AZD0530抑制wnt/β-catenin通路延缓青春期大鼠肾间质纤维化的实验研究

Inhibition of wnt/β-catenin pathway by AZD0530 for alleviating renal interstitial fibrosis in young rats

目的观察AZD0530治疗大鼠肾间质纤维化的实验效果。方法雄性2月龄SD青春期大鼠25只,分为假手术组(暴露左侧输尿管,5只)和肾间质纤维化(renal interstitial fibrosis,RIF)组(结扎左侧输尿管,20只)。实验1:假手术组和RIF组各5只大鼠,于术后4周处死,均取左侧肾脏制作组织学切片、应用Masson染色后计算胶原成分占比,应用免疫组织化学检测Fyn、α-SMA蛋白表达;应用PCR检测Fyn mRNA、α-SMA mRNA相对表达量;应用Western-blot检测Fyn、α-SMA蛋白表达。实验2:15只RIF组大鼠分为生理盐水(normal saline,NS)组(5只,NS灌胃/d)、5 mg组(5只,AZD0530灌胃5 mg·kg-1·d)、50 mg组(5只,AZD0530灌胃50 mg·kg-1·d),术后4周处死大鼠,检测项目同实验1。结果实验1,同假手术组相比,RIF组肾脏胶原成分占比高[(12.01±2.80)%比(0.79±0.27)%,t=8.91,P<0.001],免疫组织化学结果显示肾组织Fyn评分高[(4.06±0.37)分比(2.03±0.19)分,t=11.02,P<0.001]、α-SMA评分高[(4.92±0.48)分比(1.88±0.28)分,t=12.23,P<0.001],PCR检测结果显示肾组织Fyn mRNA相对表达量高[(4.64±0.66)倍比(1.00±0.02)倍,t=12.32,P<0.001]、α-SMA mRNA相对表达量高[(5.60±1.36)倍比(0.99±0.01)倍,t=7.600,P<0.001],Western-blot检测结果显示肾组织Fyn表达水平高[(0.26±0.04)比(0.10±0.03),t=7.35,P<0.001]、α-SMA表达水平高[(0.55±0.05)比(0.12±0.02),t=17.49,P<0.001]。实验2,经AZD0530治疗后,大鼠肾脏胶原成分占比降低[NS组(12.20±1.56)%,5 mg组(7.42±1.28)%,50 mg组(3.48±1.29)%,F=49.93,P<0.001],免疫组织化学结果显示肾组织Fyn评分无明显改变[NS组(4.22±0.67)分,5 mg组(4.17±0.63)分,50 mg组(4.00±0.53)分,F=0.18,P=0.84]、α-SMA评分降低[NS组(7.00±0.41)分,5 mg组(3.22±0.52)分,50 mg组(1.73±0.32)分,F=206.7,P<0.001],PCR检测结果显示肾组织Fyn mRNA相对表达量无明显变化[NS组(1.01±0.05)倍,5 mg组(1.00±0.07)倍,50 mg组(1.03±0.06)倍,F=0.14,P=0.87]、α-SMA相对表达量降低[NS组(1.00±0.02)倍,5 mg组(0.50±0.10)倍,50 mg组(0.24±0.04)倍,F=170.7,P<0.001],Western-blot检测结果显示肾组织Fyn表达水平无明显变化[NS组(0.36±0.04),5 mg组(0.37±0.07),50 mg组(0.32±0.06),F=0.78,P=0.48],α-SMA表达水平降低[NS组(1.27±0.15),5 mg组(0.70±0.08),50 mg组(0.15±0.04),F=149.6,P<0.001],均以50 mg组为著。结论RIF大鼠肾脏组织中wnt/β-catenin通路过度激活。AZD0530可通过阻止Fyn对β-catenin的磷酸化过程抑制wnt通路的激活,最终缓解肾间质的纤维化。

Objective To observe the experimental effect of AZD0530 on renal interstitial fibrosis(RIF)in rats.Methods Twenty-five male 2-month-old Sprague-Dawley(SD)rats were divided into two groups of sham operation(exposed left ureter,n=5)and RIF(RIF;left ureteraI obstruction,n=20).Experiment 1:5 rats in sham operation group and 5 rats in RIF group were sacrificed at Week 4 post-operation:left kidney was harvested for histological section and proportion of collagen was calculated after Masson staining.The protein levels of Fyn andα-SMA were detected by immunohistochemistry(IHC)and Western blot.Polymerase chain reaction(PCR)was utilized for detecting the relative levels of Fyn mRNA andα-SMA mRNA.Experiment 2:15 RIF rats were divided into normal saline group(NS;NS gavage/d,n=5),5 mg group(AZD0530 gavage 5 mg/kg·d,n=5),50mg group(AZD0530 gavage 50 mg/kg·d,n=5).The animals were sacrificed at Week 4 post-operation.Test items were the same as experiment 1.Results In experiment 1,as compared with sham group,proportion of collagen was higher in kidney of RIF group[(12.01±2.80)%vs(0.79±0.27)score,t=12.23,P<0.001]scores of renal tissue was high in RIF group.PCR revealed that the relative folds of Fyn mRNA[(4.64±0.66)vs(1.00±0.02)fold,t=12.32,P<0.001]andα-SMA mRNA[(5.60±1.36)vs(0.99±0.01)fold,t=7.600,P<0.001]were high in kidney tissue.Western blot indicated that the levels of Fyn[(0.26±0.04)vs(0.10±0.03),t=7.35,P<0.001]andα-SMA[(0.55±0.05)vs(0.12±0.02),t=17.49,P<0.001]were high in kidney tissue.In experiment 2,after AZD0530 treatment,proportion of collagen dropped in rat kidney[NS(12.20±1.56)%,5 mg(7.42±1.28)%,50 mg(3.48±1.29)%,F=49.93,P<0.001].IHC results showed no significant change in Fyn score of renal tissue[NS(4.22±0.67)score,5 mg(4.17±0.63)score,50 mg(4.00±0.53)score,F=0.18,P=0.84].IHC results showed thatα-SMA[NS(7.00±0.41)score,5 mg(3.22±0.52)score,50 mg(1.73±0.32)score,F=206.7,P<0.001]score of renal tissue decreased.PCR results showed no significant change in relative fold of Fyn mRNA in renal tissue[NS(1.01±0.05)fold,5 mg(1.00±0.07)fold,50 mg(1.03±0.06)fold,F=0.14,P=0.87].PCR results indicated that the relative fold ofα-SMA[NS(1.00±0.02)fold,5 mg(0.50±0.10)fold,50 mg(0.24±0.04)fold,F=170.7,P<0.001]decreased markedly in renal tissue.Western blot showed no significant change in the level of Fyn in renal tissue[NS(0.36±0.04),5 mg(0.37±0.07),50 mg(0.32±0.06),F=0.78,P=0.48].Western blot indicated that the level ofα-SMA[NS(1.27±0.15),5 mg(0.70±0.08),50 mg(0.15±0.04),F=149.6,P<0.001]declined significantly in renal tissue.The treatment outcome was better in 50 mg group.Conclusions Wnt/β-catenin pathway is over-activated in renal interstitial fibrosis of RIF rats.And AZD0530 may inhibit the activation of wnt/β-catenin pathway through preventing Fyn from phosphorylatingβ-catenin and ultimately alleviating RIF.