The Mechanism And Impact Of Autophagy In Palmitate-induced Apoptosis Of Bone Marrow Mesenchymal Stem Cells

Objective: Osteoporosis is an important public health issue,especially for postmenopausal women and the elderly,and is caused by unequilibrated bone remodeling resulting from decreased bone formation and/or accelerated bone resorption,increased bone fracture and increased risk of fracture.With the social development and scientific progress,human life expectancy,the incidence of osteoporosis is gradually rising.In recent years,the relationship between fat and osteoporosis has attracted more and more attention.In patients with osteoporosis,bone marrow fat increased,bone mass decreased.Previous studies have confirmed that fat can produce toxic effects on bone cells through the secretion of fatty acids,resulting in bone loss.As the most common saturated fatty acid,palmitate has been widely used to study the effect of fatty acids on various types of cells.In bone cells,it has been confirmed that palmitic acid can induce osteotoxicity in osteoblasts and induce apoptosis.Autophagy is an evolutionarily conserved process for the catabolism of damaged proteins and organelles in the cell to maintain intracellular environmental homeostasis by "selfclearing".Studies suggest that moderate autophagy is required for coping with cellular stress,inhibition of apoptosis,and is conducive to cell survival;however,the persistence of adverse factors can lead to excessive autophagy,which induces cell death.Autophagy can be found in all types of bone cells,and plays an important role in osteogenesis.Bone marrow mesenchymal stem cells(BMSCs)are multipotent cells that can differentiate into osteoblasts and other cell types such as adipocytes and chondrocytes.Previous studies have shown that PA can cause apoptosis in human MSCs;however,it is still unknown whether PA can cause autophagy,and the effects of autophagy on PA-induced apoptosis remain unclear.The main purpose of this study is to further understand the pathogenesis of osteoporosis by studying the role of autophagy in the process of palmitate-induced apoptosis of bone marrow mesenchymal stem cells and its related molecular mechanisms and provide a novel approach for the prevention and treatment of osteoporosis.Methods: In this study,BMSCs derived from Sprague-Dawley rats with multi-directional differentiation potential were used as research objects.The first part: Cells were treated with PA(0.125,0.25,0.5 m M)for 24 h.Cell viability was measured using an MTT assay.The viability of BMSCs treated with PA(0.5 m M)at different times(0,3,6,12,18,24 h)was detected using an MTT assay.Western blot to detect the cleaved caspase-3 levels in BMSCs treated with PA(0.125,0.25,0.5 m M)for 24 h.BMSCs treated with PA(0.125,0.25,0.5m M)for 24 h were stained with annexin V/PI and measured using flow cytometry and the apoptosis ratio of PA-treated BMSCs was obtained by flow cytometry.Then,autophagosomes in BMSCs treated with PA(0.5 m M)for 6 h were observed by transmission electron microscopy.Fluorescence microscopy was used to observe internal LC3 and nuclei in BMSCs treated with PA(0.5m M)for 6 h with or without 3-MA(5m M)pretreatment for 2 h.Western blot was also used to detect the expression of LC3 and p62 in BMSCs treated with PA(0.125,0.25,0.5m M)for 12 h and quantitative analysis of LC3 II and p62 protein expression.Meanwhile,the expression of LC3 and p62 in BMSCs treated with PA(0.5m M)at different times(0,3,6,12,18,24 h)was detected by western blotting.The second Part: To investigate the role of autophagy in PA-induced apoptosis of BMSCs,we used 3-methyladenine(3MA),a classical inhibitor of autophagy,to inhibit autophagy.Additionally,rapamycin(RA),a classical autophagy inducer,was used to induce autophagy.We measured the viability of PA-treated BMSCs with or without pretreatment with 3-MA or RA.And the changes of apoptosis-related proteins caspase-3 and caspase-9 were detected by spectrophotometry.The changes of apoptosis after autophagy were detected by MTT assay and Annexin-V FITC / PI double staining flow cytometry;To further study the relationship between autophagy and apoptosis,the cells were pretreated with autophagy inhibitor3-methyladenine(3MA)and autophagy agonist rapamycin(RA)and detected by Western blot to detect the LC3,p62,and cleaved caspase-3 levels in BMSCs treated with PA(0.5 m M)for 24 h with or without 3-MA(5 m M)or RA(5 ?M)pretreatment.The third part: