrial function by alleviating the elevated oxidative stress levels induced by exercise fatigue, ultimately enhancing energy metabolism and exerting its anti-fatigue effects.\u003c/p\u003e \u003cp\u003eJHP alleviated the structural damage of mitochondria and cell death induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in mouse myoblast cells\u003c/p\u003e \u003cp\u003eTo further validate our findings, we utilized an \u003cem\u003ein vitro\u003c/em\u003e model of fatigue-induced damage in mouse myoblast cells (C2C12 cells) induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Caffeine, known for its ergogenic effect [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], was used as the positive control (POS) group. Consistent with the POS group, we found that JHP was able to alleviate the elevated levels of ROS caused by oxidative damage (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA) and promote the expression levels of mitochondrial complex I and complex II (Figs. S2B and S2C). The functionality of mitochondria largely depends on the integrity of their structure [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Crucially, the ΔΨm generated by the proton pump is a crucial component of the energy storage process during oxidative phosphorylation, and plays a key role in maintaining structural and functional homeostasis of mitochondria [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The unstable changes of ΔΨm induced by high ROS level can result in unnecessary loss of cellular vitality and contribute to various pathological conditions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, we further investigated the using immunofluorescence assays. In the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, the significant increase in fluorescence intensity of J-monomers indicated a marked decrease in ΔΨm, suggesting in the disruption of mitochondrial structure and triggering the early events of cellular apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Effectively, JHP and caffeine mitigated the elevation in J-monomers fluorescence intensity, preserving the integrity of the mitochondrial structure (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, we observed that the application of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e significantly increased oxidative stress and mitochondrial damage, leading to a considerable increase in cell apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Notably, the treatment with JHP and caffeine alleviated this increase in cell apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results provide further evidence that JHP maintained energy metabolism levels and protected against cell death by lowering oxidative stress levels, mitigating mitochondrial structural damage, and restoring mitochondrial function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEffects of JHP on metabolites in fatigued mice\u003c/p\u003e \u003cp\u003e Given the promising anti-fatigue effects observed in the JHP-L group according to the aforementioned results, we conducted serum metabolomics analysis in the MOD and JHP-L groups for investigation of the metabolic changes induced by JHP in mice. A total of 612 metabolites were identified (Fig. S3A and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and the OPLS-DA results revealed significant differences in overall metabolite abundance between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Subsequent differential abundance analysis identified 142 metabolites with altered abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), including 42 upregulated and 100 downregulated metabolites. Categorization of these differentially abundant metabolites demonstrated that a majority of lipids (24 out of 26) were downregulated, while organic acids (10 out of 13) were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Organic acids are involved in energy metabolism by mediating the tricarboxylic acid (TCA) cycle, and contribute to providing redox equilibrium by participating in redox reactions and regulating the transcription of oxidase [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Additionally, lipid peroxidation is one of the important responses to oxidative stress, leading to membrane dysfunction in cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This indicates that JHP influences the abundance of metabolites closely related to oxidative stress from different class in distinct ways. Moreover, pathway enrichment analysis highlighted several pathways associated with fatigue that were enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), such as neuroactive light-receptor interaction, and metabolism of lipid, amino acid, and protein. Furthermore, in our \u003cem\u003ein vitro\u003c/em\u003e model, we observed an increased expression of ADO in taurine and hypotaurine metabolism, which was consistent with the upregulation of downstream hypotaurine between the MOD and JHP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Hypotaurine, a precursor of taurine, exhibits stronger antioxidant activity compared to taurine due to its faster reaction with superoxide radicals and hydroxyl radicals [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, it has been reported that hypotaurine plays a significant role in antioxidant activity, as it effectively scavenges oxidants released by human neutrophils, inhibits