Protective Effect Of Taraxasterol On Ulcerative Colitis And Its Mechanism

BackgroundUlcerative colitis(UC)is a kind of inflammatory bowel disease,which is immune-related with unknown cause.Currently,it is considered that the incidence of UC is related to environment?heredity?infection?intestinal flora?immunity and other factors.The drugs for ulcerative colitis include various types,such as amino salicylic acid preparation,glucocorticoid,immunosuppressive agents and biological preparation.However,these drugs are easy to produce drug resistance ?other side effects or expensive.Based on the advantages of small side effects and simple treatment methods,the use of medicinal plants and their derivatives in the treatment of UC has attracted more and more scientists' attention in recent years.Phytosterols are natural compounds found in plants.They are mainly found in all plant foods that are rich in lipids and fiber rich components.Phytosterol,similar to cholesterol in structure,has a similar structure and biological function with cholesterol.It has a protective effect on many diseases,such as cardiovascular disease,liver disease and cancer.Phytosterol also has protective effects on gastrointestinal inflammation.Taraxasterol(TS)is a kind of phytosterol,and has higher content in the root of Taraxacum mongolicum.Current studies have shown that TS has anti-inflammatory activity and has a significant inhibitory effect on the expression levels of variousinflammatory factors such as interleukin-1(IL-1),Interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-alpha).In addition,TS has protective effects on rheumatoid arthritis,acute lung injury and endotoxemia.However,there is no research about the effect of Taraxasterol on ulcerative colitis.Based on the close relationship between TS and inflammation of the body,we studied the protective effect of TS on ulcerative colitis in mice by constructing a mouse model of ulcerative colitis,and the anti-inflammatory mechanism of TS by lipopolysaccharide(LPS)induced intestinal macrophages.Part one Protective effect of Taraxasterol on ulcerative colitis model in mice ObjectiveTo study the protective effect of Taraxasterol on ulcerative colitis in mice by constructing a mouse model of ulcerative colitis.MethodsThe experimental animals were 60 Kunming mice of SPF grade,weighing18±2g,male and female half.The mice were purchased from Beijing Victoria Laboratory Animal Technology Co.,Ltd.60 mice were divided into 6 groups randomly,and they were respectively called blank control group,model group,TS low dose group,TS middle dose group,TS high dose group and positive control group.After 7 days of adaptive feeding,all rats in other groups were treated with dextran sodium sulp(DSS)for ulcerative colitis except the rats in blank control group.At the same time,rats in TS low dose group,TS middle dose group and TS high dose group were intraperitoneal injected with Taraxasterol each day respectively.The dosage of Taraxasterols were 2.5mg/kg,5mg/kg and 10mg/kg respectively.The model group received intraperitoneal injection of 40ml/kg normal saline every day.The positive control group was intraperitoneal injected with SASP,with a dose of500mg/kg.After 9 days of intervention,the mice were killed after the prohibition of food and the prohibition of water for 12 hours.The blood of eyeball was taken fromthe inner canthus of the mice before the mice were killed.The levels of interleukin-1beta(IL-1 beta),interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor-alpha(TNF-alpha)were detected in the serum of each group by ELISA kit.After killing the mice,taking out the colon,the length was measured and recorded.The colonic pathological section was made by HE staining.The pathological lesion of the colon was observed by electron microscope.The pathological score of the colon was evaluated by double blind method.The activity of superoxide dismutase(SOD)in the colon homogenate was detected by hydroxylamine method.The activity of glutathione peroxidase(GSH-Px)and myeloperoxidase(MPO)in the colon homogenate was detected by colorimetric method,and the content of malondialdehyde(MDA)in the colon homogenate was detected by TBA method.TUNEL assay was used to detect the apoptosis of colonic epithelial cells.The expression levels of bcl-2 and Bax protein witch were apoptosis-related proteins in the colon of mice were detected by western blot.Results1.The weight change of mice: During the intervention of DSS and TS,the average weight of mice in the blank control group and the positive control group increased gradually within 1 to 9 days.The average weight of mice in the model group,the TS low?middle and high dose group gradually decreased.On the fifth?seventh and ninth day,there was significant difference in the weight of mice between each group(P? 0.05).On the ninth day,compared with the blank control group,the weight of mice