Study On The Therapeutic Effect Of Indirubin On Ulcerative Colitis Model Rats And Its Mechanism

Aim:Ulcerative colitis?UC?as a non-specific inflammatory bowel disease with unclear etiology,often occurs in the nodules or rectum,.When the disease occurs,it happens a lot in patients as abdominal pain,diarrhea,urgency,and lower stools.It as a malignant tendency and has been classified as a refractory disease by WHO.For the treatment of this disease,the existing by Chemical drugs in modern medicine treatment program can not The effectively control the UC attack,especially in the prevention of recurrence is very unsatisfactory,accompanied by long treatment cycle,large drug side reaction,high treatment costs and other drawbacks.The treatment of UC has received significant efficacy in the clinic by Traditional Chinese Medicine?TCM?.Which as an active ingredient in the Qingdai,indirubin is a double indoles compound,at the same time the researchers found that indirubin can anti-tumor and anti-inflammatory The effects.The study in this paper,we studied the therapeutic The effect of Chinese traditional medicine-indirubin,on the UC model rats induced by 5%trinitrobenzene sulfonic acid?TNBS?/ethanol solution,and applied the method of molecular biology to observe the affect on MEK/ERK signaling pathway and PI3K/AKT signaling pathway in colon tissues,according to use the indirubin to treat on the UC model rat.We expect to further explore the mechanism of the way to respond well to treatment by indirubin.Method:At the beginning of the study,we got 48 clean-grade SD rats,male,body weight?200±20?g,who were randomly divided into 4 groups.For respectively,they were normal group,model group,indirubin group and sulfasalazine?SASP?group.The UC model rats were established by trinitrobenzenesulfonic acid?TNBS?/ethanol method.After 21 days of treatment,we should observe the disease activity index?DAI?score,as well as according to the naked eye to observe the difference of the gross morphology between different groups.The pathological smears of HE staining in rats were observed by electron microscope.By the technology of ELISA,we detected of the level of myeloperoxidase?MPO?,as well as the level of transforming growth factor?1?TGF-?1?in rat colonic mucosa.By the technology of RT-PCR,we detected the level of expression of intestinal trefoil factor?ITF?mRNA,as well as the level of expression of basic fibroblast growth factor?bFGF?mRNA by RT-PCR.The expression levels of MEKmRNA,ERKmRNA,PI3KmRNA,AKTmRNA and the expression levels of p-MEK,p-ERK and p-AKT protein were detected by the technology of RT-PCR or Western blot respectively.Conclusion:1.The The effect of indirubin on DAI score in UC model ratsCompared with the normal group,the DAI score of the model group was significantly increased obviously?p<0.01?.Compared with the model group,the DAI scores of the indirubin group and the SASP group were all decreased obviously?p<0.01?.There was no significant difference on the DAI score between the indirubin group and the SASP group?p>0.05?.2.The effect of indirubin on gross morphology observation and HE staining of colonic histopathology on UC model ratsThe rat of normal group were not observed significant abnormality from the rectum to the end of ileocecal.The colon tube were unobstructed,and the colonic mucosa were not observed the scar after the healing by the ulceration.There were not any hyperaemia or edema on the colonic mucosa.On the colonic mucosa of model group rats,we can see huge and deep ulceration,where were tough to touch.And the pathological changed colon tube were inelasticity,where were brownish.There were seen a small amount of excrement in the colon tube distal end of the ulceration.We can see the edema around the ulceration in the colonic mucosa.After the treatment by indirubin or SASP,we can see the scar after the healing by the ulceration,and the color of the mucosa were next to normal.We only can see hyperaemia or edema at some place,where the ulceration were occurred.HE staining of rat colon tissue showed that the colonic mucosal epithelium of the normal group was intact,the structure was clear,the glands were arranged regularly,the goblet cells in the lamina propria were abundant,and no inflammatory cell infiltration was observed.The depth can reach the mucosal muscle layer,the structure is destroyed,the crypt cells lose the epithelial cell shedding and necrosis;the lamina propria gland shrinks,the