Shouhui Tongbian Capsules induce regression of inflammation to improve intestinal barrier in mice with constipation by targeted binding to Prkaa1: With no obvious toxicity.

Constipation arising from the poor bowel movement is a rife enteric health problem. Shouhui Tongbian Capsule (SHTB) is a traditional Chinese medicine (TCM) which effectively improve the symptoms of constipation. However, the mechanism has not been fully evaluated. The purpose of this study was to evaluate the effect of SHTB on the symptoms and intestinal barrier of mice with constipation. Our data showed that SHTB effectively improved the constipation induced by diphenoxylate, which was confirmed by shorter first defecation time, higher internal propulsion rate and fecal water content. Additionally, SHTB improved the intestinal barrier function, which was manifested by inhibiting the leakage of Evans blue in intestinal tissues and increasing the expression of occludin and ZO-1. SHTB inhibited NLRP3 inflammasome signaling pathway and TLR4/NF-κB signaling pathway, reduced the number of proinflammatory cell subsets and increased the number of immunosuppressive cell subsets to relieve inflammation. The photochemically induced reaction coupling system combined with cellular thermal shift assay and central carbon metabolomics technology confirmed that SHTB activated AMPKα through targeted binding to Prkaa1 to regulate Glycolysis/Gluconeogenesis and Pentose Phosphate Pathway, and finally inhibited intestinal inflammation. Finally, no obvious toxicity related to SHTB was found in a repeated drug administration toxicity test for consecutive 13 weeks. Collectively, we reported SHTB as a TCM targeting Prkaa1 for anti-inflammation to improve intestinal barrier in mice with constipation. These findings broaden our knowledge of Prkaa1 as a druggable target protein for inflammation inhibition, and open a new avenue to novel therapy strategy for constipation injury.

Efficacy and safety of Shouhui Tongbian Capsules in the treatment of constipation: A systematic review and meta-analysis.

BACKGROUND: Constipation is a common gastrointestinal disorder, which has seriously affected the quality of people's daily life. Traditional Chinese Medicine (TCM) therapy takes syndrome differentiation and treatment as the theoretical guidance with certain advantages in treating constipation with the holistic approach. However, there are few studies on the treatment of constipation with Shouhui Tongbian Capsules (SHTB). PURPOSE: This study was aimed to evaluate the clinical effect and safety of SHTB in the treatment of constipation and provide evidence-based references for clinical application. STUDY DESIGN: A systematic review and meta-analysis of existing literature on SHTB for treating constipation. METHODS: Chinese databases (China Network Knowledge Infrastructure, Wan Fang Database and Chinese Scientific Journal Database) and English databases (PubMed, EmBase and the Cochrane Library) were thoroughly investigated through screening randomized controlled trials on SHTB for constipation from the establishment of all databases to September 26, 2022. Data extraction and quality evaluation were performed on the literature that met the inclusion criteria and a meta-analysis was performed for selected data using Review Manager 5.4, ROB 2.0 and Stata 17.0. RESULTS: A total of 14 RCTs (randomized controlled trial) including 1310 participants were included in the analysis. The results showed that the test group was superior to the control group in improving the total effective rate and curative effect, clinical symptom score, gastrointestinal peptide index and reducing adverse reactions and recurrence rate. The specific results were as follows: ① The total effective rate increased significantly (RR = 1.24, 95% CI [1.18, 1.30], Z = 8.25, p< 0.00001); ② The clinical symptom indexs, including the difficulty of defecation [SMD = -1.28, 95% CI (-1.44, -1.12), Z = 15.65, p< 0.00001], the frequency of spontaneous defecation [SMD = 1.28, 95% CI (1.01, 1.54), Z = 9.52, p< 0.00001], defecation interval [SMD = -1.47, 95% CI (-1.68, -1.26), Z = 13.79, p < 0.00001], incomplete defecation [SMD = -1.34, 95% CI (-1.57, -1.11), Z = 11.42, p < 0.00001], duration of defecation [SMD = -2.02, 95% CI (-2.39, -1.65), Z = 10.73, p < 0.00001], stool characteristics [SMD = -2.30, 95% CI (-2.60, -1.99), Z = 14.72, p< 0.00001] and TCM main syndrome scores [SMD = -1.25, 95% CI (-1.46, -1.05), Z = 11.79, p< 0.00001] increased observably; ③ The gastrointestinal peptide hormone indexs, including MTL Level [SMD = 0.43, 95% CI (0.24, 0.62), Z = 4.44, p < 0.00001] and SP Level [RR =0.57, 95% CI (0.37, 0.87), Z = 2.61, p = 0.009] were improved obviously; ④ The incidence of adverse reactions (RR = 0.57, 95% CI [0.37, 0.87], Z = 2.61, p = 0.009) and recurrence rate (RR = 0.31, 95% CI [0.18, 0.54], Z = 4.28, P <0.001) reduced significantly. Sensitivity analysis showed that there was no significant change in all outcome indicators, which suggested that the results of meta-analysis were relatively stable. Funnel plot and Egger test results showed that the literature included in the study might have publication bias. CONCLUSION: SHTB can be used to treat functional constipation, especially elderly functional constipation, constipation caused by tumor chemotherapy and disease concomitant constipation. The optimal dosage of SHTB was 0.70 g (2 capsules) each time, 3 times a day, for 28 days. Combined with basic treatment, lactose oral solution, mosaic or castor oil could improve the total effective rate, clinical symptom indicators, gastrointestinal peptide hormone indicators and reduce adverse reaction rate of patients. However, due to the limitations of the included clinical trials, high-quality clinical trials with long follow ups are needed to evaluate the effectiveness and safety of SHTB in treating different types of constipation.