lipid peroxidation, and prevents the inactivation of superoxide dismutase caused by hydrogen peroxide [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These findings indicate that the fatigue-alleviating effect of JHP in mice may be attributed to the upregulation of hypotaurine, which is achieved by promoting the expression of ADO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further explore the impact of exercise-induced fatigue on SM, we conducted a detailed analysis of the metabolic profiles in SM and SMM in the MOD and JHP groups. A total of 791 and 545 metabolites were identified in SM and SMM, respectively (Fig. S3B and S3C, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3). Consistent with the previous findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), OPLS-DA revealed notable differences in the overall abundance of metabolites in both SM and SMM between the two groups (Figs. S4A and S4B). Differential abundance analysis detected 197 and 59 metabolites with differential abundance in SM and SMM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e and S3). In both SM and SMM, the number of upregulated metabolites was higher than the number of downregulated metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In line with the findings from serum metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), a significant proportion of lipids (25 out of 30 in SM and 12 out of 12 in SMM) exhibited downregulation, while organic acids (11 out of 16 in SM and 3 out of 3 in SMM) displayed an upregulated trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Pathway enrichment analysis of the differentially abundant metabolites in SM and SMM demonstrated their involvement in pathways associated with fatigue (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Specifically, differentially abundant metabolites in SM were mainly participated in pathways related to energy and amino acid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), while those in SMM tended to involved in pathways associated with muscle contraction and metabolism of energy, lipid, amino acid and purine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The TCA cycle serves as both the energy-producing engine in cells, fueling ATP synthesis through oxidative phosphorylation, and a critical regulator in mitigating cellular stress by regulating NADH/NADPH homeostasis and scavenging ROS to control cellular function and fate across various contexts [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Notably, we observed that the enzymes CS and SDH, which are involved in the TCA cycle and corresponding to the upregulated metabolites citrate and fumarate, exhibited increased expression levels after JHP administration compared to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This finding shows the potential regulatory effects of JHP on the TCA cycle and its downstream metabolites.\u003c/p\u003e \u003cp\u003eCollectively, the improvement of fatigue by JHP may occur through two key pathways: the upregulation of organic acids and their derivatives (such as hypotaurine) and the downregulation of lipids and lipid-related metabolites. These pathways worked together to maintain cellular redox homeostasis, thereby preserving mitochondrial structure and functional integrity. This preservation facilitated energy generation through the TCA cycle, as evidenced by the increased abundance of metabolites involved in the TCA cycle, such as citrate and fumarate. Altogether, these mechanisms contributed to the alleviation of fatigue observed following JHP administration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe ingredients derived from JHP in SM demonstrated the ability to protect against oxidative stress-induced mitochondrial homeostasis imbalance and apoptosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate which bioactive ingredients of JHP enter the SM and exert their effects, we identified the ingredients derived from JHP in SM using UPLC-Q-TOF/MS (Fig. S5). By comparing with the ingredient information of PR and ASR previously established [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], we identified 4 compounds derived from PR (13-hydroxyl-9,11-hexadecane dienoic acid, disporopsin, linoleic acid and silvaticol) and 16 compounds derived from ASR (3-butylidene-7-hydroxyphthalide, dehydroligustilide-O-sulphate, dihydrophthalide-O-sulphate thiol, E-butylidenephthalide, ferulic acid, linolenic acid, linoleic acid, α-linolenic acid, ligustilide glucoside sulphate 1 (a derivative of ligustilide), phthalic acid, octadecenoic acid, stearic acid, senkyunolide D\u0026ndash;O-sulphate, senkyunolide G and trans/cis-ferulica acid-4-sulphate), resulting in a total of 19 non-redundant JHP-derived ingredients in SM (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eUsing the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated C2C12 cells model, we evaluated the anti-fatigue ability of the MM composed of main ingredients derived from JHP in SM. The proportions of each ingredient in MM were determined based on the chromatographic peak areas detected by UPLC-Q-TOF/MS (Table S4). We found that MM exhibited similar effects to caffeine in improving H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cell toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Specifically, MM significantly alleviated the elevation of