Shipi Shugan Decoction Protected against Sequela of Pelvic Inflammatory Disease via Inhibiting SIRT1/NLRP3 Signaling Pathway in Pelvic Inflammatory Disease Rats

Sequela of pelvic inflammatory disease (SPID) is a common and frequently occurring disease clinically. Traditional Chinese medicine (TCM) provided unique advantages in the treatment of SPID. In this study, we aimed to investigate the protective mechanism of Shipi Shugan Decoction (SSD), a Chinese herbal formula, on SPID using a SPID rat model. Mixed bacterial infection and mechanical injury were used for modeling. The chemical composition of SSD was analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). The inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA) and western blot techniques. We found that SSD dose-dependently inhibited the content of IL-18, IL-1β, TNF-α, and IL-6 in serum samples of SPID rats. The results from the hematoxylin and eosin (H&E) stain showed that SSD improved pathological injury of the uterus and fallopian tubes induced by a pathogen. In addition, SSD dose-dependently inhibited mitochondrial dysfunction and oxidative stress of SPID rats. The expression of SIRT1 was promoted, and NLRP3 inflammasome was deactivated by SSD gavage compared with the SPID group. Specifically, SIRT1 inhibitor EX-527 cotreatment significantly reversed the improvement effect of SSD on pelvic inflammatory disease in rats. Taken together, the results of this study suggest that Shipi Shugan Decoction may be an effective TCM for the treatment of SPID.


Introduction
Pelvic infammatory disease (PID) is a common gynecological disease of the upper genital tract infection, including endometritis, salpingitis, ovarian cysts, pelvic peritonitis, and so on [1]. PID is usually caused by ascending infection of cervicovaginal microorganisms, of which the most important pathogens are Neisseria gonorrhoeae and Chlamydia trachomatis [2,3]. Untreated PID can lead to a sequela of pelvic infammatory disease (SPID). Te clinical manifestations of the sequela of pelvic infammatory disease (SPID) are chronic pelvic pain (CPP), infertility, pelvic adhesions, ectopic pregnancy, and recurrent infammation [4]. Women with SPID sufer a lower quality of life in both physical health and psychological wellbeing. Te pathological changes in SPID are tissue destruction, adhesions, hyperplasia, and scar formation. Antibiotics have a clear efect on the elimination of pathogens in the acute phase. However, they have little efect on the improvement of clinical symptoms in the chronic phase [5]. Traditional Chinese medicine (TCM) has certain characteristics and advantages in the treatment of PID [6,7].
A Chinese herbal compound Man-Pen-Fang inhibited the infammation reaction by promoting the apoptosis of infammatory cells and downregulating the serum levels of infammatory cytokines [8]. Chinese herbal medicine Radix Paeoniae Rubra could reduce the infammatory symptoms of chronic PID by reducing the level of PTGS2 [9]. Furthermore, a meta-analysis suggested that Guizhi Fuling Wan decreased the score of traditional Chinese medicine symptoms and improved the therapeutic efect [10]. TCM believes that blood stasis is the basic pathogenesis for the occurrence and development of SPID [7,11]. Pathogenic qi combating qi and blood, qi stagnation of liver, and yang insufcient of the kidney can lead to blood stasis. Blood stasis can obstruct Chong and Conception, and the uterus has a poor operation of qi and blood, eventually forming SPID [12,13]. Tus, the principle of SPID TCM treatment is promoting blood circulation for warming and removing blood stasis, dispersing stagnated liver qi for relieving qi stagnation.
A Chinese herbal compound, Shipi Shugan Decoction (SSD), which is composed of Codonopsis Radix (Dangshen), Zingiber ofcinale Roscoe (Ganjiang), Macrocephalae Rhizoma (Baizhu), Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle (Zhigancao), Radix Bupleuri (Chaihu), Fructus Aurantii (Zhike), Paeoniae Radix Alba (Baishao), Coicis Semen (Yiyiren), Curcumae Rhizoma (Ezhu), and Astmgali Radix (Huangqi) as shown in Table 1 and Supplementary Figure 1A is efective in dispersing blood stasis and dredging collaterals. However, whether the SSD has an ameliorating efect on SPID has not yet been reported. Te present study showed that the therapy of SSD had a signifcant efect on relieving SPID in a rat model. Te efects of SSD on infammation of the uterus and fallopian tubes, oxidative stress, and the histopathological alterations, as well as SIRT1/NLRP3 signaling pathway activity in the uterus, have been addressed. Table 1 and Supplementary Figure 1A. 10 kinds of TCM were soaked for 1 hour and decocted for 2 hours. After that, the juice was concentrated at 100 mL (110 g/100 mL). Te clinically equivalent dose was used as the low-dose SSD. After the juice was concentrated 4 times, it was used as the high dose of SSD.

