N-乙酰半胱氨酸对急性酒精性肝损伤大鼠保护作用的实验研究
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摘要
目的：观察N-乙酰半胱氨酸(N-acetylcysteine，NAC)对急性酒精性肝损伤大鼠的保护作用。
方法：采用酒精灌胃法建立急性肝损伤大鼠模型。50只雄性Wistar大鼠随机分为5组(n=10)：正常对照组、模型组、乙酰半胱氨酸低剂量组(150mg/kg)、中剂量组(300mg/kg)、高剂量组(600mg/kg)。正常对照组用等量的生理盐水代替酒精灌胃和腹腔注射；模型组以56°白酒按14ml／kg体重灌胃,每日一次,连续10d；各治疗组在酒精灌胃前30min分别腹腔注射不同剂量乙酰半胱氨酸,每日一次,连续10d。治疗结束后测定各组大鼠肝脏指数,留取腹主动脉血及肝脏标本,分离血清测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)活性及肿瘤坏死因子-α(TNF-α)的含量,制备肝组织匀浆测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性与丙二醛(MDA)含量,肝组织行HE染色光镜下观察病理学改变,并行免疫组织化学染色检测肝脏中核转录因子-κB(NF-κB)与半胱氨酸蛋白酶-3(Caspase-3)的表达。
结果：①与正常对照组比较,模型组大鼠肝脏指数显著增加,血清ALT、AST活性及TNF-α含量明显升高,肝组织MDA含量及NF-κB、Caspase-3的表达增强,SOD、GSH-Px活性减低(P<0.01)；光镜下肝组织结构紊乱,肝细胞脂肪变性严重,伴炎症细胞浸润。②治疗结束后,乙酰半胱氨酸各剂量组ALT、AST活性均明显降低,肝脏指数减小,肝组织中MDA含量降低,SOD、GSH-Px活性明显升高,与模型组比较有统计学意义(P<0.01或P<0.05)。与低剂量组比较,中、高剂量组MDA含量降低,SOD、GSH-Px活性升高,(P<0.01或P<0.05)。与中剂量组比较,高剂量组SOD活性明显升高(P<0.01)。③光镜下各治疗组肝组织脂肪变性和炎症程度轻于模型组,高剂量组与正常对照组无明显差异,中剂量组轻微脂变,低剂量组脂变略重。④各治疗组肝组织中NF-κB和Caspase-3的表达均明显减少,血清TNF-α含量降低,与模型组比较差别有统计学意义(P<0.01或P<0.05)。与低剂量组比较,中、高剂量组NF-κB和Caspase-3的表达减少,高剂量组TNF-α含量降低(P<0.01或P<0.05)。与中剂量组比较,高剂量组NF-κB和Caspase-3的表达减少,TNF-α含量降低(P<0.01或P<0.05)。
结论：乙酰半胱氨酸不仅可以减少脂质过氧化产物MDA的含量,而且能提高肝组织中抗氧化酶SOD、GSH-Px活性,发挥抗氧化作用,进而改善肝功能,减轻肝细胞的脂肪堆积。同时还可以通过抑制NF-κB的激活,减少炎症因子的释放,抑制凋亡信号的传导,发挥抗炎及抗凋亡作用,从而有效防治酒精对肝脏的损伤,且其治疗作用具有一定的量效关系。
Objective:To investigate the protective effect of N-acetylcysteine(NAC) on acute alcoholic liver injury in rats.
Methods:The liver injury rat models were induced by infusing alcohol into stomach of rats. Fifty male Wistar rats were randomly divided into 5 groups(n=10):control group, acute liver injury model group, and low-(150mg/kg), middle-(300mg/kg), high-(600mg/kg) dose group of N-acetylcysteine. The control group were given the same amount of salime instead of alcohol through intraperitpneal injection and gastric infusion. The model group were infused stomach with 56°liqueur once a day for 10d. Different doses of N-acetylcysteine groups were injected NAC intraperitoneally at 30 min before infusing liqueur once a day for 10d. After treatment, the liver indices were measured and aorta blood and liver tissue were collected. Serum was isolated for measurement of alanine transaminase(ALT), aspartate transaminase(AST) activity and tumor necrosis factor-a(TNF-a) content. Liver tissue homogenate was prepared to assay superoxide dismutase(SOD), glutathione perioxidase(GSH-Px) activities and malondialdehyde (MDA) content. The liver histopathologies were explored with HE staining. The expression of nuclear factor-kappa B(NF-κB) and Caspase-3 in liver were detected by immunohistochemistry.
