Effects Of LncRNAGPRC5D-AS1 On Dexamethasone-induced Muscle Atrophy And Ferroptosis Related Proteins In Mouse C2C12 Cells

Sarcopenia is an age-related clinical syndrome of reduced skeletal muscle strength and mass and/or associated loss of physical function that seriously affects quality of life in older adults.The incidence of sarcopenia is also on the increase with limited treatment options[1-4].In recent years,it has been found that muscular dystrophy is closely related to Ferroptosis,a pattern of cell death primarily characterized by the build-up of lipid activated oxygen species(L-ROS)in iron-dependent cells[5],mainly represented by small mitochondria in the fine structure of cells and mitochondrial membrane contraction.mitochondrial cristae reduced or disappeared,and the external membrane was disrupted,but the morphological alteration of the core was not obvious.Iron death is closely related to lipid metabolism,amino acid metabolism,iron metabolism and gene regulation in vivo[6,7].In a prior study,lncRNAGPRC5D-AS1 can regulate skeletal muscle atrophy,and its downstream target genes include the iron death suppressor SLC7A11 and the regulation genes ATF4 and ARID 1 A.It can be inferred that lncRNAGPRC5D-AS1 is involved in iron death in skeletal muscle by regulating SLC7A11.Glutathione peroxidase 4(GPX4)is a key regulator of glutathione dependence.It also plays a key role in the iron death regulation pathway.By cleaning up excess free radicals and hydrolyzing lipidhydrooxides(LOP),it decreases the build-up of lipid peroxides,thereby protecting cells.The absence of GPX4 or the inhibition of GPX4 protein expression can promote the occurrence of iron death,in other words,the expression of GPX4 protein is closely related to the occurrence of iron death[5-8].However,there are few articles on iron death and skeletal muscle atrophy.Therefore,in this study,the effects of lncRNAGPRC5D-AS1 on muscle atrophy and ferroptosis related proteins were explored through dexamethasone-induced muscle atrophy model of mouse C2C12 cells.Firstly,the model of muscle atrophy induced by dexamethasone in C2C12 cells was established.The optimal dosification and aging of dexamethasone were screened by MTT,and C2C12 cells were successfully induced.The effect of lncRNAGPRC5D-AS1 on muscle atrophy in C2C12 cells was verified by cell experiments.The exposure level of lncRNAGPRC5D-AS1 in all groups was examined by qPCR.lncRNAGPRC5D-AS1 overexpression vector was constructed and the interference efficiency was detected by qPCR.The contents of GPX4 enzyme,Fe2+and Malondialdehyde(MDA)in lncRNAGPRC5D-AS 1 groups were determined by ELISA.ROS content in each group was determined by flow cytometry.The number and morphology of mitochondria in each group were observed by transmission electron microscopy.After lncRNAGPRC5D-AS1 expression was detected by WesternBlot,ferroptosis related proteins of ACSL4,SLC7A11,GPX4 and TFR4 were detected.Immunofluorescence detection of MyoD expression level in the group confirmed that lncRNAGPRC5D-AS 1 could regulate dexamethasone-induced muscle atrophy progression in mouse C2C12 cells through the ferroptosis signaling pathway.In this study,MTT method was used to screen out the best effect of dexamethasone induction of C2C12 at 100ug/ml concentration and time for 48 hours.The expression level and interference rate of lncRNAGPRC5D-AS1 in each group were detected by qPCR.The exposure level of lncRNAGPRC5D-AS1 was markedly higher in natural C2C12 cells than in the dexamethasone induced C2C12 cells(p<0.001),and the overexpression of lncRNAGPRC5D-AS1 showed a significant remission trend of muscular dystrophy,further confirming the role of lncRNAGPRC5D-AS1 in the symptoms of muscular dystrophy.GPX4,Fe2+,Malondialdehyde(MDA)contents were determined by ELISA after lncRNAGPRC5D-AS1 overexpression.Compared with normal C2C12 cells,dexamethasone-induced concentrations of Fe2+and Malondialdehyde(MDA)were significantly increased(p<0.05)and GPX4 content was significantly decreased(p<0.05).When lncRNAGRPC5D-AS1 was overexpressed,Fe2+and MDA contents were significantly decreased compared with dexamethasone-induced C2C12 cell group(p<0.05)and GPX4 content was significantly increased(p<0.05).Flow cytometry revealed that the ROS content of dexamethasone-induced C2C12 cells was obviously greater than that of regular C2C12 cells.The ROS amounts in the dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS1-OE group were distinctly lower than those in the dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS1-NC group.The expression levels of SLC7A11 and GPX4 in dexamethasone-induced C2C12 cells were significantly lower than those in normal C2C12 cells,and the expression levels of ACSL4 and TFRC were notably greater than in commercial C2C12 cells.The expression levels of SLC7A11 and GPX4 in dexamethasone-induced C2C12 cells+lncRNAGPRC-AS1-OE group were significantly higher than those in dexamethasone-induced C2C12 cells,and the expression levels of ACSL4 and TFRC were obviously inferior to those in the dexamethasone-induced C2C12 cells.MyoD expression was apparently diminished in dexamethasone-induced C2C12 cells in comparison with normal C2C12 cells.The expression of Myod in dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS 1-OE group was significantly decreased compared with dexamethasone-induced C2C12 cells+lncRNAGPRC5D-AS1-NC group.The expression of MyoD was significantly increased.This study shows that:(1)The experimental model required for this subject was established by dexamethasone-induced mouse C2C12 cells,and the survival rate of dexamethasone-induced mouse C2C12 cells was tested by MTT.It was confirmed that dexamethasone could induce mouse C2C12 cells to establish a muscular dystrophy model.(2)The expression of lncRNAGPRC5D-AS1 in muscular atrophy cells was significantly decreased,and the expression of iron death related proteins was significantly increased.(3)Overexpression of lncRNAGPRC5D-AS1 inhibited the development of muscular atrophy and inhibited the expression of related proteins that contribute to the occurrence of iron death.lncRNA GPRC5D-AS1 may play a protective role in dexamethasone-induced iron death of myoblasts by mediating SLC7A11/GPX4/ACSL4 signaling axis and promote cell repair and regeneration.