天麻素对高脂和高胆固醇饮食致小鼠非酒精性脂肪性肝炎的治疗作用

Therapeutic effect of gastrodin against high-fat and high-cholesterol diet-induced nonalcoholic steatohepatitis in mice

目的 探讨天麻素(GSTD)对高脂和高胆固醇(HFHC)饮食诱导的小鼠非酒精性脂肪性肝炎(NASH)的治疗作用及可能机制。方法 6周龄雄性C57BL/6小鼠按体重分为6组,每组8只:正常对照组、模型组、模型+GSTD 12.5,25.0和50.0 mg·kg^(-1)组及模型+二甲双胍(Met)100 mg·kg^(-1)阳性对照组。除正常对照组外,其余各组小鼠饲喂HFHC饮食24周建立NASH模型;第17周开始,治疗组同时ig给予相应分组药物,每天1次,连续8周。第24周末,测量小鼠肝湿重、体重和肝指数。HE染色观察肝组织形态,计算非酒精性脂肪性肝病活动积分(NAS),Masson染色观察肝组织胶原纤维增生,油红O染色观察肝组织脂质沉积,血糖仪测全血血糖,ELISA法检测血清胰岛素水平计算胰岛素抵抗指数;化学显色法检测血清谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性,肝组织中甘油三酯(TG)、胆固醇(TC)、游离脂肪酸(NEFA)、丙二醛(MDA)和谷胱甘肽(GSH)水平,超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性;实时定量PCR(RT-qPCR)检测肝组织脂质合成基因[脂肪酸合成酶(FASN)、血小板反应蛋白受体(CD36)、硬脂酰辅酶A去饱和酶1(SCD1)、过氧化物酶增殖物活化受体γ(PPARγ)]脂质氧化基因PPARα、肉毒碱棕榈酰基转移酶1α(CPT1-α)、炎症因子基因[肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β、趋化因子配体2(CCL2)和CCL5]和纤维化标志物基因[Ⅰ型胶原α1(ColⅠα1)、肌动蛋白α2(Actα2)、结缔组织生长因子(CTGF)和转化生长因子β1(TGF-β1)]mRNA水平;Western印迹法检测肝组织胰岛素受体底物1(IRS^(-1))、蛋白激酶B(Akt)和NF-κB蛋白磷酸化水平。结果 与正常对照组相比,模型组肝湿重和体重增加(P<0.01),肝指数升高(P<0.05);肝组织出现严重的大泡性脂肪变性,并伴有大量炎症细胞浸润和胶原纤维增生,NAS评分升高(P<0.01);血糖和血清胰岛素水平、胰岛素抵抗指数以及血清GPT和GOT活性升高(P<0.01);肝组织TG、TC、NEFA和MDA水平升高(P<0.01),GSH水平、SOD和CAT活性降低(P<0.01);脂质合成基因mRNA水平升高(P<0.01),脂质氧化基因mRNA水平降低(P<0.01);炎症因子和纤维化基因mRNA水平升高(P<0.01);肝组织IRS-1和Akt蛋白磷酸化水平降低(P<0.01),NF-κB蛋白磷酸化水平升高(P<0.01)。与模型组相比,模型+GSTD 50.0 mg·kg^(-1)组肝湿重、体重和肝指数降低(P<0.05,P<0.01)。模型+GSTD 25.0和50.0 mg·kg^(-1)及模型+Met 100 mg·kg^(-1)组肝组织大泡性脂肪变性减少,NAS评分显著降低(P<0.05,P<0.01),炎症细胞浸润和胶原纤维增生减少,同时,血糖和血清胰岛素水平、胰岛素抵抗指数以及血清GPT和GOT活性降低(P<0.05,P<0.01);此外,模型+GSTD 50.0 mg·kg^(-1)组肝组织中TG、TC、NEFA和MDA水平降低(P<0.01),GSH水平、SOD和CAT活性升高(P<0.01),脂质合成基因mRNA水平降低(P<0.01),脂质氧化基因mRNA水平增加(P<0.01);模型+GSTD 25.0和50.0 mg·kg^(-1)组肝组织中炎症因子和纤维化基因mRNA水平均显著下降(P<0.05,P<0.01);模型+GSTD 25.0和50.0 mg·kg^(-1)及模型+Met 100 mg·kg^(-1)组肝组织IRS-1和Akt蛋白磷酸化水平升高(P<0.01),NF-κB蛋白磷酸化水平降低(P<0.01)。结论 GSTD对HFHC饮食诱导的小鼠NASH具有显著的治疗作用,其作用机制可能与激活肝内IRS-1/Akt信号通路,缓解氧化应激和抑制NF-κB信号介导的炎症反应有关。

