| Background and objective The state of high free fatty acids in the blood is one of the pathological characteristics of diabetic patients.Palmitic acid(PA)is the most abundant saturated fatty acid in the body and is widely used in the study of lipotoxicity.Micro RNAs(mi RNAs)are single-stranded non-coding small RNAs with a high degree of conservation.mi RNAs use the base pairing principle to target m RNA to regulate gene expression.In recent years,studies have reported the role of mi RNAs in regulating gene expression and in the development and progression of various diseases including diabetic nephropathy.It has been reported that mi R-483-5p is related to the development of diabetes.Meanwhile,it has been reported that mi R-483-5p may be involved in the process of human cardiomyocyte apoptosis and oxidative stress induced by hypoxia.The above research results provide a basis for this study to explore the effects of mi R-483-5p on cell apoptosis and oxidative stress in diabetic nephropathy.Based on the above evidence,palmitic acid was used to simulate high-fat environment to treat mouse renal podocytes,and explored the role and potential mechanism of mi R-483-5p in palmitic acid-induced podocytes,which could provide experimental basis for clarifying the mechanism of lipid nephrotoxicity and provide a certain theoretical basis for the development of new drugs for diabetic nephropathy in the future.Methods1)Different concentrations of palmitic acid(0,0.05,0.1,0.2,0.5 and 1 mmol/L),were used to treat the mouse renal podocytes and the viability of podocytes and the expression level of mi R-483-5p in each group were detected.2)mi R-483-5p inhibitor,mi R-483-5p mimic and Kruppel-like factors-2(KLF-2)plasmids were used to treat renal podocytes from mice,and they were divided into control groups according to different treatment methods,PA group,(PA+NC)group and(PA+In)group,use CKK-8(Cell counting kit-8)experiment to detect the effect of mi R-483-5 on cell viability in each group;flow cytometry The effect of apoptosis in each group;colorimetric method to detect the effect of mi R-483-5p on the cell oxidative stress indicators(including malondialdehyde and superoxide dismutase)after palmitic acid treatment;3)The luciferase dual reporter gene verifies the relationship between mi R-483-5p and KLF2,and will be divided into four groups according to different treatment methods,namely Blank+KLF2-WT group,mi R-483-5p mimic+KLF2-WT,Blank+KLF2-MT and mi R-483-5p mimic+KLF2-MT groups,check the luciferase activity of each group.4)According to different cell processing methods,it is divided into Control group,(PA+NC)group,(PA+M)group and(PA+M+KLF2)group,using Quantitative Realtime Polymerase Chain Reaction(Quantitative Real-time Polymerase Chain)Reaction,q RT-PCR)to detect changes in the expression levels of mi R-483-5p and KLF2 m RNA in each group of cells,cell viability of each group was detected by CKK-8,the level of MDA and SOD of each group were examine by the colorimetric method,and the change of cell apoptosis in each group was tested by flow cytometry.5)Western blotting was used to detect the effects of palmitic acid and mi R-483-5p on cleaved caspase3,Nephrin,Podocin protein expression levels and oxidative stress(MDA and SOD).Results1)Control group,0.05mmol/L group,0.1mol/L group,0.2 mmol/L group,0.5 mmol/L group and 1 mmol/L podocyte viability were(97.51±10.73)% and(85.61±10.79),respectively %,(74.83±7.89)%,(64.10±7.75)%,(44.60±4.21)%,(23.86±2.69)%,compared with the control group and 0.05mol/L,the cell viability of other groups was significantly reduced(F =35.16,P<0.0001),the expression of mi R-483-5p in podocytes of Control group,0.05mmol/L group,0.1mol/L group,0.2 mmol/L group,0.5 mmol/L group and 1 mmol/L group The levels are(1.14±0.07),(1.66±0.08),(2.03±0.18),(3.05±7.75),(4.23±0.44),and(6.60±0.54)respectively.Compared with the control group and 0.05mol/L group,the level of mi R-483-5p in other groups of cells was significantly increased(F=114.40,P<0.0001).The above results showed that palmitic acid can significantly inhibit cell viability and promote the expression of mi R-483-5p in a certain concentration..When the palmitic acid concentration was 0.5 mmol/L,cell viability decreased by more than 50%,so 0.5 mmol/L was chosen as the treatment condition for subsequent experiments.2)The expression levels of mi R-483-5p in the control group,PA group,(PA+NC)group and(PA+In)group were(0.99±0.08),(4.29±0.49),(4.33±0.47)and(3.70±0.47).Compared with the control group,0.5 mmol/L palmitic acid can significantly promote the expression of mi R-483-5p,while mi R-483-5p inhibitor can reverse the inhibitory effect of palmitic acid on the expression of mi R-483-5p to a certain extent.