Shouhui Tongbian Capsule ameliorates constipation via gut microbiota-5-HT-intestinal motility axis.

Constipation has become an epidemic enteric medical problem, accompanied with increasing long-term sequelae. Gut microbiota and serotonin (5-HT) have been believed as predominant player in the treatment of constipation. In clinical practices, Shouhui Tongbian Capsule (SHTB) was found to effectively improve constipation symptoms and promote gastrointestinal motility. However, the specific mechanism of SHTB is not clearly elucidated. Our current study aims to explore the therapeutic effects of SHTB against the development of constipation and the underlying mechanisms related to gut bacterial and 5-HT. We established loperamide hydrochloride (LH)-induced experimental constipation mouse model to evaluate the effect of SHTB. 16S RNA sequencing, fecal microbiota transplants (FMT), high performance liquid chromatograph, and molecular biological analysis were performed to investigate the potential mechanisms of SHTB. Our data demonstrated that SHTB significantly ameliorated LH-induced experimental constipation and accelerated enteric motility via promoting 5-HT biosynthesis in enterochromaffin cells and enteric neuron growth of the enteric nervous system (ENS) in both the small intestine and colon. Additionally, SHTB significantly modulated gut microbiota dysbiosis and potentially altered microbiota metabolites to enhance intestinal 5-HT production. Finally, FMT study confirmed that the effects of SHTB on 5-HT production and constipation are dependent on modulating intestinal microbiota dysbiosis. In conclusion, our current study deciphered therapeutic mechanism of SHTB in the treatment of experimental constipation from perspectives of gut microbiota-5-HT-intetinal motility axis and provides novel insights into the appropriate and safe application of SHTB in the clinic.

[Identification of intestine direct targets of Shouhui Tongbian Capsules using "target fishing" strategy].

"Target fishing" strategy was used to investigate the direct targets and mechanism of Shouhui Tongbian Capsules on relaxing bowel. Magnetic beads cross-linked with the chemical constituents from Shouhui Tongbian Capsules were prepared. The potential target proteins were captured from the total protein lysates of rat intestine using the beads. The captured proteins were further identified by LC-MS/MS, and the associated pathways were analyzed by Cytoscape. RESULTS:: showed that 138 potential target proteins were identified, which were involved in eight signaling pathways, including tricarboxylic acid cycle, pyrimidine metabolism, sulfur metabolism, fatty acid degradation, alanine/aspartate/glutamate metabolism, arginine/proline metabolism, valine/leucine/isoleucine degradation, and ß-alanine metabolism. Taken together, Shouhui Tongbian Capsules may exert relaxing bowel effect by acting on multiple signaling pathways to promote intestinal gurgling, inhibit inflammation, as well as improve intestinal barrier function, intestinal water secretion, and intestinal flora.

[Mechanism of Shouhui Tongbian Capsules in treating constipation based on network pharmacology and molecular docking].

