| Background:Diarrhea-predominant irritable bowel syndrome(IBS-D)is classified as a functional disorder,characterized by recurring symptoms without apparent organic changes.Despite the absence of structural alterations,the cyclic manifestation of symptoms greatly troubles patients,profoundly affecting their quality of life.As a result,research on IBS has garnered significant attention.Although modern medicine has yet to discover definitive curative methods for managing IBS-D,a range of treatment approaches has been proposed for this condition.For patients with milder symptoms,non-pharmacological treatments are typically employed.Additionally,symptomatic drug treatments are often tailored to patients with more pronounced clinical manifestations.However,current treatments often target individual symptoms,leading to the need for a combination of multiple drug formulations in patients with complex clinical presentations.The efficacy of such polypharmacy regimens is often inconsistent and associated with high relapse rates.In contrast,traditional Chinese medicine(TCM)has a rich history in the treatment of IBS-D.TCM possesses advantages such as multitarget effects,significant efficacy,and high safety.These characteristics suggest that TCM might hold potential for developing complementary and alternative therapies for IBS-D.While modern medicine struggles to provide a comprehensive solution,TCM’s holistic approach could offer a promising avenue for addressing the multifaceted nature of IBS-D.Objective:This study aims to investigate the clinical efficacy of the traditional Chinese herbal compound "Liver and Spleen Harmonizing Formula" in the treatment of diarrhea-predominant irritable bowel syndrome(IBS-D).The study employs network pharmacology methods to predict and analyze the potential targets and molecular mechanisms of the formula in treating IBS-D.The findings are then validated through animal experiments.The study focuses on exploring the mechanisms through which the formula affects the colonic mucosal barrier function in IBS-D and assessing the potential effectiveness and feasibility of traditional Chinese medicine in treating IBS-D.The goal is to shed light on the role of traditional Chinese medicine in IBS-D treatment,aiming to provide better therapeutic options for IBS-D patients and improve their quality of life and overall health.Methods:1.A total of 60 IBS-D patients with liver-spleen deficiency type were randomly divided into an observation group and a control group,with 30 cases in each group.The control group received oral administration of the Western medicine pinaverium bromide,while the observation group received oral administration of the "Liver and Spleen Harmonizing Formula." The treatment duration for both groups was 4 weeks,with visits scheduled every 2 weeks.The study aimed to observe and compare the general conditions,individual Traditional Chinese Medicine(TCM)symptom scores,overall symptom scores,anxiety and depression scores,stool conditions,inflammatory markers,and intestinal mucosal barrier indicators between the two groups.2.Network pharmacology methods were employed to predict potential target points and molecular mechanisms of the "Liver and Spleen Harmonizing Formula" for treating IBS-D.Intersection target points between the formula and IBS-D were obtained.A "drug-one target-one disease" protein interaction network was constructed to identify the core target points of the formula’s treatment for IBS-D.GO and KEGG enrichment analyses were conducted.3.Fifty male Sprague-Dawley rats were divided into five groups,each consisting of 10 rats:the normal group(SHAM group),the model group(MG group),the low-dose "Liver and Spleen Harmonizing Formula" group(L-TGHPF),the medium-dose "Liver and Spleen Harmonizing Formula" group(M-TGHPF),and the high-dose "Liver and Spleen Harmonizing Formula" group(H-TGHPF).The model was induced using senna leaf decoction combined with restraint stress.The study aimed to observe changes in fecal water content,body weight,abdominal withdrawal reflex(AWR)scores,and evaluate intestinal function and sensitivity in the rat model.Additionally,measurements were taken for D-lactic acid content,inflammatory factors TNF-a and IL-6,expression of tight junction proteins occludin and claudin-1,as well as the NF-κB/MLCK signaling pathway protein expression.The study also involved electron microscopy observation and comparison of intestinal mucosal microstructure to assess treatment effects and explore potential mechanisms of action.Results:1.In the clinical study,there were no significant differences in gender,age,disease duration,height,weight,blood pressure(systolic/diastolic),axillary temperature,respiration,and pulse between the herbal treatment group and the Western medicine control group(p>0.05),indicating comparability.After 2 weeks of treatment,the herbal treatment group showed a significant reduction in bowel movements,alleviation of abdominal distension,and increased appetite(p<0.05).These improvements further improved after 4 weeks of treatment compared to the baseline and the 2-week mark(p<0.05).After 4 weeks of treatment,symptoms such,as abdominal pain,epigastric fullness,fatigue,and irritability showed improvement(p<0.05).The improvement in symptoms of abdominal pain,decreased appetite,and irritability was more pronounced after 4 weeks compared to 