Research Of STAT1-P53-P21 Pathway On Stress Aging In Adriamycin Nephrosis Mice

Objective: By detecting the expression of STAT1,P53 and P21 in the kidney of adriamycin induced nephropathy in mice,the effect of stress aging in adriamycin nephropathy mice was studied.Whether the expression of STAT1-p53-p21 pathway further intervention can regulate aging mechanism,thus delaying the progression of adriamycin nephropathy in mice.Method: Ninety male BALB/c mice aged 6 weeks were randomly divided into control,mode(ADR),drug intervention(ADR + ARB)groups,respectively.The mode and drug intervention groups were subjected to the single tailvein injection of adriamycin(10mg/kg)to establish adriamycin nephropathy models.the control group were injected physiological saline as the same method.Feeding into the metabolic cage,free feeding,drinking water.The urine protein 2+ was determined by paper strip method,and the model was reproduced successfully three days later.The mice of drug intervention groups were respectively gastric-perfused with valsartan(20mg/kg)every day for 12 weeks.Other two groups were using the same method replaced with saline.In the 5 period of 0,2,4,6,8 weeks in the different groups were randomly selected 6 mice were collected 24 h urine,serum and renal tissue.Detection of 24 h urinary protein with Coomassie brilliant blue method,biochemical indicator detection automatic biochemical analyzer:serum albumin,total cholesterol and creatinine content.The kidney STAT1?P53 and P21 m RNA expressions were assayed by RT-PCR.The renal paraffin-embedded sections of 3?m,HE.PAS staining was performed to observe the degree of glomerular sclerosis and renal tubular damage,and the expression of STAT1,P53 and P21 were detected by immunohistochemistry.All data were analyzed by SPSS 21 statistical analysis software.The normal distribution test measurement data,accord with the standard deviation of normal distribution data using(+ s)said that the application of comparative test of homogeneity of variance between the two groups,using t or T '-test,analysis and comparison of application of single factor variance between groups,using LSD test between groups the average number of 22,P<0.05 said there was statistical significance.Results:1 The general condition of mice after modeling:Model and intervention group mice at first weeks after injection of adriamycin were lack of energy,dry sparse fur,eating and activities reduced,there are different degrees of diarrhea and weight loss,Body weight decreased to the lowest in second weeks,and then gradually recovered,but there was still a significant difference compared with the normal group.During the experiment,4 mice died because of diarrhea and weight loss in experimental group;and 3 mice died of gastrointestinal bleeding after intragastric administration.and a total of 7 died,with a mortality rate of about 7.8%.2replication models failed.The control group mice survived.2 Adriamycin in the different periods of 24 hours urinary protein and blood biochemical index:The model group significantly increased urinary protein in second weeks and reached the peak,fourth,6,8 and 2 weeks compared to urine protein decreased slightly,compared with the same period in 2W[group(16.78±1.35)vs(2.21±0.79),4 W[(13.88±0.89)vs(2.20.0.70),6 W[(12.67 ±1.47)vs(2.13±0.42)and 8 W[(10.35±0.92)vs(2.25±1.01)was statistically significant difference(P<0.05).The intervention group urine protein by valsartan after intragastric administration was significantly lower than the same period model group: 2W[(11.62±1.96)vs(16.78±1.35)],4W[(9.96±1.31)vs(13.88±0.89)],6W[(6.73±1.87)vs(12.67±1.47)],8W[(4.10±0.87)vs(10.35±0.92)],there was significant difference(P<0.05),but still higher than the normal control group,and there was significant difference(P<0.05).There was no significant difference between the control group(P>0.05).(see Table 1)ALB decreased gradually with time,reached the lowest value at 2 weeks(19.77±1.50),and was slightly higher in the 4week(21.73±3.43),the 6week(24.33±3.14)and the 8 week(28.05±3.22)than that in the 2 week,but the difference was statistically compared with the control group,(P<0.05),the intervention group ALB was higher than the model group at the same period,2W[(23.8±2.73)vs(19.77±1.50)],4W[(25.90±1.71)vs(21.73±3.43)],6W[(27.23±1.06)vs(24.33±3.14)]?? 8W[(32.93±3.73)vs(28.05±3.22)],but still lower than the normal group,compared with the model group was statistically significant(P<0.05).(see Table 2)CHOL gradually increased with time and reached the peak at 2 weeks(6.46±1.47),4(4.60±0.64),6(3.90±0.80),8(3.56±0.43)weeks compared with second weeks down,but still in high level,compared with the control group the difference was statistically significant(P<0.05);at the same time in the intervention group CHOL lower than the model group,but still higher than the normal group,there was statistical significance compared with the model group(P<0.05).(see Table 2)Scr in the control group,model group and intervention group,the difference was not statistically significant(P>0.05).