| Part Ⅰ Expression and mechanism of miR-223-3p in endothelial cell injury in vitro Objective:High glucose-inducd Human Umbilical Vein Endothelial Cells(HUVECs)and Human Renal Glomerular Endothelial Cells(HRGECs)were widely used as in vitro models of diabetic kidney disease(Diabetic Kidney Disease,DKD).The relationship between miR-223-3p and endothelial injury was initially explored,and the regulatory role of miR-223-3p on high glucose-caused endothelial injury was investigated.To better clarify the mechanism of miR-223-3p as a regulatory factor in high glucose stimulated endothelial injury,the regulation of miR-223-3p expression on IL6 ST and its pathway downstream target gene STAT3 was verified by HUVECs and HRGECs.Methods:1.HUVECs were treated with normal glucose(1 g/L)or high glucose(6.5 g/L)or mannitol(6.5 g/L)for 6,12 and 24 hours,with mannitol as the control variable for experimental osmolality;real-time fluorescence quantitative PCR(Quantitative Realtime PCR,RT-qPCR)was used to detect the relative expression levels of endothelial damage factors endothelial vasoconstrictor peptide-1,vascular hemophilia factor and vascular endothelial growth factor(Et-1,Vwf,Vegfa)and miR-223-3p m RNA.2.Knockdown or overexpression of miR-223-3p adenovirus-transfected HUVECs or HRGECs were treated with normal glucose or high glucose.RT-qPCR detect the relative expression levels of miR-223-3p,endothelial damage factor Et-1,Vwf,Vegfa and inflammatory factor interleukin-6(Il-6)m RNA in each group.3.HUVECs were treated with high glucose(6.5 g/L)for 0,6,12,and 24 h.IL6 ST protein expression levels were detected by Western blotting;Knockdown or overexpression of miR-223-3p HUVECs or HRGECs were treated with normal glucose or high glucose,RT-qPCR was performed to detect the relative expression levels of Il6 st m RNA in HUVECs and HRGECs,WB to detect IL6 ST,p-STAT3 and STAT3 protein expression levels in HUVECs.Results:1.The relative expression levels of endothelial damage factors Et-1,Vwf,and Vegfa were elevated(P< 0.05)and the relative expression level of miR-223-3p was significantly decreased(P< 0.05)in the high glucose-stimulated HUVECs compared to the control groups,while,the relative expression levels of endothelial damage factors Et-1,Vwf,and Vegfa and miR-223-3p expression didn’t show statistically significant.2.The relative m RNA expression levels of miR-223-3p in HUVECs and HRGECs cells treated with normal sugar or high sugar were significantly downregulated(P<0.01);the expression of Et-1,Vwf,Vegfa were significantly decreased,and Il-6expression was significantly increased in the high glucose group after high stimulation compared with control group(P< 0.001).In miR-223-3p knockdown cells,the endothelial damage factors Et-1,Vwf,Vegfa and inflammatory factor Il-6 was higher compared with the control group(P< 0.01),while was further upregulated in the cells of the adenovirus group with knockdown of miR-223-3p under high glucose treatment compared with control group(P< 0.05).3.RT-qPCR was carry out to detect the relative m RNA expression levels in miR-223-3p overexpressing HUVECs and HRGECs cells(P< 0.01).Under normal sugar condition,the relative expression levels of Et-1,Vwf and Il-6 in the cells of the miR-223-3p adenovirus group,indeed,these factors was down-regulated compared with control group(P< 0.05).the expression of Et-1,Vwf,Vegfa and Il-6 in the cells of the miR-223-3p adenovirus group under high sugar treatment(p < 0.01)was also downregulated compared with that of the adenovirus group.4.The protein expression levels of IL6 ST in HUVECs in response to high sugar stimulation.5.RT-qPCR was carry out to detect the relative m RNA expression levels of Il6 st in HUVECs and HRGECs cells,and the expression of Il6 st was significantly higher in the high glucose group compared with the normal glucose group in control group(P<0.05).Under normal glucose treatment,the expression of Il6 st was higher in miR-223-3p knockdown cells compared with control group(P< 0.05),and the expression of Il6 st in the empty group was further upregulated than that in high glucose treatment(P<0.05).6.RT-qPCR