Preventing effects of Pen Yan Jing Tablets on sequelae of pelvic inflammatory disease by inhibiting Akt/NF-κB pathway

Preventing effects of Pen Yan Jing Tablets on sequelae of pelvic inflammatory disease by inhibiting Akt/NF-κB pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Preventing effects of Pen Yan Jing Tablets on sequelae of pelvic inflammatory disease by inhibiting Akt/NF-κB pathway Ping Tang, Zhongrui Wu, Juan Lin, Yong Jiang, Weipeng Luo, Yuxin Ye, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1495843/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Pen Yan Jing tablets (PYJ), a Chinese patent medicine, has been used for sequelae of pelvic inflammatory disease (SPID) effectively. However, the underlying anti-SPID mechanisms of PYJ remain unknown. The Akt/NF-κB pathway plays an important role in promoting inflammation. This study was designed to investigate whether PYJ has preventing effects on SPID by inhibiting Akt/NF-κB pathway. Methods: A rat model of mixed bacteria liquid plus mechanical damage-induced SPID and a cell model of lipopolysaccharide (LPS)-activated RAW 264.7 macrophages were performed. After PYJ treatment, the morphology of uteri and extent of pelvic adhesion were observed. The pathological changes were evaluated by hematoxylin-eosin (HE) staining. The protein expressions of CD68, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) were identified by immunohistochemistry. Additionally, the viability and NO level of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were calculated by CCK-8 and Griess method, respectively. The tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected by ELISA. Protein kinase B (Akt)/nuclear factor kappa-B (NF-κB) pathway-related protein expressions were assayed by western blot. Results: PYJ not only remarkably alleviated morphological changes of uteri, pelvic adhesion and histological change of chronic inflammation in uteri, but also down-regulated protein expressions of ICAM-1, VCAM-1, MCP-1, COX-2, p-Akt, p65, p-p65 and p-IκBα in uteri. Moreover, PYJ medicated serum inhibited cell viability, NO release, levels of TNF-α and IL-6, and protein expressions of p-Akt, p-p65 and p-IκBα in LPS-activated RAW 264.7 macrophages. Conclusion: Taken together, the preventing effect of PYJ on SPID attributes to the anti-inflammatory activity via inhibiting Akt/NF-κB pathway. Pen Yan Jing tablets SPID Akt NF-κB Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Pelvic inflammatory disease (PID) is a multiple bacterial infection-induced inflammation disorder in the upper female genital tract, typically involving uteri, fallopian tubes, ovaries and pelvic peritoneum. Without adequate and effective treatments, PID has a great risk of severe sequelae of pelvic inflammatory disease (SPID), such as infertility, ectopic pregnancy and chronic pelvic pain [ 1 ]. According to a self-reporting USA poll, PID incidence is up to 4.4% [ 2 ]. Besides, after follow-up of 84 months, about 8% of women with PID are infertile, 19.0% are categorized as ectopic pregnancy, 42.7% have chronic pelvic pain, and 21.3% have recurrent PID [ 3 ]. Hence, SPID is considered to be a major threat to women at reproductive age all over the world. The pathogenesis of SPID remains poorly understood. Inflammation is reported as a core element accompanying the occurrence and development of SPID [ 1 ]. Inadequate treatment contributes to constant inflammation of uteri, fallopian tubes, ovaries and pelvic, which results in tissue hyperplasia as well as pelvic adhesion [ 4 ]. Therefore, chronic inflammation in tissues is the main pathological manifestation of SPID. Proinflammatory mediators such as adhesion molecules, chemokines and inflammatory cytokines play important roles in regulating the recruitment of leucocytes [ 5 ]. These proinflammatory mediators are produced and formed an inflammatory storm through a positive feedback cycle when inflammatory pathways are activated [ 5 ]. Consequently, inhibition of proinflammatory mediators may be a therapeutic strategy for treating SPID. Nuclear factor kappa-B (NF-κB) was reported as an important transcription factor of proinflammatory mediators [ 6 ]. Akt, also known as protein kinase B, participates in inflammation through regulating NF-κB. Strong evidences confirm that the inhibition of Akt significantly reversed NF-κB activation and binding to DNA of inflammatory genes, suppressing the generation of proinflammatory mediators [ 7 , 8 ]. Thus, we speculated that suppressing Akt/NF-B pathway may be important for treating SPID by inhibiting the generation of proinflammatory mediators. Furthermore, proinflammatory macrophages markedly contribute to chronic inflammation and adhesion formation [ 9 – 11 ]. Bacterial lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. LPS induces macrophage infiltration into peritoneal cavity in mice and proinflammatory macrophage activation in vitro [ 12 ] (Zhou et al. , 1999). Accordingly, targeting LPS-induced inflammatory responses in macrophages may be another potential strategy for ameliorating SPID. Antibiotics are the main treatments for PID [ 13 ]. Unfortunately, they are not effective for tissue adhesion and hyperplasia in SPID and exhibit several severe adverse effects [ 14 ]. Traditional Chinese medicines with the characteristics of multitargets, multi-channels, coordination and synergism have been paid more and more attention to treating SPID. Pen Yan Jing tablets (PYJ), a Chinese patent medicine approved by the China Food and Drug Administration (CFDA), is composed of eight Chinese herbal medicines, including Lonicera japonica Thunb., Spatholobus suberectus Dunn., Cibotium barometz (L.) J.Sm., Taraxacum mongolicum Hand.-Mazz., Leonurus japonicus Houtt., Plantago asiatica L., Paeonia lactiflora Pall. and Ligusticum chuanxiong Hort. at a mass ratio of 25: 25: 25: 12: 12: 12: 6: 6. PYJ and other formulae of Pen Yan Jing have been used to treat SPID in clinic [ 15 – 17 ]. Our previous study shows that PYJ is effective in rats with SPID induced by phenolic mucilage [ 18 ]. However, the underlying anti-SPID mechanisms of PYJ are still unknown. Therefore, we focused on the Akt/NF-B pathway to investigate the underlying anti-SPID mechanisms of PYJ in a rat model of mixed bacteria liquid plus mechanical damage-induced SPID and a cell model of LPS-activated RAW 264.7 macrophages. Materials And Methods Chemical profile analysis of PYJ by high performance liquid chromatography (HPLC) 1 g of PYJ (CFDA approval number: Z20090070, produced by Hutchison Whampoa Baiyunshan Laida Pharmaceutical (Shantou) Co., Ltd, Shantou, China, 190601) powder was added into 25 mL methanol. The mixture was treated with an ultrasonic instrument (200 W, 53 kHZ) for 30 min and then filtered. Mixed standards including protocatechuic acid, catechin, chlorogenic acid and paeoniflorin (Weikeqi, Chengdu, China) were dissolved in methanol. All solutions were filtered through 0.22 µm filter membrane. The HPLC analysis was performed by a Shimadzu HPLC system (LC-20AT, Shimadzu, Kyoto, Japan) equipped with a Diamonsil-C18 column (4.6 mm × 250 mm, 5 µm, DiKMA, Beijing, China). The mobile phase was composed of acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B). The gradient elution program was as follows: 0–25 min, 98 − 90% B; 25–35 min, 90 − 88% B; 35–45, 88–88% B; 45–65 min, 88 − 85% B. The injection volume was 10 µL and the flow rate was 1.0 mL/min. The column temperature was maintained at 35°C and UV detection wavelength was 220 nm. Bacterial strains and animals Escherichia coli (CMCC44102), Staphyloccocus aureus (ATCC25923) and β-hemolytic streptococcus (CMCC32210) were purchased from Beijing Baiou Bowei Biotechnology Co., Ltd. Female Sprague-Dawley rats, weighing 180–220 g, were obtained from the Guangdong Medical Laboratory Animal Center (certificate No. SCXK 2018-0002) and were housed at 24.0 ± 0.5°C and a 12 h light/dark cycle lighting schedule in the Biosafety Level 2 Laboratory of the Experimental Animal Center, Guangzhou University of Chinese Medicine (certificate No. SYXK 2018-0001). Rats were allowed free access to food and water. The in vivo research was reviewed and approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine. The establishment of SPID model The SPID model of rats was established as described previously with slight modifications [ 19 , 20 ]. Rats were anesthetized with an intraperitoneal injection of 36 mg/kg pentobarbital sodium (Merck, Darmstadt, Germany). A 0.5 cm × 0.5 cm piece of gelatin sponge was inserted into the vagina. Then a 1.0-1.5 cm incision along the midline of the lower abdomen was made to expose the uteri. Both sides of uterine cavities were injected with bacteria mixture (6×10 10 cfu/mL) of Escherichia coli, Staphylococcus aureus and Beta-hemolytic streptococcus at a volume ratio of 2:1:1 in 0.2 mL normal saline with an insulin syringe (Braun, Melsungen, Germany) and the endometrium was scratched by a syringe needle four times. The uteri of rats in the sham group received an injection of 0.2 mL normal saline without scratch. Animals treatment Rats were randomly divided into 8 groups ( n = 8): normal group, sham group, model group, three dosages of PYJ groups (388, 775 and 1550 mg/kg/d, p.o.), positive control groups of Fuke Qianjin tablets group (FKQJ; 656 mg/kg/d, p.o.; Zhuzhou QianJin Pharmaceutical Co., Ltd, Zhuzhou, China) and dexamethasone tablets group (DEX; 0.31 mg/kg/d, p.o.; Guangdong Nanguo Pharmaceutical Co., Ltd., Zhanjiang, China). Based on the body surface area conversion between humans and rats, the medium dose of PYJ in rats was calculated by clinical dose of 7.74 g/d in human. The tablets were crushed and dissolved in a 0.5% CMC-Na solution. The rats in the normal group, sham group and model group received a 0.5% CMC-Na solution. One week after modeling, all rats were orally administered corresponding drugs or vehicle solutions once per day for four consecutive weeks. Evaluation of uterus appearance and pelvic adhesion Rats were anesthetized with pentobarbital sodium. The appearance of uteri was observed. The extent of pelvic adhesion was evaluated by the Philips scoring criteria [ 21 ]. Then, the left uteri were harvested and stored at -80℃, and the right ones were immersed in 10% neutral buffered formalin (Yongjin, Guangzhou, China). Histopathologic analysis The right uteri fixed in formalin were placed in processing cassettes, dehydrated through a serial alcohol gradient, embedded in paraffin wax, and sliced into 4 µm thick sections. Before staining, the sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol. Then they were stained with hematoxylin and eosin (HE; Leagene, Beijing, China). The histopathology of uteri was observed and pictured by a microscope (BX53, Olympus, Tokyo, Japan). Immunohistochemistry analysis The uterus paraffin sections were dewaxed and hydrated. The antigen was restored with citrate buffer (pH 6.0; Yongjin, Guangzhou, China) or EDTA buffer (pH 9.0; Zhong Shan-Golden Bridge, Beijing, China). After washed with phosphate buffered saline (PBS), the slices were put into 3% hydrogen peroxide solution, incubated at room temperature and hidden from light for 25 min, and then washed with PBS. The uterine tissues were blocked with 10% normal goat serum (Zhong Shan-Golden Bridge, Beijing, China) for 30 min. Then the slices were incubated with different primary antibodies against CD68 (1:5000; Abcam, Cambridge, USA), intercellular cell adhesion molecule-1 (ICAM-1; 1:100; Huabio, Hangzhou, China), vascular cell adhesion molecule-1 (VCAM-1; 1:500; Abcam, Cambridge, USA), monocyte chemotactic protein-1 (MCP-1; 1:400; Abcam, Cambridge, USA) and cyclooxygenase-2 (COX-2; 1:1600; Abcam, Cambridge, USA) at 4℃ overnight. After washing, the second antibody (1:250; Jackson Immuno Research, West Grove, USA) was added, incubating for 1 h at room temperature. After washing, the tissue sections were covered with diaminobenzidine chromogenic solution until the positive solution became brown. The nuclei were stained with haematoxylin for 3 min and