Investigation On The Mechanism Of Melatonin-induced Cell Apoptosis In The Human Fetal Osteoblastic Cell Line HFOB 1.19

Subjective: To investigate the mechanism and the protective factors of the melatonin-induced cell apoptosis of cell apoptosis in the human fetal osteoblastic cell line h FOB 1.19.Method: The human normal fetal osteoblastic cell line h FOB 1.19 were stably cultured and treated in different concentrations of melatonin for different durations of action.Apoptosis was assessed quantitatively using flow cytometric analysis.Expression levels of MTNR1,MTNR2,GRP78 and GRP94 were assessed using western blotting to demonstrate the occurrence of endoplasmic reticulum stress(ERS).m RNA levels and protein expression levels of unfolded protein response(UPR)-related proteins(ATF4,ATF6α,XBP1 and p-e IF2α)were assessed by performing RT-q PCR and western blotting.Expression levels of Periostin(POSTN)and Septin7(SEPT7)were assessed after the human normal fetal osteoblastic cell line h FOB 1.19 were treated in different concentrations of melatonin for different durations of action.The ERS-related proteins and apoptosis-related proteins were assessed after transfected with POSTN/SEPT7 overexpression plasmids and/or POSTN/SEPT7 si RNAs.Results: Firstly,results exhibited that the quantity of early apoptotic cells in the Q4(early apoptotic)region are significantly increased in 4 m M melatonin treated group than that in 2 m M or 6 m M melatonin treated groups;however,the number of late apoptotic cells in the Q2 region in 2 m M or 6 m M melatonin treated groups were increased compared with that in 4 m M melatonin treated group.Following treatment with melatonin for 48 h,the number of late apoptotic cells increased for all concentrations of melatonin,results also demonstrated that the total quantity of apoptotic cells was increased after treatment with 2 m M melatonin,which consistent with the expression patterns of GRP78 and GRP94.Following comprehensive consideration,4 m M melatonin for 24 h was selected as the appropriate experimental condition for subsequent experiments as this group exhibited the greatest number of early apoptotic cells(Q4)and the least number of late apoptotic cells(Q2).After treatment with different concentrations of melatonin,the expression level of MTNR2 obvious decreased compared to that of MTNR1.After the osteoblasts were treated with 4 m M melatonin for24 h,proteins associated with the signaling transduction pathways,including PERK,IRE1 and ATF6α,activated by UPR were also assessed.The protein and m RNA expression levels of p-e IF2α,ATF4,X-box binding protein 1(XBP1)and ATF6α were measured using western blot and RT-q PCR analyses,and the results showed that the expression levels of p-e IF2α and ATF4 were increased significantly among the groups.To determine the transduction pathways through which ERS mediate melatonin-induced apoptosis,the expression levels of CHOP and caspase-3 were detected using western blot analysis.It was observed that 4 m M melatonin significantly increased the expression levels of ATF4,p-e IF2α,CHOP and caspase-3 following treatment for 24 h,compared with those in the control groups.The levels of p-JNK were also markedly increased.The expression of POSTN/SEPT7 in the groups treated with different concentrations of melatonin for 24 h was assessed using western blot and RT-q PCR analyses.The results showed that the expression of POSTN/SEPT7 was positively correlated with the concentration of melatonin.RT-q PCR analysis was performed to assess the m RNA level of POSTN following transfection with the POSTN/SEPT7 overexpression plasmids or POSTN/SEPT7 si RNAs.The results showed that the transfections were efficient.Finally,either POSTN/SEPT7 si RNA or control si RNAs were transfected into osteoblasts using Lipofectamine ? 2000 according to the manufacturer’s protocol.There were three control groups: Blank control,transfection reagent control and scramble si RNA control.A rescue experiment was also performed via transfection with the POSTN/SEPT7 overexpression plasmid.Western blot analysis was then performed to assess the protein levels of CHOP,ATF4,p-e IF2α,pro caspase-3,cleaved caspase-3,POSTN and SEPT7.The results demonstrated that the levels of CHOP,ATF4,p-e IF2α and cleaved caspase-3 in the group treated with melatonin in combination with POSTN/SEPT7 si RNAs were increased significantly,compared with those in the control groups.Following transfected with POSTN/SEPT7 si RNAs,the expression level of POSTN/SEPT7 decreased significantly.The POSTN/SEPT7 overexpression plasmids was then transfected in order to perform the rescue experiment.The results demonstrated that following transfection with the POSTN/SEPT7 overexpression plasmids,the expression level of POSTN or SEPT7 was increased to a level similar to what was observed in the 4 m M melatonin-treated group comparing with the control group.Conclusions: In this research,it was for the first time we demonstrated that melatonin can induce cell apoptosis by activating the PERK-p-e IF2α-ATF4 signal transduction pathway,and ERS mediated this process by triggering the cascade reactions of CHOP,caspase-3 and p-JNK.The upregulated expression of POSTN/SEPT7 had protective effects against apoptosis of the h FOB 1.19 human osteoblastic cell line.