Xuanhuang Runtong Tablets Relieve Slow Transit Constipation in Mice by Regulating TLR5/IL-17A Signaling Mediated by Gut Microbes

This study aims to investigate the regulation effects of Xuanhuang Runtong tablets (XHRTs) on intestinal microbes and inflammatory signal toll receptor 5 (TLR5)/interleukin-17A (IL-17A) in STC mice. First, high-performance liquid chromatography (HPLC) was used to verify the composition of XHRT and quality control. Then, the defecation ability of STC mice was evaluated by measuring fecal water content and intestinal transit function. The pathological examination of colonic mucosa was observed by Alcian Blue and periodic acid Schiff (AB-PAS) staining. 16S ribosomal DNA (16S rDNA) genes were sequenced to detect the fecal microbiota. Western blotting, immunofluorescence, and real-time fluorescence quantitative PCR (qRT-PCR) were applied to detect the expression of aquaporin 3 (AQP3), connexin 43 (Cx43), TLR5, and IL-17A. The defecation function of the STC mice was significantly decreased. The amount of mucus secretion and the thickness of the colonic mucus layer were decreased, and the number of microbial species in the intestinal wall, such as Firmicutes/Bacteroidetes, anaerobic bacteria, and Alistipes, were also decreased. In addition, the expression of AQP3 and Cx43 was disordered, and the inflammatory factorsTLR5 and IL-17A were activated in the colon. The changes in the above indicators were significantly reversed by XHRT. This study demonstrates that XHRT provides a new strategy for the treatment of slow transit constipation by regulating the activation of the intestinal inflammatory signal TLR5/IL-17A mediated by gut microbes.


Introduction
Slow transit constipation (STC) is a chronic gastrointestinal disease characterized by intestinal transit dysfunction. Te main clinical manifestations of patients include prolonged defecation, reduced defecation frequency, and abdominal distension and pain [1]. Te global prevalence of STC is estimated to be as high as 14%, particularly 36% among the elderly. Under severe conditions, STC can cause acute intestinal obstruction or intestinal necrosis perforation and has been identifed as a high-risk factor for some gastrointestinal diseases, such as colorectal tumors [2]. Te pathogenesis of STC includes infammation, secretory dysfunctions, gastrointestinal innerves changes, and various abnormalities in gastrointestinal microstructures, the interaction of various factors makes drug treatment difcult [3]. Currently, volumetric, osmotic, or secretory laxatives are commonly used to relieve constipation, but these therapies often fail or have only short-term efcacy and induce side efects [4]. In addition, previous studies indicate that STC surgery has a variable success rate (39-100%), with a high recurrence rate [5]. Terefore, it is very essential to research on a drug that can efectively alleviate and treat STC.
Traditional Chinese medicine (TCM) has a long history of understanding constipation, which was frst recorded as "difculty to defecate" in "Neijing." STC belongs to the category of "constipation" in TCM, which is one of the dominant diseases treated by TCM. Based on TCM theory, the body's internal heat and a defciency of body fuid trigger STC. Treatment mainly focuses on invigorating Qi and nourishing Yin, moistening the intestines, and relieving constipation [6]. Various academic publications have reported on the Chinese herbal extracts'efects in treating intractable diseases [7][8][9], and the efects in treating constipation have also been confrmed [10].
Xuanhuang Runtong tablets (XHRTs) is a prescription composed of 9 Chinese herbal medicines, including Rehmannia glutinosa Libosch, Scrophulariae radix, Angelicae sinensis radix, Persicae semen, Cistanches herba, Cannabis fructus, Cassiae semen, and Aurantii fructus immaturus. Te laxative efect of these Chinese herbal medicines has long been reported in the literature [11][12][13][14]. XHRT has a positive curative efect on functional constipation caused by yin defciency, blood stasis, and senile functional constipation with reasonable safety and few side efects. However, the exact mechanism of action has not been determined.
An increasing number of studies have shown that gastrointestinal dysfunction is closely related to disorders of the host microbiota; a change has been observed in the microbial structure of STC patients, and clinical symptoms have been improved signifcantly after fecal transplantation [16,17]. Deng et al. also found that controlling intestinal microbial disorders through a mixture of bacteria could improve loperamide-induced slow transit constipation in rats [18]. Several immunologically active structures protect the mucosal surface of the gastrointestinal tract from antigens and microorganisms [19]. If the intestinal microbiota is disturbed, it can damage intestinal mucosal cells, destroy mechanical and immune barriers, and afect intestinal immunity [20]. One of the key factors of STC may cause abnormal information exchange between the intestinal microbiota and the intestinal mucosal immune system. Intestinal epithelial cells can express a variety of pattern recognition receptors, including toll-like receptors (TLRs) and nucleotide oligomerization domains (NODs). Tese stimulate intestinal epithelial T17 cells to produce proinfammatory cytokines, thus directly afecting the intestinal microbiota [21].
Tus, elucidation of the role of intestinal microorganisms in the intestinal immune barrier would provide insights into the mechanism whereby XHRT might be of value in the treatment of STC.  [22], a mouse STC model induced by loperamide hydrochloride was established with slight modifcations. We randomly divided ICR mice into the control group (Control), slow transit constipation (STC) model group, and XHRT high-, medium-, and low-dose (XHRT-H, XHRT-M, and XHRT-L) groups. For 14 days, mice in all groups were gavaged with loperamide hydrochloride solution (10.0 mg/kg) twice a day except for the control group. Diferent doses of XHRT solution (4.056, 2.028, and 1.014 g/kg, respectively) were administered for 7 days following each administration of loperamide hydrochloride for 1 h in the XHRT-H, XHRT-M, and XHRT-L groups.

