To Explore The Effect Of Sinomenine On The Expression Of DC2.4 Inflammatory Factors In Mice Based On TLR2 And TLR4 Gene Silencing

Objective:The transfection technique of cell plasmids was used in this experiment.It is used to selectively inhibit the expression of TLR2 and TLR4 receptor proteins on dendritic cells,finding the key target of sinomenine on the regulation of Dendritic cell in mice.To explore the anti-inflammatory mechanism of sinomenine on rheumatoid arthritis.It provides experimental basis for the experimental research of rheumatoid arthritis and the development of new drugs.Methods:1.TLR2 and TLR4 receptor proteins from murine dendritic cells were used as experimental objects to design and synthesize recombinant plasmids.Selective inhibition of TLR2 and TLR4 receptor protein expression Selectively suppressing the expression of TLR2 and TLR4 receptor protein,using cell plasmid transfection technology.The protein expression of TLR2 and TLR4 was detected by Western blot method,and the purpose was to select the best inhibitory plasmids of TLR2 and TLR4 protein.2.According to single and combined inhibition of TLR2 and TLR4 protein expression,we named DC2.4 as blank group,single inhibition of TLR2 blank group,single inhibition of TLR4 blank group,the inhibition of TLR2+4 blank group,the model group,single inhibition of TLR2 model group,single inhibition of TLR4 model group,the inhibition of TLR2+4 model group,sinomenine group,single inhibition of TLR2 sinomenine group,single inhibition of TLR4 sinomenine group,the inhibition of TLR2+4 sinomenine group and methotrexate group.In addition to the blank group,single inhibition of TLR2 blank group,single inhibition of TLR4 blank group and the inhibition of TLR2+4 blank group,each group was added 1g/ml R848 solution to cultivate 24 h.The purpose is to induce DC2.4 maturation of immune response.Next step is that inomenine group,single inhibition of TLR2 sinomenine group,single inhibition of TLR4 sinomenine group and inhibition of TLR2+4 sinomenine group were added 40μg/ml sinomenine solution to cultivate 48 h.At the same time,methotrexate group was added 25 ng/ml methotrexate solution to cultivate 48 h.The contents of IL-1,IL-6,IL-10 and TNF-alpha in the supernatant of each group were detected by ELISA method.The protein expression of IRAK-4,P65 and P38 in each group was detected by Western blot method.Results:1.Detection of protein expression after single and co silencing of TLR2 and TLR4 by Western blot(1)Detection of TLR2 and TLR4 protein expression to screen out the best silence group by Western blotCompared with blank group,there was no significant change in the gray value of TLR2/β-action and TLR4/β-action in the transfection reagent blank group and the control plasmid transfection group.TLR2-16# transfection group,TLR2-17# transfection group,TLR2-18# transfection group,TLR4-19# transfection group,TLR4-20# transfection group and TLR2-21# transfected group were all lower than the blank group in the gray value of TLR2/β-action and TLR4/β-action.Compared with TLR2-16# transfection group,the gray value of TLR2/ β-action in TLR2-17# transfection group was higher,and the gray value of TLR2/β-action in TLR2-18# transfected group was lower.In these transfection groups,the gray value of TLR2/β-action in TLR2-18# transfection group was the lowest.Compared with the TLR4-19# transfection group,the gray value of TLR4/β-action of TLR4-20# transfected group and TLR4-21# transfected were significantly lower.Compared with the TLR4-20# transfection group,the gray value of TLR4/β-action of TLR4-21# transfection group was higher.In these transfection groups,The gray value of TLR4/ β-action of TLR4-20# transfection group was the lowest.(2)Detection of protein silencing after TLR2 and TLR4 silencing by Western blotCompared with the TLR4-20# transfection group,the gray value of TLR2/β-action of TLR2-18# transfection group and TLR2,4-18,20# transfection group were significantly lower.Compared with the TLR2-18# transfection group,the gray value of TLR4/β-action of TLR4-20# transfection group and TLR2,4-18,20# transfection group were significantly lower.2.Effects of sinomenine on the expression of inflammatory cytokines in DC2.4 cells after silencing of TLR2 and TLR4 genesThe experimental results show that R848 can stimulate the secretion of IL-1β,IL-6 and TNF-α in dendritic cells,reduce the content of IL-10,and promote the expression of IRAK-4,P65 and P38 proteins in the cells.Sinomenine can reduce the contents of IL-1β,IL-6 and TNF-α in the supernatant of dendritic cells,increase the content of IL-10,and decrease the expression of IRAK-4,P65 and P38 proteins in cells.After single inhibition of TLR2 or TLR4,the inhibitory effect of sinomenine on IL-1β,IL-6 and TNF-α was weakened,and the secretion of anti-inflammatory factors IL-10 decreased,and the effect of inhibiting the protein expression of IRAK-4,P65 and P38 was weakened.Compared with the single inhibitory TLR2 or TLR4,the effect of sinomenine was further reduced after the proteins expression of TLR2 and TLR4 were inhibited,but it could still regulate the secretion of IL-1β,IL-6,TNF-α and IL-10 in dendritic cells and the proteins expression of IRAK-4,P65 and P38.Conclusion:1.Sinomenine can reduce the secretion of proinflammatory cytokines IL-1β,IL-6 and TNF-α in the supernatant of DC2.4 in mice,promote the secretion of anti-inflammatory factor IL-10 and reduce the protein expression of IRAK-4,P65,and P38.Therefore,it can reduce inflammatory reaction and play an anti-inflammatory role.2.This experiment preliminarily confirmed sinomenine can act on TLR2 and TLR4 receptor protein in DC2.4.The corollary of the research group was clarified that sinomenine acts on TLR2 and TLR4 receptor proteins in DC cells,and plays an anti-inflammatory role.3.Sinomenine not only regulates DC2.4 through TLR2 and TLR4 receptor protein,but also regulates DC2.4 in other ways or proteins,it reduces inflammatory response and plays an anti-inflammatory role.