ROS levels induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and restored the reduced levels of mitochondrial complex I and complex II induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C). Moreover, immunofluorescence assays showed that MM effectively reduced the increased expression of J-monomers caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and mitigated cellular apoptosis induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). These results indicated that MM possesses antioxidant abilities similar to JHP and can prevent necrosis by reducing the oxidative stress level to protect the integrity and stability of mitochondria, suggesting that these JHP-derived ingredients play a crucial role in the anti-fatigue effects of JHP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eJHP-derived ingredients in SM potentially regulated key genes involved in pathways related to energy production\u003c/h2\u003e \u003cp\u003eTo elucidate the pathways through which the JHP-derived ingredients in SM exert their anti-fatigue effects, we identified 12 potential fatigue-related target genes corresponding to the 9 ingredients according to the top10 KEGG pathways enriched by metabolites with differential abundance in SM after JHP administration (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, Table S5). These potential fatigue-related target genes were found to be involved in metabolism pathways related to metabolism of energy and amino acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), which is consistent with the pathways associated with differentially abundant metabolites in SM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Interestingly, some target genes were found to be associated with the upregulated metabolites observed in SM following JHP administration, including 6 organic acids and derivatives (citrate, fumaric acid, L-argininosuccinate, phenylacetylglycine, spermine and spermidine) and phosphoric acid belongs to homogeneous non-metal compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). These findings suggest that the ingredients derived from JHP may regulate the upregulation of metabolites, particularly organic acids and derivatives, by binding to and modulating the expression of genes involved in the respective pathways in SM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we employed molecular docking to further validate the results obtained from network pharmacology analysis. It is generally believed that a lower binding affinity between a receptor and a ligand is associated with a more stable receptor-ligand complex, indicating a stronger potential activity of the ligand [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In our results, most of the protein-ligand interactions exhibited low binding energies (\u0026thinsp;\u0026lt;\u0026thinsp;\u0026minus;\u0026thinsp;5 kcal/mol), indicating a strong binding potential between these 9 JHP-derived ingredients and their potential target genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Importantly, 7 out of 9 ingredients derived from JHP, as candidate bioactive components, exhibited strong binding potentials with proteins involved in lipid oxidation, oxidative stress regulation and mitochondrial homeostasis. Specifically, ligustilide had a binding energy of \u0026minus;\u0026thinsp;7.9 kcal/mol with aldehyde dehydrogenase 2 (ALDH2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), which is a non-cytochrome P450 mitochondrial aldehyde oxidizing enzyme and acts as a protector against oxidative stress by oxidizing toxic aldehydes derived from lipid peroxidation under oxidative stress [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Silvaticol, 13-hydroxyl-9,11-hexadecane dienoic acid and octadecenoic acid showed binding energies of \u0026minus;\u0026thinsp;7.6, \u0026minus;\u0026thinsp;5.8, \u0026minus;\u0026thinsp;5.8 kcal/mol, respectively, with 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, S6A and S6B), which maintains cellular cholesterol homeostasis by serving as the rate-limiting step in the synthesis of cholesterol and other isoprenoids, and exerts regulatory functions over mitochondria metabolism [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Ligustilide and silvaticol potentially bound to monoamine oxidase A and B (MAOA and MAOB) with binding energies of \u0026minus;\u0026thinsp;8.4, \u0026minus;\u0026thinsp;6.9, \u0026minus;\u0026thinsp;8.1 kcal/mol, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, S6C and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). The activity of MAO is commonly utilized as an indicator to evaluate the impact of oxidative stress on mitochondrial functions [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. And the downregulation of MAOA, which is involved in fat metabolism, has been associated with a reduction in \u003cem\u003ein vivo\u003c/em\u003e fat oxidation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Peroxisome proliferator activated receptor alpha (PPARA), acting as a crucial regulator of mitochondrial homeostasis by participating in lipid metabolism to modulate oxidative stress levels [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], was potentially bound by 3 ingredients including linolenic acid, stearic acid and linoleic acid with binding energies of \u0026minus;\u0026thinsp;7.9, \u0026minus;\u0026thinsp;6.8, \u0026minus;\u0026thinsp;5.5 kcal/mol, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, S6D and S6E). Together, these findings suggest that JHP-derived ingredients in SM may potentially enhance overall energy metabolism by modulating key genes involved in lipid oxidation, oxidative stress regulation, and mitochondrial homeostasis, ultimately leading to improvements in fatigue.