Quantitative Analysis of the Main Active Ingredients of XHRT.
XHRT was extracted with methanol and fltered through a 0.45 μm microporous membrane. Aloin (0.9235 mg/mL) was dissolved in the mobile phase methanol-0.1% glacial acetic acid aqueous solution (39 : 61). Naringin, neohesperidin, echinacoside, and aloe rhubarb were dissolved in methanol and formulated into reference solutions, with mass concentrations of 0.7561, 0.7510, 0.5196, and 0.5488 mg/mL, respectively.

Intestinal Transit Function Analysis.
On days 0 and 7th of the experiment, the feces of mice were collected and the dry weight of feces was measured to determine whether the model was successfully replicated. Te intestinal transit function of the mice was evaluated by a defecation experiment and a small intestine exercise experiment as previously mentioned [22]. On the 14th day, we recorded the time of the frst black fecal mass, the number of fecal particles, and the water content within 6 h of the mice (fecal water content � (wet feces weight-dry feces weight)/wet feces weight × 100%). On the 15th day, the small intestine from the pylorus to the ileocecal area of the stomach was collected. Both the length of the small intestine and that of ink advancement were measured to quantify the intestine's transport capacity.

Alcian Blue and Periodic Acid Schif (AB-PAS) Staining.
Te proximal colon tissue was fxed in a 4% paraformaldehyde solution, dehydrated by gradient ethanol, and cut into 5 μm thick parafn sections. It was stained with AB-PAS and sealed with neutral resin. Among them, glycogen-and polysaccharide-positive cells were purple-red and the nucleus was blue. Under an optical microscope (Olympus, Tokyo, Japan), we observed positively stained colonic mucosal epithelial cells and analyzed mucus thickness and secretion.
2.6. 16S rDNA Gene Testing. A 16S rDNA gene sequencing technique was used to analyze the community structure of the mouse gut microbiota, as previously described with some modifcations [23]. Microbial community genomic DNA was extracted from fecal samples using the E. Z. N.
Te sequencing depth was assessed with the dilution curve generated by OTU clustering. OTU-level alpha diversity indices, such as Chao1, observed species, and Shannon indices, were calculated and analyzed to compare abundance and diversity between samples. A beta diversity analysis was used to examine the structural variation of microbial communities across samples. Based on species with relative abundance >1%, the relative abundance of intestinal mucosal bacteria in mice of diferent groups was compared at the phylum, family, and genus levels.

