Study On The Serum Inflammatory Factor Levels In Patients With IBS-D With Spleen And Kidney Yang Deficiency And The Efficacy Mechanism Of The Wenshen Jianpi Prescriptio

BackgroundsIrritable bowel syndrome（IBS）is one of the common functional gastrointestinal disorders（FGIDs）in clinical practice.The global prevalence rate is about 11.2%.It is characterized by complex mechanism,easy recurrence,and serious impact on patients’ quality of life.IBS with predominant diarrhea（IBS-D）is the most common subtype of IBS,accounting for 23.4%to 40.0%of all IBS.The current conventional treatment of IBS-D is mainly symptomatic treatment with limited efficacy.Traditional Chinese medicine treatment of IBS-D is based on the overall concept and has a systematic and unique view on the etiology,pathogenesis and diagnosis and treatment of IBS-D.Through syndrome differentiation and treatment,clinical symptoms of patients can be significantly improved and life quality can be improved.Spleen and kidney Yang deficiency syndrome is one of the main syndromes of IBS-D in traditional Chinese medicine,and it is the key link of refractory IBS-D.The preliminary clinical study of the team found that Wenshen-Jianpi prescription had clinical advantages of high efficacy and low recurrence rate in the treatment of IBS-D with spleen and kidney Yang deficiency syndrome.The total effective rate was 92.24%,while the placebo group was only 42.5%.The pathogenesis of IBS-D is complex and has not been determined yet.The low-grade state of inflammation,abnormal intestinal barrier function,and dysbiosis of intestinal flora play an important role in the occurrence and development of IBS-D,and the relationship among the three is complex,which both restrict and promote each other,and act on IBS-D together.The effect of Wenshen-Jianpi prescription on IBS-D with spleen and kidney Yang deficiency syndrome is clear,but the mechanism is still unclear.Preliminary studies have preliminarily verified that Wenshen-Jianpi prescription can regulate the imbalance of inflammatory cytokines in IBS-D model rats.Therefore,based on IBS-D low-grade inflammation and changes in intestinal mucosal permeability in spleen and kidney Yang deficiency syndrome,this study explored the intervention effects of Wenshen-Jianpi prescription on intestinal microflora,intestinal mucosal barrier and TLR4/MyD88/NF-KB signaling pathway.Objectives1.To determine the changing trend of serum inflammatory factor expression levels in IBS-D patients with spleen and kidney Yang deficiency syndrome2.To evaluate and compare three animal models of IBS-D with spleen and kidney Yang deficiency syndrome.3.To explore the mechanism of Wenshen-Jianpi prescription on intestinal mucosal barrier function,TLR4/MyD88/NF-κB signaling pathway and intestinal flora of IBS-D model rats with spleen and kidney Yang deficiency syndrome.Methods1.Serum inflammatory factor levels in IBS-D patients with spleen and kidney Yang deficiency syndrome.Thirty IBS-D patients with spleen and kidney Yang deficiency syndrome treated in Wangjing Hospital,China Academy of Chinese Medical Sciences from June 2021 to December 2021 were selected,and 10 healthy control group were selected.Gender,age and BMI were matched between the two groups.Clinical data were collected and serum samples were collected to detect the levels of inflammatory cytokines IL-1β,IL-4,IL-6,IL-10,TNF-α and IFN-y by ELISA.2.Establishment of animal model of IBS-D with spleen and kidney Yang deficiency syndrome.A total of 28 SD rats were selected.They were randomly divided into 4 groups with 7 infants in each group:mother and child separation combined with Senna leaf gavage group,rectal dilation combined with senna leaf gavage group,water avoidance stress combined with Senna leaf gavage group and normal group.Corresponding modeling intervention was given respectively,and senna leaf was given intragastric administration for 6 weeks.At 0,2,4,and 6 weeks of gavage,the daily food intake,daily water intake,fecal granule number,fecal Bristol score,visceral sensitivity,anal temperature,grip strength,and weight-bearing swimming time of the rats were evaluated.After 6 weeks of intragastric administration,the levels of serum D-xylose,adrenocorticotropic hormone（ACTH）,corticosterone（Cort）and the pathological changes of colon were detected.3.Intervention mechanism of Wenshen-Jianpi prescription on intestinal mucosal barrier function,TLR4/MyD88/NF-κB signaling pathway and intestinal flora.Sixty SD suckling rats were randomly divided into normal group,model group,Wenshen-Jianpi prescription