OBJECTIVE To investigate the therapeutic effects of gastrodin(GSTD) against nonalcoholic steatohepatitis(NASH) in mice induced by a high-fat and high-cholesterol(HFHC) diet and to explore its underlying mechanism. METHODS Six-week-old male C57BL/6 mice were randomly divided into six groups by body weight: the normal control group, model group, model+GSTD 12.5 mg·kg^(-1),model+GSTD 25.0 mg·kg^(-1), model+GSTD 50.0 mg·kg^(-1), and model+Met 100 mg·kg^(-1). Except the normal control group, the mice were fed with HFHC for 24 weeks to establish a NASH model. Starting at 17th week, the treatment group was given GSTD or Met by ig once a day for eight weeks. At the end of the24thweek, the blood glucose and body weight of mice in each group were monitored. Histopathological changes, collagen fiber proliferation and lipid deposition in liver tissues were observed by using hematoxyline-eosin(HE), Masson′s trichrome and oil red O staining. The level of insulin was detected by ELISA and insulin resistance(HOMA-IR) was evaluated. The activities of serum glutamic pyruciv transaminase(GPT) and glutamic oxaloacetic transaminase(GOT), the levels of triglyceride(TG), cholesterol(TC), nonestesterified fatty acid(NEFA), malondialdehyde(MDA) and glutathione(GSH), and the activities of superoxide dismutase(SOD) and catalase(CAT) in liver tissues were measured. Real-time quantitative PCR(RT-qPCR) was used to detect the mRNA levels of lipid synthesis genes—fatty acid synthase(FASN), thrombospondin receptor(CD36), stearoyl-coenzyme A desaturase 1(SCD1), and peroxisome proliferator activated receptor γ(PPARγ), lipid oxidation genes—PPARα, carnitine palmitoyltransferase 1α(CPT1α);inflammatory factors gene: tumor necrosis factor α(TNF-α), interleukin-6(IL-6),IL-1β, C-C motif chemokine ligand 2(CCL2), and CCL5 and fibrotic marker genes—collagen type 1alpha 1(Col1α1), actin alpha2(Actα2), connective tissue growth factor(CTGF), and transforming growth factor beta 1(TGF-β1) in liver tissues. Western blotting was used to detect the phosphorylation levels of insulin receptor substrate 1(IRS-1), protein kinase B(Akt) and NF-κB. RESULTS Compared with the normal control group, the weights of the liver and body in the model group were increased(P<0.01), the liver index was elevated(P<0.05), severe bullous steatosis was shown in liver tissues, and the NAS score was increased(P<0.01), accompanied by inflammatory cell infiltration and collagen fiber accumulation. Blood glucose, serum insulin levels, insulin resistance indexes, and the activities of GPT and GOT in serum were increased(P<0.01). The levels of TG, TC, NEFA and MDA were increased(P<0.01), while the levels of GSH, the activities of SOD and CAT were decreased in liver tissues(P<0.01). The mRNA level of lipid synthesis-related genes was increased(P<0.01), while that of lipid oxidation genes was decreased(P<0.01). The mRNA levels of inflammatory factors and fibrosis-related genes were increased(P<0.01). Moreover, the phosphorylation levels of IRS-1 and Akt protein were decreased(P<0.01), and that of NF-κB protein was increased in liver tissues(P<0.01). Compared with the model group, the wet weight of the liver, body weight and liver indexes were decreased in the model+GSTD 50.0 mg·kg^(-1)group(P<0.01, P<0.05). Bullous steatosis was reduced, the NAS score was significantly decreased(P<0.05, P<0.01), and inflammatory cell infiltration and collagen fiber accumulation were reduced in model+GSTD 25.0, 50.0 mg·kg^(-1)and model+Met 100 mg·kg^(-1)groups(P<0.05, P<0.01). At the same time, blood glucose, serum insulin levels, insulin resistance indexes and the activities of GPT and GOT in serum were decreased(P<0.05, P<0.01). The levels of TG, TC, NEFA and MDA were significantly decreased(P<0.01), while that of GSH and the activities of SOD and CAT were increased(P<0.01). The levels of mRNA of lipid synthesis and oxidation genes in liver tissues of the model+GSTD 50.0 mg·kg^(-1)group were decreased and increased, respectively(P<0.01). Moreover, in model+GSTD 25.0, 50.0 mg·kg^(-1)groups, the mRNA levels of inflammatory factors and fibrotic biomarkers were decreased in liver tissues(P<0.05, P<0.01). The phosphorylation levels of IRS-1 and Akt were increased(P<0.01), and that of NF-kB was decreased in liver tissues of model+GSTD 25.0,50.0 mg·kg^(-1)and model+Met 100 mg·kg^(-1)groups(P<0.01). CONCLUSION GSTD has significant therapeutic effects against HFHC-induced nonalcoholic fatty liver disease in mice, and the mechanism may be related to activation of IRS-1/Akt signaling pahtway, alleviation of oxidative stress and inhibition of NF-κB-mediated inflammatory responses.