Western Blotting Analysis.
Protein expressions of AQP3 and Cx43 were detected using western blotting. Total proteins were extracted from colon tissues using RIPA lysis bufer by following the manufacturer's instructions. Te protein concentration was determined by using the BCA protein quantifcation kit. 50 μg of protein was boiled at 100°C for 5 min, separated by SDS-PAGE, and transferred onto PVDF membranes (Millipore). Ten, 5% lipid-free milk was used to block the membrane, and the primary antibody diluted with TBST was incubated overnight at 4°C. Primary antibodies targeting AQP3 (A2838; ABclonal), Cx43 (26980-1-AP; Proteintech), and actin (66009-1-Ig; Proteintech) were applied. Te membrane was washed with TBST, and the secondary antibody was incubated for 1 h. Te hybrid flm was developed in a cassette with X-flm and exposed for 1-30 min. Ten, the protein bands were analyzed by using Quantity One gel analysis software.
2.9. Immunofuorescence Analysis. Te colon was fxed in 4% paraformaldehyde for 48 h, embedded in parafn, and cut into 5 mm sections. Te sections were baked at 60°C for 12 h and blocked with 10% normal serum/5% BSA, followed by incubation overnight at 4°C in solution with TLR5 (19810-1-AP; Proteintech) and IL-17A (ab79056; Abcam) polyclonal rabbit antimouse antibodies (Proteintech, Wuhan, China), respectively. After washing, the sections were incubated with the goat antirabbit IgG antibody. Te sections were then washed again, incubated in the sDAPI working solution at 37°C for 10 min, and were sealed and observed under a laser confocal microscope (TermoFisher, New York, USA). Tree felds (×400) were selected from each section for the positive cell count.
2.11. Statistical Analysis. Statistical analysis was performed using SPSS 22.0 software (Chicago, IL, USA). Te results were expressed as the mean ± standard deviation (SD). Oneway ANOVA was used to compare the results (GraphPad Prism 5.0, GraphPad Software, USA); P < 0.05 indicated that there were signifcant diferences.

Quality Control and Identifcation of XHRT.
Te chemical components of XHRT samples were generated by HPLC fngerprints and common peaks by using the similarity evaluation system of traditional Chinese medicine chromatographic fngerprints. Five components, including peak 2 for echinacoside, peak 4 for naringin, peak 5 for neohesperidin, peak 9 for aloin, and peak 15 for aloeemodin, were analyzed ( Figure 1). Te contents of echinacoside, naringin, neohesperidin, and aloin were 0.44%, 1.66%, 1.77%, and 1.76%, respectively. Te obtained results have shown that our research established a suitable and reproducible chromatographic fngerprint for the quality control of XHRT.

Efect of XHRTon Intestinal Transit Function of STC Mice.
We compared the dry weight of feces in the control group and the STC model group at days 0 and 7 and found that the feces of STC mice were dry-knotted and in the form of separated clumps or hard masses of feces. Te dry weight of feces at 24 h had a signifcant reduction (Figure 2(a)), indicating that the model was successfully replicated. After the mice were given diferent doses of XHRT solution (4.056, 2.028, and 1.014 g/kg), there was no signifcant diference in body weight (Figure 2(b)). Tis indicated that XHRT had no efect on the basal metabolism of mice. Compared with the STC model group, the time of the frst black stool was signifcantly reduced and the total amount of black stool and the water content of stool increased within 6h in the XHRT group (Figures 2(c)-2(e)). Te small intestine propulsion rate of the STC model group was signifcantly lower than that of the control group, showing that XHRT signifcantly improved small intestinal transport (Figures 2(f)-2(h)). Tese results indicated that XHRT could present a gradient dosedependent manner to enhance intestinal transit and promote defecation in STC mice.

