| ObjectivesTo investigate a new mechanism of prostaglandin El(PGE1)in the treatment of diabetic kidney disease:reducing the apoptosis of renal tubular epithelial cells,and its possible pathway.Materials and methodsPart one:Wistar ratswere used to establish diabetic kidney disease(DKD)models through unilateral nephrectomy combined with streptozotocin(STZ).Four groups were involved into the assay:sham operation group(Sham group),unilateral nephrectomy group(Unx group),unilateral nephrectomy+STZ(DKD group)and unilateral nephrectomy+STZ+PGE 1 intervention group(PGE1 group).The experiment lasted 48 days.The urine was collected and urine albumin was measured in each group of rats before the operation,4 weeks after diabetes mellitus and after 10-day PGE1 treatment.In addition,the hematoxylin and eosin(HE)staining,schiff periodic(PAS)and periodic acid silver methanamine(PASM)were introduced to check the renal tissues changes.The pathological changes of kidney were observed to verify the establishment of DKD models.After 10-day PGE1 treatment through tail vein injection,the animals were sacrificed.The renal pathology was evaluated by PAS.TUNEL was used to observed apoptosis of renal tubular epithelial cells.Bim expression was detected by immunofluorescence.Immunohistochemical staining was introduced to detect the Bim,Bax and Bcl-2 expressions in renal cortex.The expressions of apoptosis related proteins Bim,Bax and cleavedcaspase-3 were detected with western blot.Part two:Human renal tubular epithelial cell line(HK-2 cell)was cultured.CCK-8 method was used to screen out the best PGE1 concentration and intervention time.The HK-2 cells were divided into three groups:Mannitol and glucose group(MG group),high glucose group(HG group)and PGE1 group.After cultured for 48 hours,flow cytometry and TUNEL methods were used to detect the apoptosis of renal tubular epithelial cells.Immunofluorescence was introduced to examine Bim protein expression.Western blot was used to detect the apoptosis related proteins Bim,Bax,cleaved caspase-3,p-JNK and JNK expressions.What's more,the expressions of p-JNK,JNK and Bim proteins were detected cultivated in high glucose at different time points(1h,6h,12h,24h,48h).The p-JNK,JNK and Bim proteins expressions were detected respectively after using JNK agonist anisomycin(AM)and JNK inhibitor SP600125.HK-2 cellwas divided into five groups:MG group,HG group,HG+PGE1 group,HG+AM group,HG+AM+PGE1 group.After the corresponding stimulation was given for 48h,p-JNK,JNK and Bim proteins expressions were detected.Statistical analysis:All data were presented as mean ? SE,and SPSS 21.0 statistical software was used for statistical data.The comparison of data between the two groups were evaluated with t-test.The data of multiple groups were assessed by one-way ANOVA,and the P boundary value with statistical significance was 0.05.ResultsPart one:1.Typical diabetic symptoms,namely,polypodities,polypodities and polyuriawere observed in rats after three days of unilateral nephrectomy plus streptozotocin(STZ),and the blood glucose levels were over 16.7mmol/L,suggesting that diabetes model was established.The amount of urine was over 1.5 times than the original amount of urine,and the 24-hour microalbuminuria level was 10 times higher than that in the contemporaneous control group.At the same time,HE,PAS and PASM showed more serious glomerular basement membrane and mesangial thickening,renal tubular basement membrane thickening,and dilatation,exfoliation,and loss of villus of the epithelial cells in DKD group,in comparison with the control groups after 4 weeks of diabetes modeling.Together these evidence,the DKD models were considered successfully established.2.The animals' kidney tissues were harvested after 10-day PGE1 treatment.?PAS showed that the thickening of glomerular basement membrane and mesangial membrane and renal tubular basement membrane of PGE1 group were significantly improved compared with that in the DKD group.?TUNEL showed that there was no significant difference between the Sham group and Unx group.The apoptosis of renal tubular epithelial cells increased in DKD group than that in the control groups(P<0.05 vs Sham group and Unx group).The apoptosis in PGE1 group was lower than that of the DKD groupwith statistical significances(P<0.05).?Immunofluorescence showed that Bim protein expression was the higher in the DKD group compared with that of the control groups.The expression of Bim protein in the PGE1 group was less than that in the DKD group.?Immunohistochemical results confirmed that Bim