Li Yue, Ren Zhuhai, Xu Yong, Wu Shurong
Department of General Surgery, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2020 Jul 28;45(7):766-773. doi: 10.11817/j.issn.1672-7347.2020.190064.
To investigate the role of transient receptor potential cation channel subfamily M member 2 (TRPM2) in hepatic ischemia-reperfusion injury of mouse (HIRI) and the possible mechanisms.
Sixty adult male C57BL/6 mice were randomly divided into 4 groups: a sham group (S group), a HIRI model group (M group), a TRPM2 adenovirus interference vector group (T group), and a TRPM2 adenovirus control vector group (C group) (=15 in each group). The liver tissues of mice before perfusion were obtained. The efficiency of adenovirus infection was detected by fluorescence microscopy, and the silencing efficiency of adenovirus against TRPM2 was detected by real-time PCR.The abdominal aorta blood and liver tissues were collected from mice at 2, 4 and 8 h after reperfusion. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were detected. Hepatic pathological changes were examined by hematoxylin-eosin (HE) staining. The protein expression of TRPM2 and Rac family small GTPase 1 (RAC1) in liver tissues was detected by Western blotting. Changes of malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) activities in liver tissues were detected by enzyme-linked immunosorbent assay.
A strong signal of green fluorescence was observed in the liver tissues of mice in the T and C groups compared to the S or M group. Compared with the S, M or C group, the expression of TRPM2 mRNA in liver tissue in the T group was significantly down-regulated (all <0.05). The morphology of hepatocytes was normal in the S group under light microscope.Hepatic sinus dilatation, congestion, hepatocyte degeneration, central necrosis of lobule, and massive inflammatory granulocyte infiltration were observed in the M and C group, respectively. The degree of hepatocyte damage in the T group was significantly reduced compared with that in the M and C group, respectively. Compared with the S group, the serum ALT and AST activities in the M, T and C groups were significantly increased at 2, 4 and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum ALT and AST activities in the T group were significantly lower in serum of mice at 2, 4, and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum SOD activity in the T group was significantly increased at 2, 4, and 8 h after reperfusion (all <0.05), while the serum MDA and MPO activities were significantly decreased (all <0.05). The protein expression of TRPM2 and RAC1 in liver tissues in the T group were significantly lower than those in the M and C groups at 2, 4 and 8 h after reperfusion (all <0.05).
Pretreatment with TRPM2 adenovirus interference vector can effectively silence TRPM2 gene expression in liver tissues of mice and attenuate HIRI, which may be related to inhibiting oxidative stress and reducing the expression of RAC1 protein.
探讨瞬时受体电位阳离子通道M亚家族成员2（TRPM2）在小鼠肝脏缺血再灌注损伤（HIRI）中的作用及可能机制。
将60只成年雄性C57BL/6小鼠随机分为4组：假手术组（S组）、HIRI模型组（M组）、TRPM2腺病毒干扰载体组（T组）和TRPM2腺病毒对照载体组（C组）（每组n = 15）。获取灌注前小鼠的肝脏组织。通过荧光显微镜检测腺病毒感染效率，通过实时PCR检测腺病毒对TRPM2的沉默效率。再灌注后2、4和8小时收集小鼠腹主动脉血和肝脏组织。检测小鼠血清中丙氨酸氨基转移酶（ALT）和天冬氨酸氨基转移酶（AST）的活性。采用苏木精-伊红（HE）染色检查肝脏病理变化。通过蛋白质免疫印迹法检测肝脏组织中TRPM2和Rac家族小GTP酶1（RAC1）的蛋白表达。采用酶联免疫吸附测定法检测肝脏组织中丙二醛（MDA）、超氧化物歧化酶（SOD）和髓过氧化物酶（MPO）活性的变化。
与S组或M组相比，T组和C组小鼠肝脏组织中观察到强绿色荧光信号。与S组、M组或C组相比，T组肝脏组织中TRPM2 mRNA表达显著下调（均P < 0.05）。光镜下S组肝细胞形态正常。M组和C组分别观察到肝窦扩张、充血、肝细胞变性、小叶中央坏死和大量炎性粒细胞浸润。与M组和C组相比，T组肝细胞损伤程度明显减轻。与S组相比，再灌注后2、4和8小时M组、T组和C组小鼠血清ALT和AST活性显著升高（均P < 0.05）。与M组或C组相比，再灌注后2、4和8小时T组小鼠血清ALT和AST活性显著降低（均P < 0.05）。与M组或C组相比，再灌注后2、4和8小时T组小鼠血清SOD活性显著升高（均P < 0.05），而血清MDA和MPO活性显著降低（均P < 0.05）。再灌注后2、4和8小时T组肝脏组织中TRPM2和RAC1蛋白表达明显低于M组和C组（均P < 0.05）。
用TRPM2腺病毒干扰载体预处理可有效沉默小鼠肝脏组织中TRPM2基因表达并减轻HIRI，这可能与抑制氧化应激和降低RAC1蛋白表达有关。