Preparation of the Drug. Te composition of SSD was shown in
Fuke Qianjin Capsules, as positive control drugs, were purchased from Zhuzhou Qianjin Pharmaceutical Co., Ltd., (Z30020024; Zhuzhou, China). Te equivalent dose for rats was 0.22 g/kg • day based on the calculation of animal dose and human equivalent dose for the drug [14].

Animals Grouping and Experimental
Design. 80 Sprague-Dawley (SD) female rats (SPF grade, aged 12 weeks, body weight from 200 to 220 g) were purchased from Chengdu Dossy Experimental Animals CO., LTD. (Chengdu, Sichuan; SCXY (Chuan) 2020-034) and raised in the Hospital of Chengdu University of Traditional Chinese Medicine. Te feeding environment was 21 to 23°C, with a relative humidity of 40%-60% and a 12 h light-dark cycle. SD rats are allowed to eat and drink freely. All rats were adaptively fed for 7 days before the experiments. All experiments were approved according to the Ethics Committee of the Hospital of Chengdu University of Traditional Chinese Medicine (2022DL-004).
For the frst part of the experiment, 40 SD rats were divided into 5 groups using a random number table, with 8 rats in each group. 5 rats died (mortality rate 12.5%) in the process of the experiment probably due to low activity and poor diet, and 35 rats have remained. Te number of rats used for follow-up experiments was as follows: sham group (n � 8), SPID group (n � 6), high-dose SSD group (n � 8), low-dose SSD group (n � 6), and Fuke Qianjin Capsules (FQC) group (n � 7). After the SPID model was established, the rats in the high-dose SSD group and low-dose SSD group were intragastrically administered with 6.64 g/kg • day and 1.66 g/kg • day SSD, respectively. Te rats in the FQC group were intragastrically administered with 0.22 g/kg • day after the suspension of Fuke Qianjin Capsules. Te rats in the sham group and SPID group were given the same amount of normal saline. For the second part of the experiment, 40 SD rats were divided into 5 groups using a random number table, with 8 rats in each group. 7 rats died (mortality rate 17.5%) in the process of the experiment probably due to low activity and poor diet, and 33 rats have remained. Te number of rats used for follow-up experiments was as follows: sham group (n � 8), SPID group (n � 6), SSD group (n � 6), SIRT1 inhibitor (EX527) group (n � 6), and SSD + EX527 group (n � 7). After the SPID model was established, the rats in the EX527 group were intragastrically administered with a 5 mg/kg/day SIRT1 inhibitor (Sigma, Munich, Germany; No. E7034). Te rats in SSD + EX527 group were intragastrically administered with 5 mg/kg/day and 6.64 g/kg • day SSD. Te drug interventions in all rats were continued for 14 days. Ten, all rats fasted for 12 h. Blood samples, uterus, and fallopian tube tissues were collected for analysis.

Rat SPID Model Construction.
Mixed bacterial infection and mechanical injury were used for modeling. Rats were anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital sodium. Regarding mechanical injury, the endometrial tissue was injured using a blunt needle-tipped syringe, which entered the uterine cavity and was pulled back and forth twice along the uterine wall. Regarding simultaneous bacterial infection, the mixed bacterial solution was composed of Staphylococcus aureus (S. aureus, No. 44152) and pathogenic Escherichia coli (E. coli, No. 26002). 0.1 mL (1 × 10 8 CCU/mL; 1 : 1) of the bacterial solution was injected into the uterine cavity, and the rats were placed upside down for 3 minutes. Te rats in the sham group were treated with the same volume of normal saline without damaging the endometrial tissue. Te whole blood cell count in the rats after modeling was assessed every week. Te SPID model was formed when the number of total leukocytes and total lymphocytes returned to normal.

