沉默信息调节因子1介导的脂肪酸氧化在肾脏缺血再灌注导致的肾纤维化中的作用

The role and mechanism of Silence information regulator 1 in renal fibrosis induced by renal ischemia reperfusion

目的:探讨沉默信息调节因子1(Sirt1)在肾缺血再灌注损伤导致的肾纤维化中的作用。方法:C57小鼠25只I/R,其中10只为通过尾静脉注射空载腺相关病毒(AAV9-Ctrl)小鼠,15只为注射Sirt1过表达腺相关病毒(AAV9-Sirt1)小鼠。按照简单随机法进行分组,将AAV9-Ctrl小鼠随机分为假手术(AAV9-Ctrl-Sham)组和缺血再灌注(AAV9-Ctrl-I/R)组,每组各5只;AAV9-Sirt1小鼠随机分为假手术组(AAV9-Sirt1-Sham)、缺血再灌注组(AAV9-Sirt1-I/R)和CPT1抑制剂(HY-136408)干预组(AAV9-Sirt1-I/R+HY-136408),上述每组各5只。I/R组经肾蒂结扎30 min后开放造模,28 d后切除肾脏。检测AAV9-Ctrl小鼠血清肌酐(Cr)、尿素氮(BUN)水平。苏木精-伊红(HE)染色评估肾小管损伤,原位缺口末端标记法(TUNEL)染色评估肾脏细胞凋亡情况。马松(Masson)染色检测肾脏胶原纤维沉积情况,免疫组织化学(IHC)染色检测α-平滑肌肌动蛋白(α-SMA)表达;蛋白质印迹法(Western blot)检测,纤连蛋白、α-SMA和Ⅰ型胶原蛋白(Collagen1)表达水平。两组间数据比较使用非配对t检验,多组差异比较采用单因素方差分析(ANOVA)。结果:AAV9-Ctrl组血清Cr、BUN水平I/R组高于Sham组[(68.2±8.4)μmol/L比(36.9±3.5)μmol/L,t=9.59,P<0.01、(46.4±7.1)mmol/L比(23.8±5.2)mmol/L,t=5.74,P<0.01];HE染色显示AAV9-Ctrl-I/R组小鼠肾脏损伤评分高于AAV9-Ctrl-Sham组(10.23±1.03比2.32±0.91,t=5.35,P<0.05);小鼠肾脏细胞凋亡(TUNEL染色)和肾纤维沉积水平(Masson染色)同样高于AAV9-Ctrl-Sham组(9.38±0.43比1.22±0.41,t=4.64,P<0.05);AAV9-Sirt1-I/R组损伤评分低于AAV9-Ctrl-I/R组(4.53±1.64比9.38±0.43,t=5.54,P<0.05)。Masson染色结果表示AAV9-Ctrl小鼠中I/R组肾脏纤维化显著高于Sham组(13.15±0.56比1.52±0.61,t=4.24,P<0.05);而AAV9-Sirt1-I/R组损伤评分低于AAV9-Ctrl-I/R组(6.33±1.13比10.23±1.03,t=3.43,P<0.05)。小鼠肾脏细胞凋亡(TUNEL染色)和肾纤维沉积水平(Masson染色)同样低于AAV9-Ctrl-I/R组(4.53±1.64比9.38±0.43,t=5.53,P<0.05、6.15±1.89比13.15±0.56,t=3.43,P<0.05)。IHC显示AAV9-Ctrl-I/R组小鼠α-SMA的表达水平高于AAV9-Ctrl-Sham组(14.88±1.87比3.76±1.21,t=4.35,P<0.05);AAV9-Sirt1-I/R组α-SMA的表达水平低于AAV9-Ctrl-I/R组(8.33±1.66比14.88±1.87,t=3.43,P<0.05)。Western blot显示AAV9-Ctrl-I/R组小鼠Fibronectin表达水平高于AAV9-Ctrl-Sham组(2.75±0.09比1.21±0.07,t=30.20,P<0.01);α-SMA、Collagen1表达水平高于AAV9-Ctrl-Sham组(1.06±0.07比0.38±0.05,t=13.34,P<0.01、1.04±0.06比0.49±0.05,t=15.75,P<0.01)。然而,AAV9-Sirt1-I/R组Fibronectin表达水平低于AAV9-Ctrl-I/R组(1.80±0.05比2.75±0.09,t=20.63,P<0.01);α-SMA、Collagen1表达水平同样低于AAV9-Ctrl-I/R组(0.81±0.08比1.06±0.07,t=5.59,P<0.01、0.85±0.04比1.04±0.06,t=5.89,P<0.01)。AAV9-Sirt1-I/R组CPT1(3.08±0.32比2.11±0.25,t=5.34,P<0.01)和ACOX(1.89±0.19比1.22±0.10,t=6.98,P<0.01)表达水平高于AAV9-Ctrl-I/R组。然而AAV9-Sirt1-I/R+HY-136408组Fibronectin、α-SMA、Collagen1表达水平高于AAV9-Sirt1-I/R组(2.01±0.09比1.80±0.05,t=4.56,P<0.05、0.95±0.07比0.81±0.08,t=2.95,P<0.05和0.98±0.09比0.85±0.04,t=2.95,P<0.05)。结论:Sirt1可能通过调控CPT1参与缓解肾缺血再灌注导致的肾纤维化。

