| Objective1.To establish a model of human renal tubular epithelial cells(HK-2)damage induced by uric acid.2.By observing the changes in HK-2 cell inhibition rate and transforming growth factor-β1(TGF-β1)and B-cell lymphoma-2 gene(Bcl-2),to investigate whether losartan has a protective effect on uric acid-induced HK-2 damage and whether its protective effect is enhanced with increasing losartan concentration.MethodHK-2 was used as the research object.1.To establish a model of HK-2 damage induced by uric acid and to screen the concentration of losartan required for the experiment.Grouping:(1)Blank group(complete medium added,no HK-2);(2)Control group(complete medium added);(3)Uric acid group A(uric acid concentration in the complete medium was 400umol/L);(4)Uric acid group B(uric acid concentration in the complete medium was 800umol/L);(5)Uric acid group C(uric acid concentration in the complete medium was1600umol/L);(6)Losartan group A(Losartan concentration in the complete medium was 1ng/ml);(7)Losartan group B(Losartan concentration in the complete medium was 10ng/ml);(8)Losartan group C(Losartan concentration in the complete medium was 100ng/ml);(9)Losartan group D(Losartan concentration in the complete medium was 1000ng/ml).Six replicate wells were set up for each group,and the experiment was repeated three times to take the average value.The cell inhibition rate of each group was detected at 24 h,48h and 72 h,and the drug concentration and action time were screened by CCK8.2.According to the above experimental results,the concentration of uric acid was800umol/L,the concentration of losartan was 1ng/ml,10ng/ml and 100 ng /ml,the cell treatment time was 48 h,and a new experimental group was set:(1)Group A(uric acid group,the complete medium with uric acid concentration of 800umol/L and losartan concentration of 0ng/ml);(2)Group B(uric acid + losartan 1 group,the complete medium with uric acid concentration of 800umol/L and losartan concentration of1ng/ml);(3)Group C(uric acid + losartan 2 group,the complete medium with uric acid concentration of 800umol/L and losartan concentration of 10ng/ml);(4)Group D(uric acid + losartan 3 group,the complete medium with uric acid concentration of800umol/L and losartan concentration of 100ng/ml);(5)Group E(control group,the complete medium with uric acid concentration of 0umol/L and losartan concentration of 0ng/ml).Six replicate wells were set up for each group of CCK8 experiments,and the experiments were repeated three times to obtain the average of the cell inhibition rate of each group.Three replicate wells were set up for each group of PCR experiments,and the experiments were repeated three times to take the average value to detect the expression of TGF-β1 and Bcl-2 in HK-2.Results1.Among different groups which treated with different time and different concentration of uric acid and losartan,when the uric acid concentration was 400umol/L,HK-2 was mildly inhibited at 24 h,48h,and 72 h compared with the control group,with statistically significant difference(P<0.05);When the uric acid concentration was 1600umol/L,HK-2 was significantly inhibited at 24 h compared with the control group,with the highest cell inhibition rate of 93.78% at 72h;The cell inhibition rate was closest and not higher than 50% when the concentration of uric acid was 800umol/L and the duration of action was 48 h,so this concentration and time were chosen to establish the model of HK-2 damage caused by uric acid.HK-2 was not significantly altered by inhibition at concentrations of 1ng/ml,10ng/ml and 100ng/ml and duration of action of 24 h,48h and 72 h compared to the control group,and the difference was not statistically significant(P>0.05);When the concentration of losartan was 1000ng/ml,there was significant inhibition at 24 h,48h and 72 h compared with the control group,and the difference was statistically significant(P<0.05),with the highest cell inhibition rate of 96.73% at 72 h,so this concentration was excluded in the experiment.2.When HK-2 was treated under different uric acid concentration conditions for24 h,48h and 72 h,the overall analysis(two-way repeated measures analysis of variance)found that time comparison,concentration comparison and the interaction between time and concentration,difference was statistically significant(P<0.05),suggesting that the cell inhibition rate varies greatly with time and uric acid concentration,and the change with concentration was affected by time.Comparison between groups(one-way analysis of variance + multiple comparisons): the differences between the three groups were significant(P<0.05)at each time after treatment with different concentrations of uric acid,and the rate of cell inhibition varied from low to high with increasing concentrations of uric acid.Intra-group comparison(paired t-test): uric acid concentrations were statistically significant(P<0.05)in all three groups at all time points,and the rate of cellular inhibition increased with time.3.When HK-2 was treated with the same concentration of uric acid and different concentrations of losartan for 48 h,the cells were significantly inhibited at the concentration of 800umol/L of uric acid.The absorbance at 450 nm of HK-2 increased with the increase of losartan concentration,and the cell inhibition rate decreased with the increase of losartan concentration,and the difference was statistically significant(P<0.05).Losartan at the concentration of 1ng/ml could improve the inhibition of HK-2induced by uric acid,and the improvement effect of cell inhibition increased with the increase of losartan concentration within a certain range.4.The relative expression of TGF-β1 was higher and the relative expression of Bcl-2 was lower at 48 h and at a uric acid concentration of 800umol/L.With the increase of losartan concentration in uric acid,the relative expression of TGF-β 1decreased,while the relative expression of Bcl-2 increased,and the difference was statistically significant(P<0.05).A low concentration of losartan could improve the fibrosis and apoptosis of HK-2 induced by uric acid,and this improvement was enhanced with the increase of losartan concentration in a certain range.Conclusion1.When the concentration of uric acid is 800umol/L and the action time is 48 h,the cell inhibition rate is close to 50%,and the model of HK-2 damage caused by uric acid can be prepared by choosing this concentration and time.2.The cell inhibition rate varies greatly with time and uric acid concentration,and the variation with concentration is affected by time.3.A low concentration of losartan can improve the inhibition,fibrosis and apoptosis of HK-2 cells induced by uric acid,and the improvement effect varies with losartan concentration within a certain range. |
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