BMSCs were treated with PA(0.5 m M)for 6 h with or without NAC(1 m M)and incubated with DCFH-DA.Fluorescence microscopy was used to measure intracellular ROS levels;After BMSCs were exposed to PA(0.125,0.25,0.50 m M)for 6h,intracellular ROS levels were determined.To further study the relationship between ROS production and autophagy,fluorescence microscopy was used to observe internal LC3 and nuclei in BMSCs treated with PA(0.5 m M)for 6 h with or without NAC(1 m M).The expression of phospho-JNK,JNK,phospho-p38 MAPK,and p38 MAPK levels in BMSCs were detected by Western-blot and the signal pathway of PA-induced autophagy was studied.To further study the relationship between ROS and the JNK/p38 MAPK pathways,BMSCs were divided into control group,NAC(1m M)group,PA group(0.5m M),PA + NAC group.After incubation for 6h,Western blotting was used to detect LC3,phospho-JNK,JNK,phospho-p38 MAPK,and p38 MAPK levels in BMSCs.In order to further verify the role of ROS-JNK / p38 MAPK signaling pathway in PA-induced autophagy of bone marrow mesenchymal stem cells,BMSCs were pretreated with the inhibitor of JNK—SP600125—or the inhibitor of p38 MAPK—SB203580—for 1 h,then treated with PA(0.5 m M)for 6 h.Western blot to detect LC3 II and p62 levels in different groups.Results: The first part: PA can induce bone marrow mesenchymal stem cell apoptosis in a concentration-dependent and time-dependent manner;at the same time PA can increase the number of intracellular autophagosomes and increase the clustering of LC3 clustering The effect of this increase in the number of clustered LC3 clusters within the cell can be blocked by 3-MA.PA can increase the expression of LC3 II protein in bone marrow mesenchymal stem cells and decrease the expression of p62 protein in a concentration-dependent manner.In addition,with the same concentration of PA acting on cells at different times,LC3B-II protein levels gradually increased from 3 h,peaked at 12 h,while p62 protein levels decreased significantly at 3 h and reached a minimum at 12 h,beginning at 18 h increase.The second Part: After pretreatment of cells with 3MA,cell viability was significantly decreased compared to cells treated with PA alone;whereas,after pretreatment of cells with autophagy agonist RA,cells were treated with PA alone The results of flow cytometry showed that the cell apoptosis rate increased with the pretreatment of 3MA and the apoptosis rate decreased with RA treatment.The changes of the apoptosis markers caspase-3 and caspase-9 activity Flow cytometry.Compared with cells treated with PA alone,pretreatment of cells with 3MA could reduce the expression of LC3 II protein and increase the expressions of p62 and cleaved caspase-3 in bone marrow mesenchymal stem cells.The expression of LC3 II protein in mesenchymal stem cells increased,while the expression of p62 and cleaved caspase-3 protein decreased.The third part: PA can upregulate intracellular ROS content in a concentration-dependent manner;the addition of antioxidant NAC can significantly reduce the production of ROS;PA can increase the number of intracellular clusters of LC3 point LC3 This increase in intracellular LC3 Cluster-like clustering can be blocked by NAC;PA can increase the expression of P-JNK and P-p38 MAPK proteins in bone marrow mesenchymal stem cells in a concentration-dependent manner;compared with cells treated with PA alone,antioxidants NAC could effectively inhibit the expression of LC3B-II,P-JNK and Pp38 MAPK in cells.Pretreatment of cells with JNK blocker SP600125 and p38 MAPK blocker SB203580,respectively,compared with the cells treated with PA alone,Pretreatment of cells with blockers can significantly reduce the expression of LC3 II protein in bone marrow-derived mesenchymal stem cells and promote the expression of p62 protein.Conclusion: The first part: PA induced apoptosis of bone marrow mesenchymal stem cells in a dose-and time-dependent manner.PA induced autophagy of BMSCs in a concentration-dependent manner.When PA was exposed to PA for a long time,the autophagy of the cells began to weaken.The second Part: Autophagy plays a protective role in PA-induced apoptosis of bone marrow-derived mesenchymal stem cells,inhibition of autophagy can promote apoptosis,while enhance autophagy can inhibit apoptosis.The third part: PA can be a significant increase in intracellular ROS production,activation of ROS-JNK / p38 MAPK signaling pathway,to promote bone marrow mesenchymal stem cell autophagy.