| BackgroundOsteoporosis is a systemic metabolic bone disease characterized by osteopenia,destruction of bone microstructure,reduction of bone strength and susceptibility to fracture.Recently,a great deal of researches showed that chronic inflammation played an important role in the pathogenesis of osteoporosis,like diabetes mellitus and cardiovascular disease.Fatty acids played an important role in the development of osteoporosis.As an important biological molecule,fatty acid not only was an energy resource in the body,but also a signal molecule regulating metabolism and inflammatory process.Recently,studies showed that a group of G protein coupled receptors(GPCRs)were on the surface of cells,and GPR120,which was a long chain polyunsaturated fatty acid receptor,has become a hot topic.Beta-arrestin2 is a multifunctional adaptor protein,which have been discovered in recent years.It played a role in regulating the desensitization and internalization of GPCRs and inhibiting inflammation.Previous researches have been focused on the effects of Omega-3 on bone metabolism by the GPR120/ beta-arrestin2/p-TAK1 pathway,and have shown that dietary fatty acids,monounsaturated fatty acids,polyunsaturated fatty acids have different effects on the immune and inflammatory state of the body,resulting in different effects on bone turnover.We hypothesiz that the PUFA has different effects on the inflammatory pathway of GPR120/beta-arrestin2/p-TAK1,which may be one of the mechanisms of the inconsistent effects of PUFA on osteoporosis.Like omega-3,Omega-6(linoleic acid),Omega-9(oleic acid)belong to long chain polyunsaturated fatty acids,and aslo are natural ligands for GPR120.The research of Omega-6and omega-9 on the inflammatory pathway of osteoblasts by GPR120 are less.In this study,we investigated the effects of Omega-6 and omega-9 on GPR120 on inflammatory pathway in mouse MC3T3-E1 cells.ObjectiveLinoleic acid(LA),oleic acid(OA)act on mice MC3T3-E1 cells in different concentrations and time to investigate the effects of LA and OA on GPR120/ beta-arrestin2/p-TAK1 pathway,by observing the levels of beta-arrestin2,p-TAK1 which are in GPR120/ beta-arrestin2/p-TAK1 pathway,and also the levels of inflammatory factors downstream.Methods1.After tarvation treatment for 12 h,MC3T3-E1 cells,which are in logarithmic phase,are treated with different concentrations of linoleic acid and oleic acid(0?mol/L,100?mol/L,200?mol/L,300?mol/L,400?mol/L,500?mol/L),and different time(6h,12 h,24h),then testing the relative expression level of beta-arrestin2 and p-TAK1 protein by Western blot2.The levels of inflammatory cytokines in each group are tested by ELISA.Results1.MC3T3-E1 cells were treated with different concentrations of linoleic acid,then the results of relative expression of beta-arrestin2 were as follow: 1)when treated for 6h,the relative expression of beta-arrestin2 protein in 400umol/L,500umol/L group were lower than that of 0umol/L group(P < 0.05);2)when treated for 12 h,the relative expression of beta-arrestin2 protein showed a downward trend with the change of concentration and began at 200umol/L group(P<0.05),the relative expression of beta-arrestin2 protein in 400umol/L,500umol/L group were significantly lower than that of 0 umol/L group(P<0.001),and the relative expression of beta-arrestin2 protein in 500umol/L group was lower than that of 400umol/L group(P<0.05);3)when treated for 24 h,the relative expression of beta-arrestin2 protein showed a downward trend with the change of concentration and began at 300umol/L group(P<0.05),the relative expression of beta-arrestin2 protein in 400umol/L,500umol/L group were significantly lower than that of 0 umol/L group(P<0.001).2.MC3T3-E1 cells were treated with different concentrations of linoleic acid,then the results of relative expression of p-TAK1 were as follow: 1)when treated for 6h,the relative expression of p-TAK1 protein in 300umol/L,400umol/L,500umol/L group were higher than that of 0umol/L group(P<0.05);2)when treated for 12 h,the relative expression of p-TAK1 protein showed a upward trend with the change of concentration and began at 300umol/L group(P<0.05),the relative expression of p-TAK1 protein in 400umol/L,500umol/L group were significantly higher than the 0 umol/L group(P<0.001),and the relative expression of p-TAK1 protein in 500umol/L group was higher than that of 400umol/L group(P<0.05);3)when treated for 24 h,the relative expression of p-TAK1 protein showed a upward trend with the change of concentration and began at 300umol/L group(P<0.05),the relative expression of p-TAK1 protein in 400umol/L,500umol/L group were significantly higher than that of 0 umol/L group(P<0.001).3.MC3T3-E1 cells were treated with different concentrations of oleic acid,then the results of