| Objective:To observe the therapeutic effect of Kuikeling modified prescription(KKL)with different doses on the mouse colitis model induced by DSS,and to explore whether KKL modified prescription alleviates colitis by inhibiting TLR4/MyD88/NF-kB signaling pathway,and to lay a theoretical foundation for its clinical application.Methods:(1)50 SPF C57BL/6J mice were adaptive fed for a week,and randomly divided into 5 groups with 10 mice in each group,that is:normal control(NC)group,DSS control(DSS)group,Kuikeling modified prescription high dose(KKL H)group,Kuikeling modified prescription medium dose(KKL M)group,Kuikeling modified prescription low dose group(KKL L).NC group free drinking deionized water,the rest of the four groups are free to drink 4%DSS solution for building,building 2-8 days.NC group and DSS group was given 0.5 ml of normal saline and the remaining three groups were given the same volume but different doses of KKL granules suspension by gavage.At the end of intragastric gavage on the 7th day,mice in each group were fasted without water and treated and collected 24h later.(2)During the experiment,the general condition of mice was observed every day,the body weight of mice was measured,the stool character and color were observed,the food intake and water intake were recorded,the activity status and sensitivity of mice were observed,and the disease activity index(DAI)was used to evaluate the disease status of mice.(3)After the end of the experiment,blood was collected from the orbit of the mice,and the colon was intercepted and measured.After the partial colon tissue was fixed with paraformaldehyde fixative solution,the pathological conditions of the colon were observed by HE staining and the pathological score was made.(4)ELISA method to detect serum myeloperoxidase(MPO)in mice and the content of inflammatory cytokines TNF-α,Western Blot(WB)method and Real Time quantitive PCR(RT-qPCR)method to detect TLR4/MyD88/NF-kB protein involved in the signaling pathway and gene expression and the expression of inflammatory cytokines to evaluate different doses of KKL in the treatment of colitis effects and discuss whether its by inhibiting TLR4/MyD88/NF-kB the signaling pathways and play a role of ease of colitis.Results:(1)Compared with the NC group,mice in the DSS group began to lose weight on the 4th day of modeling(P<0.05),and significant weight loss at the end of the experiment(P<0.01),visible naked blood stools,listlessness of mental state,slow reaction and laziness,significantly reduced mobility,hair drying and losing luster,reduced intake of food and water,DAI was significantly increased(P<0.01),the colon length of mice was significantly shortened(P<0.01),the histopathological score of mice was significantly increased by HE staining(P<0.01),indicating successful modeling.(2)Compared with DSS group,KKL H,M,L groups could relieve the symptoms and slow down the weight loss of mice(P<0.05),KKL M group also can reduce DAI score(P<0.05),the colon length of mice in KKL intervention groups were prolonged(P<0.01),HE staining showed that histopathological score of KKL H,M groups were significantly decreased(P<0.01),suggesting that KKL had therapeutic effect on mouse colitis,among which KKL M had the best effect.(3)ELISA results showed that the serum MPO,TNF-α content of DSS group was significantly higher than that of NC group(P<0.01),compared with DSS group,As for MPO,only the serum MPO content in KKL M group decreased(P<0.05),As for TNF-α,the serum TNF-α content of KKL H、M mice decreased(P<0.01).(4)WB results showed that the protein expression levels of TLR4,MyD88 and NF-kB p65 in colon tissue of mice in DSS group were significantly higher than those in NC group(P<0.01),compared with DSS group,the expression levels of the above proteins were decreased in KKL M group(P<0.05).(5)RT-qPCR results showed that compared with the NC group,the gene expression levels of TLR4,MyD88,NF-kB p65 and TNF-α,IL-6 in colon tissue of mice in DSS group-were significantly increased(P<0.01),compared with DSS group,the expression levels of the above genes were decreased in KKL intervention group(P<0.05).Conclusion:KKL has a good th erapeutic effect on DSS-induced colitis in mice,among which KKL M has the best effect.It can improve the general state of mice,slow down the weight loss of mice,inhibit the pathological damage of mice colon tissue,and down-regulate the level of inflammatory factors in serum of mice.Its mechanism may be to achieve the purpose of treating colitis by inhibiting the activation of TLR4/MyD88/NF-kB signaling pathway. |
PDF Full Text Request