分心木提取物对大鼠肝缺血-再灌注损伤的防护作用及机制

Protective Effect and Mechanism of Extract of Diaphragma Juglandis Fructus on Hepatic Ischemia-Reperfusion Injury in Rats

目的探讨分心木体积分数40%乙醇提取物乙酸乙酯萃取部位(DJFE)对大鼠肝缺血-再灌注损伤(hepatic ischemia-reperfusion injury,HIRI)的防护作用及机制。方法将32只大鼠随机分为对照组、模型组、DJFE低、高剂量组(62.5、125 mg·kg^(-1)),每组各8只。分心木组给予相应DJFE溶液,其余两组给予等体积的生理盐水,采用夹闭肝脏的左外侧叶、中叶的静脉和门静脉90 min、再灌注24 h的方法建立HIRI模型。建模后进行取血,取肝脏。对大鼠肝脏组织进行HE染色,观察肝脏组织的病理学变化。生化试剂盒检测肝功能指标[谷草转氨酶(AST)、谷丙转氨酶(ALT)]和氧化应激指标[超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-PX)、黄嘌呤氧化酶(XOD)]的水平。qPCR法检测肝脏组织中NF-κBp65和TGF-β1 mRNA的表达水平。Western blot法检测各组大鼠肝脏组织中NF-κB p65和TGF-β1蛋白的表达水平。结果HE染色及生化指标测定结果表明与模型组相比较,分心木组能够明显减轻HIRI后的肝脏组织损伤、明显降低血清中AST和ALT的水平、明显降低肝脏组织中XOD、MDA的水平、明显提升SOD和GSH-PX的活性。运用Western blot法和qPCR法对肝脏组织中的NF-κBp65、TGF-β1因子的蛋白与mRNA表达水平进行测定,发现HIRI后NF-κBp65、TGF-β1的mRNA和蛋白表达水平均上调,而DJFE对两者均显示出一定程度的抑制作用。结论DJFE可通过减少氧自由基产生和降低体内脂质氧化程度;抑制NF-κBp65和TGF-β1的表达,减轻炎症反应,从而起到保护肝脏的功效。

OBJECTIVE To investigate the protective effect and mechanism of ethyl acetate soluble portion of 40%ethanol extract of Diaphragma Juglandis Fructus(DJFE)on hepatic ischemia reperfusion injury(HIRI)model in rats.METHODS Thirty-two rats were divided randomly into four groups with eight rats for each,including control group,model group,low dose group(62.5 mg·kg^(-1))and high dose group(125 mg·kg^(-1))of DJFE extract.Diaphragma Juglandis Fructus(DJF)groups were given the corresponding extract solution of DJFE while the other two groups were given equal volume of normal saline,and HIRI model was established by clamping the left lateral lobe,middle lobe vein and portal vein of the liver for 90 min and reperfusion for 24 h.After modeling,blood and liver were taken.The pathological changes of rat liver were observed by HE staining.The levels of liver function indexes(AST,ALT)and oxidative stress indexes(SOD,MDA,GSH-Px,XOD)were detected by biochemical kits.The mRNA expression levels of NF-κBp65 and TGF-β1 in liver of rats were detected by qPCR.The protein expression levels of NF-κBp65 and TGF-β1 in liver of rats were detected by Western blot.RESULTS Compared with the model group,the results of HE staining and biochemical indexes showed that DJF could significantly reduce the liver tissue injury,the levels of AST and ALT in serum,and the levels of XOD and MDA in liver tissue,and significantly increase the activities of SOD and GSH-Pxafter HIRI.And after HIRI,the expression levels of mRNA and proteins of NF-κBp65 and TGF-β1 were all increased.However,DJFE showed a certain degree of inhibition effect on the expression levels of mRNA and proteins of NF-κBp65 and TGF-β1.CONCLUSION DJFE can reduce the production of oxygen free radicals,decrease the degree of lipid oxidation,inhibit the expressions of NF-κBp65 and TGF-β1,and reduce the inflammatory reaction,thus it can be used to protect the liver.