铁皮石斛叶发酵液对酒精性肝炎小鼠干预的效果及机制

Interventional effect and mechanism of fermentation liquid of Dendrobium officinale leaves on alcoholic hepatitis mice

目的探讨铁皮石斛叶发酵液对酒精性肝炎(AH)小鼠的干预效果及机制。方法选取70只6~8周龄近交品系C57BL/6J雄性小鼠,按随机数字表法分为正常组(NG)、模型组(MG)、液体饲料对照组(CG)、水飞蓟宾组(SI)及铁皮石斛叶发酵液低剂量组(DL)、中剂量组(DM)、高剂量组(DH),每组10只。NG组给予普通饲料喂养,CG组给予对照饲料(LB酒精液体对照饲料),SI组给予LB酒精液体饲料及水飞蓟宾灌胃,DL、DM和DH组分别给予LB酒精液体饲料及25%、50%、100%浓度的铁皮石斛叶发酵液灌胃。LB酒精液体饲料喂养8周建立AH模型。第8周取小鼠眼球血,检测丙氨酸转氨酶(ALT)、甘油三酯(TG)、转铁蛋白(TRF)、白细胞介素(IL)-6、IL-10、IL-1β、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)等含量;并取肝组织行HE、油红O、普鲁士蓝和免疫荧光ROS染色;肝组织匀浆检测谷胱甘肽(GSH)、丙二醛(MDA)含量;PCR及Western印迹法检测腺苷酸活化蛋白激酶(AMPK)、AMPKβ1、磷酸化AMPKβ1(p-AMPKβ1)、抑癌基因p53(p53)、溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GXP4)的mRNA及蛋白相对表达量,以分析铁皮石斛叶发酵液对AH小鼠的干预效果及机制。结果与MG组相比,DL、DM、DH组小鼠血清ALT和TG均降低[ALT分别为(45.94±19.85)、(45.73±22.62)、(41.68±7.13)比(75.51±17.76)U/L;TG分别为(0.90±0.23)、(0.69±0.22)、(0.41±0.20)比(1.28±0.19)mmol/L,均P<0.05];IL-6、IL-1β、TNF-α、IFN-γ均降低(均P<0.05);DM、DH组小鼠血清TRF、IL-10均升高(均P<0.05)。与模型组相比,DL、DM、DH组小鼠肝组织MDA均降低[分别为(0.41±0.05)、(0.40±0.03)、(0.43±0.14)比(0.64±0.06)μmol/g],GSH均升高(均P<0.05)。与模型组相比,DL、DM、DH组小鼠肝组织AMPK(分别为1.36±0.11、1.61±0.17、1.68±0.11比0.80±0.12)、SLC7A11(分别为0.91±0.12、0.97±0.12、0.99±0.13比0.60±0.14)、GPX4(分别为0.51±0.11、0.63±0.17、0.83±0.15比0.42±0.14)的mRNA表达量均增加(均P<0.05)。与模型组相比,DL、DM、DH组小鼠肝组织AMPKβ1、p-AMPKβ1、SLC7A11和GPX4的蛋白相对表达量均增加,p53蛋白相对表达量降低(均P<0.05)。与模型组相比,DL、DM、DH组小鼠肝组织脂肪变性、小叶内炎症程度减轻,肝组织铁染色、ROS染色变浅。结论铁皮石斛叶发酵液可减轻小鼠AH的严重程度,其机制可能与上调AMPK抑制p53/SLC7A11/GPX4介导的铁死亡通路有关。

Objective To explore the intervention effect and mechanism of Dendrobium officinale leaf fermentation liquid on alcoholic hepatitis(AH)mice.Methods Seventy inbred C57BL/6J male mice aged 6-8 weeks were selected and randomly divided into normal group(NG),model group(MG),liquid feed control group(CG),silybum group(SI),low-dose group(DL),medium-dose group(DM),and high-dose group(DH)of Dendrobium officinale fermentation liquid,with 10 mice in each group.NG group was given common feed,CG group was given control feed(LB alcoholic liquid control feed),SI group was given LB alcoholic liquid feed and silybum by gavage,DL,DM and DH groups were given LB alcoholic liquid feed and 25%,50%and 100%concentration of Dendrobium officinale leaf fermentation liquid by gavage.An AH model was established by feeding LB alcoholic liquid feed for 8 weeks.At week 8,alanine Transaminase(ALT),triglyceride(TG),transferrin(TRF),interleukin(IL)-6,IL-10,and IL-1β,tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ)were detected in eye blood of mice.Liver tissues were stained with HE,Oil Red O,Prussian blue and immunofluorescence ROS.The contents of glutathione(GSH)and malondialdehyde(MDA)in liver tissue homogenate were detected.To analyze the intervention effect and mechanism of Dendrobium officinale leaf fermentation solution on AH mice,the mRNA and protein relative expression levels of adenylate activated protein kinase(AMPK),AMPKβ1,phosphorylated AMPKβ1(p-AMPKβ1),tumor suppressor gene p53(p53),solsolic vector family 7 member 11(SLC7A11),glutathione peroxidase 4(GXP4)were detected by polymerase chain reaction(PCR)and Western blot.Results Compared with MG group,the serum ALT and TG levels in the DL,DM,and DH groups were all reduced[ALT:(45.94±19.85),(45.73±22.62),and(41.68±7.13)vs(75.51±17.76)U/L,respectively;TG:(0.90±0.23),(0.69±0.22)and(0.41±0.20)vs(1.28±0.19)mmol/L,respectively,all P<0.05];IL-6,IL-1β,TNF-α,IFN-γwere decreased(all P<0.05).The serum TRF and IL-10 levels in the DM and DH groups were increased(all P<0.05).Compared with MG group,the liver tissue MDA of mice in DL,DM and DH groups was decreased[(0.41±0.05),(0.40±0.03),and(0.43±0.14)vs(0.64±0.06)μmol/g,respectively],GSH was increased(all P<0.05).Compared with MG,mRNA expression levels of AMPK(1.36±0.11,1.61±0.17,1.68±0.11 vs 0.80±0.12,respectively),SLC7A11(0.91±0.12,0.97±0.12,0.99±0.13 vs 0.60±0.14,respectively)and GPX4(0.51±0.11,0.63±0.17,0.83±0.15 vs 0.42±0.14,respectively)in the liver tissue of DL,DM and DH groups were all increased(all P<0.05).Compared with MG group,DL,DM and DH groups showed the relative expression levels of AMPKβ1,p-AMPKβ1,SLC7A11 and GPX4 were increased in the liver tissue of mice,while the relative expression levels of p53 protein were decreased(all P<0.05).Compared with MG group,DL,DM and DH groups reduced the degree of hepatic steatosis and inflammation in the lobules,while the iron and ROS staining in the liver tissue became lighter.Conclusion Dendrobium officinale leaf fermentation liquid can alleviate the severity of AH in mice,and its mechanism may be related to the up-regulation of AMPK to inhibiting the p53/SLC7A11/GPX4 mediated Ferroptosis pathway.