2 weeks of treatment(p<0.05).In the Western medicine control group,abdominal pain,bowel movements,abdominal distension,epigastric fullness,and decreased appetite improved after 4 weeks of treatment compared to baseline(p<0.05).Abdominal pain,bowel movements,and abdominal distension showed significant relief compared to 2 weeks of treatment(p<0.05).The herbal treatment group showed a more significant improvement in bowel movements and abdominal distension after 2 weeks of treatment compared to the Western medicine control group(p<0.05).After 4 weeks of treatment,the herbal treatment group demonstrated a significantly higher relief of bowel movements,abdominal distension,decreased appetite,and irritability compared to the Western medicine control group(p<0.05).The herbal treatment effectively improved anxiety and depression in patients after 4 weeks of treatment(p<0.01).The Western medicine control group showed effective relief of depression after 4 weeks of treatment.Comparatively,the herbal treatment group exhibited a greater improvement in depression compared to the Western medicine control group(p<0.05),The herbal treatment significantly improved stool characteristics(p<0.05),while the Western medicine treatment showed no significant improvement in stool characteristics(p>0.05).After 4 weeks of treatment,both groups showed a decrease in IL-6、TNF-α and CRP levels compared to before treatment(p<0.05).The reduction in inflammatory levels was more significant in the herbal treatment group compared to the control group(p<0.05).After 4 weeks of treatment,both the herbal treatment group and the control group showed a significant decrease in D-lactic acid levels(p<0.05).There was no significant difference in D-lactic acid levels between the two groups after treatment(p>0.05).2.Through target prediction using TCMSP,a total of 275 target points for the herbal formula for Tiaogan Hepi Decoction were obtained.Using GeneCards and OMIM databases,3274 disease target points for IBS-D were identified.By mapping these targets,167 potential target points for the herbal formula in treating IBS-D were obtained.These 167 intersecting target genes were subjected to protein-protein interaction predictions using the String database,and core target points such as IL6,TNF,AKT1,TP53,STAT3,JUN,MAPK3,CASP3,EGFR,and IL1B were identified.GO and KEGG enrichment analysis revealed protein binding as the main biological function,with the NF-κB signaling pathway being one of the important signaling pathways.3.Inthe experimental study:The experiment involved measuring and calculating the fecal water content of experimental rats at different time points:before modeling(T0),after modeling(T1),and after treatment(T2).The collected data was subjected to statistical analysis.At T0,there were no significant statistical differences in fecal water content among the rat groups(p>0.05).At T1 and T2,significant differences in fecal water content were observed among the groups.At T1,the fecal water content of all groups(MG group,L-TGHPF group,M-TGHPF group,H-TGHPF group)after modeling was significantly higher than that of the control group(p<0.01).At T2,the fecal water content of the MG group and L-TGHPF group was higher than that of the SHAM group(p<0.05),indicating statistical significance.There were no significant differences in fecal water content between the M-TGHPF group,H-TGHPF group,and SHAM group rats(p>0.05).Additionally,at T2,the fecal water content of the M-TGHPF group and H-TGHPF group rats significantly improved compared to the MG group,and the H-TGHPF group rats had significantly lower fecal water content than the L-TGHPF group rats(p<0.05),indicating that the improvement in fecal water content was associated with the dosage of the herbal medicine.At T0,there were no significant statistical differences in body weight among the rat groups(p>0.05).After intervention,significant differences in body weight increase were observed among the groups:at T1,the body weight of SD rats in the MG group,L-TGHPF group,M-TGHPF group,and H-TGHPF group increased significantly less compared to the SHAM group rats,and the differences were statistically significant(p<0.01).Among these,the body weight of rats in the L-TGHPF group,M-TGHPF group,and H-TGHPF group was not significantly different from that of the MG group(p>0.05),confirming the success of the model;at T2,the body weight of the MG group rats was significantly lower than that of rats in the other groups,with statistical significance(p<0.01),and the body weight of rats in the H-TGHPF group was significantly higher than that of the SHAM group rats,also with statistical significance(p<0.05).There were no significant differences in body weight between the L-TGHPF group,M-TGHPF group,and SHAM group rats at T2(p>0.05).At T0,there were no significant statistical differences in intestinal sensitivity among the groups(p>0.05)compared to the control group.However,at T1 and T2,there were significant differences in abdominal withdrawal reflex(AWR)scores among the rat groups.At T1,the intestinal sensitivity of the MG group,L-TGHPF group,M-TGHPF group,and H-TGHPF group rats was all higher than that of the SHAM group rats(p<0.05).At T2,compared to the SHAM group,there were no significant differences in intestinal sensitivity for the M-TGHPF group and H-TGHPF group rats(p>0.05),while the MG group and