Effect Of LPS On Autophagy And Apoptosis In MC3T3-E1 And Protective Effect Of Aurantiamide Acetate

Lipopolysaccharide(LPS),the main component of cellular wall derived from gram-negative bacteria,can cause bone inflammatory diseases,e.g.rheumatoid arthritis,osteomyelitis,and traumatic arthritis.Unfortunately,the mechanism of LPS on autophagy and apoptosis of osteoblasts remain unclear.Lycii cortex derived from root bark of Lycium chinense has the highest content of aurantiamide acetate(AA)which resists inflammation and regulates bone metabolism.However,the mechanism of AA on osteoblastic autophagy and apoptosis has been rarely reported.The present study aims to explore the mechanism of LPS-mediated autophagy and apoptosis in MC3T3-E1.Alizarin red staining,Western Blot,flow cytometry,transmission electron microscope(TEM),and high content analysis system were applied to investigate the protective role of AA on LPS-mediated osteoblastic autophagy and apoptosis.The results will provide a novel orientation for the development of new drugs to treat bone inflammatory diseases.1.Effects of LPS on autophagy and apoptosis of MC3T3-E1To explore the effects of LPS on osteoblastic autophagy and apoptosis,MC3T3-E1 was treated with 1 ?g/mL LPS for different time(0,3,6,12,and 24h),or with different concentrations of LPS(0,0.5,1,and 5?g/mL)for 6 h or 21 d.The mineralized nodules,autophagy and apoptosis-related proteins,macrophage of nucleus,and apoptosis rate and mitochondrial membrane potential was detected by alizarin red staining,Western Blot,Hoechst 33258 staining and flow cytometry,respectively.The results showed that the area of mineralized nodules was significantly decreased(P<0.01)after the treatment with LPS in a dose-dependent manner.Compared with the control group,the expression level of ATG5,Beclinl and LC3-II was significantly(P<0.05 or P<0.01)increased after 1 ?g/mL LPS treatment for 6,12,and 24h.Similarly,the expression level of ATG7,p62,BAX/Bcl-2 and Cleaved Caspase-3/Caspase-3 was significantly(P<0.05 or P<0.01)increased at 3,6,12,and 24 h.In addition,the expression of ATG5,ATG7,LC3-II,Beclinl,BAX/Bcl-2 were significantly(P<0.05 or P<0.01)increased after treating with different concentration of LPS(0.5,1,and 5 ?g/mL)for 6 h,and the expression of p62 was significantly(P<0.05 or P<0.01)increased after treating with 1,5 ?g/mL LPS for 6 h.The expression level of Cleaved Caspase-9/Caspase-9 and Cleaved Caspase-3/Caspase-3 was significantly(P<0.01)increased after the treatment with LPS(1,5?g/mL)for 6 h.The number of abnormal nucleus stained by Hoechst 33258 were increased after treating with LPS.The early and late status of apoptosis rates detected by flow cytometry were significantly(P<0.01)increased after treatment with LPS for 3,6,12 and 24 h.The apoptosis rate was increased in a concentration-dependent manner treated with LPS for 6 h(P<0.01).The mitochondrial membrane potential(MMP)was significantly(P<0.05 or P<0.01)decreased after treating with LPS(1 and 5 ?g/mL)for 6 h.All these results showed that the level of autophagy and apoptosis of cell was increased and osteoblastic mineralization ability was decreased after treatment with LPS(1 ?g/mL)for 6 h.2.Role of AMPK/mTOR/ULK1 signaling pathway in autophagy and apoptosis of MC3T3-El induced by LPSTo explore the role of AMPK/mTOR/ULK1 signaling pathway in LPS-induced autophagy and apoptosis of MC3T3-E1,the cells was pre-treated with 1 ?g/mL Compound C(CC)for 1 h,and then treated with LPS for 6 h.The expression of proteins related with AMPK/mTOR/ULK1 signaling,autophagy and apoptosis were detected by Western Blot.The results showed that the ratio of p-AMPK/AMPK was significantly(P<0.01)increased after addition with LPS(0.5,1,and 5 ?g/mL)in a dose-dependent manner,while the ratio of p-mTOR/mTOR was significantly(P<0.01)decreased,and the ratio of p-AKT/AKT has no difference.In