高良姜素减轻非酒精性脂肪肝大鼠肝脂肪变性和纤维化的机制研究

Study on the mechanism of galangin alleviating hepatic steatosis and fibrosis in nonalcoholic fatty liver rats

目的探究高良姜素(galangin)减轻非酒精性脂肪肝(NAFLD)大鼠肝脂肪变性和纤维化的机制。方法采用随机数字表法将60只大鼠分为对照组(NC组)、高脂组(H组)、高良姜素低剂量组(H+Gal L组)、高良姜素中剂量组(H+Gal M组)和高良姜素高剂量组(H+Gal H组),每组各12只。NC组大鼠喂以普通饲料,其余各组均喂以高脂饲料。连续喂养8周建立NAFLD模型。第9周开始灌胃给药,H+Gal L组、H+Gal M组、H+Gal H组分别给予含25 mg/kg、50 mg/kg、100 mg/kg 0.9%氯化钠溶液溶解的高良姜素;NC组、H组灌胃等量的0.9%氯化钠溶液,1次/d,连续4周。给药结束后,观察大鼠一般情况及检测肝功能指标;HE染色观察大鼠肝脏组织病理学变化;油红O染色观察大鼠肝脏组织脂肪沉积情况;Masson染色观察大鼠肝脏组织纤维化情况;RT-qPCR检测肝脏组织脂质合成相关基因的表达;Western-blot检测肝脏组织纤维化相关蛋白、AMPK蛋白表达。将LX-2细胞分为NC组、H组、H+Gal L组、H+Gal M组和H+Gal H组。除NC组外,其余各组采用0.6 mmol/L油酸处理8 h诱导细胞脂质沉积,NC组加入等量0.9%氯化钠溶液;随后H+Gal L组、H+Gal M组和H+Gal H组培养基中分别加入25、50、100μM的高良姜素培养24 h。造模结束后,CCK-8法检测LX-2细胞毒性;流式细胞术检测细胞凋亡水平;检测LX-2细胞甘油三酯(TG)、总胆固醇(TC)含量;油红O染色观察LX-2细胞脂肪沉积;RT-qPCR检测LX-2细胞脂质合成相关基因的表达;Western-blot检测LX-2细胞纤维化相关蛋白、AMPK蛋白表达。结果NC组大鼠毛色顺滑有光泽,精神状态好,体重稳定增加,肝细胞内未见脂滴和胶原沉积;与NC组相比,H组大鼠毛色暗淡无光泽,精神萎靡,体重快速增加,肝细胞内存在大量脂滴和胶原沉积;肝湿质量、TG、TC、AST、ALT水平,胆固醇调节元件结合蛋白-1C(SREBP-1c)、脂肪酸合酶(FAS)、乙酰辅酶A羧化酶(ACC)mRNA水平,Collagen-I、α-SMA蛋白水平明显升高,p-AMPK/AMPK蛋白水平明显降低(P<0.05);与H组相比,H+Gal L组、H+Gal M组、H+Gal H组毛色逐渐润泽,精神较充沛,体重缓慢增加,肝细胞内存在少量脂滴和胶原沉积;肝湿质量、TG、TC、AST、ALT水平,SREBP-1c、FAS、ACC mRNA水平,Collagen-I、α-SMA蛋白水平明显降低,p-AMPK/AMPK蛋白水平明显升高(P<0.05),且呈高良姜素剂量依赖性。当高良姜素浓度小于100μM时不会造成细胞毒性。NC组细胞内内未见脂滴;与NC组相比,H组细胞存在大量脂滴;细胞凋亡水平,TG、TC水平,SREBP-1c、FAS、ACC mRNA水平,Collagen-I、α-SMA蛋白水平明显升高,p-AMPK/AMPK蛋白水平明显降低(P<0.05);与H组相比,H+Gal L组、H+Gal M组、H+Gal H组细胞存在少量脂滴;细胞凋亡水平,TG、TC水平,SREBP-1c、FAS、ACC mRNA水平,Collagen-I、α-SMA蛋白水平明显降低,p-AMPK/AMPK蛋白水平明显升高(P<0.05),且呈高良姜素剂量依赖性。结论高良姜素能够减轻NAFLD大鼠肝脂肪变性和纤维化,促进大鼠肝功能恢复,其几只可能通过激活AMPK信号通路而发挥作用。