rinsed with running water. A neutral gum seal was applied. The images were captured by a microscope and five fields at 400× magnification were randomly chosen from each sample. The average number of CD68 positive staining cells of the five fields was counted. The ratio of stained intensity of density (IOD) to the stained area in each image was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, USA) to gain the mean protein expressions. Cell culture RAW 264.7 cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China), a murine macrophage cell line, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel) and antibiotics (100 units/L penicillin, 100 mg/L streptomycin; Solarbio, Beijing, China) at 37°C in an incubator with 5% CO 2 . Preparation of PYJ medicated serum for cell treatment 20 rats were randomly divided into two groups: normal group and PYJ group. The rats in the normal group and PYJ group were administered with a 0.5% CMC-Na solution or PYJ (1550 mg/kg/d, p.o.) respectively for five consecutive days. Finally, all rats were anesthetized with pentobarbital sodium after the last administration. The blood was collected from abdominal aorta and then centrifuged at 3,000 rpm for 10 min to obtain serum. The serum was incubated at 56℃ for 30 min, sterilized through 0.22 µm filter membrane and stored at -80℃ before use. The serum from rats of normal and PYJ groups was used as blank and PYJ medicated serum, respectively. The blank serum was diluted with serum-free DMEM to a final volume ratio of 10%. The PYJ medicated serum was diluted with serum-free DMEM and blank serum to final volume ratios of 1%, 5% or 10%. Cell viability assay To analyze the effect of PYJ medicated serum on viability of normal RAW 264.7 cells, the cells (2×10 4 cells per well) were seeded into 96-well plates. After overnight, the cells were incubated with 10% FBS, 10% blank serum, 1%, 5% or 10% PYJ medicated serum for 24 h. To analyze the effect of PYJ medicated serum on the viability of RAW 264.7 cells stimulated with LPS, cells (3×10 4 cells per well) were seeded into 96-well plates. After overnight, the cells were pre-treated with PYJ medicated serum or 10% blank serum for 24 h and then incubated with LPS (1.0 µg/mL; Sigma-Aldrich, St. Louis, USA) dissolved in PYJ medicated serum or 10% blank serum for 12 h. After incubation, each well was added with 10% CCK-8 solution (v/v; APE×BIO, Houston, USA) and then incubated for 1–4 h at 37°C. The absorbance at 450 nm was read using a microplate reader (Multiskan GO, Thermo Scientific, Vantaa, Finland). The cell viability was expressed as a percentage of the optical density of each treatment group relative to the control group. Determination of NO release RAW 264.7 cells were seeded into 6-well plates at a density of 5×10 5 cells per well overnight, pre-treated with PYJ medicated serum or 10% blank serum for 24 h, and then incubated with LPS (1.0 µg/mL) dissolved in PYJ medicated serum or 10% blank serum for 12 h. The culture supernatants (50 µL) were mixed with an equivalent volume of Griess reagent (Beyotime, Shanghai, China), and the absorbance of the mixture at 540 nm was measured by a microplate reader. A standard curve was constructed using sodium nitrate. Determination of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) levels RAW 264.7 cells were seeded into 96-well plates at a density of 3×10 4 cells per well overnight. The cells pre-treated with PYJ medicated serum or 10% blank serum for 24 h and then incubated with LPS (1.0 µg/mL) dissolved in PYJ medicated serum or 10% blank serum for 12 h. The supernatants were collected to determine the levels of TNF-α and IL-6 according to the instruction of commercial kits (Lianke, Shanghai, China). Western blot analysis RAW 264.7 cells were treated as the method of determination NO release and then were collected. The left uteri were homogenized. Next, the tissues and cells were lysed in an ice-cold RIPA lysis buffer (CWBIO, Beijing, China), blended with 2× SDS-PAGE sample buffer (FDbio, Hangzhou, China), boiled for 10 min, and then resolved with 10% SDS-PAGE. Then the proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, USA), blocked at 37℃ for 60 min with 5% nonfat dry milk, and reacted with diluted primary antibodies (1:1000) including inhibitory protein kappa B (IκBα, Abcam, Cambridge, USA), p65, Akt, p-p65, p-IκBα, and p-Akt (CST, Danvers, USA). After washing, the membranes were incubated with second antibodies (1:5000) at 37℃ for 1 h. The blots were visualized using an ECL reagent (Millipore, Billerica, USA) and scanned by a chemiluminescence apparatus (5200CE, Tanon, Shanghai, China). Statistical analysis Statistical analyses were performed with SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). The pelvic adhesion score data were expressed as the average rank ( \(\stackrel{-}{R}\) ) and the statistical analyses were performed by Kruskal-Wallis H test. The quantitative data were expressed as mean ± standard deviation (SD) and the statistical analyses were performed by one-way analysis of variance followed by Least-Significant Difference test (equal variances) or Dunnett’s T3 test (unequal variances). A P value of 0.05 or less than 0.05 was considered to be statistically significant. Results Chemical profile of PYJ As shown in Fig. 1 , PYJ contained protocatechuic acid, catechin, chlorogenic acid and paeoniflorin. PYJ ameliorated uterine appearance changes and pelvic adhesion The uteri of normal and sham rats were covered with smooth and pink serosa surface and their shapes were uniform, while the uteri in model rats exhibited uneven thickness, hyperemia and adhesion with pelvic tissues. These lesions were significantly attenuated by PYJ at doses of 775 and 1550 mg/kg (Fig. 2 ). In addition, compared to the model group, the pelvic adhesion score was significantly decreased by PYJ at doses of 775 ( P ≤ 0.05) and 1550 mg/kg ( P ≤ 0.01, Table 1 ). Table 1 PYJ alleviated pelvic adhesion in SPID rats( f , \(\stackrel{-}{{R}}\) , n = 10–16) Group Uteri( n ) Pelvic adhesion score \(\stackrel{-}{{R}}\) 0 Ⅰ Ⅱ Ⅲ Ⅳ Normal 16 16 0 0 0 0 28.00 Sham 16 14 2 0 0 0 33.38 Model 16 2 5 1 5 3 88.78 **$$ 388 mg/kg PYJ 16 3 5 5 2 1 78.00 775 mg/kg PYJ 16 5 8 3 0 0 59.29 # 1550 mg/kg PYJ 16 8 5 3 0 0 54.28 ## FKQJ 16 5 6 5 0 0 65.53 # DEX 10 2 1 3 4 0 86.25 The values were given as f and \(\stackrel{-}{R}\) , n = 10–16 of uteri for each group. ** P ≤ 0.01 vs . normal group; $$ P ≤ 0.01 vs . sham group; # P ≤ 0.05 and ## P ≤ 0.01 vs . model group. PYJ reduced uterine histopathologic changes and leucocytes infiltration As shown in Fig. 3 , the uteri in the normal and sham groups presented complete and clear structures and a few scattered inflammatory cells. In the model group, uterine tissues showed obvious chronic inflammatory pathological changes as follows. The uterine cavities were narrowed. The number of vessels in lamina propria and myometrium was increased, and these vessels were surrounded by a large number of leucocytes, mainly neutrophils and macrophages. Moreover, the serous layers were thickened and accompanied with enhanced mesothelial cells, fibroblasts, inflammatory cells, collagen fibers and congested capillaries. As compared with the model group, leucocytes infiltration in uteri, and collagen fiber and congestion in serous layers were reduced in 775 and 1550 mg/kg PYJ groups. Moreover, PYJ at 775 and 1550 mg/kg significantly decreased the number of CD68 + macrophages of uteri in SPID rats ( P ≤ 0.01 vs. model group, Fig. 4 ). PYJ reduced production of inflammatory mediators in uteri As shown in Fig. 5 A-D, ICAM-1, VCAM-1, MCP-1 and COX-2 were highly expressed in leucocytes and/or vascular endothelial cells in rats’ uteri of model group ( P ≤ 0.01 vs. normal and sham group). PYJ remarkably reversed these protein expressions. As compared with the model group, PYJ at 775 mg/kg remarkably decreased the protein expressions of ICAM-1, VCAM-1, MCP-1 and COX-2 by 35.74%, 30.07%, 24.02% and 24.41%, respectively ( P ≤ 0.01 vs. model group, Figs. 5 E- 5 H). PYJ down-regulated related protein expressions of Akt/NF-κB pathway in uteri As shown in Fig. 6 , the protein expressions of p-Akt, p-IκBα, p65 and p-p65 related with Akt/NF-κB pathway were enhanced in SPID rats ( P ≤ 0.01 vs. normal and sham group). And these proteins were down-regulated by 32.01%, 30.44%, 36.49% and 24.68% by 775 mg/kg PYJ, respectively. PYJ medicated serum inhibited cell viability and inflammation in macrophages stimulated by LPS As compared with 10% FBS, 10% blank serum and 1%-10% PYJ medicated serum had no effect on the cell viability of normal RAW 264.7 cells (Fig. 7 A). However, 10% PYJ medicated serum decreased the cell viability by 11.09% in LPS-stimulated RAW 264.7 cells ( P ≤ 0.05 vs. normal group, Fig. 7 B). Furthermore, the levels of NO, TNF-α and IL-6 were increased in LPS-stimulated RAW 264.7 cells ( P ≤ 0.01 vs. normal group). And 10% PYJ medicated serum dramatically reversed them by 23.56%, 55.39% and 47.77%, respectively (Fig. 7 C-E). PYJ inhibited related protein expressions of Akt/NF-κB pathway in macrophages stimulated by LPS As shown in Fig. 8 , LPS stimulation significantly increased the protein expressions of p-Akt p-IκBα and p-p65 in RAW 264.7 cells ( P ≤ 0.05 vs. normal group). 10% PYJ medicated serum significantly inhibited these proteins by 46.16%, 47.92% and 42.12%, respectively. Discussion Our previous study demonstrates that PYJ is effective for SPID induced by phenolic mucilage in rats [ 16 ]. In the present study, we established a rat model of SPID induced by a mixture of bacterial infections plus mechanical damage in the uterus to investigate the underlying anti-SPID mechanism of PYJ, because such model resembled the pathogenesis of SPID in humans more closely than the previous one. The apparent changes including severe pelvic adhesion and uterine morphology changes were observed in this study. Moreover, the uteri presented pathological changes of chronic inflammation, such as leucocytes infiltration, tissue hyperplasia, congestion and increased collagen fibers were also observed. These results suggested that the SPID model was established successfully. PYJ attenuated uterine morphology characteristics and pelvic adhesion of rats with SPID, indicating that PYJ is effective in treating SPID. What’s more, with the effects of anti-inflammation, reducing collagen deposition, and inhibiting fibrocyte DNA synthesis and granulomas proliferation, DEX was often used as a positive control in anti-SPID drug studies and was effective for SPID induced by phenolic mucilage [ 16 , 17 ]. However, DEX not only had no effect on SPID induced by a mixture of bacterial infections plus mechanical damage, but also resulted in the death of three rats. The reason may be that the immunosuppressive effect of DEX causes serious bacterial infections. Macrophages play a distinctive role in contributing to chronic inflammation and adhesion formation [ 9 – 11 ]. CD68 is a heavily glycosylated protein and has been widely employed as a macrophage marker [ 10 ]. In this study, the result showed that PYJ reduced the number of CD68 + macrophages in uteri of SPID rats. In addition, PYJ medicated serum also inhibited the cell viability of macrophages stimulated with LPS in vitro . These results indicate that PYJ decreases the infiltration and cell viability of macrophages, thus inhibiting chronic inflammation and pelvic adhesion. Adhesion molecules and chemokines are crucial to the recruitment of inflammatory cells [ 5 ]. ICAM-1 and VCAM-1 as essential adhesion molecules promote the migration of leucocytes from blood to inflammation tissues [ 23 ]. Moreover, ICAM-1 is important for the development of intestinal adhesion [ 24 ]. In a mouse model of ovalbumin-induced lung inflammation, VCAM-1 antibody inhibits the recruitments of macrophages, neutrophils and eosinophils [ 25 ]. MCP-1, also known as chemokine (C-C motif) ligand 2 (CCL2), is a ligand on the surface of monocytes and has a specific chemotactic activation effect on monocytes. MCP-1 is closely