The Inhibition Effect Of Kangfuxin Solution On Inflammation-associated Intestinal Fibrosis By Regulating TGF-?1/Smads Signaling Pathway

Background: Intestinal fibrosis is a common complication of inflammatory bowel disease.The mechanism is unknown and it is easy to cause intestinal stenosis.Surgery is the only way to treat the current stenosis.Therefore,the use of drug intervention in the early stage of intestinal fibrosis has become an important means to prevent or delay the progression of fibrosis,but currently there is no ideal drug in the clinic.KFX solution is a dried worm extract of the American cockroach.In recent years,it has been found that the drug has the effect of anti-inflammatory,anti-fibrosis and good tissue healing with low adverse reactions.In view of the fact that the clinical intestinal fibrosis is caused by long-term repeated inflammatory injury,the research has certain value on the role of KFX on the prevention and treatment of intestinal fibrosis.Objective: In this experiment,we explored the role and mechanism of KFX solution in inflammation-related intestinal fibrosis model through in vivo and in vitro experiments.Method: 1.100 female BALB/c mice were divided into normal group,TNBS model group,Kangfuxin solution(0.1 ml/20 g,0.2 ml/20 g,0.4 ml/20g)group,5-ASA group(40mg/kg).The doses of TNBS were 1.0 mg,1.5 mg and 2.0 mg,respectively.Mice were given TNBS+ 45% ethanol by intrarect injections weekly.The levels of IL-10 and IL-17 in serum were detected by El ISA method.The pathological changes of intestinal tract were observed by H&E and Massson staining.The expression of ?-SMA in intestinal tract was detected by immunohistochemistry.Real time-PCR and Western blot were used to detect the m RNA and protein expression of ?-SMA,Collagen I,TGF-?1 and its downstream molecules Smad2,Smad3,p-Smad2,p-Smad3,Smad7.2.In vitro,MTT assay showed that the concentration of KFX at 1,2,4 mg/ml were safe and effective.Real time-PCR results showed that ?-SMA and Collagen I were increased in the model group.The expression in KFX group and 5-ASA group can be reduced.The WB results showed that the expression of ?-SMA and Collagen I was increased in the model group,and the expression was decreased in the KFX group and the 5-ASA group.Results: 1.HE and Masson staining showed that there were a large number of inflammatory cell infiltration in the intestinal tissue of mice in the TNBS model group,and the gland and crypt decreased or disappeared,and the fibrin collagen was expressed in a large amount,suggesting that the intestinal fibrosis model was successful;KFX(0.2ml,0.4 ml groups and the 5-ASA group,the inflammatory cell infiltration was relatively reduced compared with the model group,the basic structure was relatively intact,and collagen expression was significantly reduced.The ELISA results showed that IL-10 level in the model group was lower than normal group.The IL-10 levels in the KFX groups and the 5-ASA group were significantly higher than model group.IL-17 level in the model group was higher than normal group,and IL-17 levels were reduced in the KFX groups and 5-ASA group compared to model group.The m RNA and protein expression of ?-SMA and Collagen I were increased in the TNBS group;the expression in the KFX(0.2 ml and 0.4 ml)groups and the 5-ASA group were decreased compared with the TNBS group.Real time-PCR and WB showed that TGF-?1,Smad2,Smad3 were increased in the model group and decreased in the KFX(0.2 ml,0.4 ml)groups and the 5-ASA group;whereas Smad7 expression was decreased in the model group,and increased in the KFX groups(0.2 ml and 0.4 ml)and the 5-ASA group.WB results also showed that expression of p-Smad2 and p-Smad3 was increased in the model group,while decreased in the KFX(0.2 ml,0.4 ml)groups and the 5-ASA group.Conclusion:KFX has an inhibitory effect on the formation of intestinal fibrosis induced by chronic intestinal inflammation in mice.The mechanism may be through regulation of TGF-?1/Smads signaling pathway and inhibition of fibroblasts.