Results:①Compared with control group, liver indices, serum ALT, AST level and TNF-αcontent increased, liver tissue MDA content and the expression of NF-κB and Caspase-3 increased, SOD, GSH-Px activities decreased(P<0.01) significantly in model group. Under light microscope, the structure of the liver tissue was disorded, with a serious liver steatosis, accompanied by the infiltration of inflammatory cells.②After treatment, serum ALT, AST level and liver indices decreased, and liver tissues MDA content decreased, SOD, GSH-Px activities increased in difference doses of NAC groups(P<0.01 or P<0.05), the difference was significant compared with model group. Compared with low-dose group, MDA content decreased, SOD, GSH-Px activity increased in middle and high-dose groups(P<0.01 or P<0.05).Compared with the middle dose group, SOD activity was significantly higher in high-dose group(P<0.01).③Under light microscope, the treatment groups were lighter in liver steatosis and the degree of inflammation than the model group. High-dose group and control group were not significantly different. The middle-dose group had a slight steatosis and the low-dose group have a more serious steatosis.④In three different doses of NAC groups, the expression of NF-κB and Caspase-3 were significantly reduced, serum TNF-a content were decreased(P<0.01 or P<0.05). Compared with model group, the difference was significant. Compared with low-dose group, the expression of NF-κB and Caspase-3 reduced in middle and high-dose group, serum TNF-a decreased in the high-dose group(P<0.01 or P<0.05). Compared with the middle-dose group, the expression of NF-κB and Caspase-3 were reduced, serumTNF-a content decreased in the high-dose group(P<0.01 or P<0.05).
Conclusions:The acute alcoholic liver injury models in rats were made successfully. N-acetylcysteine can not only reduce the lipid peroxidation product MDA content, but also improve the liver tissue antioxidant enzymes SOD, GSH-Px activity, to improve liver function, reduce the accumulation of lipid in liver cells. Also, it can reduce the release of inflammatory cytokines by inhibiting NF-κB activating, and inhibit apoptosis signal transduction, having anti-inflammatory and anti-apoptotic effects. Thus NAC might have effect of alleviating the liver injury induced by alcohol and its therapeutic effect has a certain dose-effect relationship.
方法：采用酒精灌胃法建立急性肝损伤大鼠模型。50只雄性Wistar大鼠随机分为5组(n=10)：正常对照组、模型组、乙酰半胱氨酸低剂量组(150mg/kg)、中剂量组(300mg/kg)、高剂量组(600mg/kg)。正常对照组用等量的生理盐水代替酒精灌胃和腹腔注射；模型组以56°白酒按14ml／kg体重灌胃,每日一次,连续10d；各治疗组在酒精灌胃前30min分别腹腔注射不同剂量乙酰半胱氨酸,每日一次,连续10d。治疗结束后测定各组大鼠肝脏指数,留取腹主动脉血及肝脏标本,分离血清测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)活性及肿瘤坏死因子-α(TNF-α)的含量,制备肝组织匀浆测超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性与丙二醛(MDA)含量,肝组织行HE染色光镜下观察病理学改变,并行免疫组织化学染色检测肝脏中核转录因子-κB(NF-κB)与半胱氨酸蛋白酶-3(Caspase-3)的表达。
结果：①与正常对照组比较,模型组大鼠肝脏指数显著增加,血清ALT、AST活性及TNF-α含量明显升高,肝组织MDA含量及NF-κB、Caspase-3的表达增强,SOD、GSH-Px活性减低(P<0.01)；光镜下肝组织结构紊乱,肝细胞脂肪变性严重,伴炎症细胞浸润。②治疗结束后,乙酰半胱氨酸各剂量组ALT、AST活性均明显降低,肝脏指数减小,肝组织中MDA含量降低,SOD、GSH-Px活性明显升高,与模型组比较有统计学意义(P<0.01或P<0.05)。与低剂量组比较,中、高剂量组MDA含量降低,SOD、GSH-Px活性升高,(P<0.01或P<0.05)。与中剂量组比较,高剂量组SOD活性明显升高(P<0.01)。③光镜下各治疗组肝组织脂肪变性和炎症程度轻于模型组,高剂量组与正常对照组无明显差异,中剂量组轻微脂变,低剂量组脂变略重。④各治疗组肝组织中NF-κB和Caspase-3的表达均明显减少,血清TNF-α含量降低,与模型组比较差别有统计学意义(P<0.01或P<0.05)。与低剂量组比较,中、高剂量组NF-κB和Caspase-3的表达减少,高剂量组TNF-α含量降低(P<0.01或P<0.05)。与中剂量组比较,高剂量组NF-κB和Caspase-3的表达减少,TNF-α含量降低(P<0.01或P<0.05)。
结论：乙酰半胱氨酸不仅可以减少脂质过氧化产物MDA的含量,而且能提高肝组织中抗氧化酶SOD、GSH-Px活性,发挥抗氧化作用,进而改善肝功能,减轻肝细胞的脂肪堆积。同时还可以通过抑制NF-κB的激活,减少炎症因子的释放,抑制凋亡信号的传导,发挥抗炎及抗凋亡作用,从而有效防治酒精对肝脏的损伤,且其治疗作用具有一定的量效关系。
Objective:To investigate the protective effect of N-acetylcysteine(NAC) on acute alcoholic liver injury in rats.