related with diseases characterized by monocyte infiltration such as rheumatoid arthritis [ 26 ]. Furthermore, COX-2 plays an important role in regulating inflammation and angiogenesis in the development of postoperative adhesion [ 27 ]. Therefore, ICAM-1, VCAM-1, MCP-1 and COX-2 are key proinflammatory mediators and contribute to leucocytes recruitment and adhesion formation. In the present study, PYJ remarkably decreased protein expressions of ICAM-1, VCAM-1, MCP-1 and COX-2 in uteri of SPID rats, especially in inflammation infiltration sites, suggesting that PYJ attenuates chronic inflammation and pelvic adhesion by suppressing these inflammatory mediators. In addition, proinflammatory cytokines also contribute to chronic inflammation by positive feedback loops [ 28 ]. Both IL-6 and TNF-α induce the production of ICAM-1, VCAM-1, MCP-1 and COX-2, amplifying leucocyte recruitment [ 29 – 31 ]. Besides, NO is produced from nitric oxide enzyme catalyzed by L-arginine and involved in the killing and release of proinflammatory cytokines of macrophages [ 32 ]. LPS triggers a cytokine storm characterized by myriad proinflammatory mediators in macrophages [ 33 ]. In this study, PYJ medicated serum suppressed LPS-induced secretion of NO, TNF-α and IL-6 in macrophages, which explains the inhibitory effects of PYJ on macrophage inflammation and pelvic adhesion. Physiologically, NF-κB, a heterodimer commonly composed of p50 and p65, forms a complex with IκBα and locates in cytoplasm [ 6 ]. After pattern recognition receptors are activated by pathogens, IκB undergoes phosphorylation and then dissociates with the complex of NF-κB and finally degrades. NF-κB activates and translocates into nuclear, ultimately promoting the transcription of genes involved in inflammatory responses such as IL-6, TNF-α, ICAM-1, VCAM-1, MCP-1 and COX-2 [ 34 ]. Akt activates NF-κB by phosphorylating IκB kinase (IKK). Accumulating studies have demonstrated that Akt participates in the regulation of inflammatory mediator production, chemotaxis, migration and survival of macrophages by regulating NF-κB [ 7 , 8 ]. In the present study, the protein expressions of p-IκB, p65, p-p65 and p-Akt were increased in uteri of SPID rats and LPS-stimulated RAW 264.7 cells, indicating that Akt/NF-κB pathway activates. Nevertheless, these increased protein expressions were attenuated by PYJ. These results reveal that PYJ inhibits the activation of Akt/NF-κB pathway, suppressing infiltration of macrophages and production of pro-inflammation mediators. PYJ contained four bioactive components including protocatechuic acid, catechin, chlorogenic acid and paeoniflorin. These components attenuate inflammatory pain and neuroinflammation by inhibiting Akt and NF-κB [ 35 – 38 ]. They may contribute to the antiinflammation activity of PYJ. Conclusion In conclusion, PYJ down-regulated protein expressions related with Akt/NF-κB pathway, and decreased proinflammatory mediator production as well as infiltration and activation of macrophages. Therefore, the preventing effect of PYJ on SPID attributes to the anti-inflammatory activity via inhibiting Akt/NF-κB pathway (Fig. 9 ). Abbreviations Akt: protein kinase B; CCL2: chemokine (C-C motif) ligand 2; COX-2: cyclooxygenase-2; DEX: dexamethasone; DMEM: Dulbecco’s modified Eagle’s medium; FBS: fetal bovine serum; FKQJ: Fuke Qianjin tablets; HE: hematoxylin-eosin; ICAM-1: intercellular cell adhesion molecule-1; IκBα: inhibitory protein kappa B; IKK: IκB kinase; IOD: intensity of density; IL-6: interleukin-6; LPS: lipopolysaccharide; MCP-1: monocyte chemotactic protein-1; NF-κB: nuclear factor kappa-B; PBS: phosphate buffered saline; PYJ: Pen Yan Jing tablets; SPID: sequelae of pelvic inflammatory disease; TNF-α: tumor necrosis factor-α; VCAM-1: vascular cell adhesion molecule-1. Declarations Acknowledgments The authors thank the financial support of Guangzhou Science and Technology Basic and Applied Basic Research Project. (No. 202002030108). Authors’ contributions Tang Ping: conception and design of the work, experimental operation; data analysis and interpretation, figure preparation and original draft writing. Wu Zhongrui: experimentation performance; data analysis and interpretation, figure preparation. Lin Juan: conception and design of the work, data analysis and interpretation. Jiang Yong: data analysis and interpretation. Luo Weipeng, Ye Yuxin, Yang Xinrong, Ding Qi and Wang Yiting: experimental operation. Lin Baoqin and Wang Deqin: conception and design of the work, data interpretation and curation, project administration and funding acquisition. All authors revised the manuscript and approved the final version. Funding This work was supported by Guangzhou Science and Technology Basic and Applied Basic Research Project (No. 202002030108). Availability of data and materials The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request. Ethics approval and consent to participate Study procedures involving experimental animals were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals. all methods were carried out in accordance with relevant guidelines and regulations. All studies involving animals are reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Brunham RC, Gottlieb SL, Paavonen J. Pelvic inflammatory disease. N Engl J Med. 2015;372:2039–48. https://doi.org/10.1056/NEJMra1411426 . Kreisel K, Torrone E, Bernstein K, Hong J, Gorwitz R. Prevalence of pelvic inflammatory disease in sexually experienced women of reproductive age-United States, 2013–2014. MMWR-Morb Mortal Wkly Rep. 2017;66(3):80–3. https://doi.org/10.15585/mmwr.mm6603a3 . Trent M, Bass D, Ness RB, Haggerty C. Recurrent PID, subsequent STI, and reproductive health outcomes: findings from the PID evaluation and clinical health (PEACH) study. Sex Transm Dis. 2011;38(9):879–81. https://doi.org/10.1097/OLQ.0b013e31821f918c . Yeung YT, Aziz F, Guerrero-Castilla A, Arguelles S. Signaling pathways in inflammation and anti-inflammatory therapies. Curr Pharm Des. 2018;24(14):1449–84. https://doi.org/10.2174/1381612824666180327165604 Navarro-González JF, Mora-Fernández C, Muros de Fuentes M, García-Pérez J. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol. 2011;7(6):327–40. https://doi.org/10.1038/nrneph.2011.51 . Mulero MC, Huxford T, Ghosh G. NF-κB, IκB, and IKK: integral components of immune system signaling. Adv Exp Med Biol. 2019;1172:207–26. https://doi.org/10.1007/978-981-13-9367-9_10 . Lee YG, Lee J, Byeon SE, Yoo DS, Kim MH, Lee SY, et al. Functional role of Akt in macrophage-mediated innate immunity. Front Biosci. 2011;16: 517–30. https://doi.org/10.2741/3702 . Li T, Gao D, Du M, Cheng X, Mao X. Casein glycomacropeptide hydrolysates inhibit PGE2 production and COX2 expression in LPS-stimulated RAW 264.7 macrophage cells via Akt mediated NF-κB and MAPK pathways. Food Funct. 2018;9(4):2524–32. https://doi.org/10.1039/c7fo01989k . Sindrilaru A, Peters T, Wieschalka S, Baican C, Baican A, Peter H, et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J Clin Invest. 2011;121(3):985–97. https://doi.org/10.1172/JCI44490 . Brochhausen C, Schmitt VH, Mamilos A, Schmitt C, Planck CN, Rajab TK, et al. Expressin of CD68 positive macrophages in the use of different barrier materials to prevent peritoneal adhesions-an animal study. J Mater Sci Mater Med. 2017;28(1):15. https://doi.org/10.1007/s10856-016-5821-3 . Herrick SE, Allen JE. Surgical adhesions: A sticky macrophage problem. Science. 2021;371(6533):993–4. https://doi.org/10.1126/science.abg5416 . Zhou Y, Yang Y, Warr G, Bravo R. LPS down-regulates the expression of chemokine receptor CCR2 in mice and abolishes macrophage infiltration in acute inflammation. J Leukoc Biol. 1999;65(2):265–9. https://doi.org/10.1002/jlb.65.2.265 . Duarte R, Fuhrich D, Ross JD. A review of antibiotic therapy for pelvic inflammatory disease. Int J Antimicrob Agents. 2015;46(3):272–7. https://doi.org/10.1016/j.ijantimicag.2015.05.004 . Zhang YH, Xie YM, Luo SP, Wei SB, Ma K, Zhao RH, et al. Interpretation and prospect of clinical practice guideline on traditional Chinese Medicine therapy alone or combined with antibiotics for pelvic inflammation. Global Tradit Chin Med. 2018;11(10):1545–7. https://doi:10.3969/j.issn.1674-1749.2018.10.009 . Clinical efficacy observation for Penyanjing tablets combined with antibiotics in the treatment of 90 cases of chronic pelvic inflammatory disease. Clin Res Prac.2016;1(2):55. https: doi.org/10.19347/j.cnki.2096-1413.2016.02.041 . Lu HY. Pen Yan Jing granules treats 50 cases of chronic pelvic inflammation. J Chengdu Univ Tradit Chin Med. 1999;22(4):55 – 6. https://doi.org/10.3969/j.issn.1004-0668.1999.04.030 . Sun JY, Lu Y, Wang L, Wang N, Qi H. Therapeutic effect of using Penyanjing oral liquid on sequelae of pelvic inflammatory disease. Contem Med. 2020;26(25):50–2. https://doi.org/10.3969/j.issn.1009-4393.2020.25.020 . Tang P, Zhang CP, Lu ZQ, Lin J, Lin BQ. Effect of Pen Yan Jing Tablets on SPID induced by phenolic mucilage in rats. Tradi Chin Drug Res Clin Pharmacol. 2021;32(3):332–7. https://doi.org/10.19378/j.issn.1003-9783.2021.03.005 . Liang C, Wang ZP, Li F, Hang Y, Ye ML, Feng Q, et al. Pharmacodynamics of Baijin granules on model rats with sequelae of pelvic inflammatory disease. Chin Tradit Pat Med. 2017;39(7):1329–35. https://doi.org/10.3969/j.issn.1001-1528.2017.07.001 . (in Chinese) Zhang LJ, Zhu JY, Sun MY, Song YN, Rahman K, Peng C, et al. Anti-inflammatory effect of Man-Pen-Fang, a Chinese herbal compound, on chronic pelvic inflammation in rats. J Ethnopharmacol. 2017;208:57–65. https://doi.org/10.1016/j.jep.2017.06.034 . Zhang JY, Xu LM. Effects of Kunfukang capsule on red blood cell immune function in SPID rats. Chin Tradit Pat Med. 2016;38(10):2253–6. https://doi.org/10.3969/j.issn.1001-1528.2016.10.032 . Song LY, Tian LW, Ma Y, Xie Y, Feng HX, Qin F, et al. Protection of flavonoids from Smilax china L. rhizome on phenol mucilage-induced pelvic inflammation in rats by attenuating inflammation and fibrosis. J Funct Food. 2017;28:194–204. https://doi.org/10.1016/j.jff.2016.11.015 . González-Amaro R, Díaz-González F, Sánchez-Madrid F. Adhesion molecules in inflammatory diseases. Drugs. 1998; 56: 977–88. https://doi.org/10.2165/00003495-199856060-00003 . Zhang Y, Li X, Zhang Q, Li J, Ju J, Du N, et al. Berberine hydrochloride prevents postsurgery intestinal adhesion and inflammation in rats. J Pharmacol Exp Ther. 2014;349(3):417–26. https://jpet.aspetjournals.org/content/349/3/417 . Lee JH, Sohn JH, Ryu SY, Hong CS, Moon KD, Park JW. A novel human anti-VCAM-1 monoclonal antibody ameliorates airway inflammation and remodelling. J Cell Mol Med. 2013;17(10):1271–81. https://doi.org/10.1111/jcmm.12102 . Rana AK, Li Y, Dang Q, Yang F. Monocytes in rheumatoid arthritis: Circulating precursors of macrophages and osteoclasts and, their heterogeneity and plasticity role in RA pathogenesis. Int Immunopharmacol. 2018;65:348–59. https://doi.org/10.1016/j.intimp.2018.10.016 . Alpay Z, Saed GM, Diamond MP. Postoperative adhesions: from formation to prevention. Semin Reprod Med. 2008;26:313–21. https://doi.org/10.1055/s-0028-1082389 . Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014;1843(11):2563–82. https://doi.org/10.1016/j.bbamcr.2014.05.014 . Liang J, Yuan S, Wang X, Lei Y, Zhang X, Huang M, et al. Attenuation of pristimerin on TNF-α-induced endothelial inflammation. Int Immunopharmacol. 2020;82:106326. https://doi.org/10.1016/j.intimp.2020.106326 . Romano M, Sironi M, Toniatti C, Polentarutti N, Fruscella P, Ghezzi P, et al. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity. 1997;6(3):315–25. https://doi.org/10.1016/s1074-7613(00)80334-9 . Oh JW, Van Wagoner NJ, Rose-John S, Benveniste EN. Role of IL-6 and the soluble IL-6 receptor in inhibition of VCAM-1 gene expression. J Immunol. 1998;161(9):4992–9. https://www.jimmunol.org/content/161/9/4992 . Bogdan C, Röllinghoff M, Diefenbach A. Reactive oxygen and reactive Nitrogen intermediates in innate and specific immunity. Curr Opin Immunol. 