expressed only in the renal tubules,and Bcl-2 and Bax expressed not only in glomeruli but renal tubules.Bim and Bax protein expressions were higher in DKD group compared with the control groups.Bim and Bax expressions in the PGE1 group were less than that in the DKD group.The expression of Bcl-2 was just the opposite of the two proteins.?Western blot results confirmed that the expressions of Bim,Bax and cleaved caspase-3 increased in the renal cortex homogenate of the DKD group(P<0.05 vs control groups).The expressions of Bim,Bax and cleaved caspase-3 decreased in the PGE1 group(P<0.05 vs DKD group).Part two:1.CCK8 results showed that the best intervention concentration and optimal intervention time of PGE1 were 10uM and 48h,respectively.2.Flow cytometry result showed that the apoptosis of HK-2 cells in HG group was higher(P<0.05 vs control group).The cell apoptosis in PGE1 group was lower(P<0.05 vs HG group).3.TUNEL results showed that the number of cell apoptosis in HG group was larger(P<0.05 vs control group).The PGE1 group had a significant decrease cell apoptosis rate(P<0.05 vs HG group).4.Immunofluorescence results showed that Bim expressed most in HG group.While Bim expression was significantly lower in PGE1 group than that of the HG group.5.Western blot?HK-2 cells were divided into the MG group,HG group and PGE1 group.The expressions of p-JNK,Bim,Bax and cleaved caspase-3 increased in the HG group(P<0.01,0.05,0.05,0.01 vs MG group).The expressions of the four proteins in PGE1 group were reduced after cultured for 48 hours(P<0.01,0.05,0.05,0.05 vs HG group).?It showed that the p-JNK and Bim expressions of HK-2 cells increased at different time(1h,6h,12h,24h and 48h)under high glucose.The trend of the two changes was consistent.The expressions were the highest at 48 hour and the expression of JNK remained unchanged.?After adding JNK agonist AM,HK-2 cells were divided into MG group,HG group and HG+AM group.The p-JNK and Bim protein expressionsin HG group increased(P<0.05,0.05 vs MG group).The expressions of p-JNK and Bim proteins in HG+AM group were up-regulatedfurther(P<0.01,0.05 vs HG group).?After adding JNK inhibitor SP600125,HK-2 cells were divided into group MG,HG and HG+ SP600125.The expressions of p-JNK and Bim in HG group increased(P<0.05,0.05 vs MG group).The expressions of these two proteins decreased in HG+ SP600125 group(P<0.05,0.05 vs HG group).?In addition,the HK-2 cells were divided into the MG group,HG group,HG+PGE1 group,HG+AM group and HG+AM+PGE1 group.There were obvious increased expressions of p-JNK and Bim in the HG group(P<0.01,0.01 vs MG group).The expressions of p-JNK and Bim were elvated further in HG+AM group(P<0.01,0.01 vs HG group)and decreased in HG+PGE1 group(P<0.055 0.01vs HG group).The expressions of p-JNK and Bim in HG+PGE1+AM group were higher than that of HG+PGE1 group(P<0.01,0.05).There was no significant difference in p-JNK expression between the HG+AM+PGE1 group and HG+AM group(P>0.05),while Bim expression decreased in HG+AM+PGE1 group(P<0.01 vs HG+AM group).Conclusions1.Both in vivo and in vitro experiments showed that high glucose increased the apoptosis of renal tubular epithelial cells,and elevated the expressions of Bim,Bax and cleaved caspase-3 proteins in renal tubules.2.Both in vivo and in vitro experiments showed that PGE1 decreased the apoptosis of renal tubular epithelial cells and downgrulated the expressions of Bim,Bax and cleaved caspase-3 proteins in renal tubules.3.In vitro experiments showed that p-JNK and Bim protein increased under high glucose with the time gradient.When JNK agonist was added in high glucose environment,the expression of p-JNK and Bim increased further.After the JNK inhibitor was added,the two proteins decreased at the same time,indicating that JNK could regulate Bim.4.The p-JNK expression increased treated with PGE1 combined with JNK agonist than single use of PEG 1,but Bim expression slightly increased.The agonist could not completely reverse the Bim decline induced by PGE1.The p-JNK expression was almost unchanged,but the expression of Bim was obviously downregulated cultured with PGE1 combined with JNK agonist than single use of JNK agonist.JNK was sustained excited,but there was still a decrease in Bim after PGE1 was given.It suggested that PGE1 could partly slow down the expression of Bim through JNK.There may be other molecules involved in PGE1 eased Bim expression.That was,PGE1 did not entirely depend on JNK/Bim pathway to mediate the alleviated tubular apoptosis. |
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