high-dose,medium-dose and low-dose groups,western medicine group,with 10 rats in each group.Except the normal group,the other groups were treated with mother-child separation combined with senna leaf gavage for modeling,and corresponding drugs were given for 2 weeks after modeling.After treatment,the symptoms and behavioral changes of rats in each group were evaluated,and the levels of ACTH,Cort,D-xylose,IL-6,TNF-α,IL-1β and LPS in serum were detected,and the protein and mRNA expression levels of TLR4,NF-κB P65,MyD88,Claudin-1,Occludin and ZO-1 in colon tissues were detected.HE staining was used to detect the pathological changes of colon.After treatment,feces of rats in the normal group,model group and Wenshen-Jianpi prescription medium-dose group were collected and compared based on 16S rRNA gene sequencing.Results1.Serum inflammatory factor levels in IBS-D patients with spleen and kidney Yang deficiency syndrome.Compared with the healthy control group,serum inflammatory factors IL-1β,IL-6,TNF-α,and IFN-γ were increased in IBS-D patients（P<0.05）.Il-4 and IL-10 showed a decreasing trend in IBS-D group,but no significant difference was found between IBS-D group and normal group（P>0.05）.IL-1β,IL-6,TNF-α,IFN-γ were not correlated with IBS-SSS score or Bristol score.2.Establishment of animal model of IBS-D with spleen and kidney Yang deficiency syndrome.After 2 weeks of senna leaf gavage,compared with the normal group,rats in model groups lost weight,increased daily water intake,fecal grain number,Bristol score and AWR score（P<0.05）,daily food intake in mother-child separation group decreased（P<0.05）,and anal temperature in mother-child separation group and rectal dilation group decreased（P<0.05）.After 4 weeks of senna leaf intragastric administration,compared with the normal group,the body weight,daily food intake and daily water intake of rats in all model groups were decreased（P<0.05）,fecal granule number,Bristol score and AWR score were increased（P<0.05）,and anal temperature was decreased（P<0.05）.After 6 weeks of senna leaf gavage,compared with normal group,all model groups lost weight,increased daily water intake,Bristol score,AWR score,anal temperature and grip strength（P<0.05）.The daily food intake of mother and child separation group and rectal dilation group were decreased,the number of defecation grains was increased（P<0.05）,and the weight-bearing swimming time of rectal dilation group was decreased（P<0.05）.Serum D-xylose and ACTH in mother-child separation group and rectal dilation group were decreased（P<0.05）,and serum Cort in mother-child separation group was decreased（P<0.05）.No obvious abnormality was observed in the intestinal mucosa histopathology of rats in each group.3.Intervention mechanism of Wenshen-Jianpi prescription on intestinal mucosal barrier function,TLR4/MyD88/NF-κB signaling pathway and intestinal flora.In terms of behavioral performance,compared with model group,Wenshen-Jianpi prescription at high,medium and low doses could reduce the number of defecation particles and Bristol score in IBS-D model rats（P<0.05）,Bristol score in the Western medicine group was significantly lower than that in the model group,but there was no significant difference in the number of fecal particles at 4h（P<0.05）.After treatment,AWR scores of rats in each group were decreased,and Wenshen-Jianpi prescription high-dose and medium-dose groups were lower than those in low-dose group and Western medicine group（P<0.05）.Compared with the low-dose group and western medicine group,Wenshen-Jianpi prescription high-dose group could better increase the grip strength and anal temperature of rats（P<0.05）.No obvious abnormality was observed in the intestinal mucosa histopathology of rats in each group.The results of biochemical tests on the relevant indicators show that,①The contents of ACTH,Cort and D-xylose in serum of model group were lower than those of normal group（P<0.001）.The levels of ACTH,Cort and D-xylose in serum of Wenshen-Jianpi prescription at high,medium and low doses were significantly higher than those in low dose and western medicine groups（P<0.05）.②The protein and mRNA expressions of Claudin-1,Occludin and ZO-1 in colon of model group were lower than those of normal group（P<0.001）,the protein and mRNA contents of model group were increased by high,medium and low dose of Wenshen-Jianpi prescription and western medicine（P<0.01）.Claudin-1,Occludin and ZO-1 proteins in high and medium dose groups were significantly higher than those in low dose group and western medicine group（P<0.05）.The expression of Claudin-1 mRNA,Occludin mRNA and ZO-1 mRNA increased in high and medium dose groups and western medicine group,which was significantly higher than that in low dose group（P<0.05）.