Role of transient receptor potential cation channel subfamily M member 2 in hepatic ischemia-reperfusion injury in the mouse and the underlying mechanisms

Objectives: 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.

Methods: 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) (n=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.

Results: 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 P<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 P<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 P<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 P<0.05), while the serum MDA and MPO activities were significantly decreased (all P<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 P<0.05).

Conclusions: 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.

目的: 观察瞬时受体电位阳离子通道亚家族M成员2(transient receptor potential cation channel subfamily M member 2，TRPM2)在小鼠肝缺血再灌注损伤(hepatic ischemia-reperfusion injury，HIRI)模型中的作用及可能的机制。方法: 60只成年雄性C57BL/6小鼠随机分为假手术组(S组)、模型组(M组)、TRPM2腺病毒干扰载体预处理组(T组)和TRPM2腺病毒对照载体预处理组(C组)(均n=15)。取各组小鼠灌注前肝组织，采用荧光显微镜观察腺病毒感染效率，利用real-time PCR检测腺病毒对TRPM2的沉默效率。各组小鼠分别于再灌注2，4和8 h抽取腹主动脉血并获取肝组织，分别检测血清中谷丙转氨酶(alanine aminotransferase，ALT)和谷草转氨酶(aspartate amino-transferase，AST)活性。采用HE染色观察肝组织病理学变化；蛋白质印迹法检测肝中TRPM2和Rac家族小GTP酶1(Rac family small GTPase 1，RAC1)蛋白质的表达量变化；并通过酶联免疫吸附试验检测肝中丙二醛(malondialdehyde，MDA)、超氧化物歧化酶(superoxide dismutase，SOD)及髓过氧化物酶(myeloperoxidase，MPO)活性的变化。结果: 与S组和M组相比，T组和C组小鼠肝组织可见大量绿色荧光。与S组、M组及C组相比，T组小鼠肝组织中TRPM2 mRNA的表达量显著降低(均P<0.05)。光镜下S组小鼠肝细胞形态正常；M组和C组小鼠肝窦扩张和淤血、肝细胞变性、小叶中央坏死及大量炎症细胞浸润；与M组和C组相比较，T组小鼠肝细胞受损程度明显减轻。与S组相比较，M组、T组及C组小鼠血清中ALT及AST活性在再灌注2，4和8 h均显著升高(均P<0.05)；与M组和C组相比较，T组小鼠血清中ALT及AST活性在再灌注2，4和8 h均显著降低(均P<0.05)。与M组和C组相比较，T组小鼠中SOD活性在再灌注2，4和8 h均显著升高(均P<0.05)，而MDA和MPO活性均显著降低(均P<0.05)。T组小鼠肝组织中TRPM2和RAC1蛋白质表达量在再灌注2，4和8 h较M组和C组均显著降低(均P<0.05)。结论: TRPM2腺病毒干扰载体预处理能有效沉默小鼠肝组织中TRPM2基因表达，并能减轻HIRI，该机制可能与抑制氧化应激和降低RAC1蛋白质表达水平有关。.

Keywords: Rac family small GTPase 1; hepatic ischemia-reperfusion injury; mechanism; oxidative stress; transient receptor potential cation channel subfamily M member 2.