To explore the mechanism of Shouhui Tongbian Capsules in treating constipation by means of network pharmacology and molecular docking approach. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Bioinfoematics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN) were applied to obtain chemical components and potential targets of eight herbs in Shouhui Tongbian Capsules according to the screening principles of oral availability(OB)≥30% and drug-like property(DL)≥0.18. Disease targets relating to constipation were screened out through GeneCards, PharmGkb and other databases, drug targets were integrated with disease targets, and intersection targets were exactly the potential action targets of Shouhui Tongbian Capsules for treating constipation; PPI network of potential targets was constructed using STRING platform, and GO(gene ontology) analysis and KEGG(Kyoto encyclopedia of genes and genomes) pathway data were obtained to conduct enrichment analysis and predict its mechanism of action. Cytoscape 3.6.1 was used to construct a network of "medicinal materials-chemical components-drug targets", and the network topology analysis was carried out on the PPI network to obtain its main components and key targets. Molecular docking between components and key targets of Shouhui Tongbian Capsules verified the accuracy of network pharmacological analysis results. The PPI network analysis showed 92 chemical components, including quercetin, stigmaste-rol, aloe-emodin, rhein, and key targets for instance AKT1, MAPK1, IL6, JUN, TNF and TP53. The enrichment analysis of KEGG screened out 157 signal pathways(P<0.01), mainly involving interleukin 17 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, thyroid hormone signaling pathway. Quercetin, resveratrol and lysine with top degree value had a rational conformation in docking site of protein crystal complexes. This study preliminarily showed that various active ingredients in Shouhui Tongbian Capsules could regulate multiple signaling pathways, increase intestinal smoothness and peristalsis function, ensure smooth intestinal lumen, and play a role in treating constipation by acting on key targets, such as AKT1, MAPK1, IL6 and JUN.

[Therapeutic effect and mechanism of Shouhui Tongbian Capsules on slow transit constipation model mice].

Shouhui Tongbian Capsules was used to explore the therapeutic effect and potential mechanism on slow transit constipation model mice induced by loperamide hydrochloride. In the experiment, loperamide hydrochloride-induced ICR mice were used as the model of slow transit constipation. Fifty ICR mice were divided into the blank group, model group and high, medium and low dose groups of Shouhui Tongbian Capsules extract(100, 200 and 400 mg·kg~(-1)). The model group and the administration groups were then modeled using loperamide hydrochloride intragastrically to obtain slow transit constipation. After successful modeling, high, medium and low doses of drugs were given to each drug group by intragastric administration. After 14 days of administration, the first defecation time, 6 h defecation grain number, 6 h defecation wet weight and dry weight, black feces discharged within 6 h and the fecal water content were measured. Intestinal tissues were taken for c-Kit and SCF immunohistochemical sections to detect the expression of c-Kit and SCF in the blank group, model group and high, medium and low dose groups of the medicinal extract of Shouhui Tongbian Capsules. The tissue changes in the intestinal wall of mice were detected by HE staining. At the same time, partial intestinal tissues were taken to test the activity of ATP synthase and isocitrate dehydrogenase in intestinal tissues of mice. RESULTS:: showed that Shouhui Tongbian Capsules effectively improved the symptoms of slow transit constipation in ICR mice and promoted intestinal movement. Shouhui Tongbian Capsules obviously shortened the time of discharging black stool for the first time, improved the intestinal propulsion rate, increased the water content and amount of feces, and improved the constipation symptoms. Mechanism study revealed that Shouhui Tongbian Capsules increased ATP synthase activity and mitochondrial isocitrate dehydrogenase activity in intestinal tissue, and up-regulated c-Kit/SCF signaling pathway to promote interstitial Cajal cells proliferation, intestinal nerve transmission, intestinal motility and transport capacity.

[Molecular mechanism study of Shouhui Tongbian Capsules on promoting energy metabolism of gastrointestinal stromal cells].

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 µg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.

[Metabonomics study of Shouhui Tongbian Capsules in slow transit constipation based on UPLC-ESI-QE-Orbitrap-MS].