Efect of XHRT on the Morphological Structure of the Colonic Mucosa in STC Mice.
By staining the colonic epithelial cells with AB-PAS (Figure 3(a)), we found abundant acidic mucins with sulfur mucin and densely distributed goblet cells on both sides of the crypt, with a full and round morphology and a large amount of secreted mucus in the control group. In the STC model group, the mucosal epithelial cells of the colon tissue of the mice showed diferent degrees of atrophy. Tere was a reduction in the number of goblet cells, and the acidic mucous layer on the mucosal surface became thinner. In the XHRT-H group, the thickness of the mucous layer on thecolonic mucosa was signifcantly increased (Figure 3(b)) and the number of goblet cells and mucus secretion signifcantly increased (Figure 3(c)).

Microbial Abundance Change Analysis.
To explore the potential participation of the gut microbiota in the efects of the treatment of STC mice with XHRT, we analyzed the community structure of intestinal microbiota in mouse feces using 16S rDNA microbiota profling. An analysis of the number of operational taxonomic units (OTUs) within the bacterial colony and their crossover between groups was performed using a Venn diagram, and there were 1226 OTUs in the control group, 1151 OTUs in the STC model group, 1228 OTUs in the XHRT-H group, 1241 OTUs in the XHRT-M group, and 1215 OTUs in the XHRT-L group (Figure 4(a)). Te sparse curve fattened as the Chao1 index increased, indicating the preferred sequencing depth and high species coverage (Figure 4(b)). Tere was a signifcant diference in the abundance of fecal intestinal fora Evidence-Based Complementary and Alternative Medicine between the STC model group and the control group. After diferent doses of XHRT treatment, the abundance of intestinal fora in the mice was signifcantly increased. PLS-DA analysis was performed to estimate the percentage diference in bacterial abundance between samples, and the results showed obvious diferences among each group (Figure 4(c)).

Microbial Community Diversity
Analysis. Te alpha diversity index was used to analyze species diversity in feces, taking into account two factors: the richness and uniformity of species composition. Te observed species index of the STC model group was signifcantly lower than that of the control group. XHRT substantially increased the observed species index, Shannon index, and Chao1 index of the intestinal microbes of STC mice (Figures 5(a)-5(c)), which indicated that XHRTcould efectively restore the intestinal microbe alpha diversity of STC mice. Beta diversity evaluated the diferences between microbial communities by comparing the composition of microbial communities. Te diference between the samples was analyzed using the unweighted pair group method with arithmetic mean (UPGMA). Te similarity between the samples was observed in the distance of branches and the distance of clusters. Tis resulted in a signifcant diference between the bacterial communities of the control group and the STC model group (Figure 5(d)). Deviating from the STC model group, the XHRT group was close to the control group. Tese results indicated that XHRT could improve the composition and diversity of intestinal fora in STC mice.

Microbiota Species Annotation and Diference Analysis.
At the phylum level, this study identifed Firmicutes, Bacteroides, Actinomycetes, Campylobacter, and Desulfobacterota from 40 samples. Te relative abundance of Firmicutes and Bacteroides was relatively high, and the ratio of Firmicutes/Bacteroidetes (F/B) refected the health status of the body. In comparison with the control group, its relative abundance signifcantly decreased in the STC model group but recovered with XHRT treatment (Figures 6(a) and 6(d)). At the order level, Lactobacillus abundance in the STC model group was signifcantly lower than that in the control group. In contrast, Lactobacillus abundance in the XHRT group was higher than that in the STC group ( Figure 6(b)). At the genus level, we found that the relative abundance of    Lactobacillus and norank_f_Muribaculaceae in the STC model group decreased and increased after XHRT intervention compared with the control group (Figures 6(c), 6(e), and 6(f )). We obtained a total of 135 bacterial genera from the samples, mainly Lactobacillus, norank_f_Muribaculaceae, Bacteroides, Faecalibaculum, and Staphylococcus ( Figure 6(g)), covering more than 70% of all microbial species. Tese results suggested that the changes in the intestinal microbial community structure during the pathogenesis of STC may be reversed by XHRT treatment.

