Research On Mechanism Of Si Shen Wan Combined With Tong Xie Yao Fang In Treating Ulcerative Colitis With Spleen-kidney Yang Deficiency And Liver Stagnation

1 Background and objectiveUlcerative Colitis(UC)is an inflammation of the intestinal mucosa that persists after mucosal damage has healed.The continuous development of UC course will have a variety of adverse effects on patients,such as the increased permeability of the intestinal,increased risk of tumor development,and increased risk of other complications.Commonly used drugs for the clinical treatment of UC include amino salicylic acids,glucocorticoids,immunosuppressants,biologic agents,etc.,but most of them are expensive and accompanied by side effects,and patients’ sensitivity to drugs decreases with the development of the disease.In this case,traditional Chinese medicine has attracted much attention in the treatment of UC due to its multi-pathway,multi-target effect and small side effects.According to the symptoms of UC,TCM doctor classified it as diarrhea,abdominal pain,blood in the stool,etc.In the theory of traditional Chinese medicine,spleen deficiency is the root of UC,which may leads to dysfunction of liver and kidney.Clinically,patients with dampness-heat type of large intestine,liver stagnation with spleen deficiency,spleen-kidney Yang deficiency type are common.Clinical data survey showed that more than 30%of UC patients were manifested as spleen-kidney Yang deficiency or liver stagnation with spleen deficiency or multiple syndroms.Si Shen Wan from the "Zheng Zhi Zhun Sheng",can warm the kidney and spleen;Tong Xie Yao Fang is found in "Dan Xi Xin Fa",which has the function of tonifying the spleen and soothing the liver.Both of them are effective in treating diarrhea,in clinical cases,the combination of Si Shen Wan and Tong Xie Yao Fang for UC patients has achieved good efficacy,but the mechanism of action of the combination of the two prescriptions for UC has not been explored.In this study,based on the clinical status of UC,a rat model of ulcerative colitis with spleen-kidney Yang deficiency and liverstagnation was constructed,and the indicators were selected to evaluate the molding effect.In order to clarify the mechanism of action of TCM compound,reveal the scientific connotation of TCM treatment of UC,and better guide clinical drug use,the therapeutic effect and mechanism of Si Shen Wan with Tong Xie Yao Fang on ulcerative colitis with spleen-kidney Yang deficiency and liver-depression was explored from three aspects:inhibiting inflammatory,repairing intestinal mucosal barrier and regulating water transport.2 Methods and results2.1 Establishment of rat model of ulcerative colitis with spleen-kidney Yang deficiency and liver-depression and the effect of Si Shen Wan with Tong Xie Yao Fang on the syndromeSixty male SD rats were randomly divided into normal group,model group,mesalazine group,Si Shen Wan combined Tong Xie Yao Fang(SSW&TXYF)highdose,medium-dose and low-dose groups.The rats except the normal group were given senna leaf decoction by intragastric administration,hydrocortisone by intramuscular injection,the fight was triggered by tail clamping method.All stimulation modes lasted for 14 days to make the rat model of spleen-kidney Yang deficiency and liver stagnation.On the 14th day,rats were given enema with TNBS-ethanol solution to cause UC.The drug was administered on the 15th day and continued for 14 days.Behavioral indicators of the rats were observed and recorded.After modeling,compared with the normal group,the body weight of rats in the model group decreased significantly,and the manifestations of spleen-kidney Yang deficiency and liver stagnation were observed.The model rats showed loose stool,fear of cold,lazy movement,easy to anger,and anal temperature decreased.The content of D-xylose in serum decreased and GAS content increased(p<0.01);the proportion of cAMP/cGMP in serum was unbalance,and the contents of ACTH and CORT were reduced(p<0.01);the proportion of sugar water drinking in rats decreased,and the content of 5-HT in hippocampus decreased(p<0.01).After enema with TNBS,the rats began to develop bloody stools and sticky purulent stools,and shortening of colon length was observed by anatomy.HE staining shows that a large number of inflammatory cells infiltrated in the colon tissue of the model group,and the structure of the colon tissue was severely damaged.The model had the syndrome manifestations and evaluation indications of spleen-kidney Yang deficiency and liver stagnation,as well as the symptoms and histopathological characteristics of ulcerative colitis,indicating that the rat model of ulcerative colitis with spleen-kidney Yang deficiency and liver stagnation was successfully induced.Mesalazine can repair the pathological damage of colon in rats,but it couldn’t improve the state of spleen-kidney Yang deficiency and liver stagnation.The high,medium,and low dose of SSW&TXYF groups could significantly improve the related indicators of the syndrome(p<0.05,p<0.01),restore the length of colon,reduce inflammatory infiltration,restore the structure of gland crypt,and repair the pathological damage of the tissue.2.2 Effects of Si Shen Wan with Tong Xie Yao Fang on inflammatory cytokines and MAPK inflammatory pathway of ulcerative colitis rats with spleen-kidney Yang deficiency and liver stagnation2.2.1 Serum inflammatory cytokines were detected by ELISABlood was collected from abdominal aorta and