③In model group,the serum LPS content was increased,and the protein and mRNA expressions of TLR4,NF-κB P65 and MyD88 in colon were increased（P<0.001）.The protein contents of TLR4,MyD88,NF-κB P65 and LPS levels in colon of high,medium and low dose groups and western medicine group were significantly lower than those in model group（P<0.01）,and those in high and medium dose groups were lower than those in low dose group and western medicine group（P<0.05）.The expression of TLR4 mRNA,MyD88 mRNA and NF-κB p65 mRNA in high and medium dose groups and western medicine group were significantly lower than that in model group（P<0.05）.④The serum content of IL-6,TNF-and IL-1 was increased in the model group（P<0.001）.The serum content of IL-6,TNF-and IL-1 in the high,medium and low dose group and western medicine groups were significantly reduced（P<0.01）,and the high,medium dose and western medicine groups were lower than the low dose groups（P<0.001）.The results of 16S rRNA sequencing of intestinal flora show that,①ACE index,Chao 1 index,Simpson index and Shannon index were not abnormal among the normal group,model group and Wenshen-Jianpi prescription group（P>0.05）.②PCoA analy sis,NMDS analysis and Lefse analysis showed that Wenshen-Jianpi prescription group was closer to the normal group in species composition,while the difference between model group and the normal group and Wenshen-Jianpi prescription was greater.③At phylum level,compared with the normal group,the abundance of Firmicutes and Prote obacteria increased in the model group,while the abundance of Bacteroidetes decrease d.The abundance of Firmicutes and proteobacteria was down-regulated and the abunda nce of Bacteroidetes was up-regulated by Wenshen-Jianpi prescription,but no significa nt difference was found in the phylum level.The ratio of Firmicutes to Bacteroidetes（F/B）in model group was higher than that in normal group,and the F/B value in We nshen-Jianpi prescription group could be reduced.④Eleven different genera were scree ned at the genus level.In the model group,Bacteroides,Phascolarctobacterium,Alloba culum,Romboutsia,Helicobacter,Blautia,Christensenellaceae_R-7_group,Clostri Dium increases significantly（P<0.01）,and the relative abundance of the above bacteria c ould be decreased by Wenshen-Jianpi prescription（P<0.05）.Ruminiclostridium₆ in model group was significantly lower than that in normal group（P<0.05）,ruminiclost ridium 6（P<0.05）.⑤LEfSe analysis was used to analyze the characteristic species at phylum,class,order,family,genus and species level.At the genus level,the main characteristic bacteria in the normal group were Ruminococcaceae_UCG₀14,and the main characteristic bacteria in the model group were Bacteroidetes,Brucella and Helic obacter.The main characteristic bacteria of Wenshen-Jianpi prescription group were Pre votellaceae_NK3B31_group and uncultured_bacterium_oMollicutes_RF39,which were basically consistent with the previous results.⑥Further analysis of the correlation bet ween different bacteria and LPS,The results showed that Bacteroides,Phascolarctobact erium,Allobaculum,Romboutsia,Helicobacter,Blautia,Dubosiella,Clostr Idium was p ositively correlated with LPS content（P<0.05）,Ruminiclostridium₆ was negatively correlated with LPS（P<0.05）.Wenshen-Jianpi prescription may affect the changes of serum LPS level by regulating intestinal flora structure.Conclusions1.The IBS-D patients with spleen and kidney Yang deficiency syndrome have a low-grade inflammatory state of imbalance between pro-inflammatory factors and anti-inflammatory factors,however,no significant correlation was found between the levels of inflammatory factors and the severity of symptoms in IBS-D patients in this study.2.The separation of mother and child combined with senna leaf gavage for 6 weeks can better establish the IBS-D rat model of disease and syndrome combined with spleen and kidney Yang deficiency syndrome.3.Wenshen-Jianpi prescription can improve symptoms and visceral hypersensitivity of IBS-D model rats with spleen and kidney Yang deficiency syndrome.The mechanism of therapeutic effect may be related to restoring intestinal mucosal barrier function,inhibiting TLR4/MyD88/NF-κB P65 signaling pathway,regulating intestinal microflora,reducing the expression of inflammatory factors IL-6,TNF-α and IL-1β downstream of the pathway,and thus improving low-grade inflammation.