鸢尾素后处理对大鼠肝缺血再灌注的影响

Effect of irisin postconditioning on hepatic ischemia reperfusion injury in rats

目的观察鸢尾素后处理对大鼠肝缺血再灌注损伤的影响并探讨其机制。方法健康雄性SD大鼠(武汉大学实验动物中心提供)36只,采用随机数字表法分为假手术组(S组)、缺血再灌注组(IR组)和鸢尾素组(Ir组),每组12只。IR组和Ir组大鼠采用夹闭左、中叶肝蒂建立大鼠肝脏70%缺血再灌注模型(缺血60 min,再灌注6 h);Ir组于灌注开始时静脉注射鸢尾素10μg/kg;S组仅游离肝脏周围血管及韧带。再灌注6 h后下腔静脉采血并留取肝脏组织标本。检测血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、白细胞介素(IL)-6、IL-1β、肿瘤坏死因子-α(TNF-α)含量及肝脏组织丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPX)含量;苏木精-伊红(HE)染色,光镜观察肝脏组织病理学的变化。用蛋白质印迹法检测磷酸化信号转导与转录激活因子3(p-STAT3)和磷酸化Janus激酶2(p-JAK2)的表达水平及B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)、半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3含量。采用SPSS软件对3组大鼠肝脏指标进行方差分析,并进行两两比较。结果与S组比较,IR组肝脏组织病理学损伤较重;S组、I/R组和Ir组的ALT含量分别为(75.24±2.65)、(705.33±4.02)、(385.46±3.58)U/L;AST含量分别为(122.33±6.76)、(1357.54±5.23)、(738.26±3.98)U/L;IL-6含量分别为(104±16)、(586±86)、(275±35)ng/L;TNF-α含量分别为(92±10)、(1165±102)、(511±73)ng/L;IL-1β含量分别为(98±12)、(610±79)、(312±41)ng/L;MDA含量分别为(174±38)、(1900±460)、(1055±266)ng/L;SOD含量分别为(124±10)、(46±8)、(70±10)ng/L;GPX含量分别为(106±12)、(42±5)、(62±11)ng/L;Caspase-3含量分别为(0.22±0.06)、(0.86±0.14)、(0.57±0.11)ng/L;p-JAK2含量分别为0.44±0.05、0.91±0.07、0.62±0.11;p-STAT3含量分别为0.35±0.04、0.86±0.08、0.57±0.07。I/R组及Ir组大鼠血清ALT、AST、IL-6、TNF-α、IL-1β及肝组织MDA、Caspase-3升高(t=445.551、219.362、21.700、7.700、36.182、14.132、24.191、10.111、13.753、7.023、14.456、6.556,P<0.01),差异有统计学意义,SOD和GPX含量降低(t=20.374、14.104、15.945、10.965,P<0.01),差异有统计学意义,JAK2和STAT3磷酸化上调(t=14.289、5.479、19.054、8.224,P<0.01),差异有统计学意义;与IR组比较,Ir组大鼠肝脏损伤减轻,ALT、AST、IL-6、IL-1β、TNF-α下降(t=226.183、14.000、14.081、22.052,P<0.01),差异有统计学意义,肝组织MDA和Caspase-3浓度降低(t=6.730、7.906,P<0.01),差异有统计学意义,SOD和GPX浓度升高(t=6.274、4.985,P<0.01),差异有统计学意义,JAK2和STAT3磷酸化下调(t=8.819、10.834,P<0.01),差异有统计学意义。结论外源性鸢尾素后处理可减轻大鼠肝脏缺血再灌注损伤,其机制可能与抑制JAK2/STAT3表达,减轻氧化应激,减少细胞凋亡有关。

Objective To investigate the effect and mechanism of irisin postconditioning on hepatic ischemia reperfusion injury in rats.Methods Totally 36 SD rats were randomly divided into sham group(S),model group(I/R)and irisin group(Ir),with 12 rats in each group.Hepatic ischemia was constructed in model group and irisin group by ligating left middle lobe of liver vascular branches for 60 min.Irisin was administered at the beginning of reperfusion by intravenous injection at the concentration of 10μg/kg for rats in irisin group.We only dissected the hepatic duodenal ligament of rats in the S group.Serum and liver tissues were collected at 6 h after reperfusion through the inferior chamber.The serum levels of alanine aminotransferase(ALT),glutamic oxalacetic transaminase(AST),interleukin(IL)-6,IL-1βand tumor necrosis factor-α(TNF-α)were examined.The contents of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GPX)in liver tissue were measured.The pathological changes of liver tissue were observed by hematoxylin and eosin(HE)staining.The phosphorylations of janus kinase 2(JAK2),signal transducer and activators of transcription 3(STAT3)and B cell lymphoma/leukemia-2(bcl-2),bcl-2 associated X protein(bax),cysteinyl aspartate-specific protease(Caspase)-3 were assessed by Western blotting.Results The ALT levels in S group,I/R group and irisin group were as follows:(75.24±2.65),(705.33±4.02),(385.46±3.58)U/L;The AST levels were as follows:(122.33±6.76),(1357.54±5.23),(738.26±3.98)U/L;The IL-6 levels were as follows:(104±16),(586±86),(275±35)ng/L;The TNF-αlevels were as follows:(92±10),(1165±102),(511±73)ng/L;The IL-1βlevels were as follows:(98±12),(610±79),(312±41)ng/L;The MDA contents were as follows:(174±38),(1900±460),(1055±266)ng/L;The SOD contents were as follows:(124±10),(46±8),(70±10)ng/L;The GPX contents were as follows:(106±12),(42±5),(62±11)ng/L;The Caspase-3 levels were as follows:(0.22±0.06),(0.86±0.14),(0.57±0.11)ng/L;The p-JAK2 levels were as follows:0.44±0.05,0.91±0.07,0.62±0.11;The p-STAT3 levels were as follows:0.35±0.04,0.86±0.08,0.57±0.07.As compared with the sham group,the levels of ALT,AST,IL-6,TNF-α,IL-1β,MDA and Caspase-3 were significantly increased(t=445.551,219.362,21.700,7.700,36.182,14.132,24.191,10.111,13.753,7.023,14.456 and 6.556,P<0.01).The activities of GPX and SOD in model group and irisin group were significantly decreased(t=20.374,14.104,15.945 and 10.965,P<0.01),and the pathological damage worsened and the expression levels of activated p-JAK2 and p-STAT3were up-regulated in other groups(t=14.289,5.479,19.054 and 8.224,P<0.01);As compared with model group,the levels of ALT,AST,IL-6,TNF-α,IL-1β,MDA and Caspase-3 in irisin group were significantly decreased(t=226.183,14.000,14.081,22.052 and 7.906,P<0.01),and pathological damage was alleviated and the expression levels of activated SOD and GPX significantly increased(t=6.274 and 4.985,P<0.01).The p-JAK2 and p-STAT3 were down-regulated in irisin group(t=8.819 and 10.834,P<0.01).Conclusion Exogenous irisin postconditioning can significantly decrease hepatic ischemia-reperfusion injury in rats by suppressing oxidative stress and cell apoptosis,and the mechanism may be associated with the suppression of JAK2/STAT3 signaling activation.