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Protective effect and mechanism of dioscin on TNF-?induced FLS injury through 5-LO/LTB4 pathway Background and Objective:RA is an autoimmune disease characterized by abnormal hyperplasia of the synovial membrane which was related to the imbalance of apoptosis and proliferation of synovial cells.As the main cells of pannus formation,FLS is actively involved in the process of RA because of its insufficient apoptosis.The FLS in RA patient often proliferate like tumor cells,and play a key role in the pannus invasion,cartilage and bone erosion.TNF-? produced in the early stage of RA can induce the production of IL-6,GM-CSF,IL-8,VEGF,COX-2 and MMP,which can promote the hyperplasia of FLS.Dio has promising application,such as immune regulation,anti-inflammatory,anti-tumor,anti-platelet aggregation.In our study,using TNF-?-induced FLS cells as an in vitro model of RA,we explored the effect of dioscin(Dio)on TNF-?-induced FLS and its underlying mechanism.Methods:1.After incubated with 1.25ng/ml?2.5ng/ml?5ng/ml?10ng/ml?20ng/ml?40ng/ml?80ng/ml?160ng/ml TNF-? for 24h?48h?72h,cell viability of CIA rat FLS was determined by MTT.2.After incubated with 0.5?g/ml?1?g/ml?1.5?g/ml?2?g/ml?2.5?g/ml?3?g/ml?3.5?g/ml?4?g/ml?4.5?g/ml?5?g/ml Dio for 24h?48h?72h,cell viability of TNF-?induced FLS was determined by MTT.3.The content of LTB4 in the supernatant of the control group,TNF-? group,Dio 1?g/ml,Dio 2?g/ml,Dio 3?g/ml and Dio 4?g/ml groups was determined by ELISA.4.The changes of 5-LO,LTA4 H,BLT1,BLT2 mRNA expression in the control group,TNF-? group,Dio 1?g/ml,Dio 2?g/ml,Dio 3?g/ml and Dio 4?g/ml groups was determined by quantitative real time PCR.5.The protein expressions of 5-LO and LTA4 H protein in the control group,TNF-? group,Dio 1?g/ml,Dio 2?g/ml,Dio 3?g/ml and Dio 4?g/ml groups were detected by western blot.6.The expressions of LTA4 H in the control group,TNF-? group,Dio 1?g/ml,Dio 2?g/ml,Dio 3?g/ml and Dio 4?g/ml groups were detected by immunofluorescence assay.Result:1.After incubated with 5ng/ml?10ng/ml?20ng/ml TNF-? for 24h?48h?72h,cell viability was significantly increased,especially the 10ng/ml TNF-?(P<0.05).Therefor,in the later experiments we use the 10ng/ml TNF-? for 24 h to induce FLS.2.After incubated with 1?g/ml?1.5?g/ml?2?g/ml?2.5?g/ml?3?g/ml?3.5?g/ml?4?g/ml?4.5?g/ml?5?g/m l Dio for 24h?48h?72h,cell viability of TNF-? induced FLS was significantly decreased in a time-dose dependent manner(P<0.05).When incubated with 3?g/ml Dio for 24 h,the viability of TNF-? induced FLS was49.46±1.33.Therefore,we chose 1?g/ml?2?g/ml?3?g/ml?4?g/ml Dio to treat TNF-?induced FLS in the later experiments.3.After incubated with 10ng/ml TNF-? for 24 h,the content of LTB4 in the supernatant was significantly higher than that in the control group(p<0.05),and1?g/ml?2?g/ml?3?g/ml?4?g/ml Dio can significantly reduce the increased LTB4 which caused by TNF-?(p<0.05).4.Compared with the control group,the expressions of 5-LO and LTA4 H mRNA and protein were significantly increased in TNF-? group(p<0.05).Compared with TNF-? group,the expressions of 5-LO and LTA4 H m RNA and protein were significantly decreased in 1?g/ml?2?g/ml?3?g/ml?4?g/ml Dio groups(p<0.05).5.The mRNA expressions of BLT1 and BLT2 was significantly higher compared with control group(P<0.05).The mRNA expressions of BLT1 and BLT2 in TNF-? group was significantly decreased when incubated with 1?g/ml?2?g/ml?3?g/ml Dio,but 4?g/ml Dio can only reduced the expression of BLT2 mRNA(p<0.05).Conclusion:1.When incubated with 10ng/ml TNF-? for 24 h,cell proliferation of FLS wassignificantly increased,which indicate that FLS incubated with 10ng/ml TNF-? for24 h can be used as an in vitro RA model2.After incubated with TNF-? induced FLS,1?g/ml?2?g/ml?3?g/ml?4?g/ml Dio can inhibit the proliferation of FLS,suggesting its protective effect on TNF-?induced FLS injury.3.Dio can reduce the content of LTB4 in supernatant of TNF-? induced FLS,which may be related to its protective effect on FLS injury.4.Dio can reduce the mRNA and protein expressions of 5-LO and LTA4 H in TNF-? induced FLS.It may be the reason why LTB4 was reduced.5.Dio can reduce the expression of BLT1 and BLT2 in TNF-? induced FLS.It demonstrated that Dio may restrain TNF-? induced FLS injury by its decreasing LTB4 receptors.Part ? Effects and mechanisms of dioscornin