relative expression of beta-arrestin2 were as follow: 1)when treated for 6h,the relative expression of beta-arrestin2 protein in 300umol/L,400umol/L,500umol/L group were higher than that of 0umol/L group(P<0.05);2)when treated for 12 h,the relative expression of beta-arrestin2 protein showed a upward trend with the change of concentration and began at 300umol/L group(P<0.05),the relative expression of beta-arrestin2 protein in 400umol/L,500umol/L group were significantly higher than that of 0 umol/L group(P<0.001),and the relative expression of beta-arrestin2 protein in 500umol/L group was higher than that of 400umol/L group(P<0.05);3)when treated for 24 h,the relative expression of beta-arrestin2 protein showed a upward trend with the change of concentration and began at 200umol/L group(P<0.05),the relative expression of beta-arrestin2 protein in 400umol/L,500umol/L group were significantly higher than that of 0 umol/L group(P<0.001);4)the relative expression of beta-arrestin2 protein in 6h,24 h group were significantly lower than that of 12 h group in 500umol/L.4.MC3T3-E1 cells were treated with different concentrations of oleic acid,then the results of relative expression of p-TAK1 were as follow: 1)when treated for 6h,the relative expression of p-TAK1 protein in 300umol/L,400umol/L,500umol/L group were lower than that of 0umol/L group(P<0.05);2)when treated for 12 h,the relative expression of p-TAK1 protein showed a downward trend with the change of concentration and began at 200umol/L group(P<0.05),the relative expression of p-TAK1 protein in 400umol/L,500umol/L group were significantly lower than that of 0 umol/L group(P<0.001),and the relative expression of p-TAK1 protein in 500umol/L group was lower than that of 400umol/L group;3)when treated for 24 h,the relative expression of p-TAK1 protein in 200umol/L,300umol/L,400umol/L group were lower than that of 0umol/L group(P<0.05),500umol/L group were significantly lower than that of 0 umol/L group.5.MC3T3-E1 cells were treated with different concentrations of linoleic acid,then the results of expression of IL-6 and TNF-? were as follow: 1)when treated for 6h,the relative expression of IL-6 and TNF-? in 300umol/L,400umol/L,500umol/L group were higher than that of 0umol/L group(P<0.05);2)when treated for 12 h,the relative expression of IL-6 and TNF-? showed a upward trend with the change of concentration and began at 300umol/L group(P<0.05),the relative expression of IL-6 and TNF-? in 400umol/L,500umol/L group were significantly higher than that of 0 umol/L group(P<0.001),and the relative expression of IL-6 and TNF-? in 500umol/L group was higher than that of 400umol/L group(P<0.05);3)when treated for 24 h,the relative expression of IL-6 and TNF-? showed a upward trend with the change of concentration and began at 300umol/L group(P<0.05),the relative expression of IL-6 and TNF-? in 400umol/L,500umol/L group were significantly higher than that of 0 umol/L group(P<0.001),and the relative expression of IL-6 and TNF-? in 500umol/L group was higher than that of 400umol/L group.4)the relative expression of IL-6 and TNF-? in 6h,24 h group were significantly lower than that of 12 h group in 500umol/L.6.MC3T3-E1 cells were treated with different concentrations of oleic acid,then the results of expression of IL-6 and TNF-? were as follow: 1)when treated for 6h,the relative expression of IL-6 and TNF-? in 300umol/L,400umol/L,500umol/L group were lower than that of 0umol/L group(P<0.05),500umol/L group were significantly lower than the 400 umol/L group(P<0.001);2)when treated for 12 h,the relative expression of IL-6 and TNF-? showed a downward trend with the change of concentration and began at 200umol/L group,the relative expression of IL-6 and TNF-? in 400umol/L,500umol/L group were significantly lower than that of 0 umol/L group(P<0.001),and the relative expression of IL-6 and TNF-? in 500umol/L group was lower than that of 400umol/L group;3)when treated for 24 h,the relative expression of IL-6 and TNF-? showed a downward trend with the change of concentration and began at 200umol/L group,the relative expression of IL-6 and TNF-? in 400umol/L,500umol/L group were significantly lower than that of 0 umol/L group(P<0.001),and the relative expression of IL-6 and TNF-? in 500umol/L group was lower than that of 400umol/L group.4)the relative expression of IL-6 and TNF-? in 6h,24 h group were significantly higher than that of 12 h group in 500umol/L.Conclusions1.linoleic acid could induce the inflammatory response of MC3T3-E1 cells in mice,and the effect was in a concentration dependent.2.oleic acid could inhibit the inflammatory response of MC3T3-E1 cells in mice,and the effect was in a concentration dependent. |
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