Hematoxylin and Eosin (H&E) Stain.
After fxation in 3% glutaraldehyde and 1% osmium tetroxide for 2 hours, uterus and fallopian tube tissues were used for H&E stain. Uterus and fallopian tube tissues were embedded in epoxy resins to prepare 5 μm thick parafn sections under electron microscopy. After deparafnization with diferent concentrations of acetone, the sections were stained with hematoxylin and eosin. Six image area visual felds were randomly selected and assessed using a Panthera Upright Compound Microscope (Motic, Xiamen, China) under 100× and 400× magnifcations.
2.6. Transmission Electron Microscopy (TEM). 5 μm thick parafn sections of the uterus and fallopian tube tissues were collected on a 300-mesh copper-mesh TEM grid. Te sections were stained with uranyl acetate for 10∼15 min and lead citrate for 1∼2 min. A transmission electron microscope (JEM-1400FLASH, JEOL, Japan) was used to collect images of the TEM grid. Each TEM grid was observed and photographed at 6000× magnifcation to observe the specifc lesions.

Oxidative Stress Biomarker Assay.
Te contents of glutathione peroxidase (GSH-Px, No. A005-1-2) in serum samples were detected by the colorimetry method using a microplate reader according to the kit instructions (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). Superoxide dismutase (SOD) content in serum samples was measured using the SOD assay Kit-WST1 (No. A001-3-2). Malondialdehyde (MDA) content in serum samples was detected by the method of thiobarbituric acid (TBA) according to the manufacturer's instructions (No. A003-1-2).

Mitochondrial Membrane Potential (ΔΨm) Assay.
A mitochondrial-specifc fuorescence probe JC-1 (Beyotime, Shanghai, China; No. C2006) was used to test ΔΨm. Briefy, uterine tissue sections were incubated with a mixture of JC-1 stain at 37°C for 20 min. A FACS Calibur Flow Cytometer (BD Biosciences) was used to analyze the fuorescence intensity of JC-1aggregates (red) and monomers (green). 21175D-96) in serum samples were quantifed according to the instructions of the ELISA kits. In brief, uterus and fallopian tube tissues were placed in precooled phosphatebufered saline (PBS) and homogenized. Te supernatant was collected by centrifugation at 4000 r/min for 10 min at 4°C. Te BCA assay kit (Termo Fisher Scientifc, USA) was used to determine the protein content of the supernatant. 15 ng/μL of the protein samples was used for incubation with corresponding antibodies. Te absorbance was measured at 450 nm wavelength and was estimated using an enzymelinked immune monitor (Termo Fisher Scientifc, USA). Te concentration of these proteins in the sample was calculated from the standard curve.

SSD Dose-Dependently Inhibited Mitochondrial Dysfunction and Oxidative Stress of SPID Rats.
It is well documented that mitochondrial dysfunction can lead to a decline in energy production, generation of ROS, and induction of stress-induced apoptosis. Oxidative damage is a major cause leading to infammation response. Tus, we studied the efect of SSD on mitochondrial oxidative stress of uterine tissues. Te mixed bacterial solution treatment signifcantly promoted mitochondrial injury in uterine tissues, which was reversed by the positive control drug FQC and diferent concentrations of SSD (Figure 2(a)). Meanwhile, the ΔΨm was detected by JC-1 fuorescence stain. Te decreased level of ΔΨm was observed in uterine tissues of SPID rats, and this increase was hindered by SSD administration in a dose-dependent manner (Figures 2(b) and 2(c)). We then confrmed that SSD and positive control drug FQC suppressed ROS and MDA expression, as well as increased GSH-Px and SOD expression compared with that in the SPID rat group (Figures 2(d)-2(g)).
Tese results implied that the partial recovery of mitochondrial biogenesis was achieved by SSD administration.