Objective:The purpose of this study was to investigate the role of silence information regulator 1(Sirt1)in kidney fibrosis caused by ischemia-reperfusion injury.Methods:Twenty-five C57 mice.Ten of them were empty-loaded adeno-associated virus(AAV9-Ctrl)mice injected through the tail vein,and 15 were Sirt1 overexpressing adeno-associated virus(AAV9-Sirt1)mice injected.The AAV9-Ctrl mice were randomly divided into the sham-operated(AAV9-Ctrl-Sham)group and the ischemia-reperfusion(AAV9-Ctrl-I/R)group,with 5 mice in each group;the AAV9-Sirt1 mice were randomly divided into the sham-operated group(AAV9-Sirt1-Sham),the ischemia-reperfusion group(AAV9 Sirt1-I/R)and CPT1 inhibitor(HY-136408)groups(AAV9-Sirt1-I/R+HY-136408),with 5 mice in each group.The I/R group separated the left renal artery and vein ligation for 30 min and then unclamped,and the kidneys were resected 28 d later to complete the modeling.Serum creatinine(Cr)and urea nitrogen(BUN)levels of AAV9-Ctrl mice were measured.HE staining was used to assess renal tubular damage,and TUNEL staining was used to assess the level of apoptosis in the kidney cells.Masson staining was used to detect renal collagen fiber deposition,and immunohistochemical staining was used to detectα-smooth muscle actin(α-SMA)expression;protein immunoblotting(Western blotting)was used to detect Fibronectin,α-SMA and collagen typeⅠ(Collagen1)expression levels.Comparison of data between two groups was performed using unpaired t-test,and comparison of differences between multiple groups was performed using one-way analysis of variance(ANOVA).Results:Cr and BUN levels in AAV9-Ctrl mice were higher in I/R group than in Sham group,which were[(68.2±8.4)μmol/L vs.(36.9±3.5)μmol/L,t=9.59,P<0.01,(46.4±7.1)mmol/L vs.(23.8±5.2)mmol/L,t=5.74,P<0.01];HE staining showed that the kidney injury scores of mice in the AAV9-Ctrl-I/R group were higher than those in the AAV9-Ctrl-Sham group(10.23±1.03 vs.2.32±0.91,t=5.34,P<0.05);apoptosis(TUNEL staining)and renal fiber deposition levels(Masson staining)of the kidneys of the mice were similarly higher than those in the AAV9-Ctrl-Sham group(9.38±0.43 vs.1.22±0.41,t=4.63,P<0.05);the damage score of AAV9-Sirt1-I/R group was lower than that of AAV9-Ctrl-I/R group(4.53±1.64 vs.9.38±0.43,t=5.53,P<0.05).Masson staining results indicated that the level of renal fibrosis of mice in AAV9-Ctrl-I/R group was higher than that of AAV9 Ctrl-Sham group(13.15±0.56 vs.1.52±0.61,t=4.24,P<0.05);However,the damage score of AAV9-Sirt1-I/R group was lower than that of AAV9-Ctrl-I/R group(6.33±1.13 vs.10.23±1.03,t=3.43,P<0.05).Mouse kidney apoptosis(TUNEL staining)and renal fiber deposition levels(Masson staining)were similarly lower in the AAV9-Ctrl-I/R group(4.53±1.64 vs.9.38±0.43,t=5.53,P<0.05;6.15±1.89 vs.13.15±0.56,t=3.43,P<0.05).IHC showed that the expression level ofα-SMA in mice in the AAV9-Ctrl-I/R group was higher than that in the AAV9-Ctrl-Sham group(14.88±1.87 vs.3.76±1.21,t=4.35,P<0.05);and the expression level ofα-SMA in the AAV9-Sirt1-I/R group was lower than that in the AAV9-Ctrl-I/R group(8.33±1.66 vs.14.88±1.87,t=3.43,P<0.05).Western blotting showed that the expression level of Fibronectin was higher in the AAV9-Ctrl-I/R group of mice than that in the AAV9-Ctrl-Sham group(2.75±0.09 vs.1.21±0.07,t=30.20,P<0.01);similarly theα-SMA,Collagen1 expression level was higher than that of AAV9-Ctrl-Sham group(1.06±0.07 vs.0.38±0.05,t=13.34,P<0.01;1.04±0.06 vs.0.49±0.05,t=15.75,P<0.01).However,the expression level of Fibronectin was lower in the AAV9-Sirt1-I/R group than in the AAV9-Ctrl-I/R group(1.80±0.05 vs.2.75±0.09,t=20.63,P<0.01);the expression levels ofα-SMA,Collagen1 were similarly lower than in the AAV9-Ctrl-I/R group(0.81±0.08 vs.1.06±0.07,t=5.59,P<0.01;0.85±0.04 vs.1.04±0.06,t=5.89,P<0.01).The expression levels of CPT1 in the AAV9-Sirt1-I/R group(3.08±0.32 vs.2.11±0.25,t=5.34,P<0.01)and ACOX(1.89±0.19 vs.1.22±0.10,t=6.98,P<0.01)expression levels were higher than those of the AAV9-Ctrl-I/R group.However,the expression levels of Fibronectin,α-SMA,and Collagen1 were higher in the AAV9-Sirt1-I/R+HY-136408 group than in the AAV9-Sirt1-I/R group(2.01±0.09 vs.1.80±0.05,t=4.56,P<0.05;0.95±0.07 vs.0.81±0.08,t=2.95,P<0.05 and 0.98±0.09 vs.0.85±0.04,t=2.95,P<0.05).Conclusion:Sirt1 may be involved in alleviating renal fibrosis caused by renal ischemia-reperfusion by regulating CPT1.