(F=32.90,P<0.0001);the cell viability of each group were(101.01±14.21)%,(42.34±3.21)%,(43.20±2.84)% and(74.22±8.24)%,respectively.The results showed that mice renal podocytes were treated with 0.5 mmol/L palmitic acid for 24 hours,the cell viability was significantly reduced,and mi R-483-5p inhibitor can reverse the inhibitory effect of palmitic acid on cell viability and partially restore cell viability.The results of apoptosis experiment showed that the apoptosis rate of each group was(2.76±0.19)%,(35.44±7.86)%,(34.99±7.64)%,(16.92±2.30)%,compared with control group,the apoptosis rates in other groups were significantly increased(F=23.74,P=0.000).The results showed that palmitic acid can significantly promote cell apoptosis,and mi R-483-5p inhibitor partially reversed the effect of palmitic acid on cell apoptosis.The expression levels of malondialdehyde(MDA)in the cells of each group were(4.99±0.26)nmol/L,(24.77±3.26)nmol/L,(25.31±3.23)nmol/L,(10.17±1.98)nmol /L,compared with control,the expression levels of MDA in other groups were significantly increased(F=50.86,P<0.0001),and the expression levels of superoxide dismutase(SOD)in each group were(31.52± 3.91)U/mg,(9.04±0.93)U/mg,(9.21±1.21)U/mg and(22.83±2.90)U/mg,compared with control group,the expression level of MDA in other groups was significantly increased(F=42.96,P<0.0001).The results show that palmitic acid can significantly promote the expression of malondialdehyde and inhibit the expression of superoxide dismutase(SOD),while mi R-483-5p inhibitor partially reverses palmitic acid The effect on cell MDA and SOD expression.3)mi R-483-5p has a potential binding site on the 3'-UTR(3'-Untranslated Region)of the KLF2 gene.The results of the dual luciferase reporter gene experiment show that the relative activities of each group of luciferase are(0.99±0.16),(0.26±0.08),(1.00±0.19)and(0.94±0.13),compared with Blank+KLF2-WT group,mi R-483-5p mimic+KLF2-WT group significantly inhibited cell fluorescence Compared with the mi R-483-5p mimic+KLF2-MT group,the Blank+KLF2-MT group has no significant difference in fluorescence activity(t=7.324,P=0.002).4)The relative expression levels of KLF2 m RNA in the cells of the control group,(PA+NC)group,(PA+M)group and(PA+M+KLF2)group were(0.99±0.17),(0.64±0.08),(0.61±0.07)and(0.84±0.15).Compared with the control group,the expression level of KLF2 m RNA in the cells of the PA group,(PA+NC)group and(PA+In)group was significantly lower(F=6.269,P=0.017),q RT-PCR detection of KLF2 expression in mouse renal podocytes showed that the palmitic acid group was significantly lower than the control group's KLF2 expression,mi R-483-5p inhibitor partially reversed the inhibitory effect of palmitic acid on KLF2 expression in cells.The cell viability of each group was(98.67±10.02)%,(45.67±3.06)%,(25.33±1.53)% and(72.67±7.09)%,respectively.mi R-483-5p mimic further enhanced the inhibitory effect of palmitic acid on cell viability,And this enhancement was partially reversed by KLF2 overexpression.5)The expression levels of MDA in the cells of the Control group,(PA+NC)group,(PA+M)group and(PA+M+KLF2)group were(5.13±0.39)nmol/L and(25.40±2.24)nmol,respectively /L,(38.43±4.02)nmol/L and(20.82±2.69)nmol/L,the expression levels of SOD in each group were(34.16±4.50)U/mg,(9.36±2.42)U/mg,(5.91±1.76)U/mg and(15.68±3.01)U/mg.Palmitic acid significantly increased the expression level of MDA and inhibited the expression level of SOD.mi R-483-5p mimic further enhanced the regulation of palmitic acid on the expression levels of MDA and SOD,and this enhancement was partially reversed by overexpression of KLF2.The expression levels of Nephrin and Podocin in each group of cells were [(0.97±0.09),(1.75±0.20),(2.68±0.30),(1.37±0.22)],[(0.99±0.06),(0.56±0.10),(0.19±0.20),(0.55±0.12)] and[(1.01±0.05),(0.26±0.07),(0.14±4.02),(0.53±0.09)],respectively.Palmitic acid can significantly increase the expression of MDA and cleaved caspase3 and inhibit the expression of SOD,Nephrin and Podocin.mi R-483-5p mimic can enhance the palmitic acid's regulatory effect on MDA,SOD,cleaved caspase3,Nephrin and Podocin,and the effect was partially reversed by KLF2 overexpression.Conclusion mi R-483-5p can affect palmitic acid-induced podocyte damage by regulating the expression of KLF2.This regulatory mechanism is helpful to the study of the prognosis of patients with diabetic nephropathy. |
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