2000;12(1):64–76. https://doi.org/10.1016/s0952-7915(99)00052-7 . Hamidzadeh K, Christensen SM, Dalby E, Chandrasekaran P, Mosser DM. Macrophages and the recovery from acute and chronic inflammation. Annu Rev Physiol. 2017;79:567–92. https://doi.org/10.1146/annurev-physiol-022516-034348 . Mussbacher M, Salzmann M, Brostjan C, Hoesel B, Schoergenhofer C, Datler H, et al. Cell type-specific roles of NF-κB linking inflammation and thrombosis. Front Immunol. 2019;10:85. https://doi.org/10.3389/fimmu.2019.00085 . Nam YJ, Lee CS. Protocatechuic acid inhibits Toll-like receptor-4-dependent activation of NF-κB by suppressing activation of the Akt, mTOR, JNK and p38-MAPK. Int Immunopharmacol. 2018;55:272–81. https://doi.org/10.1016/j.intimp.2017.12.024 . Hu B, Xu G, Zhang X, Xu L, Zhou H, Ma Z, et al. Paeoniflorin attenuates inflammatory pain by inhibiting microglial activation and Akt-NF-κB signaling in the central nervous system. Cell Physiol Biochem. 2018;47(2):842–50. https://doi.org/10.1159/000490076 . Syed Hussein SS, Kamarudin MN, Kadir HA. (+)-Catechin attenuates NF-κB activation through regulation of Akt, MAPK, and AMPK signaling pathways in LPS-induced BV-2 microglial cells. Am J Chin Med. 2015; 43(5), 927–52. https://doi.org/10.1142/S0192415X15500548 . Chen J, Luo Y, Li Y, Chen D, Yu B, He J. Chlorogenic acid attenuates oxidative stress-induced intestinal epithelium injury by co-regulating the PI3K/Akt and IκBα/NF-κB signaling. Antioxidants (Basel). 2021; 10(12): 1915. https://doi.org/10.3390/antiox10121915 . Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additionalfile2.pdf Additionalfile3.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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-1495843","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":96800477,"identity":"81a10b19-3a36-416f-a6be-8c7af39a8510","order_by":0,"name":"Ping Tang","email":"","orcid":"","institution":"First Affiliated Hospital of Guangzhou University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Tang","suffix":""},{"id":96800478,"identity":"62a55ee2-bc86-4ace-8ca0-80e6ec5f315e","order_by":1,"name":"Zhongrui Wu","email":"","orcid":"","institution":"First Affiliated Hospital of 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05:14:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1495843/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1495843/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20122269,"identity":"00338e29-d262-4333-9aba-d464780df121","added_by":"auto","created_at":"2022-04-08 14:57:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64176,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC profile of (A) PYJ and (B) standards. (1) protocatechuic acid, (2) catechin (3) chlorogenic acid and (4) paeoniflorin.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/d2c554927d576de3ac7cfa5a.png"},{"id":20123494,"identity":"6bfeee88-6d0b-442e-afe8-9c886a219cce","added_by":"auto","created_at":"2022-04-08 15:07:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1424871,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ ameliorated uterine appearance changes. White arrows pointed to pelvic adhesion.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/9f69f18981e47866f939b0e8.png"},{"id":20123786,"identity":"6c566db8-c638-470a-b149-d14a7d0272d9","added_by":"auto","created_at":"2022-04-08 15:12:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1356131,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ reduced uterine histopathologic changes and leucocytes infiltration. SCEp, simple columnar epithelium; LPM, lamina propria mucosa; Mm, myometrium; Ss, serosa. Yellow arrows pointed to macrophages. Blue arrows pointed to neutrophils.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/9674bd61d99d35724aee211d.png"},{"id":20122846,"identity":"a2a988aa-c474-4cc9-a9fb-a1786276527b","added_by":"auto","created_at":"2022-04-08 15:02:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":411623,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ reduced CD68\u003csup\u003e+\u003c/sup\u003e macrophages of uteri. (A) Representative immunostaining images of CD68\u003csup\u003e+\u003c/sup\u003e macrophages in uteri. Red arrows pointed to CD68\u003csup\u003e+\u003c/sup\u003e macrophages. (B) The number of CD68\u003csup\u003e+\u003c/sup\u003e macrophages in uteri. Data were expressed as mean ± SD, \u003cem\u003en\u003c/em\u003e=7-8. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. normal group; \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. sham group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. model group.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/26b44e59483aa0c1624ab6b4.png"},{"id":20123495,"identity":"7288dd52-6d66-4169-af8e-12cb8803c0fd","added_by":"auto","created_at":"2022-04-08 15:07:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1515245,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ reduced production of inflammatory mediators in uteri. (A-D) Representative immunostaining images of ICAM-1, VCAM-1, MCP-1 and COX-2 in uteri. Black arrows pointed to vascular endothelia cells. Blue arrows pointed to neutrophils. Red arrows pointed to macrophages. (E-H) The ratio of IOD/area of ICAM-1, VCAM-1, MCP-1 and COX-2 positive staining in uterine sections. Data were expressed as mean ± SD, \u003cem\u003en\u003c/em\u003e=7-8. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. normal group; \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. sham group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. model group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/75a846fa661f82fe0ce89078.png"},{"id":20122279,"identity":"109368ee-59b4-4b14-b9f1-e77e3a8e68cc","added_by":"auto","created_at":"2022-04-08 14:57:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":104558,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ decreased protein expressions related with Akt/NF-κB pathway in uteri. Representative western blots and densitometric quantification of p-Akt (A), p-IκBα (B), p65 (C), p-p65 (D). Data were expressed as mean ± SD, \u003cem\u003en\u003c/em\u003e=6. **\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs.\u003c/em\u003e normal group; \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs.\u003c/em\u003e sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. model group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/9380fb79e99c651b6c4b407a.png"},{"id":20122276,"identity":"e1e565cf-1d0b-4988-9eee-4e172122bb83","added_by":"auto","created_at":"2022-04-08 14:57:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":108186,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ inhibited cell viability and inflammation in macrophages stimulated by LPS. (A) PYJ medicated serum showed no effect on the cell viability of normal RAW 264.7 cells. (B) PYJ medicated serum inhibited cell viability of RAW 264.7 cells stimulated by LPS. Data were expressed as mean ± SD, \u003cem\u003en\u003c/em\u003e=6. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. normal group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.05 \u003cem\u003evs\u003c/em\u003e. model group. (C) PYJ medicated serum inhibited NO release of RAW 264.7 cells stimulated by LPS. Data were expressed as mean ± SD, \u003cem\u003en\u003c/em\u003e=3. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. normal group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. model group. (D-E) PYJ medicated serum inhibited NO release and levels of TNF-α and IL-6 of cell supernatant in RAW 264.7 cells stimulated by LPS. Data were expressed as mean ± SD, \u003cem\u003en\u003c/em\u003e=6. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. normal group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. model group. FBS, fetal bovine serum; BS: blank serum; PYJMS, PYJ medicated serum.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/e819fbcafe593f5caba8a0f4.png"},{"id":20122273,"identity":"abce2740-c0b5-498f-aac3-bec2b9acf7e1","added_by":"auto","created_at":"2022-04-08 14:57:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":90644,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ inhibited protein expressions related with Akt/NF-κB pathways in macrophages stimulated by LPS. Representative western blots and densitometric quantification of p-Akt (A), p-IκBα (B), p-p65 (C). Data were expressed as mean ± SD, \u003cem\u003en\u003c/em\u003e=3. **\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs.\u003c/em\u003e normal group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e≤0.01 \u003cem\u003evs\u003c/em\u003e. model group.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/ca84d1391a7e4f912d06941a.png"},{"id":20122851,"identity":"f7be1b80-eac9-4dac-bcc1-db42e9f69436","added_by":"auto","created_at":"2022-04-08 15:02:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":306617,"visible":true,"origin":"","legend":"\u003cp\u003ePYJ prevents SPID via inhibiting Akt/NF-κB pathway.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/69c323e2a8b2b42e5b818377.png"},{"id":26328857,"identity":"c0a2b4e4-8d01-445f-90e9-3f96c3c5a38b","added_by":"auto","created_at":"2022-09-12 08:59:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5631144,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/56c09675-33e3-4ea5-8d3e-483724a38a0b.pdf"},{"id":20122271,"identity":"99e2c976-156d-462c-86dc-fd3cfea673bb","added_by":"auto","created_at":"2022-04-08 14:57:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12893,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/dd07f480d16ab589814e1961.docx"},{"id":20122274,"identity":"b0b6f53c-ebfc-4f80-b79c-69309dda885c","added_by":"auto","created_at":"2022-04-08 14:57:01","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":446872,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/4072fc85329ee185e7e8946e.pdf"},{"id":20122848,"identity":"87d06b4f-9b09-4714-bae3-1b0099ae3e9c","added_by":"auto","created_at":"2022-04-08 15:02:01","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":462599,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1495843/v1/aa00ea7c3ce7cebd305541c2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Preventing effects of Pen Yan Jing Tablets on sequelae of pelvic inflammatory disease by inhibiting Akt/NF-κB pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePelvic inflammatory disease (PID) is a multiple bacterial infection-induced inflammation disorder in the upper female genital tract, typically involving uteri, fallopian tubes, ovaries and pelvic peritoneum. Without adequate and effective treatments, PID has a great risk of severe sequelae of pelvic inflammatory disease (SPID), such as infertility, ectopic pregnancy and chronic pelvic pain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to a self-reporting USA poll, PID incidence is up to 4.4% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Besides, after follow-up of 84 months, about 8% of women with PID are infertile, 19.0% are categorized as ectopic pregnancy, 42.7% have chronic pelvic pain, and 21.3% have recurrent PID [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Hence, SPID is considered to be a major threat to women at reproductive age all over the world.\u003c/p\u003e \u003cp\u003eThe pathogenesis of SPID remains poorly understood. Inflammation is reported as a core element accompanying the occurrence and development of SPID [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Inadequate treatment contributes to constant inflammation of uteri, fallopian tubes, ovaries and pelvic, which results in tissue hyperplasia as well as pelvic adhesion [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, chronic inflammation in tissues is the main pathological manifestation of SPID.