锌转运体ZIP13在小鼠肝脏缺血再灌注损伤中的作用

Role of zinc transporter ZIP13 in hepatic ischemia/reperfusion injury in mice

目的:探讨锌转运体ZIP13(SLC39A13)在肝脏缺血再灌注损伤中的作用及机制。方法:通过结扎肝左叶和肝中叶的门静脉及肝动脉共干,建立小鼠在体肝脏缺血再灌注模型,将小鼠分组如下:(1)ZIP13^(fl/fl)-Sham组、ZIP13^(fl/fl)+I1R12组和ZIP13^(LKO)+I1R12组。(2)ZIP13^(fl/fl)-Sham组、ZIP13^(fl/fl)+I1R24组和ZIP13^(LKO)+I1R24组。(3)Vector-Sham组、Vector+I1R12组和ZIP13^(OE)+I1R12组。(4)Vector-Sham组、Vector+I1R24组和ZIP13^(OE)+I1R24组,上述每组小鼠3~4只。采用电感耦合离子发射光谱仪(ICP-OES)测量肝组织的锌含量;试剂盒检测丙氨酸转氨酶(ALT)和门冬氨酸转氨酶(AST)水平;HE染色观察病理学改变;原位末端转移酶标记法(TUNEL法)检测细胞凋亡;Western印迹检测CHOP、GRP78和凋亡蛋白表达。结果:与ZIP13^(fl/fl)小鼠相比,ZIP13^(LKO)小鼠肝脏中ZIP13表达明显下降(t=6.26,P<0.01),而与Vector感染对照小鼠相比,ZIP13^(OE)小鼠肝脏ZIP13蛋白表达明显增加(t=4.17,P<0.05)。与ZIP13^(fl/fl)-Sham组相比,ZIP13^(fl/fl)+I1R12组血清ALT、AST水平升高(t=11.43、13.70,均P<0.001),ZIP13^(fl/fl)+I1R24组小鼠肝组织锌含量明显降低(t=13.49,P<0.001),内质网应激蛋白CHOP和GRP78表达增强(t=4.76、4.54,均P<0.05),凋亡蛋白Cleaved Caspase9和Cleaved Caspase3表达升高(t=4.56、3.73,均P<0.05)。与相应的ZIP13^(fl/fl)缺血再灌注组相比,ZIP13^(LKO)+I1R12组血清ALT、AST水平进一步升高(t=2.95、3.20,均P<0.05),ZIP13^(LKO)+I1R24组肝组织中锌含量显著降低(t=3.29,P<0.05),内质网应激蛋白表达上调(t=2.60、2.98,均P<0.05),凋亡蛋白表达也明显升高(t=3.44、2.49,均P<0.05)。与相应的Vector感染缺血再灌注组相比,ZIP13^(OE)+I1R12组血清ALT、AST水平明显下降(t=3.69、4.26,均P<0.05),ZIP13^(OE)+I1R24组小鼠肝组织中锌含量增加(t=3.88,P<0.05),内质网应激蛋白表达下降(t=3.47、2.88,均P<0.05),凋亡蛋白的表达水平也呈现降低(t=3.02、2.96,均P<0.05)。结论:肝脏缺血再灌注时,ZIP13通过维持肝脏锌稳态,减轻内质网应激和细胞凋亡。

Objective:To investigate the role of zinc transporter ZIP13(SLC39A13)in hepatic ischemia/reperfusion(I/R)injury and the underlying mechanism.Methods:Mouse liver I/R model in vivo was established by ligating the common trunk of portal vein and hepatic artery in the left and middle lobes of the liver.Mice were grouped as follows:(1)ZIP13^(fl/fl)-Sham group,ZIP13^(fl/fl)+I1R12 group and ZIP13^(LKO)+I1R12 group.(2)ZIP13^(fl/fl)-Sham group,ZIP13^(fl/fl)+I1R24 group and ZIP13^(LKO)+I1R24 group.(3)Vector-Sham group,Vector+I1R12 group and ZIP13^(OE)+I1R12 group.(4)Vector-Sham group,Vector+I1R24 group and ZIP13^(OE)+I1R24 group,with 3-4 mice in each group.Hepatic zinc levels were measured by inductively coupled plasma-optical emission spectrometer(ICP-OES).The levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by the assay kits.HE staining was adopted to observe the structure changes of liver tissues.TUNEL staining was performed to assess apoptosis.Western blotting analysis was conducted to detect the expressions of CHOP,GRP78,and apoptotic proteins in the liver.Results:Compared with ZIP13^(fl/fl)mice,the expression of ZIP13 protein in the liver of ZIP13^(LKO)mice was obviously decreased(t=6.26,P<0.01),while its protein level was clearly increased in the liver of ZIP13^(OE)mice(t=4.17,P<0.05).Compared with the ZIP13^(fl/fl)-Sham group,the levels of ALT and AST in ZIP13^(fl/fl)+I1R 12 group were increased(t values were 11.43 and 13.70,P<0.001),and hepatic zinc content in the ZIP13^(fl/fl)+I1R24 group was decreased(t=13.49,P<0.001).Additionally,the expressions of endoplasmic reticulum stress proteins CHOP and GRP78 were enhanced(t=4.76,4.54,both P<0.05),and the expressions of apoptosis proteins Cleaved Caspase9 and Cleaved Caspase3 were also increased(t=4.56,3.73,both P<0.05).Compared with the corresponding ZIP13^(fl/fl)mice subjected to I/R,the ZIP13^(LKO)+I1R12 group increased ALT and AST levels(t values were 2.95 and 3.2,both P<0.05),and the ZIP13^(LKO)+I1R24 group showed a further decrease in hepatic zinc content(t=3.29,P<0.05).Moreover,the expressions of endoplasmic reticulum stress proteins were upregulated(t=2.6,2.98,both P<0.05),and the expressions of apoptotic proteins were obviously increased(t=3.44,2.49,both P<0.05).In contrast,compared with the corresponding vector-infected I/R control group,the levels of ALT and AST in the ZIP13^(OE)+I1R12 group were significantly reduced(t=3.69,4.26,both P<0.05).In the ZIP13^(OE)+I1R24 group,the increase in hepatic zinc content was observed(t=3.88,P<0.05),the expressions of reticulum stress proteins were decreased(t=3.47,2.88,both P<0.05),and the expression levels of apoptotic proteins were also reduced(t=3.02,2.96,both P<0.05).Conclusion:During liver I/R,ZIP13 alleviates endoplasmic reticulum stress and apoptosis by maintaining liver zinc homeostasis.