Qushi Huayu Decoction (祛湿化瘀方) Inhibits Protein and Gene Expression of Cathepsin B in HepG2 Cells Induced by Free Fatty Acids

Objective： To study the experimental efficacy of Qushi Huayu Decoction （祛湿化瘀方,QHD） on protein and gene expression of cathepsin B （ctsb） in HepG2 cells induced by free fatty acids （FFAs）.Methods： The model of HepG2 steatosis and tumor necrosis factor-α （TNF-α） secretion was induced by long-chain FFAs.HepG2 cells were divided into 4 groups： control group （group C）,model group （group M）,low-dose QHD group （group L） and high-dose QHD group （group H ）.Long-chain FFAs were added to groups M,L and H.The 10% blank-control serum was added to group C and M,while 5% and 10% QHD-containing sera were added to group L and H,respectively.The levels of serum TNF-α and cellular triglyceride （TG） were detected.Cellular p-IκB and ctsb expression were detected using Western blot and PCR.The expression and distribution of ctsb were observed by immunofluorescence.Results： After incubating with FFA for 24 h,TG deposition in HepG2,TNF-α content in cell supernatant,the protein expression of cellular ctsb and P-IκB,as well as mRNA expression of ctsb increased markedly in group M compared with group C （P〈0.05,P〈0.01）.Compared with group M,TG deposition,the expression of cellular ctsb,P-IκB and ctsb mRNA in groups L and H,as well as TNF-α content in group H,decreased significantly （P〈0.05）.Cell immunochemical fluorescence studies showed that ctsb was released from lysosomes and distributed in the cytoplasm extensively and diffusedly after being stimulated with FFA.In this study,these above-mentioned changes were inhibited markedly in groups L and H.Conclusion： QHD might have a direct inhibitory effect on the ctsb target in the FFA-ctsb-TNFα pathway of hepatic lipotoxicity.

祛湿化瘀方对游离脂肪酸诱导的HepG2细胞脂肪沉积和TNF-α分泌的抑制作用

目的研究祛湿化瘀方对体外肝脂毒性模型脂肪沉积和肿瘤坏死因子α(tumor necrosis factorα,TNF-α)分泌的影响,以进一步探究该方防治脂肪肝的作用机理方法复制游离脂肪酸(free fatty acid,FFA)诱导的HepG2细胞脂肪变性和TNF-α分泌脂毒性模型。设正常组、模型组和不同浓度药物血清组。分别观察上清中TNF-α含量,细胞内甘油三酯(triglyceride,TG)含量,细胞油红染色和电镜超微结构变化,细胞TNF-α蛋白表达及其基因表达。结果FFA刺激24 h后,模型组细胞内脂肪沉积明显,其TG含量显著高于正常组(P<0.01),同时上清中TNF-α含量、细胞内TNF-α的蛋白表达及其mRNA表达显著增强。而10%药物血清组细胞内TG含量及上清中TNF-α含量显著低于模型组(均P<0.01),其脂肪沉积、细胞内TNF-α的蛋白表达及其mRNA表达也明显轻于模型组。

祛湿化瘀方通过调节AMPK活性改善高脂饮食诱导的实验性脂肪肝肝脏脂肪代谢

目的:研究祛湿化瘀方对高脂饮食诱导的大鼠脂肪肝AMPK蛋白活性及其相关脂肪代谢靶蛋白活性的影响,以探讨该方防治实验性脂肪肝的作用机制。方法:采用高脂饲料饮食诱导大鼠脂肪肝模型,造模大鼠给予高脂饮食4周后,按随机数字表随机分为模型组及祛湿化瘀方组,分别灌胃给予饮用水及中药祛湿化瘀方4周。实验8周末取材后观察:1)肝组织三酰甘油(Triglyceride,TG)、游离脂肪酸(Free Fatty Acid,FFA)含量,2)肝组织病理变化(HE染色、油红染色),3)肝组织腺苷酸活化的蛋白激酶(AMP-Activated K inase,AMPK)及磷酸化AMPK、肝组织总蛋白及核蛋白固醇调节元件结合蛋白-1c(Sterol Regulatory Element Binding Protein-1c、SREBP-1c)、肝组织总蛋白及核蛋白碳水化合物反应元件结合蛋白(Carbohydrate Response Element Binding Protein、ChREBP)含量、肝组织乙酰辅酶A羧化酶(Acety1 Co A Carboxylase,ACCase)及磷酸化ACC蛋白含量,4)肝组织AMPKα1、AMPKα2、SREBP-1、ACCα、SREBP-1c及ChREBP基因表达水平。结果:1)模型组肝组织TG、FFA含量显著升高,肝组织出现明显大泡样脂肪变性;模型组肝组织AMPK蛋白磷酸化水平降低、核蛋白SREBP-1与ChREBP表达增加、ACC蛋白磷酸化水平降低蛋白活性升高。2)祛湿化瘀方组肝组织TG、FFA含量较模型组显著降低,肝组织炎症及脂肪变性程度减轻;祛湿化瘀方能显著升高肝组织AMPK、ACC蛋白磷酸化水平、降低核蛋白SREBP-1及ChREBP含量。结论:祛湿化瘀方通过调节AMPK活性及其相关靶蛋白活性改善高脂饮食诱导的大鼠脂肪肝肝脂肪代谢,这可能是该方有效防治实验性脂肪肝的重要作用机制之一。

祛湿化瘀方对非酒精性脂肪肝患者脂质代谢、肝功能及肝纤维化的影响

目的:探讨祛湿化瘀方治疗非酒精性脂肪性肝病(NAFLD)的疗效及其对NAFLD患者血脂、肝功能及肝纤维化的影响。方法:80例NAFLD患者随机分为两组(各40例),对照组予常规治疗,观察组加用祛湿化瘀方口服,疗程3个月。在治疗前后分别行血脂(TC、TG)、肝功能(ALT、AST)及肝纤维化指标(HA、LN、PCⅢ、CⅣ)水平检测。结果:治疗后两组血脂、肝功能、肝纤维化指标均较治疗前降低(P<0.05),且观察组ALT、AST、HA、LN、PCⅢ、CⅣ改善程度明显优于对照组(P<0.05)。观察组治愈率、显效率和有效率分别为10%、55%、35%,而对照组为2.5%、32.5%和45%,观察组疗效优于对照组(P<0.05)。结论:祛湿化瘀方明显改善NAFLD患者肝功能和肝纤维化程度,能有效治疗NAFLD。