# Yinchenhao Decoction Protects Against Intrahepatic Cholestasis During Pregnancy Through the miR-370-3p/TM9SF4/KIT Axis

## TL;DR

This study shows that Yinchenhao Decoction helps treat intrahepatic cholestasis during pregnancy by regulating miR-370-3p and its target genes TM9SF4 and KIT.

## Contribution

The novel finding is the identification of the miR-370-3p/TM9SF4/KIT axis as a mechanism through which Yinchenhao Decoction protects against intrahepatic cholestasis of pregnancy.

## Key findings

- Exosomal miR-370-3p is upregulated in intrahepatic cholestasis of pregnancy.
- TM9SF4 and KIT are direct targets of miR-370-3p, showing a negative regulatory relationship.
- Yinchenhao Decoction reverses the altered expression of miR-370-3p, TM9SF4, and KIT in an animal model of ICP.

## Abstract

Objective: The objective is to explore the potential pathogenesis and therapeutic mechanism of Yinchenhao decoction (YCHD) in intrahepatic cholestasis of pregnancy (ICP) by focusing on the regulatory role of exosomal miR-370-3p on target genes TM9SF4 and KIT.

Methods: Exosomes were isolated from the serum samples of normal pregnant women (control), patients with ICP, HTR-8/SVneo cells, and Sprague–Dawley (SD) pregnant rats via differential centrifugation. Characterization of these exosomes was performed using electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. Quantitative reverse transcription PCR (qRT-PCR) and the bioinformatics tool starBase were used to identify miR-370-3p as a candidate miRNA. Dual-luciferase reporter assays were used to confirm that TM9SF4 and KIT are direct targets of miR-370-3p. An in vitro ICP cell model was established using HTR-8/SVneo cells to investigate the interactions between miR-370-3p and its targets. An animal model was established to validate the targeted regulation of miR-370-3p on TM9SF4 and KIT, as well as the therapeutic effect of YCHD in vivo.

Results: The exosomal miR-370-3p expression was significantly upregulated, whereas the TM9SF4 and KIT expressions were downregulated as demonstrated by qRT-PCR and western blot analyses. RNA pull-down assays confirmed a direct negative regulatory relationship between miR-370-3p and both TM9SF4 and KIT at the molecular level. Finally, the therapeutic potential of YCHD was verified by its ability to reverse the altered expression patterns of miR-370-3p, TM9SF4, and KIT in the animal ICP model.

Conclusion: Our study demonstrates that YCHD protects against ICP through the miR-370-3p/TM9SF4/KIT axis, suggesting miR-370-3p as a potential therapeutic target for ICP.