芳香烃受体核转录蛋白样1通过抑制细胞外调节蛋白激酶磷酸化改善肾纤维化

Aryl hydrocarbon receptor nuclear translocator like protein 1 improves renal fibrosis by inhibiting phosphorylation of extracellular regulated protein kinase

目的探讨芳香烃受体核转录蛋白样1(BMAL1)对肾纤维化的影响及机制。方法将HK-2细胞简单随机分组:对照组(A组)、转化生长因子-β1(TGF-β1)-5 ng/ml组(B组)、TGF-β1-10 ng/ml组(C组)、TGF-β1+si-BMAL1组(D组)、TGF-β1+BMAL1-OE组(E组)、TGF-β1+si-BMAL1+PD98059组(F组), 各组给予相应的干预措施, 用蛋白质印迹法(Western blot)检测BMAL1、纤维连接蛋白(Fibronectin)、Ⅰ型胶原纤维蛋白(COL-Ⅰ)、细胞外信号调节激酶(ERK)、磷酸化细胞外信号调节激酶(p-ERK)、E-钙黏蛋白(E-cadherin)蛋白的表达量。将24只SD雄性大鼠简单随机分组:假手术组(G组)、UUO-3 d组(H组)、UUO-7 d组(I组)、UUO-7 d+sh-BMAL1组(J组), 各组给予相应干预措施, 用马松(Masson)染色观察肾间质胶原纤维表达, 用Western blot检测BMAL1、Fibronectin、COL-Ⅰ、E-cadherin、ERK、p-ERK蛋白的表达量。两组间比较采用t检验。结果随着TGF-β1的浓度增加, HK-2细胞BMAL1、Fibronectin、COL-Ⅰ表达量逐渐升高;随着UUO时间延长, 大鼠的BMAL1、Fibronectin、COL-Ⅰ表达量、Masson染色肾间质蓝色胶原纤维面积比值逐渐升高;D组的Fibronectin、COL-Ⅰ、p-ERK表达量高于C组(4.94±0.74、3.46±0.28、3.18±0.48比3.14±0.52、2.42±0.20、2.05±0.31, t=3.455、5.269、3.400, P<0.05), BMAL1、E-cadherin表达量低于C组(0.28±0.11、0.25±0.10比2.69±0.27、0.58±0.08, t=-14.179、-4.586, P<0.05);E组的Fibronectin、COL-Ⅰ、p-ERK表达量低于C组(1.76±0.21、1.69±0.12、1.52±0.11比3.14±0.52、2.42±0.20、2.05±0.31, t=-4.032、-5.375、-2.788, P<0.05), BMAL1、E-cadherin表达量高于C组(3.80±0.36、0.79±0.05比2.69±0.27、0.58±0.08, t=4.244、3.875, P<0.05);J组的Fibronectin、COL-Ⅰ、p-ERK表达量高于I组(16.48±1.42、3.68±0.31、2.36±0.44比8.93±2.35、2.65±0.31、1.55±0.14, t=6.258、5.141、4.440, P<0.05), BMAL1、E-cadherin表达量低于I组(0.51±0.21、0.22±0.07比5.26±0.48、0.55±0.09, t=-19.928、-6.841, P<0.05);F组Fibronectin、COL-Ⅰ、p-ERK表达量低于D组(4.03±0.29、2.51±0.24、0.85±0.09比4.94±0.74、3.46±0.28、3.18±0.48, t=-1.979、-4.475、-8.196, P<0.05), E-cadherin表达量高于D组(0.43±0.05比0.25±0.10, t=2.828, P<0.05)。结论 BMAL1可通过抑制ERK磷酸化改善肾纤维化。

Objective To explore the effects and mechanisms of Aryl hydrocarbon receptor nuclear translocator like protein 1(BMAL1)regulating extracellular signal-regulated kinase(ERK)on renal fibrosis.Methods HK-2 cells were simply randomly divided into control group(group A),transforming growth factor-β1(TGF-β1)-5 ng/ml group(group B),TGF-β1-10 ng/ml group(group C),TGF-β1+si-BMAL1 group(group D),TGF-β1+BMAL1-OE group(group E),TGF-β1+si-BMAL1+PD98059 group(group F).All the rats were given the corresponding intervention measures.The protein expression levels of BMAL1,fibronectin,collagen typeⅠ(COL-Ⅰ),ERK,phosphorylated ERK(p-ERK),and E-cadherin were detected by Western blotting.A total of 24 male SD rats were simply randomly divided into sham operation group(group G),UUO-3 d group(group H),UUO-7 d group(group I),and UUO-7 d+sh-BMAL1 group(group J).After receiving the corresponding intervention measures,the expression of renal interstitial collagen was observed by Masson staining,and the expression levels of BMAL1,fibronectin,COL-Ⅰ,E-cadherin,ERK and p-ERK proteins were detected by Western blotting.SPSS 25.0 software was used for statistical analysis,and t test was used for comparison between the two groups.Results With the increase of TGF-β1 concentration,the expression levels of BMAL1,fibronectin and COL-Ⅰin HK-2 cells increased gradually.With the prolongation of UUO time,the expression levels of BMAL1,fibronectin and COL-Ⅰin rats and the area ratio of blue collagen fibers in the renal interstitium stained by Masson staining were gradually increased.The expression levels of fibronectin,COL-Ⅰand p-ERK in group D were higher than those in group C(4.94±0.74,3.46±0.28,3.18±0.48 vs.3.14±0.52,2.42±0.20,2.05±0.31,t=3.455,5.269,3.400,P<0.05).The expression levels of BMAL1 and E-cadherin were lower in group D than those in group C(0.28±0.11,0.25±0.10 vs.2.69±0.27,0.58±0.08,t=-14.179,-4.586,P<0.05).The expression levels of fibronectin,COL-Ⅰand p-ERK in group E were lower than those in group C(1.76±0.21,1.69±0.12,1.52±0.11 vs.3.14±0.52,2.42±0.20,2.05±0.31,t=-4.032,-5.375,-2.788,P<0.05).The expression levels of BMAL1 and E-cadherin in group E were higher than those in group C(3.80±0.36,0.79±0.05 vs.2.69±0.27,0.58±0.08,t=4.244,3.875,P<0.05).The expression levels of fibronectin,COL-Ⅰand p-ERK in group J were higher than those in group I(16.48±1.42,3.68±0.31,2.36±0.44 vs.8.93±2.35,2.65±0.31,1.55±0.14,t=6.258,5.141,4.440,P<0.05).The expression levels of BMAL1 and E-cadherin in group I were lower than those in group I(0.51±0.21,0.22±0.07 vs.5.26±0.48,0.55±0.09,t=-19.928,-6.841,P<0.05).The indexes of fibronectin,COL-Ⅰand p-ERK in the group F were lower than those in the group D(4.03±0.29,2.51±0.24,0.85±0.09 vs.4.94±0.74,3.46±0.28,3.18±0.48,t=-1.979,-4.475,-8.196,P<0.05).The index of E-cadherin in the group D was higher than that in the group D(0.43±0.05 vs.0.25±0.10,t=2.828,P<0.05).Conclusion BMAL1 can improve renal fibrosis by inhibiting ERK phosphorylation.