Objective To explore the mechanism of galangin in alleviating liver steatosis and fibrosis in nonalcoholic fatty liver disease(NAFLD)rats.Methods Animal experiment:60 rats were randomly divided into control group(NC group),high-fat group(H Group),low-dose galangin group(H+Gal L group),medium dose galangin group(H+Gal M group)and high-dose galangin group(H+Gal H Group),with 12 rats in each group.In NC group,rats were given normal diet,while in other groups,rats were given high-fat diet.NAFLD model was established after continuous feeding for 8 weeks.At the 9th week,the drug was administered by gavage.The H+Gal L group,H+Gal M group and H+Gal H group were given galangin dissolved in 0.9%sodium chloride solution containing 25 mg/kg,50 mg/kg and 100 mg/kg respectively;NC group and H group were gavaged with the same amount of 0.9%sodium chloride solution,once a day,for 4 weeks.After the administration,the general conditions of rats was observed and the indicators of liver function were detected;HE staining was used to observe the structural changes of rat liver;Oil red O staining was used to observe the fat deposition in rat liver;Masson staining was used to observe the fibrosis of rat liver tissue;T-qPCR was used to detect the expression of genes related to lipid synthesis in liver tissue;Western blot was used to detect the expression of fibrosis related protein and AMPK protein in liver tissue.Cell experiment:LX-2 cells were divided into NC group,H group,H+Gal L group,H+Gal M group and H+Gal H group.Except the NC group,the other groups were treated with 0.6 mmol/L oleic acid for 8 hours to induce cell lipid deposition,and the NC group was added with the same amount of 0.9%sodium chloride solution;Then 25,50 and 100μM galangin were added to the culture medium of H+Gal L group,H+Gal M group and H+Gal H group respectively and cultured for 24 hours.After modeling,CCK-8 method was used to detect the cytotoxicity in LX-2 cells;Flow cytometry was used to detect the level of apoptosis;The content of triglyceride(TG)and total cholesterol(TC)in LX-2 cells was detected;Oil red O staining was used to observe the fat deposition in LX-2 cells;RT-qPCR was used to detect the expression of genes related to lipid synthesis in LX-2 cells;Western blot was used to detect the expression of fibrosis related protein and AMPK protein in LX-2 cells.Results Animal experiment:NC group rats had smooth and glossy hair,good mental state,stable weight gain,and no lipid droplets and collagen deposition in hepatocytes;Compared with NC group,rats in group H had dull hair color,listless spirit,rapid weight gain,and a large number of lipid droplets and collagen deposition in hepatocytes;Liver wet mass,TG,TC,AST,ALT levels,cholesterol regulatory element binding protein-1c(SREBP-1c),fatty acid synthase(Fas),acetyl-CoA carboxylase(ACC)mRNA level,collagen-I,α-SMA protein level were significantly increased,and p-AMPK/AMPK protein level was significantly decreased(P<0.05);Compared with H group,the hair color of H+Gal L group,H+Gal M group and H+Gal H group was gradually moist,energetic,weight increased slowly,and there were a small amount of lipid droplets and collagen deposition in hepatocytes;The levels of liver wet mass,TG,TC,AST,ALT levels,SREBP-1c,Fas,ACC mRNA,collagen-I,α-SMA protein were significantly decreased,and p-AMPK/AMPK protein was significantly increased(P<0.05);And it was dose-dependent on galangin.Cell experiment:when the concentration of galangin was less than 100μM,it will not cause cytotoxicity.No lipid droplets were found in NC group;Compared with NC group,there were a large number of lipid droplets in H group;The levels apoptosis,TG,TC,AST,ALT,SREBP-1c,Fas,ACC mRNA,Collagen-I,α-SMA protein were significantly increased,and p-AMPK/AMPK protein level was significantly decreased(P<0.05);Compared with H group,there were a few lipid droplets in H+Gal L group,H+Gal M group and H+Gal H group;The levels of apoptosis,TG,TC,AST,ALT,SREBP-1c,Fas,ACC mRNA levels,Collagen-I,α-SMA protein were significantly decreased,and p-AMPK/AMPK protein level was significantly increased(P<0.05);And it was dose-dependent on galangin.Conclusion Galangin can reduce liver steatosis and fibrosis in NAFLD rats and promote the recovery of liver function,which may play a role by activating AMPK signaling pathway.