L-TGHPF group rats showed a significant increase in intestinal sensitivity compared to the SHAM group rats(p<0.01).Furthermore,compared to the MG group,the intestinal sensitivity of the L-TGHPF group,M-TGHPF group,and H-TGHPF group rats improved significantly(p<0.05).After different doses of herbal medicine treatment,the improvement in intestinal sensitivity in the H-TGHPF group was more pronounced than in the L-TGHPF group and M-TGHPF group(p<0.05),showing statistical significance.After intervention,compared to the SHAM group,the levels of TNF-α and IL-6 increased significantly in the MG group,L-TGHPF group,and M-TGHPF group rats,with statistical significance(P<0.05),while the TNF-α and IL-6 levels in the H-TGHPF group rats were not significantly different from those in the SHAM group(P>0.05).Compared to the MG group,the levels of TNF-α and IL-6 in the L-TGHPF group,M-TGHPF group,and H-TGHPF group rats decreased significantly,with statistical significance(P<0.01).After treatment with different doses of herbal medicine,the levels of inflammatory cytokines were significantly lower in the H-TGHPF group compared to the L-TGHPF group and M-TGHPF group(P<0.05).After treatment,compared to the SHAM group,the levels of D-lactic acid significantly increased in the MG group,L-TGHPF-group,and M-TGHPF group rats(P<0.01).Compared to the MG group,the levels of D-lactic acid decreased significantly in all herbal medicine intervention groups(P<0.01),with the most significant decrease observed in the H-TGHPF group compared to the L-TGHPF group and M-TGHPF group(P<0.05).Compared to the SHAM group,the protein expression of Occludin and Claudin-1 decreased in the MG group,L-TGHPF group,M-TGHPF group,and H-TGHPF group rats(P<0.01).Compared to the MG group,the protein expression of Occludin and Claudin-1 increased significantly in the L-TGHPF group,M-TGHPF group,and H-TGHPF group rats,with statistical significance(P<0.01),indicating that the herbal medicine can upregulate the expression of tight junction proteins and repair mucosal damage.After intervention with the herbal medicine,the level of Occludin was significantly higher in the high-dose group compared to the low-dose and medium-dose groups(P<0.05),while there was no significant difference in Claudin-1 protein expression between different doses of herbal medicine treatment groups(P>0.05),indicating that high-dose herbal medicine had the strongest ability to increase the expression level of Occludin in rat colon tissue.Compared to the SHAM group,the protein expression levels of NF-κB/MLCK were significantly elevated in the MG group,L-TGHPF group,M-TGHPF group,and H-TGHPF group rats(P<0.05).When compared to the MG group,the expression levels of NF-κB/MLCK in the L-TGHPF group,M-TGHPF group,and H-TGHPF group rats decreased significantly(P<0.01).When comparing different herbal medicine treatment groups,the H-TGHPF group exhibited a significant decrease in NF-κB/MLCK protein expression level compared to the L-TGHPF group and M-TGHPF group(P<0.01),and the M-TGHPF group showed a significantly lower NF-κB/MLCK protein expression level than the L-TGHPF group(P<0.01).These results indicate that the extent of NF-κB/MLCK downregulation by the herbal medicine is dose-dependent,with the high-dose herbal medicine having a stronger ability to lower NF-κB/MLCK protein levels.Under electron microscope observation,it was noted that in the control group of rats,the colonic mucosal epithelium displayed a normal morphology.The microvilli were neatly arranged,tightly adhered,and distinct,with consistent lengths,and no detachment was observed.In the model group of rats,the colonic mucosa exhibited irregular and disorderly microvilli of varying lengths,with instances of local breakage.In comparison to the model group rats,the high-dose herbal medicine group demonstrated a denser and more consistent arrangement of colonic microvilli,with uniform lengths.The low-dose and medium-dose herbal medicine groups displayed similar improvements,albeit to a lesser extent.Conclusion:1.The traditional Chinese herbal formula "Tiao Gan He Pi Fang" has demonstrated potential clinical advantages in the treatment of patients with Irritable Bowel Syndrome with Diarrhea(IBS-D).It exhibits a positive impact on improving clinical symptoms,emotional states,stool characteristics,inflammation levels,and protecting the intestinal mucosal barrier.This offers valuable insights for comprehensive management of IBS-D.2.The core action targets of "Tiao Gan He Pi Fang" in IBS-D treatment involve inflammatory factors such as IL-6,TNF,with the NF-κB signaling pathway identified as a significant route.These findings provide valuable cues for further research endeavors.3."Tiao Gan He Pi Fang" holds therapeutic potential in IBS-D rat models,achieved through modulation of intestinal sensitivity,reduction of inflammation levels,intervention in mucosal tight junction proteins Occludin and Claudin-1 expression,and protection of intestinal mucosal barrier.These mechanisms are likely associated with the modulation of the NF-κB/MLCK signaling pathway by "Tiao Gan He Pi Fang".A deeper understanding of the mechanisms underlying this signaling pathway will facilitate the development of more precise and targeted treatment strategies,thereby better meeting the needs of IBS-D patients. |
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