was performed to detect the relative m RNA expression levels of Il6 st in miR-223-3p overexpression HUVECs and HRGECs cells,and the expression of Il6 st was reduced in the miR-223-3p overexpression cells compared with the empty group under normal glucose treatment(P< 0.05).Under high glucose treatment,the expression of Il6 st in the cells of the miR-223-3p overexpression group was also downregulated compared with that of the controls group under high-sugar treatment conditions(P< 0.01).7.WB results showed that the relative expression levels of IL6 ST and p-STAT3 proteins were downregulated in miR-223-3p overexpressing HUVECs cells treated with normal sugar or high sugar(P< 0.01).WB results showed that the relative expression levels of IL6 ST and p-STAT3 proteins were significantly increased in miR-223-3p knocked down HUVECs cells treated with normal sugar or high sugar(P< 0.05).Conclusions:1.High glucose stimulation successfully established an endothelial injury HUVECs model in which,the relative miR-223-3p m RNA expression was significantly reduced and the IL6 ST protein expression was up-regulated.2.In HUVECs and HRGECs,knockdown of miR-223-3p expression could promote high glucose induced endothelial injury and inflammatory response;Overexpression of miR-223-3p could improve endothelial cell injury and inflammatory response.Taken together,all these results indicated that miR-223-3p is a key regulator in high glucose caused endothelial injury and inflammatory response.3.In vitro studies,knockdown of miR-223-3p promoted the expression of target gene IL6 ST and its downstream target protein p-STAT3.Overexpression of miR-223-3p decreased the expression of IL6 ST and p-STAT3,indicating miR-223-3p can regulate IL6ST/STAT3 signaling,thus participating in the regulation of endothelial injury caused by high glucose stimulation.Part Ⅱ Mechanisms of miR-223-3p in an in vivo DKD mice model Objective:In vitro findings demonstrate that miR-223-3p is a key regulator in the progression of high-glucose-stimulated endothelial injury.To verify the regulatory effect of miR-223-3p on glomerular endothelial injury in DKD in vivo,we explored whether miR-223-3p acts as a protective factor in DKD glomerular endothelial injury and verified its regulation of IL6ST/ STAT3 pathway expression in DKD mice.Methods:1.Detection the fasting blood glucose levels and body weight in 15-week-old Wild type(WT)and db/db mice,(n=5 in each group);H&E and PAS staining of kidney tissue sections to observe glomerular pathological changes and glomerular glycogen deposition to clarify the DKD mouse model.2.RT-qPCR was used to determine the relative m RNA expression levels of miR-223-3p,Il6 st and endothelial cell injury factors(Et-1,Vegfa,Vwf)in the renal cortex of WT and DKD mice,(n=5 in each group);WB detected the relative expression of IL6 ST,p-STAT3 and STAT3 protein in the renal cortex of WT and db/db mice levels,(n=3 in each group).Results:1.db/db mice showed a significant increase in body weight and blood glucose compared with WT(P< 0.001);H&E staining of kidney tissue sections suggested that db/db mice had increased glomerular volume and significant inflammatory infiltration compared with control mice;PAS staining of kidney tissue sections showed thickened glomerular basement membrane and significant glycogen deposition.2.RT-qPCR results showed that the relative expression levels of miR-223-3p in the renal cortex of DKD mice were decreased(P< 0.001),and the relative m RNA expression levels of Il6 st and endothelial cell injury factors Et-1,Vegfa and Vwf were increased(P< 0.01);WB results showed that the relative expression levels of IL6 ST and p-STAT3 proteins were significantly increased in the renal cortex of WT and db/db mice(P< 0.001).Conclusions:In vivo study,miR-223-3p expression was significantly decreased in the renal cortex of DKD mice,and IL6 ST and p-STAT3 expression levels were up-regulated,suggesting that miR-223-3p is involved in the regulation of DKD progression and may regulate glomerular endothelial injury through IL6ST/STAT3 signaling. |
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