# Neferine Ameliorates Slow-Transmitting Constipation by Inducing PINK1/Parkin-Mediated Mitophagy in Protective Enteric Glial Cells

## TL;DR

Neferine helps treat slow-transmitting constipation by promoting mitophagy in gut nerve support cells.

## Contribution

Shows neferine improves constipation via PINK1/Parkin-mediated mitophagy in enteric glial cells.

## Key findings

- Neferine reduced inflammation and cell damage in constipated rats.
- Neferine increased mitophagy markers like PINK1, Parkin, and LC3II/I.
- PINK1 silencing blocked neferine's protective effects on mitochondrial health.

## Abstract

The enteric glial cells (EGCs) are the main components of the enteric nervous system (ENS) and contribute to the development of slow transit constipation (STC). In this study, we aimed to explore the effects of neferine (Nef) on EGCs based on PINK1/Parkin-mediated mitophagy. In vivo, 7 days of loperamide feeding was conducted to model STC rats, which were then treated with 2.5, 5, 10 mg/kg/d Nef, and 2 mg/kg/d mosapride for 14 days. In vitro, a CCK-8 assay was performed to detect EGC viability. EGCs were then stimulated by 400 μM H2O2, transfected with si-PINK1, and treated with Nef or mitochondrial division inhibitor 1 (Mdivi-1). Colon tissue was observed by H&E staining, TEM, ELISA (to quantify SOD, MDA, GDNF, and NGF expression), and immunofluorescence (to count the number of mitochondria). In addition, flow cytometry was used to quantify cell apoptosis, ROS, and mitochondrial membrane potential (MMP). Finally, the p62, PINK1, Parkin, and LC3II/I expression levels were measured by western blotting. Nef was shown to significantly improve STC in rats and reduce mucosal epithelial cell loss, inflammatory cell infiltration, and fibrous proliferation. Moreover, Nef reduced ROS and MDA levels while increasing SOD, GDNF, and NGF. Nef treatment also increased the LC3II/I ratio, as well as p62, PINK1, and Parkin expression, which helped mitigate mitochondrial expansion. However, PINK1 silencing shared the same function as Mdivi-1 in the STC+Nef group, inhibiting EGC viability, oxidative stress, and PINK1/Parkin signaling activation. Additionally, mitophagy was exacerbated by si-PINK1 in the STC+Nef group EGCs. In short, Nef ameliorates STC by inducing PINK1/Parkin-mediated mitophagy in EGCs.