tablets oncollagen-induced arthritis mice Background and Objective:Rheumatoid Arthritis(RA)is one of the most common autoimmune disease characterized by chronic inflammation of the join synovium.In clinical,RA often seriously causes the declince in the life quality of patients by joint stiffness,swelling,deformity,even disability.As the most common animal model of RA,CIA is an ideal to screen and exploiture new drug.Chinese medicine believes that dioscin has a "activate blood and relax tendons,promote digestion and water retention,expectorant,recurrence of malaria" efficacy.Modern research also confirmed that it is a potential traditional Chinese medicine to treat RA with the pharmacological function of anti-inflammatory,anti-tumor,immune regulation,anti-platelet aggregation.In the current studies,bovine collagen ? was used to induce CIA mice model in Kunming mice to study the therapeutic effect and underlying mechanism of Dioscornin tablets on CIA mice.Methods:1.Male Kunming mice(6-8 weeks,25±2g)were randomly divided into 7groups,named as control group,CIA group,Dioscornin tablets(DT)(15.6 mg/(kg·d),31.2 mg/(kg·d),62.4 mg/(kg·d)?124.8mg/(kg·d),249.6 mg/(kg·d))groups(n=12).From 21 day after initial immunization,the mice in control and CIA group were lavaged with 0.9% NaCl,the mice in DT group were lavaged with DT(15.6 mg/(kg·d)?31.2 mg/(kg·d)?62.4 mg/(kg·d)?124.8mg/(kg·d)?249.6 mg/(kg·d)).The effects of DT on CIA mice were observed by mice body weight,arthritis index,paw swelling,thymus and spleen index,as well as the spleen and joint pathology.2.ELISA was use to detect the change of LTB4 in serum of control group,CIA model group,DT low,DT middle,DT high group mice.3.Reverse transcriptional PCR was used to detect the changes of 5-LO,LTA4 H,BLT1 and BLT2 mRNA in spleen of control group,CIA model group,DT low,DT middle,DT high group mice.4.Using western blot and immunohistochemical to check the discrepancy of5-LO,LTA4 H protein expressions in spleen of control group,CIA model group,DT low,DT middle,DT high group mice.Result:1.The body weight,arthritis index,paw swelling,thymus and spleen index of the CIA group was significantly higher than the control group(p<0.05).The DT(31.2mg/(kg·d)?62.4 mg/(kg·d)?124.8mg/(kg·d))groups mice recovery their body weight,joint index,paw swelling by day 12 after lavaged(p<0.05).And recovery their spleen index by day 21 after lavaged.The thymus index,spleen and joint pathology was also changed after lavaging with 62.4 mg/(kg·d)DT for 21day(p<0.05).2.The content of LTB4 in CIA mice serum was significantly increased,while DT significantly decreased the content of LTB4 in CIA mice serum(p<0.05).3.The mRNA and protein expressions of 5-LO and LTA4 H in the spleen of the CIA model group were significantly higher compared with control group(p<0.05).While DT low,DT middle,DT high significantly decreased the expressions of 5-LOand LTA4 H protein in the spleen of CIA mice(p<0.05).DT low and DT middle also significantly decreased the expression of 5-LO mRNA in the spleen of CIA mice(p<0.05).4.The mRNA expressions of BLT1 and BLT2 in the spleen of the CIA mice was significantly higher compared with control group.After lavaged with DT low and DT middle can decrease the expressions of BLT1 and BLT2 mRNA in the spleen of CIA mice,while DT high only decreased the mRNA expression of BLT2 in the spleen of CIA mice(p<0.05).Conclusion:1.DT(31.2 mg/(kg·d)?62.4 mg/(kg·d)?124.8mg/(kg·d))can treat CIA mice by recovering body weight,arthritis index,paw swelling,thymus and spleen index,and improving the thymus and spleen pathological damage,which indicated DT have a protective effect on CIA model mice.2.The content of LTB4 level in serum of CIA group was significantly higher than that of control group.Different concentrations of DT could decrease the level of LTB4 in serum of CIA mice,which indicated that the abnormal increase of LTB4 might be related to the pathogenesis of RA,and the therapeutic effect of DT on CIA mice related to its reducing the LTB4 content in CIA mice.3.DT can reduce content of LTB4 level by decreasing the expression of 5-LO and LTA4 H.It may be the reason why LTB4 was reduced.4.DT can reduce the expressions of BLT1 and BLT2 in spleen of CIA mice.It demonstrated that DT may treat CIA mice by decreasing LTB4 receptors. |
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