Efect of XHRT on the Expression of Protein AQP3 and
Cx43 in STC Mice. Western blot analysis showed that the relative gray value of Cx43 protein was signifcantly reduced in the colon tissue of the STC model group, while that of AQP3 protein was signifcantly increased compared with the control group. After XHRT treatment, the relative gray values of AQP3 and Cx43 protein were reversed (Figures 7(a) and 7(b)). As a result of XHRT, colon feces in STC contained more water and promoted defecation, which was closely related to the expression of water secretion protein AQP3 and grassroot-related protein Cx43.

Efect of XHRT on the Expression of Intestinal Mucosal
Immune Barrier Regulation-Related Proteins and Genes in STC Mice. Using immunofuorescence (IF), we determined the expression of mouse colon tissue protein TLR5 and T17 cellrelated factor IL-17A to further investigate the molecular mechanism of XHRT on defecation and intestinal mucosal immune function in STC mice. Using laser confocal microscopy, we observed the localization of each protein and the fuorescence intensity (Figure 8(a)). Compared with the control group, the fuorescence intensity of TLR5 and IL-17A proteins in the colon tissue of the STC model group was increased. Te relative fuorescence intensity of TLR5 and IL-17A proteins in posterior colon tissue was signifcantly reduced by XHRT administration (Figure 8(b)). At the same time, we detected the expression of TLR5 and IL-17A mRNA in colon tissue by qRT-PCR (Figure 8(c)) and found that the relative expression of TLR5 and IL-17AmRNA in the STC model group signifcantly increased compared with that in the control group. Based on the abovementioned protein detection trend, the relative expression of TLR5 and IL-17A mRNA in the colon tissues of mice in the XHRT-H was signifcantly reduced. Tese results revealed that XHRT could effectively modulate the immune function of the intestinal mucosa  Figure 6: In STC mice treated with XHRT, the relative abundance and family-level composition of the phylum (a), order (b), and genus (c) were assessed. Te signifcance of diferences between multiple groups at the phylum level through species diferences was analyzed (d). Signifcant comparison of the diference in bacteria between the two groups and classifcation levels of intestinal microbes at the genus level (e-g).
by regulating the expression of the intestinal TLR5/IL-17A signaling pathway.

