褪黑素对大鼠肝纤维化的影响及部分机制研究

目的探讨褪黑素(melatonin,MEL)对大鼠肝纤维化的影响及部分机制。方法 111只雄性SD大鼠随机分为6组:正常对照组、模型对照组、N-乙酰-L-半胱氨酸组(N-ac-etyl-L-cysteine,NAC)组、褪黑素低、中、高剂量组(剂量分别为2.5、5、10 mg.kg-1)。采用四氯化碳(carbon tetrachloride,CCl4)制备大鼠肝纤维化模型,同时腹腔注射褪黑素,HE染色和VG胶原纤维染色观察肝脏病理改变;生化法测定肝脏丙二醛(malondialdehyde,MDA)含量和谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)、超氧化物歧化酶(superox-ide dismutase,SOD)活性;RT-PCR法测定肝组织中α1(I)前胶原(α1(I)procollagen)mRNA表达;原位灌注加密度梯度离心法分离正常SD大鼠肝脏星状细胞,在培养的肝星状细胞中加入脂多糖(lipopolysaccharide,LPS)进行刺激,用MTT法测定不同浓度的MEL对肝星状细胞增殖的抑制作用。结果和模型组比较,MEL(10 mg.kg-1)组肝纤维化评分较低(P<0.05),MEL能明显降低肝匀浆MDA含量,升高SOD、GPx活性,MEL(10 mg.kg-1)组、NAC组大鼠肝组织α1(I)型前胶原mRNA表达明显减少(P<0.05);培养的肝星状细胞经LPS刺激后A值增大,与LPS组相比,LPS+NAC组和LPS+MEL(0.1 mmol·L-1)组A值明显减小(P<0.05)。结论 MEL对大鼠肝纤维化具有改善作用,其机理可能与抗氧化、抑制前胶原基因转录和抑制肝星状细胞增殖有关。

Effects of melatonin on hepatic fibrosis induced by carbon tetrachloride in rats and its mechanisms

Aim To investigate the effects of melatonin on hepatic fibrosis induced by carbon tetrachloride in rats as well as its possible mechanisms.Methods All rats were randomly divided into normal control group,model control group treated with CCl4 for 12 wk,N-acetyl-L-cysteine(NAC) group treated with CCl4+NAC(100 mg·kg-1,i.p.) for 12 wk,and melatonin(MEL) groups(2.5 mg·kg-1,5 mg·kg-1,10 mg·kg-1) treated with CCl4 and different MEL doses for 12 wk.Hematoxylin and eosin(HE) staining and Van Gieson(VG) staining were used to examine changes in liver pathology.Malondialdehyde(MDA),superoxide dismutase(SOD) and glutathione peroxidase(GPx) levels in liver homogenates were assayed by spectrophotometry.The expression of α1(I) procollagen mRNA in liver tissue was detected by RT-PCR.Hepatic stellate cells(HSCs) were isolated from the liver of normal SD rat by perfusion of collagenase and pronase,followed by centrifugation over Nycodenz.With LPS,NAC and various concentrations of MEL,the proliferation of HSC was determined by MTT method.Results Fibrosis score showed that the fibrogenesis was much less severe in MEL(10 mg·kg-1) group than in model control group(P<0.05).Treatment with melatonin significantly reduced the MDA content and increased the SOD,GPx activity in liver homogenates compared with model control group.Compared with model control group,the expression of α1(I) procollagen mRNA in liver tissue in MEL group(10 mg·kg-1) and NAC group was significantly decreased(P<0.05).Compared with the LPS group,MEL obviously inhibited the proliferation of rat HSCs stimulated with LPS at the concentration of 0.1 mmol·L-1(P<0.05).Conclusions Melatonin may have beneficial effects on hepatic fibrosis induced by CCl4 in rats.The protective effect of melatonin on hepatic fibrosis may be related to its antioxidant activities and its inhibitory action on the expression of hepatic α1(I) procollagen mRNA and the proliferation of HSC.