黄芪总黄酮(AFIF)是从AFI中提取的主要有效组分,对促进胃肠蠕动有很好的作用。本研究旨在探讨AFIF调控miR-5100通过靶向Frizzled-2(Fzd2)减轻Cajal间质细胞(ICCs)钙离子平衡和自噬凋亡改善小鼠便秘症状的作用。用抗生素悬液诱导小鼠模型,然后用AFIF治疗。RNA-seq测序,荧光素酶测定,免疫荧光染色,透射电子显微镜,ELISA,流式细胞术,定量聚合酶链反应(PCR),本研究采用Westernblot。结果表明,AFIF改善了抗生素诱导的便秘小鼠的便秘症状,并降低ICCs中自噬相关蛋白Beclin1水平和LC3-II/I比值。miR-5100及其靶基因Fzd2被筛选为与自噬相关的关键miRNA和调控因子。miR-5100下调导致Fzd2表达增加,ICCs增殖活性降低,凋亡细胞增加,增强钙离子释放和自噬信号。AFIF治疗后,miR-5100表达上调,Fzd2下调,自噬相关蛋白水平和钙离子浓度降低。此外,AFIF提高了SP的水平,5-HT,VIP,并增加了PGP9.5,Sy,和Cx43,通过改善肠神经系统网络的完整性来缓解便秘。总之,AFIF可以通过上调miR-5100的表达和靶向抑制Fzd2来减轻便秘症状,减轻ICCs的钙超载和自噬死亡。调节神经递质的含量,增强肠神经系统网络的完整性。
Aurantii Fructus Immaturus total flavonoids (AFIF) is the main effective fraction extracted from AFI, which has a good effect on promoting gastrointestinal motility. This study aimed to investigate AFIF which regulates miR-5100 to improve constipation symptoms in mice by targeting Frizzled-2 (
Fzd2) to alleviate interstitial cells of Cajal (ICCs) calcium ion balance and autophagy apoptosis. The constipated mouse model was induced by an antibiotic suspension, and then treated with AFIF. RNA-seq sequencing, luciferase assay, immunofluorescence staining, transmission electron microscopy, ELISA, flow cytometry, quantitative polymerase chain reaction (PCR), and Western blot were applied in this study. The results showed that AFIF improved constipation symptoms in antibiotic-induced constipated mice, and decreased the autophagy-related protein Beclin1 levels and the LC3-II/I ratio in ICCs. miR-5100 and its target gene
Fzd2 were screened as key miRNAs and regulator associated with autophagy. Downregulation of miR-5100 caused increased expression of
Fzd2, decreased proliferation activity of ICCs, increased apoptotic cells, and enhanced calcium ion release and autophagy signals. After AFIF treatment, miR-5100 expression was upregulated and
Fzd2 was downregulated, while autophagy-related protein levels and calcium ion concentration decreased. Furthermore, AFIF increased the levels of SP, 5-HT, and VIP, and increased the expression of PGP9.5, Sy, and Cx43, which alleviated constipation by improving the integrity of the enteric nervous system network. In conclusion, AFIF could attenuate constipation symptoms by upregulating the expression of miR-5100 and targeting inhibition of
Fzd2, alleviating calcium overload and autophagic death of ICCs, regulating the content of neurotransmitters, and enhancing the integrity of the enteric nervous system network.