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we revealed that JHP, a traditional Chinese medicine formulation falling under the category of medicine food homology, can improve exercise-induced fatigue in mice without affecting body weight or food intake. Both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments have demonstrated that JHP exerts its anti-fatigue effects by reducing oxidative stress and preserving mitochondrial structural integrity, thus maintaining mitochondrial homeostasis to protect cell death and enhance energy production. Further metabolomic analysis has shown that JHP achieves its regulatory effects on oxidative stress by upregulating the abundance of organic acids and derivatives while downregulating the abundance of lipids and lipid-like molecules. Additionally, we identified the ingredients derived from JHP in SM and verified their similarity to JHP in terms of their ability to protect against fatigue-induced oxidative stress-mediated mitochondrial damage, subsequently preventing cellular apoptosis. Through network pharmacology analysis and molecular docking, we discovered potential interactions between candidate bioactive components in JHP and key genes involved in fatigue-related pathways, such as ligustilide and ALDH2, silvaticol and HMGCR, ligustilide and MAOA, silvaticol and MAOB, linolenic acid and PPARA. Taken together, our findings suggest that JHP can serve as a therapeutic agent to alleviate exercise-induced fatigue without causing adverse effects by regulating metabolite abundance and gene expression, leading to an enhancement of energy production via the mitigation of the dysfunction caused by increased oxidative stress-induced impairment of mitochondrial structural integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJHP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eJiuzhuan Huangjing Pills\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolygonati Rhizoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eASR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAngelicae Sinensis Radix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePQR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePanacis Quinquefolii Radix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUPLC-MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eultra performance liquid chromatography-mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBUN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecreatine kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elactic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elactate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eliver glycogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSH-Px\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglutathione peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuperoxide dismutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emalondialdehyde\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATPase\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenosine triphosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebicinchoninic acid protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emitochondrial complex I\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial respiratory chain complex I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emitochondrial complex II\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial respiratory chain complex II\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecalcein-AM/PI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecalcein-acetoxymethyl ester/propidium iodide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDCFH-DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2\u0026prime;,7\u0026prime;-dichlorofluorescein diacetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJC-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e5,5\u0026prime;,6,6\u0026prime;-tetrachloro-1,1\u0026prime;,3,3\u0026prime;-tetraethyl-benzimi-dazolylcarbocyanine iodide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eΔΨm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitochondrial membrane potential\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s modified Eagle medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCON\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enormal control group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emodel group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJHP-L\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow-dose JHP-treated group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eJHP-H\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-dose JHP-treated group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eC2C12 cells\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emouse myoblast cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epositive control group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMM-L\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elow-dose