In Vitro Study On Anti-inflammatory And Anti-osteoclastic Effects Of Kaempferol On Periodontitis Through MAPK Signaling Pathway

Objective:This paper studied the proliferation and differentiation of kaempferol on mouse macrophage RAW 264.7 cells and osteoclasts in vitro,and further explored the effects of kaempferol on anti-inflammatory and anti-osteoclast of experimental periodontitis in MAPK signaling pathway,in order to understand the molecular biological mechanism of kaempferol in treating experimental periodontitis.Methods:1.The mouse macrophage cell line RAW 264.7 was cultured and treated with Kaempferol,and the experimental concentration of Kaempferol was determined by CCK-8 detection:Kaempferol with different concentrations was selected to act on RAW 264.7 cells,and after a certain period of time,CCK-8 determined the cell proliferation activity to determine the appropriate concentration of Kaempferol for subsequent experiments.2.Anti-inflammatory and bone protective effects of Kaempferol on RAW 264.7 cells:The experiment was randomly divided into blank Control group(control group),control group(PG LPS)and treatment group(PG LPS+Kaempferol 20 μM,40 μM and 80 μM).After different concentrations of Kaempferol treatment,OPG/RANKL and inflammatory factors IL-1β and IL-were detected by real-time fluorescence quantitative PCR.3.Effect of MAPK signaling pathway in the anti-inflammatory process of RAW 264.7 cells affected by kaempferol:The group was the same as above,and the expression of MAPK key proteins was detected by WB after different concentrations of kaempferol treatment.4.The differentiation of RAW264.7 cells induced by osteoclasts:RAW 264.7 cells was induced into osteoclasts by RANKL with a concentration of 50 ng/mL and identified by TRAP staining.5.Effects of kaempferol on osteoclast-related factors in RAW 264.7 cells:The experiment was randomly divided into Control group,control group(50 ng/ml Rankl)and kaempferol experimental group(50ng/mL RANKL+kaempferol 20μM,40μM,80μM).Real-time fluorescence quantitative PCR was used to detect the relative expressions of matrix metalloproteinase-9(MMP-9),activated T cell nuclear factor-1(NFATC 1),integrin αV(ITGAV),integrin subunit β 3(ITGB 3),c-Fos and OPG in the cells treated with different concentrations of Kaempferol.6.MAPK signaling pathway in the influence of kaempferol on the role of RAW264.7 cells in osteoclast formation:Group 5,and the expression of key proteins was detected by WB.Result:1.The results of CCK-8 cell activity test showed that the concentration of Kaempferol was 160 μM,which had a great influence on the cell activity.Kaempferol of 20 μM,40 μM and 80 μM was selected for the follow-up experiments.2.Anti-inflammatory effect:After the experimental periodontitis inflammation model was treated with drugs,the expression levels of pro-inflammatory factors IL-1β,TNF-α and IL-6 in cells were down-regulated,while the expression level of anti-inflammatory factor IL-1Ra was up-regulated.The expression level of p-JNK/JNK protein in MAPK signaling pathway is down-regulated.3.Bone protection:Kaempferol in the treatment group can increase the OPG/RANKL ratio,and Kaempferol in the 20 μM concentration group shows the best bone protection.4.50 ng/mL RANKL induction medium acted on RAW264.7 cells,and osteoclasts could be observed five days later.5.Anti-osteoclast effect:Kaempferol acted on the cells induced for 5 days,and the expression of osteoclast-related genes was detected by q-PCR.The expression levels of MMP-9,NFATC 1,ITGAV,ITGB 3 and c-Fos genes in osteoclasts were down-regulated,while the expression level of OPG genes was up-regulated.Compared with the control group,the phosphorylation levels of MAPK signaling pathway proteins ERK,p 38 and JNK were all down-regulated.Conclusion:1.Kaempferol of 80 μM and below has no toxic and side effects on experimental periodontitis.2.Kaempferol in a certain concentration range can improve the expression ratio of OPG/RANKL gene in cells,which has a certain bone protection effect;At the same time,by inhibiting JNK/MAPK signaling pathway,down-regulating the expression of proinflammatory factor gene,the inflammatory progress of experimental periodontitis was inhibited.3.A certain concentration of kaempferol can down-regulate the expression levels of MMP-9,NFATC1,ITGAV,ITGB3 and c-Fos in osteoclasts through MAPK signaling pathway,and up-regulate the expression level of OPG gene,thus inhibiting the formation,differentiation,proliferation and adhesion of experimental periodontitis osteoclasts.