Discussion
Slow transit constipation is one of the most common types of refractory functional constipation being studied by scholars. Te pathogenesis of STC involves multiple factors, but little is known about its pathophysiology and etiology. Among them, colonic sensory motor dysfunction and decreased sensitivity are the most widely recognized causes. As a result of these abnormal changes, the intestinal mucosal barrier is unable to maintain its normal structure and function for a prolonged period [24]. Intestinal mucosal barriers include mechanical, biological, and immune barriers, which interact to play a synergistic role. In current studies, it has been found that patients with functional constipation experience changes in intestinal mucosal permeability. Tese changes participate in the pathogenesis of constipation by infuencing intestinal motility, intestinal sensitivity, and intestinal immune status [25]. However, the underlying molecular mechanism remains unclear.
As the frst barrier of intestinal mucosal defense, the mechanical barrier consists of intestinal epithelial cells, mucins, electrolytes, and water on the surface of the intestinal lumen, lubricating the intestine and providing a habitat for intestinal commensal bacteria. In patients with constipation, there is a signifcant decrease in the secretion of goblet cells (mainly secreting mucins) in the colonic mucosa and the thickness of the mucus layer is reduced, which was also confrmed in our study.
Te structural basis of the mechanical barrier is mainly composed of junctional complexes between intestinal epithelial cells. Te role of claudin in maintaining the structure of the intestinal mucosal mechanical barrier in patients with constipation has been reported in several studies [26,27]. A class of connexins found in colonic epithelial cells is closely related to gastrointestinal slow waves and gastrointestinal motility disorders. Among them, abnormal expression of Cx43 may cause gastrointestinal diseases, such as gastrointestinal tumors, Hirschsprung's disease, and functional dyspepsia [28,29]. A study by Bhave et al. [30] revealed that the expression of Cx43 protein in the intestinal mucosa of functionally constipated patients was signifcantly reduced. Tis occurred because its abnormal expression could cause changes in the intestinal mucosa and infammation. Te regulation of intestinal water fuid is an important manifestation of mechanical barrier function. Epithelial cells of the colonic mucosal villi have aquaporin AQPs located at the top and bottom of their plasma membranes, which are involved in the transmembrane transport of water. Tis plays a pivotal role in the regulation of intestinal absorption, secretion, and water metabolism [31]. When the expression level of its critical isoform AQP3 increases, the intestinal contents absorb too much water, causing the accumulation of colonic contents, which can lead to difculty in defecation [32]. Our study shows that XHRT could signifcantly improve the structural and molecular changes in the colonic mucosal mechanical barrier in STC mice. Further research was conducted to determine whether these were related to the synergistic regulation of the biological barrier and immune barrier.
According to a literature survey, the gut bacteria B. thetaiotaomicron and Faecalibacterium prausnitzii promote goblet cell diferentiation and induce mucin glycosylation-related genes in the biological barrier [33]. Using the active substance of sennoside A metabolized by the intestinal fora, Kon et al. [34] demonstrated that macrophages secrete PGE2. Tis reduces AQP3 expression in the colonic epithelium of rats, prevents the colon from absorbing water, and promotes defecation. Based on the available evidence, we can speculate that XHRT modulates the structure and function of the mechanical barrier while altering the composition and metabolism of gut microbes symbiotic in the mucus layer, thereby afecting defecation. Gut microbes constitute a biological barrier against pathogens. In recent years, breakthroughs in the feld of intestinal microbiology have provided new strategies for the  Figure 8: XHRT regulated the expression of TLR5/IL-17A related to the intestinal mucosal immune function in the STC mice induced by loperamide hydrochloride (a).Immunofuorescence method used antibodies targeting TLR5 and IL-17A proteins, observed the protein localization and fuorescence intensity under a laser confocal microscope, and used the average fuorescence intensity of the protein to refect the relative expression level of each protein (b) (400×). (c) Te relative expression of TLR5 mRNA and IL-17A mRNA in colon tissue by realtime fuorescent quantitative PCR with GAPDH as an internal reference was detected. STC: slow transit constipation; XHRT: Xuanhuang Runtong tablet; TLR5: toll receptor 5; IL-17A: interleukin-17A; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. Values were expressed as the mean ± SD, * * P < 0.01 vs. control group; # P < 0.05, ## P < 0.01 vs. STC model group.
diagnosis and treatment of slow transit constipation. Due to changes in the modern lifestyle and diet structure, the composition and diversity of the intestinal microbiota have changed, causing symptoms such as constipation. Additionally, constipation can aggravate the imbalance of intestinal microbes and even cause colon cancer, accelerate aging, and promote a variety of intestinal diseases [2,3,35]. Studies have found that compared with healthy controls, the number and quality of benefcial bacteria (such as Lactobacillus, Bifdobacterium, and Bacteroides) in the colonic mucosal microbiota of STC patients are signifcantly reduced. Te number of potential pathogens (such as verdigris Pseudomonas, Campylobacter jejuni, and Clostridium putrefaction) increased signifcantly [25,36]. Of note, these changes in the composition of intestinal microbes may afect intestinal motility and alter local environmental homeostasis [37].
