Division of Gastroenterology & Hepatology, VA Maryland Health Care System and University of Maryland School of Medicine, Baltimore, MD, United States.
Biochem Pharmacol. 2013 Jul 15;86(2):329-38. doi: 10.1016/j.bcp.2013.05.010. Epub 2013 May 21.
Previously, we reported that azoxymethane (AOM)-induced liver injury is robustly exacerbated in M3 muscarinic receptor (M3R)-deficient mice. We used the same mouse model to test the hypothesis that selective pharmacological modulation of M3R activity regulates the liver injury response. Initial experiments confirmed that giving a selective M3R antagonist, darifenacin, to AOM-treated mice mimicked M3R gene ablation. Compared to vehicle controls, mice treated with the M3R antagonist had reduced survival and increased liver nodularity and fibrosis. We next assessed AOM-induced liver injury in mice treated with a selective M3R agonist, pilocarpine. After pilocarpine treatment, stimulation of post-M3R signaling in the liver was evidenced by ERK and AKT activation. In contrast to the damaging effects of the M3R antagonist, administering pilocarpine to AOM-treated mice significantly attenuated hepatic stellate cell activation, collagen deposition, bile ductule proliferation, and liver fibrosis and nodularity. As anticipated from these findings, livers from pilocarpine-treated mice exhibited reduced expression of key players in fibrosis (α1 collagen, α-smooth muscle actin, TGF-β1, PGDF, TGF-β1R, PGDFR) and decreased mRNA levels for molecules that regulate extracellular matrix formation (TIMP-1, TIMP-2, MMP-2, MMP-13). Cleaved caspase-3, nitrotyrosine and BrdU immunostaining provided evidence that pilocarpine treatment reduced hepatocyte apoptosis and oxidative stress, while increasing hepatocyte proliferation. Collectively, these findings identify several downstream mechanisms whereby M3R activation ameliorates toxic liver injury. These novel observations provide a proof-of-principle that selectively stimulating M3R activation to prevent or diminish liver injury is a therapeutic strategy worthy of further investigation.
先前，我们报道了在 M3 毒蕈碱受体（M3R）缺失小鼠中，氧化偶氮甲烷（AOM）诱导的肝损伤明显加重。我们使用相同的小鼠模型来检验以下假说，即选择性调节 M3R 活性可以调节肝损伤反应。初步实验证实，给予 AOM 处理的小鼠选择性 M3R 拮抗剂达非那新可模拟 M3R 基因缺失。与载体对照相比，用 M3R 拮抗剂处理的小鼠存活率降低，肝结节和纤维化增加。接下来，我们评估了用选择性 M3R 激动剂毛果芸香碱处理 AOM 诱导的肝损伤的情况。在毛果芸香碱处理后，通过 ERK 和 AKT 激活证实了 M3R 后信号转导的刺激。与 M3R 拮抗剂的损伤作用相反，给予 AOM 处理的小鼠毛果芸香碱可显著减轻肝星状细胞激活、胶原沉积、胆管增生和肝纤维化及结节形成。从这些发现可以预期，毛果芸香碱处理的小鼠肝脏中纤维化的关键参与者（α1 胶原、α-平滑肌肌动蛋白、TGF-β1、PGDF、TGF-β1R、PGDFR）的表达减少，调节细胞外基质形成的分子（TIMP-1、TIMP-2、MMP-2、MMP-13）的 mRNA 水平降低。半胱天冬酶-3 切割、硝基酪氨酸和 BrdU 免疫染色提供了证据，表明毛果芸香碱处理可减少肝细胞凋亡和氧化应激，同时增加肝细胞增殖。总的来说，这些发现确定了 M3R 激活改善毒性肝损伤的几种下游机制。这些新的观察结果提供了一个原理证明，即选择性刺激 M3R 激活以预防或减轻肝损伤是一种值得进一步研究的治疗策略。
