枳实-白术配伍调控AMPK/Drp1通路维持线粒体动力学平衡改善STC小鼠结肠动力障碍的作用机制

Zhi Shi-Bai Zhu Pair Ameliorates Colonic Motility Dysfunction in STC Mice via AMPK/Drp1-Mediated Mitochondrial Dynamics Regulation

关键词:  枳实  白术  慢传输型便秘  线粒体动力学  AMPK  DRP1

Key words:  Aurantii Fructus Immaturus  Atractylodis Macrocephalae Rhizoma  Slow transit constipation  Mitochondrial dynamics  AMPK  DRP1

摘要:

目的 观察枳实-白术配伍对慢传输型便秘(slow transit constipation,STC)小鼠结肠动力障碍及线粒体动力学相关蛋白的影响,探讨其治疗慢传输型便秘的潜在作用机制。方法 将56只雄性C57BL/6J小鼠按体质量随机分成正常、模型、自然恢复、枳实(2.7 g·kg-1)、白术(5.4 g·kg-1)、枳实-白术(8.1 g·kg-1)和莫沙必利组(2.5 mg·kg-1),每组各8只C57BL/6J小鼠。除正常组小鼠外,余48只小鼠均采用洛哌丁胺连续14 d灌胃(5 mg·kg-1·d-1)构建小鼠STC模型。与此同时,给药组均经灌胃给予不同药物,治疗周期 14 d,正常、模型及自然恢复组给与同体积蒸馏水灌胃。观察药物对STC模型小鼠粪便数量、含水率及小肠推进功能的影响；采用苏木精-伊红和阿尔新蓝-过碘酸雪夫染色法从微观层面观察并评估各组小鼠结肠组织的病理形态学变化。透射电子显微镜观察结肠组织超微结构；实时荧光定量聚合酶链式反应(Real-time PCR)检测小泛素相关修饰物1(SUMO1)、SUMO2、SUMO3、视神经萎缩蛋白 1(OPA1)、线粒体蛋白1(MFN1)、线粒体蛋白2(MFN2)、发动蛋白相关蛋白1(DRP1)、线粒体分裂蛋白 1(FIS1)、线粒体分裂因子(MFF)mRNA表达水平；蛋白免疫印迹法(Western blot)检测OPA1、MFN1、MFN2、DRP1、磷酸化动力相关蛋白1?(p-DRP1)、FIS1、MFF、过氧化物酶体增殖物激活受体γ辅激活子1α(PGC-1α)、腺苷酸激活蛋白激酶(AMPK)、磷酸化腺苷酸活化蛋白激酶(p-AMPK)的蛋白表达水平；同时采用免疫共沉淀技术检测Drp1的SUMO化修饰水平。结果 与正常组相比,模型组和自然恢复组小鼠的粪便数量、粪便含水率、肠道推进率均明显下降(P<0.05,P<0.01)；病理染色提示两组结肠肌层和黏膜表面层厚度显著降低,固有层及黏膜下层散在炎性细胞浸润；腺上皮细胞酸性黏液素含量减少,结肠隐窝深度变浅,细胞体积缩小,杯状细胞数量明显下降,肠绒毛排列紊乱。透射电镜下线粒体肿胀、嵴结构明显异常；部分线粒体出现基质稀疏降解及空泡化现象,自噬小体和自噬溶酶体数量显著增加；小鼠结肠组织SUMO1、SUMO2、SUMO3、OPA1、MFN1和MFN2 mRNA表达水平显著降低(P<0.01),而DRP1、FIS1和MFF mRNA表达升高明显(P<0.01)；FIS1、MFF、AMPK、p-AMPK、DRP1 和 p-DRP1蛋白表达显著升高(P<0.01)；而MFN1、MFN2、OPA1和PCG1α蛋白表达显著下降(P<0.01)。与模型组相比,白术组、枳实-白术组和莫沙必利组SUMO1、SUMO2、SUMO3、OPA1、MFN1、MFN2 mRNA水平明显升高(P<0.01),而枳实组、白术组、枳实-白术组和莫沙必利组DRP1、FIS1、MFF mRNA 明显降低(P<0.01)。枳实-白术组和莫沙必利组的FIS1、MFF、p-AMPK、DRP1 和 p-DRP1蛋白表达显著降低(P<0.05)；MFN1、MFN2、OPA1和PCG1α蛋白表达显著上升(P<0.01)。免疫共沉淀显示枳实-白术配伍对STC的治疗作用可能是通过减少Drp1的SUMO-1化修饰并增加其SUMO-2/3化修饰水平。结论 枳实-白术配伍能够显著增加STC小鼠粪便含水率,加快肠道传输功能,改善结肠病理形态和电镜下超微结构,其机制可能与抑制 AMPK/DRP1信号通路的过度激活,进而维持结肠线粒体动力学平衡相关。