arrangement is disordered,some glands are even completely lost;a large number of inflammatory cells infiltrate and infiltrate in the mucosa and submucosaAfter the intervention of indirubin and positive drug SASP,the colonic neutrophils and lymphocytes infiltrated in the UC model rats were less than the model group,a small number of glandular tubes were distorted,the degree of mucositis infiltration,glandular structure disorder,and epithelial cells were observed.Defects and ulcers have improved to varying degrees.3.The effect of Indirubin on the level of MPO in colon tissue of UC model ratsCompared with the normal group,the level of MPO in the colon tissue of the rats to model group was significantly increased?p<0.01?.Compared with the model group,the level of MPO in the colon tissue of the rats to indirubin group and the SASP group was significantly lower?p<0.01?,but there was no significant difference between the indirubin group and the SASP group?P>0.05?.4.The effect of indirubin on the level of TGF-?₁ in colon tissue of UC model ratsCompared with the normal group,the level of TGF-?₁ content in the colon tissue of the model group was significantly increased?p<0.01?.The content of TGF-?₁ in colon tissue of each treatment group was lower than that of the model group?p<0.01?.There was no significant difference in the content of TGF-?₁ in the colon tissue of the rats in the indirubin group compared with the SASP group?P>0.05?.5.The effect of indirubin on the level of ITF mRNA in colon tissue of UC model ratsCompared with the normal group,the expression of ITFmRNA in the colon tissue of the rats to the model group was significantly decreased?p<0.01?.Compared with the model group,the expression of ITF mRNA in the colon tissue of the rats to the Indirubin group and the SASP group was significantly higher?p<0.01?,but there was no significant difference between the indirubin group and the SASP group?P>0.05?.6.The effect of indirubin on the level of bFGF mRNA in colon tissue of UC model ratsCompared with the normal group,the expression of bFGF mRNA in the colon tissue of the rats to the model group was significantly increased?p<0.01?.Compared with the model group,the expression of bFGF mRNA in the colon tissue of the rats to the indirubin group and the SASP group was significantly higher?p<0.01?,but there was no significant difference between the indirubin group and the SASP group?P>0.05?.7.The effects of Indirubin on the expression of MEK mRNA,ERK mRNA and the expression of p-MEK and p-ERK protein in colon tissue of UC model ratsCompared with the normal group,the expression levels of MEK mRNA and ERK mRNA and the expression levels of p-MEK and p-ERK protein in the colon tissue of the rats to the model group were significantly decreased?p<0.01?.Compared with the model group,the expression levels of MEK mRNA and ERK mRNA and the expression levels of p-MEK and p-ERK protein in the colon tissue of the rats to the indirubin group and the SASP group were significantly higher?p<0.01?,while the Indirubin group and the SASP group were compared.The difference was not statistically significant?P>0.05?.8.The effects of indirubin on the expression of PI3KRNA,AKT mRNA and p-AKT protein in colon tissue of UC model ratsCompared with the normal group,the expression levels of PI3KRNA,AKT mRNA and p-AKT protein in the colon tissue of the rats to the model group were significantly increased?p<0.01?.Compared with the model group,the expression levels of PI3KmRNA,AKTmRNA and p-AKT protein in the colon tissue of the rats to the indirubin group and the SASP group were significantly lower?p<0.01?,while the difference between the indirubin group and the SASP group was not statistically significant significance?P>0.05?.Conclusion:1.Indirubin can inhibit the symptoms of intestinal inflammation in UC model ratsBy reducing the DAI score of rats,this study can prove that indirubin can alleviate the intestinal inflammatory symptoms of UC model rats,improve the quality of life of UC model rats,and verify the effectiveness of indirubin in the treatment of UC model rats.According to observe the gross morphology on the colonic mucosa of the rats.After the treatment of the indirubin,We only can see hyperaemia or edema at some place,where the ulceration were occurred.Compare with the model group,which on the colonic mucosa can be seen huge and deep ulceration,where were tough to touch,and the pathological changed colon tube were