(see Table 2)3 The pathological changes of the kidney.The control group of glomerular and tubular structure has no obvious change.At the end of the 2 week,mesangial cells and mesangial cells proliferated in the model group,and a few of them were observed;the membrane matrix and mesangial cells appeared mild and moderate hyperplasia,accompanied by podocyte injury,and the blood vessels and glomerular capsule had mild adhesion at the end of 4 and 6 week.Part of the glomerular atrophy,renal tubular protein increased,with renal interstitial inflammatory cell infiltration;At the end of the 8: segmental sclerosis appeared more than 50% glomeruli,some blood vessels narrow bureaucratic occlusion,vascular loop collapse,mesangial cell proliferation,mesangial expansion,renal tubular epithelial cell vacuole like change,showing a large number of protein casts and local fibrosis.in the intervention group,the glomerular sclerosis and tubulointerstitial lesions were effectively improved,such as the inflammation was mild,the protein tube type was rare,and glomerular congestion was reduced.The control group of each period in the normal glomerular morphology,no statistically significant difference(P > 0.05),the model group gradually increased over time in the degree of glomerular sclerosis,each time the score was significantly higher than those in the control group,such as2W[(0.42±0.03)vs(0.01±0.01)],4W[(0.59±0.04)vs(0.01±0.03)],6W[(0.78±0.05)vs(0.02±0.00)],8W[(1.19±0.63)vs(0.01±0.01)](P<0.05),The intervention group glomerulosclerosis is relatively reduced compared with model group,each time the scores were lower than the model group,2W[(0.17± 0.00)vs(0.42 ±0.03)],4W[(0.25 ± 0.04)vs(0.59± 0.04)],6W[(0.45 ± 0.04)vs(0.78 ±0.05(]8W[)0.52± 0.06)vs(1.19 ± 0.63),the difference was statistically significant(P<0.05),but still higher than the control group(P<0.05).(see Table 3)The control group of each period in the renal tubular morphology was normal,there was no statistically significant difference(P > 0.05),the model group gradually increased with time in renal interstitial damage,significantly higher than that in control group,2W[(0.01 + 0.06)VS(1.08 + 0.10)],4W[(1.35 + 0.05)vs(0.02 + 0.04)],6W[(1.55 + 0.06)vs(0.01 + 0.01)],8W[(1.93 + 0.12)vs(0.01 + 0),the difference was statistically significant(P <0.05);the intervention group kidney tubulointerstitial damage eased slightly lower than the same period,the model group,2W[(0.24±0.05)vs(1.08±0.10)],4W[(0.44±0.06)vs(1.35±0.05)],6W[(0.64±0.05)vs(1.55±0.06)],8W[(0.85±0.06)vs(1.93±0.12),the difference was statistically significant(P<0.05),but still higher than the control group(P< 0.05).(see Table 3)4 Expression of STAT1,P53 and P21 in renal tissue of mice with adriamycin nephropathy:The expression of STAT1,P53 and P21 in the normal group was not obvious.The model group STAT1,P21 and P53 protein were expressed in glomeruli with time increasing,a large increase over the next 4,6,8 weeks and second weeks,compared with the normal group,the intervention groupSTAT1,P53,P21 expression was compared with the model group were significantly reduced,The expression of STAT1: 2W[(75.40±9.65)vs(437.38±49.66)],4W[(91.99±9.28)vs(534.06±45.81)],6W[(136.31±12.03)vs(640.26±44.58)],8W[(167.21±15.55)vs(777.11±59.19)](P<0.05);the expression of P53: 2W[(24.24±2.60)vs(142.63±6.91)],4W[(44.94±3.79)vs(172.08±8.12)],6W[(55±3.38)vs(191.93±5.47)],8W[(75.99±3.97)vs(216.26±9.15)](P<0.05);the expression of P21: 2W[(50.16±4.13)vs(255.31±22.75)],4W[(65.46±5.40)vs(294.61±22.13)],6W[(76.97±5.50)vs(337.89±21.55),8W(92.61±3.77)vs(380.96±21.43)](P<0.05).There was significant difference between the normal group and the treatment group(P<0.05).(see Fig.1?2?3,Table 4)5 the expression levels of STAT1 m RNA,P53 m RNA and P21 m RNA:Compared with the control group at each time point,the expression of STAT1,P53 and P21 m RNA in model group increased significantly,the difference was statistically significant(P<0.05).Compared with the model group,the expression of the intervention group was significantly reduced,STAT1 m RNA2W[(9.2±0.81)vs(22.61±0.95)],4W[(15.90±0.77)vs(64.22±1.31)],6W[(45.05±1.16)vs(86.58±1.31)],8W[(94.76±1.91)vs(125.21±3.63)];P53m RNA:2W[(2.83±0.23)vs(3.94±0.42)],4W[(3.62±0.45)vs(6.46±0.26)],6W[(5.27±1.07)vs(7.89±0.64)],8W [(6.97 ±0.15)vs(9.75±0.73)];P21m RNA:2W[(3.89±0.23)vs(6.72±1.02)],4W[(6.51±0.49)vs(10.94±0.61)],6W[(5.82±0.29)vs(13.10±0.81),8W(7.83±0.31)vs(15.21±0.48)],the difference was statistically significant(P<0.05),but was signific-antly higher than that of the control group(P<0.05).(see Table 5)Conclusion:1 The expression of senescence associated protein STAT1,P53,P21 in adriamycin induced nephropathy increased significantly at second weeks,suggesting that stress-induced senescence may be involved in the occurrence and development of early renal changes in adriamycin nephropathy.2 The application of ARB can effectively inhibit the expression of JAK-STAT signaling pathway and down regulated genes P53 and P21,and the intervention of STAT1-P53-P21 pathway can delay the progression of adriamycin nephropathy.