饱和氢气生理盐水对大鼠肝脏冷缺血/再灌注损伤的影响

摘要

目的：探讨饱和氢气生理盐水对大鼠肝脏冷缺血/再灌注损伤（IRI）的保护作用及其机制。方法清洁级雄性SD大鼠24只，周龄为8～10周，体重220～250 g，采用随机数字表法分为3组（n＝8）：假手术组（Sham组）行开关腹操作，并游离相应血管；肝脏冷缺血/再灌注组（I/R组）于门静脉阻断前5 min经下腔静脉缓慢注射生理盐水（6 ml/kg）；饱和氢气生理盐水处理组（HS组）于门静脉阻断前5 min经下腔静脉缓慢注射饱和氢气生理盐水（6 ml/kg）。I/R组与HS组阻断全肝血流后，于肝下下腔静脉开口1 mm作为灌注液流出道，用4℃乳酸林格液经门静脉灌注肝脏。再灌注后6 h后经下腔静脉抽取血样，测定血清中丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）含量，通过酶联免疫吸附试验（ELISA）检测肿瘤坏死因子-α（TNF-α）和白细胞介素-10（IL-10）浓度，采用硫代巴比妥酸法测定肝组织丙二醛（MDA）含量，采用黄嘌呤氧化酶法测定超氧化物歧化酶（SOD）活性，苏木精-伊红（HE）染色，于光镜下观察肝组织病理学表现，荧光定量PCR检测天冬氨特异性半胱氨酸蛋白酶3（caspase-3）、Bcl-2和Bax mRNA的表达情况。结果与Sham组相比，I/R组和HS组血清中ALT、AST、TNF-α浓度及肝组织MDA含量、caspase-3和Bax/Bcl-2的mRNA水平均升高，I/R组血清IL-10浓度降低，HS组IL-10含量升高，肝组织SOD活性降低〔ALT（U/L）：467.3±203.6、275.7±74.0比73.9±30.1，AST （U/L）：929.6±442.7、581.4±143.3比194.6±72.2，TNF-α（μg/L）：0.0813±0.0298、0.0520±0.0099比0.0273±0.0044，MDA（nmol/mg）：1.13±0.10、1.00±0.11比0.84±0.06，caspase-3（2-ΔΔCt）：2.77±0.98、1.46±0.33比1.00±0.00，Bax/Bcl-2（2-ΔΔCt）：1.59±0.34、1.12±0.16比1.02±0.10，IL-10（μg/L）：0.1415±0.0233、0.3206±0.0648比0.2053±0.0497，SOD（U/mg）：194.9±20.4、218.1±16.0比240.8±16.9，均P＜0.05〕。与I/R组相比，HS组血清ALT、AST、TNF-α浓度及肝组织MDA含量、caspase-3和Bax/Bcl-2的mRNA水平均降低，血清IL-10浓度及肝组织SOD活性升高〔ALT（U/L）：275.7±74.0比467.3±203.6，AST（U/L）：581.4±143.3比929.6±442.7，TNF-α（μg/L）：0.0520±0.0099比0.0813±0.0298，MDA（nmol/mg）：1.00±0.11比1.13±0.10，caspase-3（2-ΔΔCt）：1.46±0.33比2.77±0.98， Bax/Bcl-2（2-ΔΔCt）：1.12±0.16比1.59±0.34，IL-10（μg/L）：0.3206±0.0648比0.1415±0.0233，SOD （U/mg）：218.1±16.0比194.9±20.4，均P＜0.05〕。结论饱和氢气生理盐水可通过其选择性抗氧化、抑制炎症反应及抗凋亡特性减轻大鼠肝脏冷IRI。%Objective To explore the protective effect of hydrogen-rich saline on ischemia-reperfusion induced liver injury in rats and the mechanism of this effect.Methods A total number of24 healthy male Sprague-Dawley(SD) rats age ranging from8-10 weeks weighting220-250 g were randomly divided into three groups(n=8 in each group) using a random number table:sham operated group(Sham group), cold ischemia-reperfusion induced liver injury model group(I/R group)and hydrogen-rich saline treated group(HS group). Laparotomy was performed and the related blood vessels were isolated in Sham group. The liver cold ischemia-reperfusion were induced in I/R and HS groups. Saline or hydrogen-rich saline were injected through inferior vena cava5 min before portal vein was clamped in I/R or HS group. After the total hepatic ischemic in I/R and HS group, the liver was reperfused with ringer lactate solution(pre-cooled,4℃) at a speed of6-8 ml/min and a 1 mm incision was cut on the infrahepatic vena cava(IHVC) as an outflow tract. The whole anhepatic phase lasted for30 min. At6 h of reperfusion, blood samples were obtained from the inferior vena cava to detect the levels of alanine amino-transferase(ALT), aspartate amino-transferase(AST), tumor necrosis factor-alpha(TNF-α) and interleukin-10(IL-10) in serum. Hepatic specimens were obtained for the determination of malondialdehyde (MDA) and superoxide dismutase(SOD) contents, expression of caspase-3, Bcl-2 and Bax mRNA, and microscopic examination of the pathological changes in hepatic tissues by light microscopy.Results Compared with Sham group, the levels of ALT, AST, TNF-α and content of MDA, Bax/Bcl-2, and expression of caspase-3 mRNA in serum were significantly increased, and the level of serum IL-10 and the activity of SOD were decreased in I/R and HS groups〔ALT(U/L):467.3±203.6,275.7±74.0 vs.73.9±30.1,AST(U/L):929.6±442.7,581.4±143.3 vs. 194.6±72.2,TNF-α(μg/L):0.0813±0.0298,0.0520±0.0099 vs.0.0273±0.0044, MDA(nmol/mg):1.13±0.10, 1.00±0.11 vs.0.84±0.06,caspase-3(2-ΔΔCt):2.77±0.98,1.46±0.33 vs.1.00±0.00,Bax/Bcl-2(2-ΔΔCt):1.59±0.34,1.12±0.16 vs.1.02±0.10,IL-10(μg/L):0.1415±0.0233,0.3206±0.0648 vs.0.2053±0.0497, SOD(U/mg):194.9±20.4,218.1±16.0 vs.240.8±16.9,allP<0.05〕. While compared with I/R group, the levels of ALT, AST, TNF-α in serum and content of MDA, Bax/Bcl-2, and expression of caspase-3 mRNA were decreased, the level of IL-10 and the activity of SOD were increased in HS group〔ALT(U/L):275.7±74.0 vs. 467.3±203.6,AST(U/L):581.4±143.3 vs.929.6±442.7,TNF-α(μg/L):0.0520±0.0099 vs.0.0813±0.0298, MDA(nmol/mg):1.00±0.11 vs.1.13±0.10,caspase-3(2-ΔΔCt):1.46±0.33 vs.2.77±0.98,Bax/Bcl-2(2-ΔΔCt):1.12±0.16 vs.1.59±0.34,IL-10(μg/L):0.3206±0.0648 vs.0.1415±0.0233,SOD(U/mg):218.1±16.0 vs. 194.9±20.4,allP<0.05〕.Conclusion Hydrogen-rich saline can alleviate cold ischemia reperfusion induced liver injury through its selective antioxidant, inhibition of inflammatory response and anti-apoptotic properties.