Methods:The liver injury rat models were induced by infusing alcohol into stomach of rats. Fifty male Wistar rats were randomly divided into 5 groups(n=10):control group, acute liver injury model group, and low-(150mg/kg), middle-(300mg/kg), high-(600mg/kg) dose group of N-acetylcysteine. The control group were given the same amount of salime instead of alcohol through intraperitpneal injection and gastric infusion. The model group were infused stomach with 56°liqueur once a day for 10d. Different doses of N-acetylcysteine groups were injected NAC intraperitoneally at 30 min before infusing liqueur once a day for 10d. After treatment, the liver indices were measured and aorta blood and liver tissue were collected. Serum was isolated for measurement of alanine transaminase(ALT), aspartate transaminase(AST) activity and tumor necrosis factor-a(TNF-a) content. Liver tissue homogenate was prepared to assay superoxide dismutase(SOD), glutathione perioxidase(GSH-Px) activities and malondialdehyde (MDA) content. The liver histopathologies were explored with HE staining. The expression of nuclear factor-kappa B(NF-κB) and Caspase-3 in liver were detected by immunohistochemistry.
Results:①Compared with control group, liver indices, serum ALT, AST level and TNF-αcontent increased, liver tissue MDA content and the expression of NF-κB and Caspase-3 increased, SOD, GSH-Px activities decreased(P<0.01) significantly in model group. Under light microscope, the structure of the liver tissue was disorded, with a serious liver steatosis, accompanied by the infiltration of inflammatory cells.②After treatment, serum ALT, AST level and liver indices decreased, and liver tissues MDA content decreased, SOD, GSH-Px activities increased in difference doses of NAC groups(P<0.01 or P<0.05), the difference was significant compared with model group. Compared with low-dose group, MDA content decreased, SOD, GSH-Px activity increased in middle and high-dose groups(P<0.01 or P<0.05).Compared with the middle dose group, SOD activity was significantly higher in high-dose group(P<0.01).③Under light microscope, the treatment groups were lighter in liver steatosis and the degree of inflammation than the model group. High-dose group and control group were not significantly different. The middle-dose group had a slight steatosis and the low-dose group have a more serious steatosis.④In three different doses of NAC groups, the expression of NF-κB and Caspase-3 were significantly reduced, serum TNF-a content were decreased(P<0.01 or P<0.05). Compared with model group, the difference was significant. Compared with low-dose group, the expression of NF-κB and Caspase-3 reduced in middle and high-dose group, serum TNF-a decreased in the high-dose group(P<0.01 or P<0.05). Compared with the middle-dose group, the expression of NF-κB and Caspase-3 were reduced, serumTNF-a content decreased in the high-dose group(P<0.01 or P<0.05).
Conclusions:The acute alcoholic liver injury models in rats were made successfully. N-acetylcysteine can not only reduce the lipid peroxidation product MDA content, but also improve the liver tissue antioxidant enzymes SOD, GSH-Px activity, to improve liver function, reduce the accumulation of lipid in liver cells. Also, it can reduce the release of inflammatory cytokines by inhibiting NF-κB activating, and inhibit apoptosis signal transduction, having anti-inflammatory and anti-apoptotic effects. Thus NAC might have effect of alleviating the liver injury induced by alcohol and its therapeutic effect has a certain dose-effect relationship.
引文
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