in the model group,the TS low?middle and high dose group was significantly lower.Compared with the model group,the weight of mice in the TS middle ? high dose group and the positive control group was significantly higher(P? 0.05).2.Disease activity index: Within 1 to 9 days,the disease activity index of mice in the blank control group has been maintained at a low level of less than 0.5.The disease activity index of mice in the model group,the TS low? middle and high group increased as a whole,but the increase trend of the disease activity index in the TS high dose group was slow.On the fifth ? seventh and ninth day,there wassignificant difference in disease activity index between each group(P? 0.05).On the ninth day,compared with the blank control group,the disease activity index of mice in the model group,the TS low?middle ?high dose group and the positive control group was significantly higher.Compared with the model group,the disease activity index of mice in the TS high dose group was significantly lower(P? 0.05).3.Colon length: The colon length of each group was statistically different(P? 0.05).Compared with the blank control group,the colon length of the model group was significantly lower.Compared with the model group,the colon length of the TS middle ?high dose group and the positive control group was significantly higher(P? 0.05).4.Colon injury: The blank control group has intact epithelial cells and no infiltration of inflammatory cells.In the model group,the crypt destruction and crypt deformation and disorder were arranged.Epithelial cells remain or completely destroyed and inflammatory cells infiltrate.The pathological injury of colon in each TS dose group was restored to varying degrees.The degree of damage in the positive control group was the lightest.5.The histopathological score of colon: The histopathological score of each group was statistically different(P? 0.05).Compared with the blank control group,the histopathological score of the model group was significantly higher.Compared with the model group,the histopathological score of TS middle,high dose group and the positive control group was significantly lower(P? 0.05).6.Inflammatory index: There was significant difference in the levels of IL-1??IL-6?IL-10 and TNF-alpha in mice serum of each group(P? 0.05).Compared with the blank control group,the levels of IL-1??IL-6 and TNF-alpha in the model group were significantly higher,while IL-10 was significantly lower.Compared with the model group,the IL-6 level of the TS high dose group and the positive control group was significantly lower,the IL-10 level of the TS middle?high dose group and the positive control group was significantly higher,while the TNF-alpha level of those groups was significantly lower(P? 0.05).There was no significant difference in IL-1? levels between TS each dose group and the model group(P>0.05).7.Index of oxidative stress: There were significant differences in three oxidativestress indexes(SOD?MPO and GSH-Px)in the colonic homogenate of each group(P? 0.05),and there was no significant difference in the content of MDA(P>0.05).Compared with the blank control group,the levels of SOD and GSH-Px in the model group and each TS dose group were significantly lower,and the level of MPO in those groups was significantly higher.Compared with the model group,the SOD level of the TS middle ? high dose group and the positive control group was significantly higher,the MPO level of each TS dose group and the positive control group was significantly lower,and the GSH-Px level of the TS high dose group and the positive control group was significantly higher(P? 0.05).8.The apoptosis of colonic epithelial cells: The difference of apoptosis rate of colonic epithelial cells in each group was statistically significant(P? 0.05).Compared with the blank control group,the apoptosis rate in the model group and the TS low?middle dose group was significantly higher.Compared with the model group,the apoptosis rate in the TS middle?high dose group and the positive control group was significantly lower(P? 0.05).9.Apoptosis related protein: The relative expression levels of Bax and Bcl-2protein in the colon of each group were statistically significant(P? 0.05).Compared with the blank control group,the relative expression level of Bax protein in the model group was significantly higher,and the relative expression level of Bcl-2 protein was significantly lower.Compared with the model group,the relative expression level of Bax protein in the TS middle? high dose group and in the positive control group was significantly lower,and the relative expression level of Bcl-2 protein in those groups was significantly higher(P? 0.05).ConclusionTS can significantly improve the symptoms and pathological lesions of the mice with ulcerative colitis,reduce the level of inflammation and oxidative stress in mice with ulcerative