Department of Traditional Chinese Medicine, the Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China.
Anorectal Department, Affiliated Hospital of Integrative Chinese Medicine and Western Medicine of Chengdu University of TCM, Chengdu, 610042, China.
Mol Neurobiol. 2024 Aug;61(8):5882-5900. doi: 10.1007/s12035-024-03958-3. Epub 2024 Jan 20.
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.
枳实总黄酮（AFIF）是从枳实中提取的主要有效部位，对促进胃肠动力有很好的作用。本研究旨在探讨 AFIF 通过靶向卷曲蛋白 2（Fzd2）调节 miR-5100 改善便秘症状的作用，以减轻间质细胞 Cajal（ICCs）钙离子平衡和自噬凋亡。通过抗生素混悬液诱导便秘小鼠模型，然后用 AFIF 进行处理。本研究应用了 RNA-seq 测序、荧光素酶测定、免疫荧光染色、透射电镜、ELISA、流式细胞术、实时定量聚合酶链反应（PCR）和 Western blot。结果表明，AFIF 改善了抗生素诱导的便秘小鼠的便秘症状，降低了 ICCs 中的自噬相关蛋白 Beclin1 水平和 LC3-II/I 比值。miR-5100 和其靶基因 Fzd2 被筛选为与自噬相关的关键 miRNA 和调节剂。下调 miR-5100 导致 Fzd2 表达增加，ICCs 增殖活性降低，凋亡细胞增加，钙离子释放和自噬信号增强。经 AFIF 处理后，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 增加了 SP、5-HT 和 VIP 的水平，增加了 PGP9.5、Sy 和 Cx43 的表达，通过改善肠神经网络的完整性缓解了便秘。综上所述，AFIF 可通过上调 miR-5100 的表达并靶向抑制 Fzd2，缓解 ICCs 钙离子超载和自噬性死亡，调节神经递质含量，增强肠神经网络的完整性，从而减轻便秘症状。