\u003c/p\u003e \u003cp\u003eProinflammatory mediators such as adhesion molecules, chemokines and inflammatory cytokines play important roles in regulating the recruitment of leucocytes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These proinflammatory mediators are produced and formed an inflammatory storm through a positive feedback cycle when inflammatory pathways are activated [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consequently, inhibition of proinflammatory mediators may be a therapeutic strategy for treating SPID. Nuclear factor kappa-B (NF-κB) was reported as an important transcription factor of proinflammatory mediators [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Akt, also known as protein kinase B, participates in inflammation through regulating NF-κB. Strong evidences confirm that the inhibition of Akt significantly reversed NF-κB activation and binding to DNA of inflammatory genes, suppressing the generation of proinflammatory mediators [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, we speculated that suppressing Akt/NF-B pathway may be important for treating SPID by inhibiting the generation of proinflammatory mediators.\u003c/p\u003e \u003cp\u003eFurthermore, proinflammatory macrophages markedly contribute to chronic inflammation and adhesion formation [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Bacterial lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. LPS induces macrophage infiltration into peritoneal cavity in mice and proinflammatory macrophage activation \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] (Zhou \u003cem\u003eet al.\u003c/em\u003e, 1999). Accordingly, targeting LPS-induced inflammatory responses in macrophages may be another potential strategy for ameliorating SPID.\u003c/p\u003e \u003cp\u003eAntibiotics are the main treatments for PID [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Unfortunately, they are not effective for tissue adhesion and hyperplasia in SPID and exhibit several severe adverse effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Traditional Chinese medicines with the characteristics of multitargets, multi-channels, coordination and synergism have been paid more and more attention to treating SPID. Pen Yan Jing tablets (PYJ), a Chinese patent medicine approved by the China Food and Drug Administration (CFDA), is composed of eight Chinese herbal medicines, including \u003cem\u003eLonicera japonica\u003c/em\u003e Thunb., \u003cem\u003eSpatholobus suberectus\u003c/em\u003e Dunn., \u003cem\u003eCibotium barometz\u003c/em\u003e (L.) J.Sm., \u003cem\u003eTaraxacum mongolicum\u003c/em\u003e Hand.-Mazz., \u003cem\u003eLeonurus japonicus\u003c/em\u003e Houtt., \u003cem\u003ePlantago asiatica\u003c/em\u003e L., \u003cem\u003ePaeonia lactiflora\u003c/em\u003e Pall. and \u003cem\u003eLigusticum chuanxiong\u003c/em\u003e Hort. at a mass ratio of 25: 25: 25: 12: 12: 12: 6: 6. PYJ and other formulae of Pen Yan Jing have been used to treat SPID in clinic [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our previous study shows that PYJ is effective in rats with SPID induced by phenolic mucilage [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the underlying anti-SPID mechanisms of PYJ are still unknown.\u003c/p\u003e \u003cp\u003eTherefore, we focused on the Akt/NF-B pathway to investigate the underlying anti-SPID mechanisms of PYJ in a rat model of mixed bacteria liquid plus mechanical damage-induced SPID and a cell model of LPS-activated RAW 264.7 macrophages.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemical profile analysis of PYJ by high performance liquid chromatography (HPLC)\u003c/h2\u003e \u003cp\u003e1 g of PYJ (CFDA approval number: Z20090070, produced by Hutchison Whampoa Baiyunshan Laida Pharmaceutical (Shantou) Co., Ltd, Shantou, China, 190601) powder was added into 25 mL methanol. The mixture was treated with an ultrasonic instrument (200 W, 53 kHZ) for 30 min and then filtered. Mixed standards including protocatechuic acid, catechin, chlorogenic acid and paeoniflorin (Weikeqi, Chengdu, China) were dissolved in methanol. All solutions were filtered through 0.22 \u0026micro;m filter membrane. The HPLC analysis was performed by a Shimadzu HPLC system (LC-20AT, Shimadzu, Kyoto, Japan) equipped with a Diamonsil-C18 column (4.6 mm \u0026times; 250 mm, 5 \u0026micro;m, DiKMA, Beijing, China). The mobile phase was composed of acetonitrile (A) and 0.1% phosphoric acid aqueous solution (B). The gradient elution program was as follows: 0\u0026ndash;25 min, 98\u0026thinsp;\u0026minus;\u0026thinsp;90% B; 25\u0026ndash;35 min, 90\u0026thinsp;\u0026minus;\u0026thinsp;88% B; 35\u0026ndash;45, 88\u0026ndash;88% B; 45\u0026ndash;65 min, 88\u0026thinsp;\u0026minus;\u0026thinsp;85% B. The injection volume was 10 \u0026micro;L and the flow rate was 1.0 mL/min. The column temperature was maintained at 35\u0026deg;C and UV detection wavelength was 220 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and animals\u003c/h2\u003e \u003cp\u003eEscherichia coli (CMCC44102), Staphyloccocus aureus (ATCC25923) and β-hemolytic streptococcus (CMCC32210) were purchased from Beijing Baiou Bowei Biotechnology Co., Ltd. Female Sprague-Dawley rats, weighing 180\u0026ndash;220 g, were obtained from the Guangdong Medical Laboratory Animal Center (certificate No. SCXK 2018-0002) and were housed at 24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and a 12 h light/dark cycle lighting schedule in the Biosafety Level 2 Laboratory of the Experimental Animal Center, Guangzhou University of Chinese Medicine (certificate No. SYXK 2018-0001). Rats were allowed free access to food and water. The \u003cem\u003ein vivo\u003c/em\u003e research was reviewed and approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe establishment of SPID model\u003c/h2\u003e \u003cp\u003eThe SPID model of rats was established as described previously with slight modifications [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Rats were anesthetized with an intraperitoneal injection of 36 mg/kg pentobarbital sodium (Merck, Darmstadt, Germany). A 0.5 cm \u0026times; 0.5 cm piece of gelatin sponge was inserted into the vagina. Then a 1.0-1.5 cm incision along the midline of the lower abdomen was made to expose the uteri. Both sides of uterine cavities were injected with bacteria mixture (6\u0026times;10\u003csup\u003e10\u003c/sup\u003e cfu/mL) of Escherichia coli, Staphylococcus aureus and Beta-hemolytic streptococcus at a volume ratio of 2:1:1 in 0.2 mL normal saline with an insulin syringe (Braun, Melsungen, Germany) and the endometrium was scratched by a syringe needle four times. The uteri of rats in the sham group received an injection of 0.2 mL normal saline without scratch.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnimals treatment\u003c/h2\u003e \u003cp\u003eRats were randomly divided into 8 groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8): normal group, sham group, model group, three dosages of PYJ groups (388, 775 and 1550 mg/kg/d, p.o.), positive control groups of Fuke Qianjin tablets group (FKQJ; 656 mg/kg/d, p.o.; Zhuzhou QianJin Pharmaceutical Co., Ltd, Zhuzhou, China) and dexamethasone tablets group (DEX; 0.31 mg/kg/d, p.o.; Guangdong Nanguo Pharmaceutical Co., Ltd., Zhanjiang, China). Based on the body surface area conversion between humans and rats, the medium dose of PYJ in rats was calculated by clinical dose of 7.74 g/d in human. The tablets were crushed and dissolved in a 0.5% CMC-Na solution. The rats in the normal group, sham group and model group received a 0.5% CMC-Na solution. One week after modeling, all rats were orally administered corresponding drugs or vehicle solutions once per day for four consecutive weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of uterus appearance and pelvic adhesion\u003c/h2\u003e \u003cp\u003eRats were anesthetized with pentobarbital sodium. The appearance of uteri was observed. The extent of pelvic adhesion was evaluated by the Philips scoring criteria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Then, the left uteri were harvested and stored at -80℃, and the right ones were immersed in 10% neutral buffered formalin (Yongjin, Guangzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistopathologic analysis\u003c/h2\u003e \u003cp\u003eThe right uteri fixed in formalin were placed in processing cassettes, dehydrated through a serial alcohol gradient, embedded in paraffin wax, and sliced into 4 \u0026micro;m thick sections. Before staining, the sections were dewaxed in xylene, rehydrated through decreasing concentrations of ethanol. Then they were stained with hematoxylin and eosin (HE; Leagene, Beijing, China). The histopathology of uteri was observed and pictured by a microscope (BX53, Olympus, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry analysis\u003c/h2\u003e \u003cp\u003eThe uterus paraffin sections were dewaxed and hydrated. The antigen was restored with citrate buffer (pH 6.0; Yongjin, Guangzhou, China) or EDTA buffer (pH 9.0; Zhong Shan-Golden Bridge, Beijing, China). After washed with phosphate buffered saline (PBS), the slices were put into 3% hydrogen peroxide solution, incubated at room temperature and hidden from light for 25 min, and then washed with PBS. The uterine tissues were blocked with 10% normal goat serum (Zhong Shan-Golden Bridge, Beijing, China) for 30 min. Then the slices were incubated with different primary antibodies against CD68 (1:5000; Abcam, Cambridge, USA), intercellular cell adhesion molecule-1 (ICAM-1; 1:100; Huabio, Hangzhou, China), vascular cell adhesion molecule-1 (VCAM-1; 1:500; Abcam, Cambridge, USA), monocyte chemotactic protein-1 (MCP-1; 1:400; Abcam, Cambridge, USA) and cyclooxygenase-2 (COX-2; 1:1600; Abcam, Cambridge, USA) at 4℃ overnight. After washing, the second antibody (1:250; Jackson Immuno Research, West Grove, USA) was added, incubating for 1 h at room temperature. After washing, the tissue sections were covered with diaminobenzidine chromogenic solution until the positive solution became brown. The nuclei were stained with haematoxylin for 3 min and rinsed with running water. A neutral gum seal was applied. The images were captured by a microscope and five fields at 400\u0026times; magnification were randomly chosen from each sample. The average number of CD68 positive staining cells of the five fields was counted. The ratio of stained intensity of density (IOD) to the stained area in each image was analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, USA) to gain the mean protein expressions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China), a murine macrophage cell line, were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Kibbutz Beit Haemek, Israel) and antibiotics (100 units/L penicillin, 100 mg/L streptomycin; Solarbio, Beijing, China) at 37\u0026deg;C in an incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of PYJ medicated serum for cell treatment\u003c/h2\u003e \u003cp\u003e20 rats were randomly divided into two groups: normal group and PYJ group. The rats in the normal group and PYJ group were administered with a 0.5% CMC-Na solution or PYJ (1550 mg/kg/d, p.o.) respectively for five consecutive days. Finally, all rats were anesthetized with pentobarbital sodium after the last administration. The blood was collected from abdominal aorta and then centrifuged at 3,000 rpm for 10 min to obtain serum. The serum was incubated at 56℃ for 30 min, sterilized through 0.22 \u0026micro;m filter membrane and stored at -80℃ before use. The serum from rats of normal and PYJ groups was used as blank and PYJ medicated serum, respectively. The blank serum was diluted with serum-free DMEM to a final volume ratio of 10%. The PYJ medicated serum was diluted with serum-free DMEM and blank serum to final volume ratios of 1%, 5% or 10%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eTo analyze the effect of PYJ medicated serum on viability of normal RAW 264.7 cells, the cells (2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well) were seeded into 96-well plates. After overnight, the cells were incubated with 10% FBS, 10% blank serum, 1%, 5% or 10% PYJ medicated serum for 24 h. To analyze the effect of PYJ medicated serum on the viability of RAW 264.7 cells stimulated with LPS, cells (3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well) were seeded into 96-well plates. After overnight, the cells were pre-treated with PYJ medicated