MM group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMM-H\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-dose MM group\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eskeletal muscle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eskeletal muscle mitochondria\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOPLS-DA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eorthogonal partial least squares-discriminant analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evariable importance in projection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMDB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman metabolome database\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto encyclopedia of genes and genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCMSP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etraditional Chinese medicine systems pharmacology database and analysis platform\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCMIP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etraditional Chinese medicine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBATMAN-TCM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebioinformatics analysis tool for molecular mechANism of traditional Chinese medicine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etricarboxylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-aminoethanethiol dioxygenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALDH2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealdehyde dehydrogenase 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecitrate synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMGCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3-hydroxy-3-methylglutaryl-CoA reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMAOA and MAOB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonoamine oxidase A and B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esuccinate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePPARA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperoxisome proliferator activated receptor alpha.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eCRediT\u0026nbsp;authorship contribution statement\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePan Shen\u003c/strong\u003e: Investigation, Data curation, Writing \u0026ndash; original draft.\u0026nbsp;\u003cstrong\u003eWei-mei Yu\u003c/strong\u003e: Investigation, Methodology, Resources, Writing \u0026ndash; original draft.\u0026nbsp;\u003cstrong\u003eBing Deng\u003c/strong\u003e: Investigation, Resources, Writing \u0026ndash; original draft.\u0026nbsp;\u003cstrong\u003eTing Ao\u003c/strong\u003e: Investigation, Data curation.\u0026nbsp;\u003cstrong\u003eYu-xuan Tao\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eData curation.\u003cstrong\u003e\u0026nbsp;Zhe-xin Ni\u003c/strong\u003e: Investigation, Resources.\u0026nbsp;\u003cstrong\u003eChao-ji Huang-fu\u003c/strong\u003e: Investigation.\u0026nbsp;\u003cstrong\u003eNing-ning Wang\u003c/strong\u003e: Methodology.\u0026nbsp;\u003cstrong\u003eYang-yi Hu\u003c/strong\u003e: Methodology.\u0026nbsp;\u003cstrong\u003eDe-zhi Sun\u003c/strong\u003e: Resources.\u0026nbsp;\u003cstrong\u003eZhi-jie Bai\u003c/strong\u003e: Resources.\u0026nbsp;\u003cstrong\u003eTian-tian Xia\u003c/strong\u003e: Resources. \u003cstrong\u003eJie Yu:\u003c/strong\u003e Funding acquisition.\u0026nbsp;\u003cstrong\u003eYue Gao\u003c/strong\u003e: Investigation, Funding acquisition.\u0026nbsp;\u003cstrong\u003eXing-xin Yang\u003c/strong\u003e: Conceptualization, Resources, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition.\u0026nbsp;\u003cstrong\u003eCheng Wang\u003c/strong\u003e: Supervision, Resources, Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eWei Zhou\u003c/strong\u003e: Supervision, Resources, Funding acquisition, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eDeclaration\u0026nbsp;of Competing Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (Grant No. 82060707 and 82104381), the Innovation Team and Talents Cultivation Program of the National Administration of Traditional Chinese Medicine (Grant No. ZYYCXTD-D-202207), the Young Elite Scientists Sponsorship Program by CAST (Grant No. 2021-QNRC1-03), and the application and basis research project of Yunnan China (Grant No. 202205AF150019, 202105AG070012, 202201AW070016 and 202001AZ070001-006).\u003c/p\u003e\n\u003cp\u003eAppendix\u0026nbsp;A. Supplementary material\u003c/p\u003e\n\u003cp\u003eThe supplementary material for this paper includes supplementary material and methods, Figs. S1-6, and Table S1-5.\u003c/p\u003e\n\u003cp\u003eData available\u003c/p\u003e\n\u003cp\u003eAll the data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRosenthal TC, Majeroni BA, Pretorius R, Malik K. Fatigue: an overview. Am Fam Physician. 2008;78:1173\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaldwell JA, Caldwell JL, Thompson LA, Lieberman HR. Fatigue and its management in the workplace. Neurosci Biobehav Rev. 2019;96:272\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu H, Xu W, Wang N, Jiang W, Cheng Y, Guo Y, Yao W, Hu B, Du P, Qian H. Anti-fatigue effect of Lepidium meyenii Walp. 