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Total Flavonoids of Aurantii Fructus Immaturus Regulate miR-5100 to Improve Constipation by Targeting Fzd2 to Alleviate Calcium Balance and Autophagy in Interstitial Cells of Cajal

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Abstract

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.

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Data Availability

The data used to support the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We are grateful for the support from the Affiliated Hospital of Southwest Medical University and the North Sichuan Medical College.

Funding

This study was supported by the Youth Fund of the National Natural Science Foundation of China (No. 82004173), Science Fund of the National Natural Science Foundation of China (No. 82074429), and the Science and Technology Research Special Project of Sichuan Provincial Administration of Traditional Chinese Medicine (No. 2020JC0063).

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  1. Yong Wen and Yu Zhan contributed equally to this work.

Authors and Affiliations

  1. Department of Traditional Chinese Medicine, the Affiliated Hospital of Southwest Medical University, Luzhou, 646000, China

    Yong Wen, Jun Li & Qing Long

  2. Anorectal Department, Affiliated Hospital of Integrative Chinese Medicine and Western Medicine of Chengdu University of TCM, Chengdu, 610042, China

    Yu Zhan

  3. Chengdu First People’s Hospital, Chengdu, 610042, China

    Yu Zhan

  4. Department of Integrated Traditional and Western Medicine Anorectal, Affiliated Hospital of North Sichuan Medical College, No. 1 Maoyuan South Road, Nanchong, Sichuan, China

    Taiyu Chen & Xuegui Tang

  5. People’s Hospital of Deyang City, Deyang, China

    Fan Zheng

  6. Universiti Sains Malaysia, Pulau Pinang, Malaysia

    Fan Zheng

  7. North Sichuan Medical College, Nanchong, 637000, China

    Shiyu Tang

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Contributions

All authors have contributed significantly to this work. YZ and YW contributed equally to this work. YZ, YW, and XGT conceived and designed the experiments; TYC, JL, and SYT performed most of the experiments; FZ and QL contributed to data collection and analysis; YZ and YW wrote the manuscript; XGT revised and edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Xuegui Tang.

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Wen, Y., Zhan, Y., Chen, T. et al. Total Flavonoids of Aurantii Fructus Immaturus Regulate miR-5100 to Improve Constipation by Targeting Fzd2 to Alleviate Calcium Balance and Autophagy in Interstitial Cells of Cajal. Mol Neurobiol (2024). https://doi.org/10.1007/s12035-024-03958-3

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