serum or 10% blank serum for 24 h and then incubated with LPS (1.0 \u0026micro;g/mL; Sigma-Aldrich, St. Louis, USA) dissolved in PYJ medicated serum or 10% blank serum for 12 h. After incubation, each well was added with 10% CCK-8 solution (v/v; APE\u0026times;BIO, Houston, USA) and then incubated for 1\u0026ndash;4 h at 37\u0026deg;C. The absorbance at 450 nm was read using a microplate reader (Multiskan GO, Thermo Scientific, Vantaa, Finland). The cell viability was expressed as a percentage of the optical density of each treatment group relative to the control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of NO release\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells were seeded into 6-well plates at a density of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well overnight, pre-treated with PYJ medicated serum or 10% blank serum for 24 h, and then incubated with LPS (1.0 \u0026micro;g/mL) dissolved in PYJ medicated serum or 10% blank serum for 12 h. The culture supernatants (50 \u0026micro;L) were mixed with an equivalent volume of Griess reagent (Beyotime, Shanghai, China), and the absorbance of the mixture at 540 nm was measured by a microplate reader. A standard curve was constructed using sodium nitrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) levels\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells were seeded into 96-well plates at a density of 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well overnight. The cells pre-treated with PYJ medicated serum or 10% blank serum for 24 h and then incubated with LPS (1.0 \u0026micro;g/mL) dissolved in PYJ medicated serum or 10% blank serum for 12 h. The supernatants were collected to determine the levels of TNF-α and IL-6 according to the instruction of commercial kits (Lianke, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eRAW 264.7 cells were treated as the method of determination NO release and then were collected. The left uteri were homogenized. Next, the tissues and cells were lysed in an ice-cold RIPA lysis buffer (CWBIO, Beijing, China), blended with 2\u0026times; SDS-PAGE sample buffer (FDbio, Hangzhou, China), boiled for 10 min, and then resolved with 10% SDS-PAGE. Then the proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, USA), blocked at 37℃ for 60 min with 5% nonfat dry milk, and reacted with diluted primary antibodies (1:1000) including inhibitory protein kappa B (IκBα, Abcam, Cambridge, USA), p65, Akt, p-p65, p-IκBα, and p-Akt (CST, Danvers, USA). After washing, the membranes were incubated with second antibodies (1:5000) at 37℃ for 1 h. The blots were visualized using an ECL reagent (Millipore, Billerica, USA) and scanned by a chemiluminescence apparatus (5200CE, Tanon, Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed with SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). The pelvic adhesion score data were expressed as the average rank (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{-}{R}\\)\u003c/span\u003e\u003c/span\u003e) and the statistical analyses were performed by Kruskal-Wallis H test. The quantitative data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and the statistical analyses were performed by one-way analysis of variance followed by Least-Significant Difference test (equal variances) or Dunnett\u0026rsquo;s T3 test (unequal variances). A \u003cem\u003eP\u003c/em\u003e value of 0.05 or less than 0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003eChemical profile of PYJ\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, PYJ contained protocatechuic acid, catechin, chlorogenic acid and paeoniflorin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003ePYJ ameliorated uterine appearance changes and pelvic adhesion\u003c/h2\u003e\n \u003cp\u003eThe uteri of normal and sham rats were covered with smooth and pink serosa surface and their shapes were uniform, while the uteri in model rats exhibited uneven thickness, hyperemia and adhesion with pelvic tissues. These lesions were significantly attenuated by PYJ at doses of 775 and 1550 mg/kg (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, compared to the model group, the pelvic adhesion score was significantly decreased by PYJ at doses of 775 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) and 1550 mg/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePYJ alleviated pelvic adhesion in SPID rats(\u003cem\u003ef\u003c/em\u003e, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{-}{{R}}\\)\u003c/span\u003e\u003c/span\u003e, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10\u0026ndash;16)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eUteri(\u003cem\u003en\u003c/em\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003ePelvic adhesion score\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{-}{{R}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eⅠ\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eⅡ\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eⅢ\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eⅣ\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSham\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88.78\u003csup\u003e**$$\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e388 mg/kg PYJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e78.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e775 mg/kg PYJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59.29\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1550 mg/kg PYJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.28\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFKQJ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65.53\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDEX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003eThe values were given as \u003cem\u003ef\u003c/em\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\stackrel{-}{R}\\)\u003c/span\u003e\u003c/span\u003e, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10\u0026ndash;16 of uteri for each group. **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs\u003c/em\u003e. normal group; \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs\u003c/em\u003e. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 and \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs\u003c/em\u003e. model group.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003ePYJ reduced uterine histopathologic changes and leucocytes infiltration\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the uteri in the normal and sham groups presented complete and clear structures and a few scattered inflammatory cells. In the model group, uterine tissues showed obvious chronic inflammatory pathological changes as follows. The uterine cavities were narrowed. The number of vessels in lamina propria and myometrium was increased, and these vessels were surrounded by a large number of leucocytes, mainly neutrophils and macrophages. Moreover, the serous layers were thickened and accompanied with enhanced mesothelial cells, fibroblasts, inflammatory cells, collagen fibers and congested capillaries. As compared with the model group, leucocytes infiltration in uteri, and collagen fiber and congestion in serous layers were reduced in 775 and 1550 mg/kg PYJ groups. Moreover, PYJ at 775 and 1550 mg/kg significantly decreased the number of CD68\u003csup\u003e+\u003c/sup\u003e macrophages of uteri in SPID rats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs.\u003c/em\u003e model group, Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec21\"\u003e\n \u003ch2\u003ePYJ reduced production of inflammatory mediators in uteri\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-D, ICAM-1, VCAM-1, MCP-1 and COX-2 were highly expressed in leucocytes and/or vascular endothelial cells in rats\u0026rsquo; uteri of model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs.\u003c/em\u003e normal and sham group). PYJ remarkably reversed these protein expressions. As compared with the model group, PYJ at 775 mg/kg remarkably decreased the protein expressions of ICAM-1, VCAM-1, MCP-1 and COX-2 by 35.74%, 30.07%, 24.02% and 24.41%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs.\u003c/em\u003e model group, Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE-\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec22\"\u003e\n \u003ch2\u003ePYJ down-regulated related protein expressions of Akt/NF-\u0026kappa;B pathway in uteri\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, the protein expressions of p-Akt, p-I\u0026kappa;B\u0026alpha;, p65 and p-p65 related with Akt/NF-\u0026kappa;B pathway were enhanced in SPID rats (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs.\u003c/em\u003e normal and sham group). And these proteins were down-regulated by 32.01%, 30.44%, 36.49% and 24.68% by 775 mg/kg PYJ, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec23\"\u003e\n \u003ch2\u003ePYJ medicated serum inhibited cell viability and inflammation in macrophages stimulated by LPS\u003c/h2\u003e\n \u003cp\u003eAs compared with 10% FBS, 10% blank serum and 1%-10% PYJ medicated serum had no effect on the cell viability of normal RAW 264.7 cells (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). However, 10% PYJ medicated serum decreased the cell viability by 11.09% in LPS-stimulated RAW 264.7 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 \u003cem\u003evs.\u003c/em\u003e normal group, Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). Furthermore, the levels of NO, TNF-\u0026alpha; and IL-6 were increased in LPS-stimulated RAW 264.7 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01 \u003cem\u003evs.\u003c/em\u003e normal group). And 10% PYJ medicated serum dramatically reversed them by 23.56%, 55.39% and 47.77%, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC-E).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec24\"\u003e\n \u003ch2\u003ePYJ inhibited related protein expressions of Akt/NF-\u0026kappa;B pathway in macrophages stimulated by LPS\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e, LPS stimulation significantly increased the protein expressions of p-Akt p-I\u0026kappa;B\u0026alpha; and p-p65 in RAW 264.7 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05 \u003cem\u003evs.\u003c/em\u003e normal group). 10% PYJ medicated serum significantly inhibited these proteins by 46.16%, 47.92% and 42.12%, respectively.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur previous study demonstrates that PYJ is effective for SPID induced by phenolic mucilage in rats [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the present study, we established a rat model of SPID induced by a mixture of bacterial infections plus mechanical damage in the uterus to investigate the underlying anti-SPID mechanism of PYJ, because such model resembled the pathogenesis of SPID in humans more closely than the previous one. The apparent changes including severe pelvic adhesion and uterine morphology changes were observed in this study. Moreover, the uteri presented pathological changes of chronic inflammation, such as leucocytes infiltration, tissue hyperplasia, congestion and increased collagen fibers were also observed. These results suggested that the SPID model was established successfully. PYJ attenuated uterine morphology characteristics and pelvic adhesion of rats with SPID, indicating that PYJ is effective in treating SPID.\u003c/p\u003e \u003cp\u003eWhat\u0026rsquo;s more, with the effects of anti-inflammation, reducing collagen deposition, and inhibiting fibrocyte DNA synthesis and granulomas proliferation, DEX was often used as a positive control in anti-SPID drug studies and was effective for SPID induced by phenolic mucilage [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, DEX not only had no effect on SPID induced by a mixture of bacterial infections plus mechanical damage, but also resulted in the death of three rats. The reason may be that the immunosuppressive effect of DEX causes serious bacterial infections.