addition,the ratio of p-ULK1/ULK1 was significant(P<0.01)increased after treatment with LPS(1 and 5 ?g/mL).Compared with the control group,the ratio of p-AMPK/AMPK was significantly(P<0.01)decreased after treating with CC.Compared with the LPS group,the ratio of p-AMPK/AMPK,p-mTOR/mTOR,p-ULK1/ULK1,and Bax/Bcl-2,and the expression level of LC3-? was significantly(P<0.05 or P<0.01)decreased in LPS+CC group,while the expression of p62 has no difference.The apoptosis rate of LPS+CC group significantly(P<0.01)higher than LPS group's.These results indicated that LPS could regulate autophagy of MTC3T3-E1 via the AMPK/mTOR/ULK1 signaling pathway,and inhibition of AMPK activity would decrease autophagy,promoting apoptosis.3 Protective effects of aurantiamide acetate on autophagy and apoptosis of MC3T3-E1 induced by LPSTo explore the role of AA in LPS-induced autophagy and apoptosis of MC3T3-E1,the cells was pre-treated with 2.5?g/mL AA for 1 h,and then with LPS for 6 h.The cell proliferation rate was analyzed by high-content analysis system,the formation of mineralized nodule was observed by alizarin red staining,the cellular ultrastructure was observed by TEM,the expression of autophagy and apoptosis-related proteins were detected by Western Blot,the macrophage of nucleus was observed by Hoechst 33258 staining,and the apoptosis rate was detected by flow cytometry.The results showed that LPS or AA had no significant effects on the cell proliferation of MC3T3-E1.Compared with the control group,LPS significantly decreased the areas of mineralized nodules(P<0.01),while AA had no significant effects.Compared with LPS group,the area of mineralized nodule significantly increased in LPS+AA group(P<0.01).The ultrastructure of cells was observed by TEM.The nuclear edge of the LPS group was invaded.Compared with the control group,the number of autophagosomes with double membrane increased after treatment with LPS,and the autophagosomes and autolysosomes of AA and AA+LPS groups increased.Western Blot detected the expression of autophagy,AMPK/mTOR/ULK1 and apoptosis related proteins.Compared with the control group,the expression of LC3-?,Beclinl and p-AMPK/AMPK in AA group was significantly increased(P<0.01),while p62 and p-mTOR/mTOR were significantly decreased(P<0.01).The level of Bax/Bcl-2 in LPS treated group was significantly increased(P<0.01).Compared with LPS group,the expression of p62,p-mTOR/mTOR,Bax/Bcl-2 in AA+LPS group was significantly decreased(P<0.05 or P<0.01),Beclinl,p-AMPK/AMPK and p-ULK1/ULK1 were significantly increased(P<0.05 or P<0.01).After MC3T3-E1 infected with GFP-RFP-LC3 lentivirus,the LC3 puncta were tracked for 25 h,and the number of fluorescent spots was analyzed by high-content analysis system.The number of pure red dots(per cell)in LPS group showed a downward trend,while that in AA and AA+LPS groups showed an upward trend.Compared with the control group,LPS treatment significantly increased the number of yellow dots per cell(P<0.01).Compared with the LPS treatment group,the number of yellow dots per cell in AA group was significantly decreased(P<0.01),while in LPS+AA group significantly decreased(P<0.05).The results of Hoechst 33258 staining showed that AA pretreatment significantly decreased the proportion of damaged nuclei(P<0.01)compared with LPS group.The results of flow cytometry analysis showed that apoptosis rate had no significant change in AA group.AA pretreatment significantly reduced the apoptosis rate induced by LPS(P<0.01)compared with the LPS group.These results indicated that AA could alleviate the inhibition effect of LPS on the mineralization of MC3T3-E1,enhance autophagy,and promote the transformation of autophagosomes to autophagolysosomes,reduce LPS-induced accumulation of autophagosomes.As a results,AA has a protective effect on autophagy and apoptosis induced by LPS in MC3T3-E1.