Our study analyzed the composition, structure, and diversity of intestinal microbial communities using 16S rDNA high-throughput sequencing in STC model mice. Te ratio of Firmicutes/Bacteroidetes closely associated with constipation was signifcantly reduced. Moreover, Alistipes, a bacterial anaerobic bacterium closely associated with intestinal infammation, was found to be less abundant. In addition to increasing the abundance of intestinal microorganisms, XHRT also increased the abundance of lactic acid bacteria and Alistipes. Several studies reported that patients taking probiotics for anti-infammatory purposes had higher Alistipes levels [38]. Te impact of Alistipes on intestinal immunity was researched by Shi et al. [39] in 2020, but it was rarely reported in STC patients. To date, no consensus has been reached regarding the characteristics of the gut microbiota of STC patients.
Te results of our study suggest that unregulated migration of gut microbes into the lamina propria may be caused by disruption of the intestinal mucosal mechanical barrier and increased permeability in patients with STC. Pathogenic microorganisms (such as pathogenic Escherichia coli) release surface antigens that are recognized by intestinal antigen receptors, which amplify infammation.
Currently, there are limited studies on the intestinal mucosal immune barrier in STC patients. We found that intestinal immune cells can regulate intestinal injury and infammation by recognizing indigestible dietary components such as fber decomposed by microorganisms and producing metabolites [40]. Te cell wall components of Gram-positive bacteria (Bifdobacterium or Lactobacillus) can induce an increase of important phagocytosis receptors(such as FccRIII and TLR), and then, primary T cells respond to cytokines produced in adaptive immunity to distinguish specifc CD4 + T helper subtype cells (T1, T2, or T17) [41]. Some gut microbes play protective roles against immune responses, promoting tissue repair and survival in part through toll-like receptor (TLR) activation [42]. Te goblet cells in the mechanical barrier mucous layer prevent direct contact between gut microbes and pattern recognition receptors in epithelial cells, which may trigger an immune response. XHRT can signifcantly increase the thickness of the acidic mucus layer on the surface of the colonic mucosa, which provides a stable breeding place for commensal bacteria, thus reducing the occurrence of infammatory reactions.
As a member of the TLR family, TLR5 is closely related to the immune response in the intestinal mucosa. It has high expression in the lamina propria of CD11c-positive intestinal cells and activates infammation in response to conditional pathogens such as Escherichia coli and Enterococcus [43]. In addition, fecal microbial transplantation (FMT) stimulates the intestinal adaptive immune response through the TLR pathway, thereby promoting the synthesis of immunoglobulins (such as IgA, IgG, and IgM) and protecting the intestinal mucosa [44]. Alistipes in the gut is closely associated with the production of infammatory factors induced by TLR receptors [45]. At the same time, the intestinal mucosa is the natural site for the diferentiation and maturation of helper T cells 17. T17 cells can secrete interleukin 17, and TLR5 signaling can activate the high expression of IL-17A in the mucosa, thereby triggering intestinal immunity [17]. Bifdobacterium adolescentis and segmental flamentous bacteria have been identifed as effective inducers of IL-17-producing T17 cells [46,47]. Te expansion of T17 cells is usually related to the destruction of intestinal microbiome homeostasis and the impairment of T regulatory cells. A strong induction of the intestinal microbiota of T17 cells can aggravate colonic infammation in mice [48].
Based on the interaction between intestinal microbes and the TLR5/IL-17A signaling pathways in intestinal mucosal immunity, we located and quantitatively analyzed TLR5/IL-17A protein in the colon tissue of STC mice using immunofuorescence. We found that XHRT signifcantly regulated the TLR5/IL-17A signaling pathway in colon mucosal infammation in STC mice. Additionally, the qRT-PCR method was used to perform double verifcation at the genetic level, revealing the molecular mechanism behind intestinal microbial diversity change and intestinal mucosal immunity disorders in STC. Te results of our research demonstrate how XHRT contributes to the nourishment of the intestine and defecation. Tey also provide new experimental insights into clinical medicine by diferentially regulating intestinal microbes, intestinal mucosal immunity, and signal transduction pathways. Troughout the research, multipathway and multitarget regulation have been observed in Chinese herbal medicine. Our studies will continue to clarify the direct relationship between the host metabolism of STC intestinal commensal bacteria and intestinal mucosal immune function.

Conclusions
Tis study has demonstrated that regulating the activation of intestinal infammatory signal TLR5/IL-17A mediated by gut microbes can improve slow transit constipation and that XHRT provides a new strategy for the treatment of slow transit constipation.