\u003c/p\u003e \u003cp\u003eMacrophages play a distinctive role in contributing to chronic inflammation and adhesion formation [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CD68 is a heavily glycosylated protein and has been widely employed as a macrophage marker [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this study, the result showed that PYJ reduced the number of CD68\u003csup\u003e+\u003c/sup\u003e macrophages in uteri of SPID rats. In addition, PYJ medicated serum also inhibited the cell viability of macrophages stimulated with LPS \u003cem\u003ein vitro\u003c/em\u003e. These results indicate that PYJ decreases the infiltration and cell viability of macrophages, thus inhibiting chronic inflammation and pelvic adhesion.\u003c/p\u003e \u003cp\u003eAdhesion molecules and chemokines are crucial to the recruitment of inflammatory cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. ICAM-1 and VCAM-1 as essential adhesion molecules promote the migration of leucocytes from blood to inflammation tissues [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, ICAM-1 is important for the development of intestinal adhesion [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In a mouse model of ovalbumin-induced lung inflammation, VCAM-1 antibody inhibits the recruitments of macrophages, neutrophils and eosinophils [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. MCP-1, also known as chemokine (C-C motif) ligand 2 (CCL2), is a ligand on the surface of monocytes and has a specific chemotactic activation effect on monocytes. MCP-1 is closely related with diseases characterized by monocyte infiltration such as rheumatoid arthritis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, COX-2 plays an important role in regulating inflammation and angiogenesis in the development of postoperative adhesion [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, ICAM-1, VCAM-1, MCP-1 and COX-2 are key proinflammatory mediators and contribute to leucocytes recruitment and adhesion formation. In the present study, PYJ remarkably decreased protein expressions of ICAM-1, VCAM-1, MCP-1 and COX-2 in uteri of SPID rats, especially in inflammation infiltration sites, suggesting that PYJ attenuates chronic inflammation and pelvic adhesion by suppressing these inflammatory mediators.\u003c/p\u003e \u003cp\u003eIn addition, proinflammatory cytokines also contribute to chronic inflammation by positive feedback loops [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Both IL-6 and TNF-α induce the production of ICAM-1, VCAM-1, MCP-1 and COX-2, amplifying leucocyte recruitment [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Besides, NO is produced from nitric oxide enzyme catalyzed by L-arginine and involved in the killing and release of proinflammatory cytokines of macrophages [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. LPS triggers a cytokine storm characterized by myriad proinflammatory mediators in macrophages [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, PYJ medicated serum suppressed LPS-induced secretion of NO, TNF-α and IL-6 in macrophages, which explains the inhibitory effects of PYJ on macrophage inflammation and pelvic adhesion.\u003c/p\u003e \u003cp\u003ePhysiologically, NF-κB, a heterodimer commonly composed of p50 and p65, forms a complex with IκBα and locates in cytoplasm [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. After pattern recognition receptors are activated by pathogens, IκB undergoes phosphorylation and then dissociates with the complex of NF-κB and finally degrades. NF-κB activates and translocates into nuclear, ultimately promoting the transcription of genes involved in inflammatory responses such as IL-6, TNF-α, ICAM-1, VCAM-1, MCP-1 and COX-2 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Akt activates NF-κB by phosphorylating IκB kinase (IKK). Accumulating studies have demonstrated that Akt participates in the regulation of inflammatory mediator production, chemotaxis, migration and survival of macrophages by regulating NF-κB [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the present study, the protein expressions of p-IκB, p65, p-p65 and p-Akt were increased in uteri of SPID rats and LPS-stimulated RAW 264.7 cells, indicating that Akt/NF-κB pathway activates. Nevertheless, these increased protein expressions were attenuated by PYJ. These results reveal that PYJ inhibits the activation of Akt/NF-κB pathway, suppressing infiltration of macrophages and production of pro-inflammation mediators.\u003c/p\u003e \u003cp\u003ePYJ contained four bioactive components including protocatechuic acid, catechin, chlorogenic acid and paeoniflorin. These components attenuate inflammatory pain and neuroinflammation by inhibiting Akt and NF-κB [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. They may contribute to the antiinflammation activity of PYJ.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, PYJ down-regulated protein expressions related with Akt/NF-κB pathway, and decreased proinflammatory mediator production as well as infiltration and activation of macrophages. Therefore, the preventing effect of PYJ on SPID attributes to the anti-inflammatory activity via inhibiting Akt/NF-κB pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAkt: protein kinase B; CCL2: chemokine (C-C motif) ligand 2; COX-2: cyclooxygenase-2; DEX: dexamethasone; DMEM: Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium; FBS: fetal bovine serum; FKQJ: Fuke Qianjin tablets; HE: hematoxylin-eosin; ICAM-1: intercellular cell adhesion molecule-1; I\u0026kappa;B\u0026alpha;: inhibitory protein kappa B; IKK: I\u0026kappa;B kinase; IOD: intensity of density; IL-6: interleukin-6; LPS: lipopolysaccharide; MCP-1: monocyte chemotactic protein-1; NF-\u0026kappa;B: nuclear factor kappa-B; PBS: phosphate buffered saline; PYJ: Pen Yan Jing tablets; SPID: sequelae of pelvic inflammatory disease; TNF-\u0026alpha;: tumor necrosis factor-\u0026alpha;; VCAM-1: vascular cell adhesion molecule-1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the financial support of Guangzhou Science and Technology Basic and Applied Basic Research Project. (No. 202002030108).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTang Ping: conception and design of the work, experimental operation; data analysis and interpretation, figure preparation and original draft writing. Wu Zhongrui: experimentation performance; data analysis and interpretation, figure preparation. Lin Juan: conception and design of the work, data analysis and interpretation. Jiang Yong: data analysis and interpretation. Luo Weipeng, Ye Yuxin, Yang Xinrong, Ding Qi and Wang Yiting: experimental operation. Lin Baoqin and Wang Deqin: conception and design of the work, data interpretation and curation, project administration and funding acquisition. All authors revised the manuscript and approved the final version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Guangzhou Science and Technology Basic and Applied Basic Research Project (No. 202002030108).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy procedures involving experimental animals were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine\u0026nbsp;in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals. all methods were carried out in accordance with relevant guidelines and regulations. All studies involving animals are reported in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrunham RC, Gottlieb SL, Paavonen J. Pelvic inflammatory disease. N Engl J Med. 2015;372:2039\u0026ndash;48. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMra1411426\u003c/span\u003e\u003cspan address=\"10.1056/NEJMra1411426\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKreisel K, Torrone E, Bernstein K, Hong J, Gorwitz R. Prevalence of pelvic inflammatory disease in sexually experienced women of reproductive age-United States, 2013\u0026ndash;2014. MMWR-Morb Mortal Wkly Rep. 2017;66(3):80\u0026ndash;3. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.15585/mmwr.mm6603a3\u003c/span\u003e\u003cspan address=\"10.15585/mmwr.mm6603a3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTrent M, Bass D, Ness RB, Haggerty C. Recurrent PID, subsequent STI, and reproductive health outcomes: findings from the PID evaluation and clinical health (PEACH) study. Sex Transm Dis. 2011;38(9):879\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/OLQ.0b013e31821f918c\u003c/span\u003e\u003cspan address=\"10.1097/OLQ.0b013e31821f918c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeung YT, Aziz F, Guerrero-Castilla A, Arguelles S. Signaling pathways in inflammation and anti-inflammatory therapies. Curr Pharm Des. 2018;24(14):1449\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/1381612824666180327165604\u003c/span\u003e\u003cspan address=\"10.2174/1381612824666180327165604\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavarro-Gonz\u0026aacute;lez JF, Mora-Fern\u0026aacute;ndez C, Muros de Fuentes M, Garc\u0026iacute;a-P\u0026eacute;rez J. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol. 2011;7(6):327\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrneph.2011.51\u003c/span\u003e\u003cspan address=\"10.1038/nrneph.2011.51\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMulero MC, Huxford T, Ghosh G. NF-κB, IκB, and IKK: integral components of immune system signaling. Adv Exp Med Biol. 2019;1172:207\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-981-13-9367-9_10\u003c/span\u003e\u003cspan address=\"10.1007/978-981-13-9367-9_10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee YG, Lee J, Byeon SE, Yoo DS, Kim MH, Lee SY, et al. Functional role of Akt in macrophage-mediated innate immunity. Front Biosci. 2011;16: 517\u0026ndash;30. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2741/3702\u003c/span\u003e\u003cspan address=\"10.2741/3702\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi T, Gao D, Du M, Cheng X, Mao X. Casein glycomacropeptide hydrolysates inhibit PGE2 production and COX2 expression in LPS-stimulated RAW 264.7 macrophage cells via Akt mediated NF-κB and MAPK pathways. Food Funct. 2018;9(4):2524\u0026ndash;32. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c7fo01989k\u003c/span\u003e\u003cspan address=\"10.1039/c7fo01989k\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSindrilaru A, Peters T, Wieschalka S, Baican C, Baican A, Peter H, et al. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J Clin Invest. 2011;121(3):985\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1172/JCI44490\u003c/span\u003e\u003cspan address=\"10.1172/JCI44490\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrochhausen C, Schmitt VH, Mamilos A, Schmitt C, Planck CN, Rajab TK, et al. Expressin of CD68 positive macrophages in the use of different barrier materials to prevent peritoneal adhesions-an animal study. J Mater Sci Mater Med. 2017;28(1):15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10856-016-5821-3\u003c/span\u003e\u003cspan address=\"10.1007/s10856-016-5821-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerrick SE, Allen JE. Surgical adhesions: A sticky macrophage problem. Science. 2021;371(6533):993\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.abg5416\u003c/span\u003e\u003cspan address=\"10.1126/science.abg5416\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y, Yang Y, Warr G, Bravo R. LPS down-regulates the expression of chemokine receptor CCR2 in mice and abolishes macrophage infiltration in acute inflammation. J Leukoc Biol. 1999;65(2):265\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jlb.65.2.265\u003c/span\u003e\u003cspan address=\"10.1002/jlb.65.2.265\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuarte R, Fuhrich D, Ross JD. A review of antibiotic therapy for pelvic inflammatory disease. Int J Antimicrob Agents. 2015;46(3):272\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijantimicag.2015.05.004\u003c/span\u003e\u003cspan address=\"10.1016/j.ijantimicag.2015.05.