serum was separated to detect the contents of IL-4,IFN-γ and TNF-α in serum.The results showed that compared with normal group,the serum levels of anti-inflammatory factor IL-4 in model group were decreased(p<0.01),and the concentrations of pro-inflammatory factors TNF-α and IFN-γ were increased(p<0.05,p<0.01).Compared with model group,IL-4 content in mesalazine group was increased(p<0.01),while TNF-α and IFN-γ contents were decreased(p<0.05,p<0.01).TNF-α was significantly down-regulated in SSW&TXYF high-dose group(p<0.01).Serum IL-4 content was increased whileTNF-α and IFN-γcontents were decreased in medium-dose group,but there was no significant difference compared with the model group.IL-4 content was significantly increased(p<0.01)and the contents of TNF-α and IFN-γ were significantly decreased(p<0.05,p<0.01)in the low-dose group.2.2.2 Determination of MAPK pathway protein expression in rat colon by qPCR&Western-BlotRNA and protein were extracted from rat colon to detect the expression of p3 8,ERK and JNK.The results showed that compared with the normal group,the expressions of p38,ERK and JNK in the model group were significantly increased,the mRNA and protein expression levels of p3 8 and JNK were significantly up-regulated(p<0.05,p<0.01),and the protein expression of ERK was significantly up-regulated(p<0.05).Compared with model group,JNK mRNA expression and p38 protein expression in colon of rats in mesalazine group were significantly decreased(p<0.05).The mRNA and protein expression levels of MAPK pathway protein in SSW&TXYF high-dose,medium-dose and low-dose groups were significantly down-regulated.The mRNA expressions of p38 and ERK in high-dose group were significantly decreased(p<0.05,p<0.01),and the protein expressions of ERK and JNK were significantly different compared with model group(p<0.05).The expressions of ERK and JNK genes in medium dose group were significantly decreased(p<0.05,p<0.01).The expressions of p38,ERK and JNK in the low dose group were significantly decreased at both gene level and protein level.2.3 Effects of Si Shen Wan with Tong Xie Yao Fang on intestinal mucosal barrier and water transport in ulcerative colitis rats with spleen-kidney Yang deficiency and liver stagnation2.3.1 The expression of tight junction protein in the colon of rats was detected by qPCR&Western-BlotqPCR and Western-Blot were used to detect the expression of Claudin-1,Occludin and ZO-1 in the colon tissue of rats.The results showed that compared with the normal group,mRNA and protein expression of Claudin-1,Occludin in the colon of rats in the model group significantly decreased(p<0.01),Compared with model group,mesalazine up-regulated mRNA transcription levels of Occludin and ZO-1(p<0.01).In SSW&TXYF high-dose group,the mRNA expression levels of Claudin-1 and Occludin in colon of rats were significantly up-regulated(p<0.05,p<0.01).The mRNA expression of Claudin-1,Occludin and ZO-1 in medium-dose group were all significantly increased(p<0.05,p<0.01),and the protein expression of Claudin-1 and Occludin was significantly up-regulated(p<0.05).In low-dose group the mRNA and protein expression of Claudin-1 was significantly up-regulated(p<0.05),and the content of Occludin in colon tissue was also increased(p<0.05).2.3.2 The expression of aquaporin in the colon of rats was detected by Western-BlotThe expression level of AQP4 in rat colon was detected by Westen-Blot.After modeling treatment,the expression of AQP4 in colon tissue of model group was significantly decreased compared with that of normal group(p<0.01).Compared with model group,AQP4 protein expression of rats in administration group showed an upward trend,and the difference was statistically significant(p<0.05)between SSW&TXYF medium-dose group and model group.3 ConclusionThis research proved that Si Shen Wan combined with Tong Xie Yao Fang had good therapeutic effect on on ulcerative colitis model rats with spleen-kidney Yang deficiency and liver stagnation.The rat model of ulcerative colitis with spleen-kidney Yang deficiency and liver stagnation was successfully established by daily administration of senna leaf,intramuscular injection of hydrocortisone,stimulation of tail clip for 14 days and enema with the mixture of TNBS and ethanol.It was observed that Si Shen Wan combined with Tong Xie Yao Fang significantly improved the syndrome of the model rats.After administration,serum pro-inflammatory factor content was decreased,anti-inflammatory factor concentration was increased.HE staining showed that pathological injury and inflammatory cell infiltration was reduced.The expression of the classical pathway that regulating inflammation MAPK related proteins was decreased,and the expression of Claudin-1,Occludin and ZO-1 in colon tissue increased,indicating that Si Shen Wan with Tong Xie Yao Fang can significantly repair the tight junction of intestinal mucosal barrier and enhance intestinal immunity to antigen invasion.AQP4 expression was decreased in the colon of model group rats,and was upregulated after administration,suggesting that Si Shen Wan with Tong Xie Yao Fang can improve intestinal water absorption and transport,and alleviate intestinal edema and diarrhea symptoms.In conclusion,Si Shen Wan with Tong Xie Yao Fang can improve the syndrome of spleen-kidney Yang deficiency with liver stagnation,and treat ulcerative colitis by reducing inflammatory response,repairing intestinal mucosal barrier and improving water transport.