The effect of Shouhui Tongbian Capsules(SHTB) on the endogenous metabolites of colon tissue in mice with slow transit constipation was analyzed by metabolomics methods to explore its mechanism in the treatment of constipation. ICR mice were randomly divided into normal group, model group and SHTB group according to the body weight. The mice were given diphenoxylate to establish the slow transit constipation model. Mouse carbon ink pushing rate, first defecation time and the number of defecation particles in 12 h were observed. The mouse colon tissue was separated and the mucous cells were detected by Periodic acid Schiff and Alcian blue(AB-PAS) staining. Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry(UPLC-ESI-Orbitrap-MS/MS) technology was used to characterize the differences in tissue metabolism to screen out the potential different metabolites and possible metabolic pathways in colon tissue. The results indicated that SHTB could significantly shorten the first defecation time and the number of defecations, and increase the number of intestinal peristalsis and mucous cells in the colonic mucosa compared to the model mice. Metabolomics results showed that, compared with the normal group, a total of 17 potential biomarkers, including L-kynurenine, N6,N6,N6-trimethyl-L-lysine, L-formylkynurenine, N6-acetyl-L-lysine, L-phenylalanine, phenylacetaldehyde, xanthoxin, thymidine, glycyl-L-leucine, cystathionine,(R)-1-aminopropan-2-ol, deoxycytidine, gamma-glutamyl-gamma-aminobutyraldehyde, D-galactose, L-arginine, L-proline and pyruvate, were found and identified in colon tissue. Treated with SHTB, these metabolic differences tended to return to normal levels. Therefore, it could be made a conclusion that the therapeutic effect of SHTB on chronic transit constipation may be related to regulating phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine and proline metabolism, cysteine and methionine metabolism, tyrosine metabolism, arginine biosynthesis, pyruvate metabolism, glycolysis, pyrimidine metabolism, tricarboxylic acid cycle and galactose metabolism.

Metabonomics study of Shouhui Tongbian Capsules in slow transit constipation based on UPLC-ESI-QE-Orbitrap-MS / 中国中药杂志

The effect of Shouhui Tongbian Capsules(SHTB) on the endogenous metabolites of colon tissue in mice with slow transit constipation was analyzed by metabolomics methods to explore its mechanism in the treatment of constipation. ICR mice were randomly divided into normal group, model group and SHTB group according to the body weight. The mice were given diphenoxylate to establish the slow transit constipation model. Mouse carbon ink pushing rate, first defecation time and the number of defecation particles in 12 h were observed. The mouse colon tissue was separated and the mucous cells were detected by Periodic acid Schiff and Alcian blue(AB-PAS) staining. Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry(UPLC-ESI-Orbitrap-MS/MS) technology was used to characterize the differences in tissue metabolism to screen out the potential different metabolites and possible metabolic pathways in colon tissue. The results indicated that SHTB could significantly shorten the first defecation time and the number of defecations, and increase the number of intestinal peristalsis and mucous cells in the colonic mucosa compared to the model mice. Metabolomics results showed that, compared with the normal group, a total of 17 potential biomarkers, including L-kynurenine, N6,N6,N6-trimethyl-L-lysine, L-formylkynurenine, N6-acetyl-L-lysine, L-phenylalanine, phenylacetaldehyde, xanthoxin, thymidine, glycyl-L-leucine, cystathionine,(R)-1-aminopropan-2-ol, deoxycytidine, gamma-glutamyl-gamma-aminobutyraldehyde, D-galactose, L-arginine, L-proline and pyruvate, were found and identified in colon tissue. Treated with SHTB, these metabolic differences tended to return to normal levels. Therefore, it could be made a conclusion that the therapeutic effect of SHTB on chronic transit constipation may be related to regulating phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine and proline metabolism, cysteine and methionine metabolism, tyrosine metabolism, arginine biosynthesis, pyruvate metabolism, glycolysis, pyrimidine metabolism, tricarboxylic acid cycle and galactose metabolism.

Molecular mechanism study of Shouhui Tongbian Capsules on promoting energy metabolism of gastrointestinal stromal cells / 中国中药杂志

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 μg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.

Therapeutic effect and mechanism of Shouhui Tongbian Capsules on slow transit constipation model mice / 中国中药杂志

