β-arrestin-2通过抑制自噬减轻小鼠肝脏缺血-再灌注损伤
β-arrestin-2 alleviates mouse hepatic ischemia-reperfusion injury by inhibiting autophagy
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摘要:目的 探讨β-arrestin-2对小鼠缺血-再灌注(IR)肝脏自噬的影响及其在肝脏缺血-再灌注损伤(IRI)中的作用。方法 采用β-arrestin-2野生型(WT)及基因敲除(KO)小鼠制作肝脏IR模型(70%肝脏缺血90 min后恢复肝脏血流)。实验分为4组:即WT小鼠假手术组(WT+Sham组)、WT小鼠IR组(WT+IR组)、KO小鼠假手术组(KO+Sham组)、KO小鼠IR组(KO+IR组), 每组各18只。再灌注6、12、24 h收集各组小鼠血清和肝组织标本。通过检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平、肝组织苏木素-伊红(HE)染色和病理学分析判断肝脏损伤程度, 用免疫组织化学(免疫组化)染色和蛋白免疫印迹法检测自噬关键蛋白轻链3(LC3)的表达、透射电子显微镜(透射电镜)观察肝组织中的自噬体来分析判断肝脏自噬活动情况。结果 与Sham组相比, IR组再灌注后各时间点血清ALT、AST显著升高(均为P < 0.01), KO+IR组较WT+IR组升高更明显(均为P < 0.01)。肝组织HE染色显示, WT+Sham组和KO+Sham组再灌注后各时间点肝细胞形态、肝小叶结构正常; KO+IR组和WT+IR组再灌注后6 h肝细胞轻、中度肿胀, 肝窦稍扩张, 12 h肝细胞重度肿胀, 炎症细胞浸润, 片状损伤区域明显, 24 h肝索排列趋于规则; 与WT+IR组比较, KO+IR组再灌注后各时间点的损伤程度更严重和范围更大。免疫组化染色和蛋白免疫印迹法结果显示再灌注后6、12 h自噬关键蛋白LC3的表达呈上升趋势, 24 h表达略有下降, 其中KO+IR组的表达水平高于WT+IR组。肝组织透射电镜结果显示再灌注后各时间点IR组的自噬体数量均明显高于Sham组(均为P < 0.01), KO+IR组的自噬体数量较WT+IR组明显增多(P < 0.05)。结论 β-arrestin-2可能通过抑制自噬来减轻小鼠肝脏IRI。Abstract:Objective To investigate the impact of β-arrestin-2 on hepatic autophagy after ischemia-reperfusion(IR) and its effect on mouse hepatic ischemia-reperfusion injury(IRI).Methods β-arrestin-2 wild type(WT) and knock out(KO) mice were used to build a mouse model of hepatic IR(70% hepatic warm ischemia for 90 min). Mice were divided into four groups: WT mice sham-operated group(WT+Sham group), WT mice IR group(WT+IR group), KO mice sham-operated group(KO+Sham group) and KO mice IR group(KO+IR group), 18 mice in each group. Serum and liver tissues were collected at 6, 12 and 24 h after reperfusion. The alanine aminotransferase(ALT), aspartate aminotransferase(AST) measurement and liver tissues hematoxylin-eosin(HE) staining and pathology analysis were used to estimate hepatic injury. The expression of light chain(LC)3, the key protein of autophagy, were detected by immunohistochemical(IHC) staining and western blot. Autophagosomes in liver tissues were evaluated by transmission electron microscopy(TEM).Results Compared with Sham groups, the levels of serum ALT and AST significantly increased in IR groups at each time point (all in P < 0.01). The levels of KO+IR group were higher than those of WT+IR group at each time point (all in P < 0.01). The result of liver tissue HE staining showed that liver cell morphology and lobular architecture was normal in WT+Sham group and KO+Sham group at each time point after reperfusion. Liver cells were light or moderate swelling with liver sinus expansion in KO+IR group and WT+IR group at 6 h after reperfusion. And liver cells were severe swelling with inflammatory cells infiltration, and flake damage area is obvious at 12 h after reperfusion. Liver rope arranged regularly at 24 h after reperfusion. The degree of hepatic injury in KO+IR group was more serious than WT+IR group. IHC staining and western blot analysis showed that the expression levels of LC3 increased in IR groups at 6, 12 h but slightly decreased at 24 h after reperfusion. And the expression levels of KO+IR group were higher than WT+IR group. TEM result show that autophagosomes in IR groups were obviously more than those in Sham groups(both in P < 0.01). The counts of autophagosomes in KO+IR group were more than those of WT+IR group(P < 0.05).Conclusions β-arrestin-2 may alleviate mouse hepatic IRI by inhibiting autophagy.
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