树豆酮酸A可减轻db/db小鼠非酒精性脂肪肝损伤

Cajanonic Acid Alleviates Liver Injury of Nonalcoholic Fatty Liver Disease in db/db Mice

非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是指肝中储存过多的脂肪,与肥胖、胰岛素抵抗、2型糖尿病(type-2 diabetes mellitus,T2DM)、血脂异常等代谢综合征密切相关。肝纤维化是NAFLD进一步发展为肝细胞癌(hepatocellular carcinoma,HCC)的关键过程,减少肝内的脂肪积累,从而延缓或阻止NAFLD向纤维化的进展是治疗的关键。树豆酮酸A(cajanonic acid A,CAA)在2型糖尿病中能够有效改善胰岛素抵抗,发挥降糖减脂作用。本研究旨在探究CAA对NAFLD肝损伤的保护作用。通过采用db/db自发性NAFLD小鼠模型,将db/db小鼠分为NAFLD组和CAA给药组,CAA组予50 mg/(kg·d)灌胃给药,同期C57BL小鼠作为正常对照组(NC组),每组6只。小鼠生化指标检测和组织病理染色发现,CAA干预可有效缓解db/db小鼠的糖脂代谢紊乱、肝功能损伤(P<0.05)及肝脂肪变性和减少脂质沉积(P<0.05)。ELISA法结果显示,经过CAA治疗后肝组织上清液中白细胞介素1β(Interlecukin-1β,IL-1β)的水平显著降低(P<0.05),提示CAA具有明显抗炎作用,并且CAA干预显著减轻小鼠肝的纤维化程度,天狼星红染色发现,CAA组胶原沉积显著减少(P<0.05),进一步Western印迹结果表明,CAA组纤维化相关基因纤连蛋白(fibronectin,FN)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)蛋白质水平表达降低,E-钙黏着蛋白(E-cadherin,E-ca)的表达升高(P<0.05)。E2F转录因子1(E2F transcription factor 1,E2F1)被证实在肝中是葡萄糖和脂质代谢的主要因子,在肥胖小鼠中表达增加,并且可通过PPARγ的转录调节参与脂肪组织代谢,我们猜测,E2F1是CAA改善NAFLD肝脂质沉积和纤维化的中间作用靶标。通过免疫组织化学染色观察E2F1和其同家族拮抗转录因子E2F转录因子7(E2F transcription factor 7,E2F7)在小鼠经CAA处理后在肝组织核质中均表达,通过Western印迹法和实时荧光定量PCR进一步检测蛋白质和mRNA的表达发现,在CAA组中E2F1较NAFLD组基因表达降低(P<0.05),E2F7基因表达增加(P<0.05),并且spearman秩相关分析提示,E2F1和E2F7呈显著负相关(P<0.05)。以上结果表明,树豆酮酸A可有效减轻db/db小鼠非酒精性脂肪性肝损伤,改善糖脂代谢紊乱,抑制脂质过度沉积,减轻肝纤维化以缓解NAFLD进一步发展,同时E2F1和E2F7可能参与了CAA改善脂质代谢和纤维化的过程。

Objective Nonalcoholic fatty liver disease(NAFLD)is a condition in which excess fat accumulates in the liver.It is closely associated with metabolic syndromes such as obesity,insulin resistance,type-2 diabetes mellitus(T2DM),and dyslipidemia.Liver fibrosis is a key process in the further development of NAFLD into hepatocellular carcinoma(HCC).Therefore,reducing the accumulation of fat in the liver is crucial in slowing or stopping the progression of NAFLD to fibrosis and treating the condition.Cajanonic Acid A(CAA)effectively improves insulin resistance and has glucose-reducing and lipid-lowering effects in type 2 diabetes mellitus.The objective of this study was to examine the protective effect of CAA against liver injury in NAFLD.The db/db spontaneous NAFLD mouse was used and divided into two groups:the NAFLD group and the CAA administration group.The CAA group was administered 50 mg/(kg·d)of CAA by gavage.During the same period,C57BL mice were used as the normal control group(NC group),with six mice in each group.Biochemical index tests and histopathological staining in mice revealed that CAA intervention effectively alleviated glucose-lipid metabolism disorders,hepatic function impairment(P<0.05),hepatic steatosis,and reduced lipid deposition in db/db mice(P<0.05).The level of interleukin 1β(IL-1β)in the supernatant of liver tissues was significantly reduced after CAA treatment(P<0.05).The results indicate that CAA has a significant anti-inflammatory effect.Additionally,the intervention of CAA led to a significant attenuation of fibrosis in the mouse liver.Sirius red staining revealed a significant reduction in collagen deposition in the CAA group(P<0.05).Western blotting results show a decrease in the protein expression of fibronectin andα-smooth muscle actin(α-SMA),genes related to fibrosis,and an increase in the expression of E-cadherin in the CAA group(P<0.05).Previous studies have identified E2F transcription factor 1(E2F1)as a key factor in glucose and liver lipid metabolism,with increased expression in obese mice.It may also be involved in adipose tissue metabolism through transcriptional regulation of PPARγ.Our hypothesis is that E2F1 serves as an intermediate target of CAA to improve hepatic lipid deposition and fibrosis in NAFLD.Immunohistochemical staining revealed the expression of E2F1 and its homologous antagonist,E2F transcription factor 7(E2F7),in the nucleus pulposus of liver tissues of mice treated with CAA.The protein and mRNA expression levels were further detected by Western blotting and real-time fluorescence quantitative PCR.The results showed a decrease in the protein and mRNA expression level of E2F1(P<0.05)and an increase in E2F7(P<0.05)in CAA group compared to the NAFLD group.Spearman rank correlation analysis indicated a significant negative correlation(P<0.05)between E2F1 and E2F7.The results indicated that CAA can effectively improve glucose-lipid metabolism disorder in db/db mice with NAFLD.It can also inhibit lipid overdeposition and attenuate hepatic fibrosis to alleviate the further progression of NAFLD.Additionally,it is possible that E2F1 and E2F7 are involved in the process of CAA to improve lipid metabolism and fibrosis.