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang YH, Xie YM, Luo SP, Wei SB, Ma K, Zhao RH, et al. Interpretation and prospect of clinical practice guideline on traditional Chinese Medicine therapy alone or combined with antibiotics for pelvic inflammation. Global Tradit Chin Med. 2018;11(10):1545\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.3969/j.issn.1674-1749.2018.10.009\u003c/span\u003e\u003cspan address=\"https://doi:10.3969/j.issn.1674-1749.2018.10.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClinical efficacy observation for Penyanjing tablets combined with antibiotics in the treatment of 90 cases of chronic pelvic inflammatory disease. Clin Res Prac.2016;1(2):55. https: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.19347/j.cnki.2096-1413.2016.02.041\u003c/span\u003e\u003cspan address=\"10.19347/j.cnki.2096-1413.2016.02.041\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu HY. Pen Yan Jing granules treats 50 cases of chronic pelvic inflammation. J Chengdu Univ Tradit Chin Med. 1999;22(4):55 \u0026ndash; 6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3969/j.issn.1004-0668.1999.04.030\u003c/span\u003e\u003cspan address=\"10.3969/j.issn.1004-0668.1999.04.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun JY, Lu Y, Wang L, Wang N, Qi H. Therapeutic effect of using Penyanjing oral liquid on sequelae of pelvic inflammatory disease. Contem Med. 2020;26(25):50\u0026ndash;2. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3969/j.issn.1009-4393.2020.25.020\u003c/span\u003e\u003cspan address=\"10.3969/j.issn.1009-4393.2020.25.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang P, Zhang CP, Lu ZQ, Lin J, Lin BQ. Effect of Pen Yan Jing Tablets on SPID induced by phenolic mucilage in rats. Tradi Chin Drug Res Clin Pharmacol. 2021;32(3):332\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.19378/j.issn.1003-9783.2021.03.005\u003c/span\u003e\u003cspan address=\"10.19378/j.issn.1003-9783.2021.03.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang C, Wang ZP, Li F, Hang Y, Ye ML, Feng Q, et al. Pharmacodynamics of Baijin granules on model rats with sequelae of pelvic inflammatory disease. Chin Tradit Pat Med. 2017;39(7):1329\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3969/j.issn.1001-1528.2017.07.001\u003c/span\u003e\u003cspan address=\"10.3969/j.issn.1001-1528.2017.07.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (in Chinese)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang LJ, Zhu JY, Sun MY, Song YN, Rahman K, Peng C, et al. Anti-inflammatory effect of Man-Pen-Fang, a Chinese herbal compound, on chronic pelvic inflammation in rats. J Ethnopharmacol. 2017;208:57\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jep.2017.06.034\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2017.06.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang JY, Xu LM. Effects of Kunfukang capsule on red blood cell immune function in SPID rats. Chin Tradit Pat Med. 2016;38(10):2253\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3969/j.issn.1001-1528.2016.10.032\u003c/span\u003e\u003cspan address=\"10.3969/j.issn.1001-1528.2016.10.032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong LY, Tian LW, Ma Y, Xie Y, Feng HX, Qin F, et al. Protection of flavonoids from Smilax china L. rhizome on phenol mucilage-induced pelvic inflammation in rats by attenuating inflammation and fibrosis. J Funct Food. 2017;28:194\u0026ndash;204. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jff.2016.11.015\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2016.11.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonz\u0026aacute;lez-Amaro R, D\u0026iacute;az-Gonz\u0026aacute;lez F, S\u0026aacute;nchez-Madrid F. Adhesion molecules in inflammatory diseases. Drugs. 1998; 56: 977\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2165/00003495-199856060-00003\u003c/span\u003e\u003cspan address=\"10.2165/00003495-199856060-00003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Li X, Zhang Q, Li J, Ju J, Du N, et al. Berberine hydrochloride prevents postsurgery intestinal adhesion and inflammation in rats. J Pharmacol Exp Ther. 2014;349(3):417\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jpet.aspetjournals.org/content/349/3/417\u003c/span\u003e\u003cspan address=\"https://jpet.aspetjournals.org/content/349/3/417\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee JH, Sohn JH, Ryu SY, Hong CS, Moon KD, Park JW. A novel human anti-VCAM-1 monoclonal antibody ameliorates airway inflammation and remodelling. J Cell Mol Med. 2013;17(10):1271\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jcmm.12102\u003c/span\u003e\u003cspan address=\"10.1111/jcmm.12102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRana AK, Li Y, Dang Q, Yang F. Monocytes in rheumatoid arthritis: Circulating precursors of macrophages and osteoclasts and, their heterogeneity and plasticity role in RA pathogenesis. Int Immunopharmacol. 2018;65:348\u0026ndash;59. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.intimp.2018.10.016\u003c/span\u003e\u003cspan address=\"10.1016/j.intimp.2018.10.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlpay Z, Saed GM, Diamond MP. Postoperative adhesions: from formation to prevention. Semin Reprod Med. 2008;26:313\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-0028-1082389\u003c/span\u003e\u003cspan address=\"10.1055/s-0028-1082389\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014;1843(11):2563\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbamcr.2014.05.014\u003c/span\u003e\u003cspan address=\"10.1016/j.bbamcr.2014.05.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang J, Yuan S, Wang X, Lei Y, Zhang X, Huang M, et al. Attenuation of pristimerin on TNF-α-induced endothelial inflammation. Int Immunopharmacol. 2020;82:106326. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.intimp.2020.106326\u003c/span\u003e\u003cspan address=\"10.1016/j.intimp.2020.106326\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomano M, Sironi M, Toniatti C, Polentarutti N, Fruscella P, Ghezzi P, et al. Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment. Immunity. 1997;6(3):315\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s1074-7613(00)80334-9\u003c/span\u003e\u003cspan address=\"10.1016/s1074-7613(00)80334-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOh JW, Van Wagoner NJ, Rose-John S, Benveniste EN. Role of IL-6 and the soluble IL-6 receptor in inhibition of VCAM-1 gene expression. J Immunol. 1998;161(9):4992\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.jimmunol.org/content/161/9/4992\u003c/span\u003e\u003cspan address=\"https://www.jimmunol.org/content/161/9/4992\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBogdan C, R\u0026ouml;llinghoff M, Diefenbach A. Reactive oxygen and reactive Nitrogen intermediates in innate and specific immunity. Curr Opin Immunol. 2000;12(1):64\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0952-7915(99)00052-7\u003c/span\u003e\u003cspan address=\"10.1016/s0952-7915(99)00052-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamidzadeh K, Christensen SM, Dalby E, Chandrasekaran P, Mosser DM. Macrophages and the recovery from acute and chronic inflammation. Annu Rev Physiol. 2017;79:567\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-physiol-022516-034348\u003c/span\u003e\u003cspan address=\"10.1146/annurev-physiol-022516-034348\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMussbacher M, Salzmann M, Brostjan C, Hoesel B, Schoergenhofer C, Datler H, et al. Cell type-specific roles of NF-κB linking inflammation and thrombosis. Front Immunol. 2019;10:85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2019.00085\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2019.00085\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNam YJ, Lee CS. Protocatechuic acid inhibits Toll-like receptor-4-dependent activation of NF-κB by suppressing activation of the Akt, mTOR, JNK and p38-MAPK. Int Immunopharmacol. 2018;55:272\u0026ndash;81. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.intimp.2017.12.024\u003c/span\u003e\u003cspan address=\"10.1016/j.intimp.2017.12.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu B, Xu G, Zhang X, Xu L, Zhou H, Ma Z, et al. Paeoniflorin attenuates inflammatory pain by inhibiting microglial activation and Akt-NF-κB signaling in the central nervous system. Cell Physiol Biochem. 2018;47(2):842\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1159/000490076\u003c/span\u003e\u003cspan address=\"10.1159/000490076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyed Hussein SS, Kamarudin MN, Kadir HA. (+)-Catechin attenuates NF-κB activation through regulation of Akt, MAPK, and AMPK signaling pathways in LPS-induced BV-2 microglial cells. Am J Chin Med. 2015; 43(5), 927\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1142/S0192415X15500548\u003c/span\u003e\u003cspan address=\"10.1142/S0192415X15500548\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen J, Luo Y, Li Y, Chen D, Yu B, He J. Chlorogenic acid attenuates oxidative stress-induced intestinal epithelium injury by co-regulating the PI3K/Akt and IκBα/NF-κB signaling. Antioxidants (Basel). 2021; 10(12): 1915. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox10121915\u003c/span\u003e\u003cspan address=\"10.3390/antiox10121915\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pen Yan Jing tablets, SPID, Akt, NF-κB","lastPublishedDoi":"10.21203/rs.3.rs-1495843/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1495843/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Pen Yan Jing tablets (PYJ), a Chinese patent medicine, has been used for sequelae of pelvic inflammatory disease (SPID) effectively. However, the underlying anti-SPID mechanisms of PYJ remain unknown. The Akt/NF-κB pathway plays an important role in promoting inflammation. This study was designed to investigate whether PYJ has preventing effects on SPID by inhibiting Akt/NF-κB pathway.\u003c/p\u003e\u003cp\u003eMethods: A rat model of mixed bacteria liquid plus mechanical damage-induced SPID and a cell model of lipopolysaccharide (LPS)-activated RAW 264.7 macrophages were performed. After PYJ treatment, the morphology of uteri and extent of pelvic adhesion were observed. The pathological changes were evaluated by hematoxylin-eosin (HE) staining. The protein expressions of CD68, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) were identified by immunohistochemistry. Additionally, the viability and NO level of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were calculated by CCK-8 and Griess method, respectively. The tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were detected by ELISA. Protein kinase B (Akt)/nuclear factor kappa-B (NF-κB) pathway-related protein expressions were assayed by western blot. \u003c/p\u003e\u003cp\u003eResults: PYJ not only remarkably alleviated morphological changes of uteri, pelvic adhesion and histological change of chronic inflammation in uteri, but also down-regulated protein expressions of ICAM-1, VCAM-1, MCP-1, COX-2, p-Akt, p65, p-p65 and p-IκBα in uteri. Moreover, PYJ medicated serum inhibited cell viability, NO release, levels of TNF-α and IL-6, and protein expressions of p-Akt, p-p65 and p-IκBα in LPS-activated RAW 264.7 macrophages. \u003c/p\u003e\u003cp\u003eConclusion: Taken together, the preventing effect of PYJ on SPID attributes to the anti-inflammatory activity via inhibiting Akt/NF-κB pathway.\u003c/p\u003e","manuscriptTitle":"Preventing effects of Pen Yan Jing Tablets on sequelae of pelvic inflammatory disease by inhibiting Akt/NF-κB pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-08 14:56:59","doi":"10.21203/rs.3.rs-1495843/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":"3d47b416-91b1-422d-b369-2db134ed84b9","owner":[],"postedDate":"April 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-12T08:59:12+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-08 14:56:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1495843","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1495843","identity":"rs-1495843","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}