inelasticity,where were brownish,the indirubin group and SASP group can be cured well.It can prove that indirubin has the effect to repair the damage by the ulceration.HE staining of colonic mucosa in rats showed that the inflammatory response of colonic mucosa in UC model rats showed only a small amount of neutrophil and lymphocyte infiltration compared with untreated UC model rats.The infiltration of mucositis cells in the colonic mucosa is profoundly improved to the mucosal muscle layer,glandular structure disorder,epithelial cell loss and ulceration.This can be used to visually demonstrate the repair function of indirubin on the colonic tissue of UC model rats and the function of inhibiting inflammation.2.Indirubin can down-regulate the level of TGF-?₁ in colon tissue of UC model ratsAs a member of the growth factor superfamily,TGF-?₁ regulates the proliferation and differentiation of immune cells,consistent inflammatory response.Its expression is in direct proportion to the order of severity of UC.Indirubin can down-regulate the content of TGF-?1in the colon tissue of UC model rats.On the one hand,it can inhibit the colonic injury of inflammatory cells in UC model rats,and on the other hand,it can heal the wounds of rough muscle intestinal epithelial ulcers and repair tissue.3.Indirubin can down-regulate MPO levels in colon tissue of UC model ratsIndirubin can down-regulate the MPO level in the colon tissue of UC model rats,which can be used to show that indirubin can The effectively control UC inflammatory symptoms and reduce inflammatory cell infiltration.MPO is an important part of neutrophil azurophilic granules.The index of cell aggregation is positively correlated with the level of UC intestinal inflammation.4.Indirubin can inhance the expression of ITF mRNA in colon tissue of UC model rats.By treated with indirubin,it can up-regulate the expression of ITFmRNA in colon tissue of UC model rats.On the one hand,it can be explained that indirubin has the effect of inhibiting apoptosis of intestinal mucosa cells,on the other hand,it has repaired damaged intestinal mucosa of UC model rats.And regulate the role of cellular immunity.5.Indirubin can inhance the expression of bFGFmRNA in colon tissue of UC model ratsThe expression of bFGFmRNA in the colon tissue of the model group was significantly higher than that in the normal group.The effectiveness of modeling was demonstrated from this result,because the expression of bFGFmRNA was minimal or not expressed in the colon tissue of normal rats,but due to these proinflammatory,the expression of bFGFmRNA in the colon tissue of the model group was increased by stimulating a cascade of inflammatory mediators such as NO.The treatment of indirubin can continue to up-regulate the expression level of bFGFmRNA in colon tissue of UC model rats,indicating that indirubin can increase the amount of angiogenic factors,thereby promoting new angiogenesis and restoring damaged mucosal integrity.6.Indirubin can increase the expression of MEK mRNA and ERK mRNA and the expression levels of p-MEK and p-ERK protein in colon tissue of UC model ratsAfter treated with indirubin,the expression level of MEKmRNA and ERKmRNA and the expression level of p-MEK and p-ERK protein in colon tissue of UC model rats were significantly increased,which can indicate that indirubin can activate MEK/ERK signaling pathway and indirubin inhibit mucosal cells apoptosis.Indirubin can promote the differentiation of intestinal mucosal cells and inhibit the inflammatory response.7.Indirubin can reduce the expression of PI3KRNA,AKT mRNA and p-AKT protein in colon tissue of UC model ratsBy reducing the expression level of PI3KmRNA,AKTmRNA and p-AKT protein in colonic tissues of UC model rats,indirubin can activate PI3K/AKT signaling pathway.Especially with the phosphorylation of AKT,a complete signaling pathway can be activated.The pathway can exerts its anti-apoptotic,and the effect to inhibit further infiltrate of inflammatory cells.In summary,indirubin can effectively treat UC model rats according to down-regulating the level of TGF-?1,down-regulating the level of MPO,increasing the level of bFGFmRNA,increasing the level of ITFmRNA expression,and activating MEK/ERK signal transduction pathways,as well as the PI3K/AKT signaling pathway.Through the mechanism above,indirubin can inhibits intestinal cell apoptosis,inhibits intestinal inflammation,and promotes intestinal damage caused by UC.