基于斑马鱼模型研究藏药十味蒂达胶囊对代谢功能障碍相关脂肪性肝炎的干预作用及可能机制

Interventional Effects and Potential Mechanisms of Tibetan Medicine Shiwei Dida Capsules on Metabolic Dysfunction-Associated Steatohepatitis in Zebrafish

关键词:  藏药  十味蒂达胶囊  斑马鱼  MASH  脂质代谢  炎症反应

Key words:  Tibetan medicine  Shiwei Dida Capsules  metabolic dysfunction-associated steatotic liver disease  zebrafish  lipid metabolism  inflammation

摘要:

摘要：目的 基于斑马鱼模型评价藏药十味蒂达胶囊对蛋黄粉诱导代谢功能障碍相关脂肪性肝炎(Metabolic Dysfunction-Associated Steatohepatitis，MASH)的干预作用并初步探讨可能机制。方法 采用受精后5天(days post-fertilization，dpf)的野生型AB品系斑马鱼幼鱼进行最大耐受浓度测定。将斑马鱼随机分为空白组、模型组、阳性对照组及十味蒂达胶囊低、中、高剂量组，在蛋黄粉诱导的同时进行药物干预。给药后，采用整体油红O染色评价肝区脂质沉积，HE染色观察肝组织病理变化，检测ALT、AST、TC、TG、HDL-C、LDL-C水平，并采用qRT-PCR法检测PPARG、Fasn、LPL、TNF-α和IL-1β mRNA表达。结果 十味蒂达胶囊在150 μg·mL-1及以下浓度对斑马鱼幼鱼未见明显毒性。与空白组比较，模型组斑马鱼肝区油红O染色积分光密度(integrated optical density，IOD)值及ALT、AST、TC、TG、LDL-C水平和PPARG、Fasn、TNF-α、IL-1β mRNA相对表达量均升高，HDL-C水平及LPL mRNA相对表达量均降低(P<0.001)，同时可见肝细胞肿胀、组织结构紊乱及脂滴样空泡形成。与模型组比较，十味蒂达胶囊低、中、高剂量组油红O染色IOD值、ALT、AST、TC及LDL-C水平均降低，HDL-C水平升高(均P<0.05)；中、高剂量组TG水平降低(分别P<0.05、P<0.01)，低剂量组TG水平差异无统计学意义(P>0.05)。各剂量组IL-1β mRNA表达均下调(均P<0.001)，PPARG和TNF-α mRNA表达在中、高剂量组下调(均P<0.05)，Fasn mRNA表达仅在高剂量组下调(P<0.05)，LPL mRNA表达在低、中、高剂量组均上调(分别P<0.05、P<0.001、P<0.001)。结论 十味蒂达胶囊对蛋黄粉诱导的斑马鱼MASH存在明确保护作用，其作用可能与改善脂质代谢紊乱及抑制炎症反应有关。

Abstract:

ABSTRACT: OBJECTIVE To evaluate the protective effects and potential mechanisms of the Tibetan medicine Shiwei Dida Capsules against egg yolk powder-induced metabolic dysfunction-associated steatohepatitis (MASH) in a zebrafish model. METHODS Wild-type AB strain zebrafish larvae at 5 days post-fertilization (dpf) were used to determine the maximum tolerated concentration of Shiwei Dida Capsules. Zebrafish were randomly assigned to a control group, a model group, a positive control group, and low-, medium-, and high-dose Shiwei Dida Capsule groups. Drug intervention was administered concurrently with egg yolk powder induction. After treatment, lipid accumulation in the liver region was evaluated by whole-mount Oil Red O staining, and liver histopathological changes were examined by hematoxylin-eosin staining. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured. The mRNA expression levels of PPARG, Fasn, LPL, TNF-α, and IL-1β were determined by quantitative real-time polymerase chain reaction. RESULTS Shiwei Dida Capsules showed no apparent toxicity to zebrafish larvae at concentrations of 150 μg·mL-1 or lower. Compared with the control group, the model group exhibited increased integrated optical density values of Oil Red O staining in the liver region, elevated levels of ALT, AST, TC, TG, and LDL-C, and increased relative mRNA expression levels of PPARG, Fasn, TNF-α, and IL-1β, whereas HDL-C levels and relative LPL mRNA expression were decreased (P<0.001). Hepatocellular swelling, disorganization of hepatic architecture, and lipid droplet-like vacuolation were also observed. Compared with the model group, the low-, medium-, and high-dose Shiwei Dida Capsule groups showed decreased Oil Red O staining integrated optical density values and reduced levels of ALT, AST, TC, and LDL-C, together with increased HDL-C levels (all P<0.05). TG levels were reduced in the medium- and high-dose groups (P<0.05 and P<0.01, respectively), whereas no statistically significant difference was observed in the low-dose group (P>0.05). IL-1β mRNA expression was downregulated in all three dose groups (all P<0.001). PPARG and TNF-α mRNA expression was downregulated in the medium- and high-dose groups (all P<0.05), whereas Fasn mRNA expression was downregulated only in the high-dose group (P<0.05). LPL mRNA expression was upregulated in the low-, medium-, and high-dose groups (P<0.05, P<0.001, and P<0.001, respectively). CONCLUSION Shiwei Dida Capsules exert a protective effect against egg yolk powder-induced MASH in zebrafish, which may be associated with the amelioration of lipid metabolic dysregulation and the suppression of inflammatory responses.