colitis,and inhibit the apoptosis of colonic epithelial cells of mice.Part two Anti inflammatory and anti apoptotic effects of Taraxasterol in vitro and its mechanism ObjectiveTo study the effects of TS on the inflammatory factors and apoptosis of mouse intestinal macrophages in cell experiments,and to study the effects of TS on nuclear factor-kappa B(NF-?B),p38 mitogen-activated protein kinase(p38MAPK)and c-Jun N-terminal protein kinases(JNK)was used to further explore the mechanism of TS treatment for ulcerative colitis.Methodsintestinal macrophages of mice were purchased from Shanghai Fu Heng Biotechnology Co.,Ltd.MTT was used to detect the toxicity of TS to intestinal macrophages.According to the results,the cells were divided into 5 groups,namely,blank control group,lipopolysaccharide(LPS)group(LPS l ug/ml),TS low dose group(LPS l u ug/ml +TS 5 ug/ml),TS middle dose group(LPS l ug/ml +TS 10ug/ml)and TS high dose group(LPS l ug/ml +TS 15 ug/ml).After 24 hours of intervention,the contents of TNF-?,IL-6 and Prostaglandin E2(PGE2)in the supernatant were detected by ELISA kit.Flow cytometry was used to detect cell apoptosis and cell cycle.Apoptosis related proteins(Bax,Bcl-2 and caspase-3)and signal pathway related proteins(NF-?B p65,phosphorylated I kappa B alpha,p38 MAPK,phosphorylated p38 MAPK,JNK,phosphorylation JNK)were detected by Western blot.RT-PCR detected the relative expression level of m RNA related factors(NF-kappa B p65,p38 MAPK and JNK).Results1.Inflammatory factors: Three inflammatory markers(TNF-?,IL-6 and PGE2)were significantly different(P? 0.05).Compared with the blank control group,the levels of TNF-?,IL-6 and PGE2 in the model group were significantly higher.Compared with the model group,the TNF-? in the TS high dose group was significantly lower,and the IL-6 and PGE2 of the TS middle and high dose groupwere significantly lower(P? 0.05).2.Cell cycle and apoptosis: There was significant difference in the proportion of cells in different stages of intervention(P? 0.05).Compared with the blank control group,the proportion of cells in the G0/G1 phase was higher in the model group than in the S phase and M/G2 phase(P? 0.05).Compared with the model group,the proportion of cells in G0/G1 phase of each TS dose group was lower,and the proportion was higher in S and M/G2 stages,but the difference was not statistically significant(P>0.05).The apoptosis rate of each group was statistically significant.Compared with the blank control group,the apoptotic rate in the model group was significantly higher,and compared with the model group,the apoptotic rate in the TS high dose group was significantly lower(P? 0.05).3.Apoptosis related proteins: Three apoptosis related proteins(Bax,Bcl-2 and Caspase-3)were significantly different(P? 0.05).Compared with the blank control group,the relative expression level of Bax protein in the model group was significantly higher,and the relative expression levels of Bcl-2 and Caspase-3 protein were significantly lower.Compared with the model group,the relative expression level of Bax protein in the TS middle and high dose group was significantly lower,the relative expression level of Bcl-2 protein in those groups was significantly higher,and the relative expression level of Caspase-3 protein in the TS middle dose group was significantly higher(P? 0.05).4.NF-?B signal: NF-?B p65 protein,p-I?B? and NF-kappa B p65 m RNA were significantly different(P? 0.05).Compared with the blank control group,NF-?B p65 protein,p-I?B? and NF-kappa B p65 m RNA in the model group were significantly higher.Compared with the model group,NF-?B p65 protein,p-I?B? and NF-kappa B p65 m RNA in the TS middle and high dose group were significantly lower(P? 0.05).5.MAPK signal: The relative expression levels of p38 MAPK protein and p38 MAPK m RNA in each group were not statistically different(P>0.05).The difference of relative expression level of p-p38 MAPK protein between each group had statistically difference.Compared with the blank control group,the p-p38 MAPK protein in the model group and each TS dose group was significantly higher.Compared with the model group,the p-p38 MAPK protein in the TS middle and high dose group was significantly lower(P? 0.05).6.JNK signal: The relative expression levels of JNK protein and JNK m RNA in each group were not statistically different(P>0.05).But the relative expression level of p-JNK protein was statistically different.Compared with the blank control group,the p-JNK protein in the model group was significantly higher(P? 0.05).But compared with the model group,the relative expression level of p-JNK protein in each TS dose group had not significant difference(P>0.05).ConclusionIn LPS induced mouse intestinal macrophages,TS can inhibit the inflammatory response and apoptosis,and inhibit the NF-?B and p38 MAPK signal pathway,but have no significant effect on the JNK signal pathway.