Objective: To investigate the effect of berberine on programmed necrosis of hepatocytes induced by metabolic-associated fatty liver disease (MAFLD) in mice and its related molecular mechanism. Methods: Twenty male C57BL/6N mice were randomly divided into four groups (n=5 in each group): control group (S), fatty liver group (H), berberine group(B), nuclear factor erythroid 2-related factor 2 inhibitor group (Nrf2), and all-trans-retinoic acid (ATRA) group (A). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TC), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) concentrations were detected at the end of week 12 to calculate fatty liver index (liver mass/body mass ratio). Liver tissue was stained with HE, Masson and Oil Red O, and SAF score was used to evaluate the degree of liver injury. The expression levels of hepatic programmed necrosis-related proteins, namely receptor-interacting protein kinase 3 (RIPK3), phosphorylated mixed series protease-like domain (p-MLKL) and Nrf2 were detected by Western blot method. One-way ANOVA was used for intragroup comparisons and LSD-t tests were used for intergroup comparisons. Results: Compared with S group, H group serum ALT, AST, LDH, TG, TC, TNF-α, IL-1β levels and fatty liver index were significantly increased. The liver tissue was filled with vacuolar-like changes and inflammatory cell infiltration. Numerous red lipid droplets were observed with oil red O staining. Collagen fiber hyperplasia was evident with Masson staining. SAF scores (6.60 ± 0.55 and 0.80 ± 0.45) were significantly increased. The expressions of RIPK3 and p-MLKL were up-regulated. Nrf2 level was relatively increased, and the differences were statistically significant (P < 0.05). Compared with H group, berberine intervention group liver biochemical indexes, lipid levels, pro-inflammatory mediator expression, fatty liver index, and SAF score were significantly reduced, and the expression of RIPK3 and p-MLKL were down-regulated, while Nrf2 levels were further increased, and the differences were statistically significant (P<0.05). Compared with B group, treatment with Nrf2 inhibitor had antagonized the protective effect of berberine on fatty liver. Serum ALT, AST, LDH, TG, TC and TNF-α, IL-1β levels, fatty liver index, and SAF scores were significantly increased and the expressions of RIPK3 and p-MLKL were relatively increased, and the differences were statistically significant (P < 0.05). Conclusion: Berberine can significantly improve the metabolic-associated fatty liver disease injury in mice, and its mechanism is related to activation of Nrf2 and inhibition of programmed necrosis of hepatocytes.
目的： 探讨小檗碱对小鼠代谢相关脂肪性肝病（MAFLD）致肝细胞程序性坏死的影响及其相关分子机制。 方法： 20只雄性C57BL/6N小鼠随机分为4组（每组n = 5）：对照（S）组、脂肪肝（H）组、小檗碱（B）组、核因子-E2相关因子（Nrf2）抑制剂全反式维甲酸组（ATRA，A组）。12周末检测各组血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、乳酸脱氢酶（LDH）、甘油三酯（TG）、总胆固醇（TC）及肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）浓度，计算肝指数（肝质量/体质量比值）；进行肝组织HE、Masson和油红O染色，采用SAF评分评价肝损伤程度；Western blot法检测肝组织程序性坏死相关蛋白，即受体相互作用蛋白激酶3（RIPK3）、磷酸化混合系列蛋白酶样结构域（p-MLKL）及Nrf2表达水平。组间均数比较采用单因素方差分析，组间多重比较采用LSD-t检验。 结果： 与S组相比，H组血清ALT、AST、LDH、TG、TC、TNF-α、IL-1β水平及肝指数显著升高；肝组织充满空泡样改变及炎性细胞浸润，油红O染色可见大量红色脂滴，Masson染色可见胶原纤维增生明显，SAF评分（6.60±0.55与0.80±0.45）显著增高。RIPK3及p-MLKL表达上调，Nrf2水平相对增加，差异均有统计学意义（P < 0.05）。与H组相比，小檗碱干预可明显降低小鼠肝脏生化指标与血脂指标水平、促炎介质表达、肝指数及SAF评分，RIPK3和p-MLKL也表达下调，而Nrf2水平进一步增加，差异均有统计学意义（P < 0.05）。与B组相比，给予Nrf2抑制剂处理可拮抗小檗碱对脂肪肝的保护效应，血清ALT、AST、LDH、TG、TC、TNF-α、IL-1β水平及肝指数显著升高，SAF评分升高，RIPK3和p-MLKL表达相对增加，差异均有统计学意义（P < 0.05）。 结论： 小檗碱可明显改善小鼠代谢相关脂肪性肝病损伤，其机制与激活Nrf2、抑制肝细胞程序性坏死有关。.
Keywords: Berberine; Metabolic associated fatty liver disease; Necroptosis; Nuclear-factor erythroid 2-related factor 2.