Abstract:

Objective: To investigate the effects of Zhi Shi-Bai Zhu (ZS-BZ) herb pair on colonic motility dysfunction and mitochondrial dynamics-related proteins in slow transit constipation (STC) mice, and to explore its potential therapeutic mechanism. Methods: Fifty-six male C57BL/6J mice were randomly divided into seven groups (n=8 per group): normal control, model, natural recovery, ZS (2.7 g·kg?1), BZ (5.4 g·kg?1), ZS-BZ (8.1 g·kg?1), and mosapride (2.5 mg·kg?1). The STC model was established by intragastric loperamide administration (5 mg·kg?1·d?1) for 14 days, except for the normal group. Drug treatments were administered for 14 days, while the normal, model, and natural recovery groups received distilled water. Fecal pellet count, water content, and intestinal propulsion rate were measured. Colon morphology was assessed via hematoxylin-eosin and Alcian blue-periodic acid-Schiff? staining. Ultrastructural changes were observed using transmission electron microscopy. Real-time PCR quantified mRNA levels of SUMO1, SUMO2, SUMO3, OPA1, MFN1, MFN2, DRP1, FIS1, and MFF. Western blot analyzed protein expression of OPA1, MFN1, MFN2, DRP1, p-DRP1, FIS1, MFF, PGC-1α, AMPK, and p-AMPK. Moreover, coimmunoprecipitation (Co-IP) was used to detect the binding of SUMO1 and SUMO2/3 to Drp1. Results: Compared with the normal group, the model and natural recovery groups exhibited significantly reduced fecal output, water content, and intestinal propulsion rate (P<0.05, P<0.01). Pathological staining revealed thinning of the muscle layer and acidic mucin layer, decreased goblet cells, disorganized villi, and inflammatory infiltration. TEM showed mitochondrial swelling, cristae disruption, matrix degradation, and increased autophagosomes. The model group had downregulated SUMO1, SUMO2, SUMO3, OPA1, MFN1, and MFN2 mRNA (P<0.01) but upregulated DRP1, FIS1, and MFF (P<0.01). Protein analysis confirmed elevated FIS1, MFF, AMPK, p-AMPK, DRP1, and p-DRP1 (P<0.01) and reduced MFN1, MFN2, OPA1, and PGC-1α (P<0.01). The BZ, ZS-BZ, and mosapride groups reversed these trends, increasing OPA1, MFN1 and MFN2 mRNA (P<0.01) and decreasing DRP1, FIS1, MFF (P<0.01). The ZS-BZ and mosapride groups also reduced FIS1, MFF, p-AMPK, DRP1, and p-DRP1 protein (P<0.05) while upregulating MFN1, MFN2, OPA1, and PGC-1α (P<0.01). Furthermore, ZS-BZ reduced the binding of Drp1 with SUMO1, increased the binding of Drp1 with SUMO2/3. Conclusion: The ZS-BZ pair alleviates STC by improving fecal hydration, intestinal motility, and colonic pathology, potentially via modulating the AMPK/DRP1 pathway to restore mitochondrial dynamics homeostasis.