| Objective: Diabetic Kidney disease(DKD)is a common microvascular complication of diabetes and one of the most common causes of end-stage nephropathy.As a major diabetic country,there are 100 million diabetic patients in China at present;while DKD has become an important disease that affects the quality of life and life span of Chinese.Clinically,diabetic patients are often accompanied by abnormal lipid metabolism,and lipid deposition is also detected in renal tissues of DKD patients.The disorder of lipid metabolism may be one of the causes of DKD.However,the mechanism of high glucose on intracellular lipid deposition is still unclear.In this study,HGVECs were taken as the research object,and RT-q PCR,WB,Gene Microarray and other techniques were used to explore the effect of high glucose on intracellular lipid deposition and its specific mechanism.Methods: 1.Water-soluble cholesterol was used to simulate the high-fat environment HGVECs were given cholesterol(0μg/ml;200μg/ml;400μg/ml;600μg/ml;800μg/ml).After 12 h,24h and 48 h,CCK8 assay was performed to detect the activity of the cells,and to select appropriate cholesterol intervention concentration and time.2.Nitric acid reductase was used to detect the content of NO HGVECs were given cholesterol(0μg/ml;200μg/ml;400μg/ml;600μg/ml;800μg/ml)for 24 h.The effect of cholesterol on the synthesis and secretion of NO was detected.3.Verification of high-fat model and effects of different glucose concentrations on intracellular lipid deposition HGVECs were given cholesterol(0μg/ml;200μg/ml;400μg/ml;600μg/ml;800μg/ml)for 24 h,and Oil red O staining and Cholesterol quantification assay were carried out to observe the intracellular lipid deposition.Then it was divided into the low-glucose group,the hyperglycemia group,the hypercholesterolemia group and the hyperglycemia and hypercholesterolemia group.After different interventions for 24 h,the effect of hyperglycemia on intracellular lipid deposition was observed.4.Effects of hyperglycemia and hypercholesterolemia on expression of ABCA1 and LDLR HGVECs were divided into the low-glucose group,the hyperglycemia group,the hypercholesterolemia group and the hyperglycemia and hypercholesterolemia group.After different interventions for 24 h,m RNA and protein levels of ABCA1 and LDLR were detected by RT-q PCR,WB and cellular immunofluorescence.5.Screening of transcription factors regulating the expression of ABCA1 in HGVECs HGVECs were divided into the low-glucose group,the hyperglycemia group,the hypercholesterolemia group and the hyperglycemia and hypercholesterolemia group.After different interventions for 24 h,ABCA1-related transcription factors were detected by RT-q PCR and WB.LXRs was selected for further study.6.The effect of LXRs activator-GW3965 on the expression of ABCA1 HGVECs were given GW3965(0μmol/ml;0.5μmol/ml;1μmol/ml;2μmol/ml;4μmol/ml).The expression of ABCA1 was detected by RT-q PCR and WB after 12 h,24h and 36 h of intervention cells.7.Verify that hyperglycemia affects the expression of ABCA1 through LXRs HGVECs were divided into the control group,the hypercholesterolemia group and the hyperglycemia and hypercholesterolemia +GW3965 group.After different interventions for 24 h,the expression of ABCA1 was detected by RT-q PCR.8.Looking for Lnc RNA that is involved in the regulation of ABCA1 transcription by LXRs and further exploring its mechanism of action HGVECs were divided into the control group and the GW3965 group.After 12 h,Gene Microarray was used to look for lnc RNA,and the results of Gene Microarray were verified by RT-q PCR.Using si RNA knockdown the expression of Lnc RNA,HGVECs were divided into the control group,the GW3965 group,the GW3965 group + Lnc RNA-si RNA group.After 100 n M si RNA transfection for 48 h and GW3965 intervention for 24 h,the expression of ABCA1 was detected by RT-q PCR.Results: 1.CCK8 experiment results showed that HGVECs activity decreased after cholesterol intervention compared with the control group,and this effect of cholesterol was time-dependent and concentration-dependent.2.The results of NO content determination showed that,compared with the control group,there was no statistical difference in NO content of cells in each group after cholesterol intervention.3.The results of Oil red O staining and Cholesterol quantification assay showed that intracellular lipid deposition was observed after the intervention of cholesterol for 24 h,and the amount of intracellular lipid deposition increased with the increase of cholesterol.Lipid deposition was more significant in the hyperglycemia and hypercholesterolemia group than in the hypercholesterolemia group.4.RT-q PCR and WB results show that compared with the control group,the expression of ABCA1 in the hypercholesterolemia group was obviously increased,the expression of LDLR decreased,but compared with the hypercholesterolemia group,the hyperglycemia and hypercholesterolemia group decreased ABCA1 expression,increased the expression of LDLR.5.RT-q PCR and WB results show that compared with the control group,the hypercholesterolemia group LXR-α,LXR-β,FXR,RXR-α and RXR-β were significantly increased,the expression of SREBP2 decreased,but compared with the hypercholesterolemia group,LXR-α,LXR-β,RXR-α and RXR-β expression levels in the hyperglycemia and hypercholesterolemia group were decreased,and the m RNA level of FXR showed no statistical difference.The protein level was significantly decreased,and the m RNA level of SREBP2 was increased,protein levels drop.6.RT-q PCR and WB results showed that compared with the control group,the expression level of ABCA1 in the GW3965 group increased.7.RT-q PCR and WB results showed that the expression of ABCA1 increased in the hyperglycemia and hypercholesterolemia group +GW3965 group compared with the hyperglycemia and hypercholesterolemia group.8.The results of Gene Microarray showed that the expression of ABCA1 was up-regulate in the GW3965 group.The analysis of m RNA showed that differentially expressed genes were mainly involved in regulation of lipid metabolism.9.Lnc RNAOR13C9 was significantly increased in the GW3965 group through RT-q PCR verification of lnc RNA differences in Gene Microarray.10.RT-q PCR results showed that the expression of Lnc RNAOR13C9 in the si RNA group was significantly lower than that in the control group.After GW3965 was given at the same time Lnc RNAOR13C9 knockdown,the expression level of ABCA1 was decreased compared with that of Lnc RNAOR13C9 without knockdown.Conclusion: 1.HGVECs under high cholesterol load will lead to intracellular lipids deposition,which will affect cell activity,and hyperglycemia will aggravate this process.2.Under high cholesterol load,HGVECs will excrete excessive cholesterol out of the cells by upregulation of the expression of ABCA1,while hyperglycemia will aggravate the deposition of cholesterol in the cells by interfering with this self-regulation of the cells and blocking the upregulation of ABCA1.3.The effect of hyperglycemia on ABCA1 is by affecting the transcription factor LXRs,which regulates its transcription,and Lnc RNAOR13C9 also plays an important role in this process. |
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