Shouhui Tongbian Capsules was used to explore the therapeutic effect and potential mechanism on slow transit constipation model mice induced by loperamide hydrochloride. In the experiment, loperamide hydrochloride-induced ICR mice were used as the model of slow transit constipation. Fifty ICR mice were divided into the blank group, model group and high, medium and low dose groups of Shouhui Tongbian Capsules extract(100, 200 and 400 mg·kg~(-1)). The model group and the administration groups were then modeled using loperamide hydrochloride intragastrically to obtain slow transit constipation. After successful modeling, high, medium and low doses of drugs were given to each drug group by intragastric administration. After 14 days of administration, the first defecation time, 6 h defecation grain number, 6 h defecation wet weight and dry weight, black feces discharged within 6 h and the fecal water content were measured. Intestinal tissues were taken for c-Kit and SCF immunohistochemical sections to detect the expression of c-Kit and SCF in the blank group, model group and high, medium and low dose groups of the medicinal extract of Shouhui Tongbian Capsules. The tissue changes in the intestinal wall of mice were detected by HE staining. At the same time, partial intestinal tissues were taken to test the activity of ATP synthase and isocitrate dehydrogenase in intestinal tissues of mice. RESULTS:: showed that Shouhui Tongbian Capsules effectively improved the symptoms of slow transit constipation in ICR mice and promoted intestinal movement. Shouhui Tongbian Capsules obviously shortened the time of discharging black stool for the first time, improved the intestinal propulsion rate, increased the water content and amount of feces, and improved the constipation symptoms. Mechanism study revealed that Shouhui Tongbian Capsules increased ATP synthase activity and mitochondrial isocitrate dehydrogenase activity in intestinal tissue, and up-regulated c-Kit/SCF signaling pathway to promote interstitial Cajal cells proliferation, intestinal nerve transmission, intestinal motility and transport capacity.

Mechanism of Shouhui Tongbian Capsules in treating constipation based on network pharmacology and molecular docking / 中国中药杂志

To explore the mechanism of Shouhui Tongbian Capsules in treating constipation by means of network pharmacology and molecular docking approach. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and Bioinfoematics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN) were applied to obtain chemical components and potential targets of eight herbs in Shouhui Tongbian Capsules according to the screening principles of oral availability(OB)≥30% and drug-like property(DL)≥0.18. Disease targets relating to constipation were screened out through GeneCards, PharmGkb and other databases, drug targets were integrated with disease targets, and intersection targets were exactly the potential action targets of Shouhui Tongbian Capsules for treating constipation; PPI network of potential targets was constructed using STRING platform, and GO(gene ontology) analysis and KEGG(Kyoto encyclopedia of genes and genomes) pathway data were obtained to conduct enrichment analysis and predict its mechanism of action. Cytoscape 3.6.1 was used to construct a network of "medicinal materials-chemical components-drug targets", and the network topology analysis was carried out on the PPI network to obtain its main components and key targets. Molecular docking between components and key targets of Shouhui Tongbian Capsules verified the accuracy of network pharmacological analysis results. The PPI network analysis showed 92 chemical components, including quercetin, stigmaste-rol, aloe-emodin, rhein, and key targets for instance AKT1, MAPK1, IL6, JUN, TNF and TP53. The enrichment analysis of KEGG screened out 157 signal pathways(P<0.01), mainly involving interleukin 17 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, thyroid hormone signaling pathway. Quercetin, resveratrol and lysine with top degree value had a rational conformation in docking site of protein crystal complexes. This study preliminarily showed that various active ingredients in Shouhui Tongbian Capsules could regulate multiple signaling pathways, increase intestinal smoothness and peristalsis function, ensure smooth intestinal lumen, and play a role in treating constipation by acting on key targets, such as AKT1, MAPK1, IL6 and JUN.

Identification of intestine direct targets of Shouhui Tongbian Capsules using "target fishing" strategy / 中国中药杂志

"Target fishing" strategy was used to investigate the direct targets and mechanism of Shouhui Tongbian Capsules on relaxing bowel. Magnetic beads cross-linked with the chemical constituents from Shouhui Tongbian Capsules were prepared. The potential target proteins were captured from the total protein lysates of rat intestine using the beads. The captured proteins were further identified by LC-MS/MS, and the associated pathways were analyzed by Cytoscape. RESULTS:: showed that 138 potential target proteins were identified, which were involved in eight signaling pathways, including tricarboxylic acid cycle, pyrimidine metabolism, sulfur metabolism, fatty acid degradation, alanine/aspartate/glutamate metabolism, arginine/proline metabolism, valine/leucine/isoleucine degradation, and β-alanine metabolism. Taken together, Shouhui Tongbian Capsules may exert relaxing bowel effect by acting on multiple signaling pathways to promote intestinal gurgling, inhibit inflammation, as well as improve intestinal barrier function, intestinal water secretion, and intestinal flora.