The Effect And Mechanism Of Cryptotanshinone In The Treatment Of Inflammatory Bowel Disease

ObjectiveIBD is a chronic and recurrent inflammatory disease of the intestine,which mainly includes Crohn’s disease and ulcerative colitis.In recent years,the incidence of IBD has been increasing worldwide and it has become one of the main diseases of the digestive system.The loss of intestinal mucosal barrier function is the most important pathological changes of IBD.Intestinal epithelial cells and the connections between cells constitute the epithelial barrier function.Cell connections are composed of desmosomes,adhesive connections,and tight connections.The most important component of adhesion is E-cadherin.Several studies have shown that the expression of E-cadherin is down regulated in inflammatory epithelium of IBD patients.Tight junctions are composed of occludin,claudin and ZO proteins,which directly restrict paracellular cell permeability.The intestinal epithelial barrier is damaged,leading to an increase in intestinal permeability.The treatment of IBD is currently based on hormones,immunosuppressants,molecular targeted therapy,etc.Although it has a certain effect,there are many side effects,such as poor specificity,high incidence of secondary infections and affecting children’s growth up.Therefore,it is necessary to explore new drug treatments for IBD.Cryptotanshinone is extracted from traditional Chinese medicine Salvia miltiorrhiza.It has the effect of removing blood stasis,promoting blood circulation and so on.And it can be used for the treatment of mastitis,lymphadenitis,arthritis and other inflammatory diseases.However,there is no report on whether cryptotanshinone can treat IBD.This study will explore the therapeutic effect of cryptotanshinone on IBD and explore its possible mechanism.Methods1.The intestinal barrier model was constructed by culturing human intestinal epithelial cell lines in vitro.The experiment was divided into negative control group(Control group),proinflammatory factor group(0.1 mg/ml LPS),treatment group(0.1mg/ml LPS+50 ug/ml CTN)and CTN alone treatment group(50 ug/ml CTN).The permeability of cell membrane was detected by comparing the permeability resistance and the permeability of fluorescent yellow,and the effect of CTN on intestinal barrier function was evaluated.2.Western blot was used to detect the expression of E-cadherin and ZO-1 in different concentrations of CTN and the effect of proinflammatory factors on the expression of E-cadherin and ZO-1 was detected as well.Immunofluorescence staining was used to detect the changes of E-cadherin and ZO-1.3.The expression of CREB and p-CREB was detected by WB and the expression of each protein was detected by adding CTN inhibitor(KG-501);After the cells were transfected with lentivirus interfering with CREB(Si CREB),protein samples were extracted,and the expression changes of connexin E-cadherin and ZO-1 were detected by WB.The changes of expression of E-cadherin and ZO-1,the optimal concentration of CTN for CREB activation,and the interaction of CREB with E-cadherin and ZO-1were studied by chromatin immunoprecipitation.4.3% DSS was used to model mice enteritis and 40mg/kg cryptotanshinone was given at the same time.The protective effect of cryptotanshinone on intestinal barrier function of mice was evaluated by comparing the weight change,DAI score,colon length change of mice and the expression of E-cadherin and ZO-1 by immunohistochemical staining.Results1.As the cell grows and proliferates,the transepithelial electrical resistance(TEER) value of the cell increases and reaches stability on the 19 th day,which meets the experimental requirements.Under the action of proinflammatory factors,the TEER decrease from 24 hours,continuing this effect to 96 hours after application,while the permeability of fluorescein yellow increased significantly(P<0.05).CTN alleviated the decrease of TEER induced by proinflammatory factors from 48 hours to 96 hours,and the permeability of fluorescein yellow descended(P < 0.05).In contrast,50 ug/ml CTN alone can significantly increase the TEER value and reduce the permeability of fluorescent yellow.2.Different concentrations of CTN(0-400ug/ml)enhanced tight junction protein markers including E-cadherin and ZO-1 with the optimized dose at 50ug/ml.In the intestinal barrier injury induced by proinflammatory factors,the expression of E-cadherin and ZO-1 decreased.Immunofluorescence staining showed that CTN could significantly increase the expression of E-cadherin and ZO-1.3.Further study on the mechanism of CTN ameliorating intestinal barrier injury,we found that the activation of CREB molecule was manifested by an increase in the ratio of p-CREB/CREB,while the levels of p-CREB,E-cadherin and ZO-1 were significantly decreased after the inhibition of the activity of CREB protein by the specific inhibitor of CREB protein(KG-501).The expression of E-cadherin and ZO-1was significantly decreased after transfection of CREB interfering with lentivirus,and CTN treatment at this time did not change the expression of E-cadherin and ZO-1.200ug/ml CTN had the most obvious activation effect on CREB.The results of chromatin immunocoprecipitation assays showed that CREB molecules were bound to the transcription promoters of E-cadherin and ZO-1.4.In the mouse model,the disease activation index(DAI)in the DSS group was increased,the length of intestinal tract was shortened,and CTN group was able to repair the above changes.Immunohistochemical results showed that the expression of E-cadherin and ZO-1 was significantly up-regulated in CTN treatment group compared with DSS model group.Conclusion1.CTN can inhibit the increase of intestinal epithelial barrier permeability caused by proinflammatory factors,repair the cell junctions and protect the barrier function of intestinal epithelial cells.2.CTN promotes the repair of intestinal epithelial barrier injury by promoting the expression of E-cadherin and ZO-1 in intestinal epithelial cells.3.CTN promotes the expression of E-cadherin and ZO-1 depending on CREB.