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Autoxidation and MAO-mediated metabolism of dopamine as a potential cause of oxidative stress: role of ferrous and ferric ions. Neurochem Int. 2004;45:103\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElgzyri T, Parikh H, Zhou Y, Dekker Nitert M, R\u0026ouml;nn T, Segerstr\u0026ouml;m \u0026Aring;B, Ling C, Franks PW, Wollmer P, Eriksson KF, et al. First-degree relatives of type 2 diabetic patients have reduced expression of genes involved in fatty acid metabolism in skeletal muscle. J Clin Endocrinol Metab. 2012;97:E1332\u0026ndash;1337.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim TS, Jin YB, Kim YS, Kim S, Kim JK, Lee HM, Suh HW, Choe JH, Kim YJ, Koo BS, et al. SIRT3 promotes antimycobacterial defenses by coordinating mitochondrial and autophagic functions. Autophagy. 2019;15:1356\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Z, Roth K, Agarwal M, Liu W, Petriello MC. The transcription factors CREBH, PPARa, and FOXO1 as critical hepatic mediators of diet-induced metabolic dysregulation. J Nutr Biochem. 2021;95:108633.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Zhang W, Cheng N, Zhu Y, Li H, Zhang S, Guo W, Ge G. Pectolinarigenin ameliorates acetaminophen-induced acute liver injury via attenuating oxidative stress and inflammatory response in Nrf2 and PPARa dependent manners. Phytomedicine. 2023;113:154726.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Jiuzhuan Huangjing Pills, Fatigue, Dietary supplement, Mitochondria, Metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-3866681/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3866681/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFatigue exerts a profound impact on the efficiency of work and learning, as well as overall health, in a significant portion of the global population. Unfortunately, current anti-fatigue medications have fallen short in delivering satisfactory outcomes, underscoring the imperative for extensive research into the development of therapeutic interventions to effectively manage fatigue and mitigate its associated adverse effects.\u003c/p\u003e\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eThe aim of this study was to investigate the efficacy of dietary supplement Jiuzhuan Huangjing Pills (JHP) in improving fatigue induced by exercise and to elucidate its underlying mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe weight-loaded forced swimming test was employed to establish a fatigue model in mice. C2C12 cells stimulated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were employed to establish an \u003cem\u003ein vitro\u003c/em\u003e oxidative stress model. Enzyme linked immunosorbent assays (ELISA) were conducted to measure oxidative stress, mitochondrial function, and energy metabolism-related markers in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models. Immunofluorescence assays were performed to assess mitochondrial membrane potential and cell apoptosis. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was used to identify metabolites in tissues and the JHP-derived ingredients, respectively. Network pharmacology analysis and molecular docking were applied to reveal the potential key genes and pathways targeted by the main ingredients.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eJHP significantly increased the swimming time of mice and improved abnormal changes in fatigue indicators caused by intensity exercise. Mechanistically, JHP improved fatigue by protecting against structural damage and functional disorders of mitochondria through the reduction of oxidative stress, thereby preventing cell death and enhancing energy metabolism. Consistent with JHP, the ingredients derived from JHP also displayed similar protective effects against fatigue-induced oxidative stress-mediated mitochondrial damage and cellular apoptosis. Importantly, JHP alleviated oxidative stress mainly by modulating the abundances of organic acids and lipids. The main ingredients of JHP as bioactive components exert their effects by binding to key genes involved in pathways crucial in fatigue.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTaken together, our findings demonstrated that JHP can serve as a candidate dietary supplement to improve exercise-induced fatigue without causing adverse effects, acting through the modulation at both metabolite and gene levels to ensure cellular survival and energy metabolism, ultimately enhancing overall energy production in the body.\u003c/p\u003e","manuscriptTitle":"Jiuzhuan Huangjing Pills alleviate fatigue by preventing energy metabolism dysfunctions in mitochondria.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 16:58:59","doi":"10.21203/rs.3.rs-3866681/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":"7c732f33-f0de-49c4-af93-5f9cf0697260","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-14T06:41:53+00:00","versionOfRecord":{"articleIdentity":"rs-3866681","link":"https://doi.org/10.1016/j.jff.2024.106262","journal":{"identity":"journal-of-functional-foods","isVorOnly":true,"title":"Journal of Functional Foods"},"publishedOn":"2024-08-01 06:41:53","publishedOnDateReadable":"August 1st, 2024"},"versionCreatedAt":"2024-01-19 16:58:59","video":"","vorDoi":"10.1016/j.jff.2024.106262","vorDoiUrl":"https://doi.org/10.1016/j.jff.2024.106262","workflowStages":[]},"version":"v1","identity":"rs-3866681","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3866681","identity":"rs-3866681","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}