从"激活PPARγ-调节Th17/Treg平衡"的角度阐明积雪草主要有效成分抑制溃疡性结肠炎的机制

Centella asiatica (L.) Urb. herbs, possessing efficacies of clearing away heat evil and promoting diuresis, can be used for the treatment of intestinal tract diseases resulting from wetness-heat in large intestine, such as ulcerative colitis (UC). Previously, we demonstrated that the saponin fraction from C. asiatica herbs (SFC) could significantly attenuate UC induced by dextran sulfate sodium (DSS) in mice, and increase the expression of peroxisome proliferator-activated receptor gamma (PPARγ) in colon tissues of mice. Madecassoside, one of the major constituents in SFC, could regulate cytokines produced by Th17 cells and regulatory T (Treg) cells in the intestine of rats with arthritis. In this study, we compare the inhibitory effects of the four major constituents in SFC on DSS-induced UC in mice, and identify the main active constituent. Depending on the effects on the differentiation and function of Th17 and Treg cells in colon tissues of mice, the mechanisms for this active constituent regulating Th17/Treg imbalance in UC can be elucidated. Moreover, the enhancing effects of this active constituent on expression and activation of PPARγ in both Th17 and Treg cells are observed by in vivo and in vitro studies. The close relationship between its activation of PPARγ and regulation of Th17/Treg balance and consequent anti-UC action is confirmed by using specific antagonists and RNA interference technique. Overall, this study should reveal the underlying mechanisms of the main active constituent in SFC and provide evidence for its development as an anti-UC drug. Meanwhile, it will offer a paradigm for the recognization of anti-UC mechanisms of traditional Chinese medicines.

积雪草具有清热利湿的功效，可用于治疗溃疡性结肠炎（UC）等以大肠湿热为主的肠道疾病。前期研究发现积雪草总皂苷灌胃给药可显著改善DSS诱导的小鼠UC，升高结肠组织PPARγ表达，其成分羟基积雪草苷可调节关节炎大鼠肠道Th17和Treg细胞因子。本项目在DSS模型小鼠，比较研究总皂苷四种主要成分的抗UC作用，明确主要有效成分。探索该成分对Th17、Treg细胞分化和功能的不同作用，揭示其调节UC小鼠结肠Th17/Treg失衡的原理和分子机制；体内外研究有效成分对结肠Th17和Treg细胞中PPARγ表达或活化的促进作用，采用特异性拮抗剂和RNA干扰等手段，揭示其激活PPARγ与调节Th17/Treg平衡及抗UC作用的紧密相关性。从"激活PPARγ-下调Th17/Treg比值-抑制免疫性炎症"的全新角度，阐明积雪草有效成分抗UC的机制，为其开发应用打下基础，同时为中药治疗UC机制的研究提供借鉴。

积雪草具有清热利湿的功效，可用于治疗溃疡性结肠炎（UC）等以大肠湿热为主的肠道疾病。本项目在DSS模型小鼠，探索积雪草改善UC的主要有效成分，阐明作用机制，为其进一步开发利用奠定基础。.（1）探明积雪草抑制DSS诱导小鼠结肠炎的主要活性成分和效应形式：比较研究积雪草主要三萜成分的抗结肠炎作用，发现羟基积雪草苷和羟基积雪草酸分别强于积雪草苷和积雪草酸；研究羟基积雪草苷和羟基积雪草酸的口服药动学/组织分布，并比较口服和直肠给药后的药效差异。证实羟基积雪草苷是积雪草抑制结肠炎的主要活性成分，羟基积雪草酸为其效应形式。.（2）从恢复Th17/Treg平衡角度，阐明羟基积雪草酸改善结肠炎的机制：在DSS所致结肠炎小鼠，羟基积雪草酸明显减少结肠部位Th17细胞数目，增加Treg细胞数目，而对Th1和Th2细胞没有明显影响；轻度降低结肠组织中Th17细胞特征性转录因子RORγt表达，显著升高Treg细胞特征性转录因子Foxp3表达，但不影响Th1和Th2细胞转录因子表达，表明羟基积雪草酸的抗结肠炎效应与改善Th17/Treg失衡状态相关。有趣的是，羟基积雪草酸并非通过调控经典的分化途径影响Th17和Treg细胞的分化和功能，其机制与促进Th17细胞向Treg细胞转换密切相关。.（3）揭示了羟基积雪草酸促进 Th17/Treg表型转换的机制：羟基积雪草酸显著抑制结肠炎小鼠结肠部位和Th17细胞分化条件下ACC1表达、促进AMPK活化。采用阻断剂及siRNA干扰等技术，证实其通过激活AMPK，抑制ACC1介导的脂肪酸合成，促进Th17/Treg细胞表型转换。此外，经EMSA和荧光素酶报告基因分析等，发现羟基积雪草酸为PPARγ选择性激动剂，其上述作用经由该受体介导。.（4）羟基积雪草酸激活PPARγ与调节Th17/Treg平衡、改善结肠炎的内在联系：在DSS 诱导的结肠炎小鼠，将PPARγ阻断剂GW9662与羟基积雪草酸联用，进一步证实羟基积雪草酸通过激动PPARγ，促进AMPK活化，抑制ACC1表达，促进Th17/Treg细胞表型转换，呈现抗结肠炎效应。 .综上，本课题从“激活PPARγ—恢复Th17/Treg平衡—抑制免疫性炎症”的全新角度，揭示了积雪草改善溃疡性结肠炎的有效成分和作用机制，为其开发利用提供了依据。