Modulation of SSD on SIRT1/NLRP3 Signaling.
Because SIRT1/NLRP3 signaling was the critical pathway in oxidative stress and infammation induction, we explored the alteration of SIRT1/NLRP3 signaling pathway activity in SPID rat uterine tissues before and after TCM treatment. As shown in Figures 3(a) and 3(b), the SPID rat group showed a lower protein expression of SIRT1 than the sham group.
After treatment with FQC and SSD, especially for a highdose group of SSD and FQC, the protein expression of SIRT1 was increased in uterine tissues of SPID rats (Figures 3(a)  and 3(b)). Subsequently, we measured the expression levels of NLRP3 infammation-related proteins NLRP3 and caspase-1, which were evident diferences between groups of sham and SPID model groups (Figures 3(a), 3(c), and 3(d)).
After treatment with SSD, the expression of NLRP3 and caspase-1 was decreased in a dose-dependent manner (Figures 3(a), 3(c), and 3(d)). Te data demonstrated that SSD stimulated SIRT1 and suppressed NLRP3 infammasome activation in uterine tissues of SPID rats.

SSD against Infammation and Pathological Damage of the Upper Genital Tract by Inhibiting SIRT1/NLRP3 Signaling.
We then focused on whether the SIRT1/NLRP3 signaling mediates the anti-infammatory and antipathological damage efects of SSD. Te SIRT1-specifc inhibitor EX527 was used to treat SPID rats, which decreased the protein expression of SIRT1 and increased the expression of NLRP3 and caspase-1 (Figures 3(e)-3(h)). SIRT1 inhibition resulted in increased levels of TNF-α, IL-6, IL-18, and IL-1β which were reduced by SSD treatment (Figures 4(a)-4(d)). In addition, the results of the H&E stain showed that EX527 treatment could block the ameliorative efect of SSD on endometrial tissue injury and infammation reaction in the upper genital tract (Figures 4(e) and 4(f )). Te data indicated that the activation of SIRT1/NLRP3 signaling reversely hindered the anti-infammatory and antipathological damage efects of SSD on SPID rats.

SSD against Mitochondrial Dysfunction and Oxidative
Stress by Inhibiting SIRT1/NLRP3 Signaling. TEM experiments showed that compromised mitochondria were redetected in the uterine tissue of SPID rats coprocessed with SSD and SIRT1 inhibitor EX527 ( Figure 5(a)). Furthermore, we found that after SPID rats were treated with

Discussion
Te results of this study indicated that SSD improved infammatory damage of the upper genital tract and reduced mitochondrial oxidative stress in uterine tissues of SPID rats. Te related mechanism is to stimulate the expression of SIRT1 and inhibit the activity of the NLRP3 infammasome. SSD included Codonopsis Radix, ginger (Zingiber ofcinale Roscoe), Macrocephalae Rhizoma, Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, Radix Bupleuri, Fructus Aurantii, Paeoniae Radix Alba, Coicis Semen, Curcumae Rhizoma, and Astmgali Radix. Each of them has been extensively used in the treatment of infammatory diseases. Codonopsis Radix was rich in various chemical constituents, including terpenoids, saponins, alkaloids, and phenolic compounds with anti-infammatory and immunomodulatory properties [15]. Te bioactive constituents of ginger contained terpenes, polysaccharides, lipids, and organic acids, which had antioxidant, anti-infammatory, and antimicrobial efects [16]. Another previous study reported that the application of Macrocephalae Rhizoma and   Astmgali Radix inhibited the generation of proinfammatory cytokines in the lipopolysaccharide (LPS)-induced chronic infammation model [17]. Moreover, clinical trials have shown that glycyrrhetinic acid and glycyrrhizic acid from Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle were efective against infammatory intestinal diseases [18]. Radix Bupleuri, Fructus Aurantii, and Paeoniae Radix Alba have been demonstrated to reduce the infammatory factors and increase anti-infammatory factors in the serum of depressive-like rats [19]. Coicis Semen was applied for treatment with ischemia-reperfusion injury, and it was proved to inhibit oxidative stress and promote angiogenesis [20]. Notably, TCM compounds with Curcumae Rhizoma, Astmgali Radix, Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle, and Radix Bupleuri as the core medicines had signifcant efects on patients with CPP caused by SPID [21]. In our study, we determined that SSD suppressed the generation of proinfammatory factors in serums and infammatory cell infltration in the uterus and fallopian tubes of SPID rats, implying that SSD could efectively eliminate the pelvic infammatory disease in in vivo study.
Mitochondrial dysfunction and oxidative stress injury were important pathophysiological mechanisms of PID [22,23]. It was reported that the reduced activity of mitochondrial respiratory chain complex 1 could promote ROS accumulation, leading to depolarization of mitochondrial membrane potential and changes in membrane permeability [24,25]. Superoxide radicals, hydrogen peroxide, hydroxyl radicals, and so on are collectively referred to as ROS [26,27]. Te excessive release of ROS induced cell damage and changed cell function by regulating protein activities and gene expression. A large amount of oxidative stress product MDA was produced when the local infammation waterfall was formed in the SPID rat model, which in turn induced oxidative stress injury [28]. SOD and GSH-Px, a class of antioxidant enzymes, had the efect of scavenging oxygen-free radicals [29,30]. In the present study, the results show that SSD concentration dependently protested mitochondrial dysfunction, exhibiting similar efcacy to positive drug. Additionally, SSD enhanced SOD and GSH-Px levels and reduced MDA and ROS production in serum samples of SPID rats. SIRT1 was a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that participated in a variety of physiological and pathological processes and played an important role in infammatory diseases [31,32]. Te study has shown that SIRT1 can participate in the regulation of cell activities such as infammation, oxidative stress, and mitochondrial function [33,34,35]. Specifcally, SIRT1 inhibited the occurrence and development of infammation by regulating the expression of NLRP3 [36,37]. NLRP3, apoptosis-associated speck-like protein (ASC), and pro-caspase-1 made up the NLRP3 infammasome, subsequently activating caspase-1. Activated caspase-1 further catalyzed the secretion of proinfammatory cytokines IL-1β and IL-18 and fnally induced infammatory responses and impairments [38,39,40]. A review revealed that the expression of NLRP3 infammasome was increased in the endometrium of women with unexplained recurrent pregnancy loss (RPL) [41]. A previous study found that Gardnerella vaginalis induced the expression of NLRP3 infammasome-dependent cytokines IL-1β, IL-18, as well as TNF-α in infammation of the genital tract [42]. Melatonin improved mouse endometritis by constraining the level of NLRP3 activation [43]. Meanwhile, melatonin attenuated pelvic pain caused by infammation of the prostate by downregulating SIRT1-dependent inhibition of the NLRP3 infammasome. In the present study, we found that SSD exerted a protective efect on pelvic infammatory disease through activation of the SIRT1 signaling and elimination of the NLRP3 infammasome. Our results were similar to previous reports that various components of SSD have signifcant inhibitory efects on SIRT1/NLRP3 signaling activity. 6-Gingerol, a phenolic compound extracted from ginger, signifcantly suppressed autophagy-induced NLRP3 infammasome and neuronal apoptosis [44]. Te fermented Chinese formula Shuan-Tong-Ling, including Codonopsis Radix, Macrocephalae Rhizoma, Radix Bupleuri, Paeoniae Radix Alba, Astmgali Radix, and so on, protected against cerebral ischemia/ reperfusion injury by reducing infammation and apoptosis through activation of the SIRT1 signaling pathway [45]. Te compounds from Fructus Aurantii signifcantly inhibited liver infammation in liver fbrosis mice by reducing NLRP3 expression [46]. Coixol, a plant polyphenol extracted from Coicis Semen, exerted anti-infammatory efects on LPS-induced macrophage cells and diminished NLRP3 infammasome activation [47]. In this study, the components of SDD synergistically improved the development of SPID by activating SIRT1 and inhibiting NLRP 3 infammasome.

Conclusions
Our fndings suggest that SSD reduced proinfammatory factor levels in serum and inhibited infammation injury of the uterine and fallopian tube in SPID rats. Additionally, SSD inhibited mitochondrial dysfunction, and oxidative stress, promoted SIRT1 activation, and caused downregulation in the protein expression of NLRP3 infammasome. Importantly, SIRT1 inhibitor EX527 signifcantly reversed the protective efect of SSD on SPID. Our results may provide new therapeutic approaches against SPID. Further research is needed to validate the clinical efectiveness of SSD.

Data Availability
Te datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.