# Knockout of the C4BPA Gene Promotes Mitophagy via Activation of the Pink1/Parkin Pathway and Alleviates the Inflammatory Response by Inhibiting the NF-κB Signalling Pathway in Bovine Mammary Epithelial Cells

## TL;DR

Disabling the C4BPA gene in cow mammary cells reduces inflammation and activates a process that clears damaged mitochondria, offering new ways to combat mastitis.

## Contribution

This study reveals that C4BPA gene knockout activates mitophagy and suppresses inflammation in bovine mammary epithelial cells.

## Key findings

- C4BPA knockout reduces pro-inflammatory cytokines like TNF-α, IL-1β, and IL-6 in bovine mammary epithelial cells.
- C4BPA deficiency activates Pink1/Parkin-mediated mitophagy and suppresses NF-κB signaling, reducing inflammation.
- Knockout of C4BPA increases ROS levels and alters mitochondrial morphology, promoting mitophagy.

## Abstract

Animal health and milk quality are both adversely affected by mastitis, a frequently occurring udder condition in dairy cattle. This study focused on the bovine C4BPA gene to explore whether it could be a key to combating mastitis. We found that deactivating C4BPA in bovine mammary epithelial cells reduces inflammatory factor levels. Additionally, deactivating this gene alters mitochondrial structure and morphology and increases the levels of reactive oxygen species. These changes trigger a cellular process called mitophagy, which results in the clearance of damaged mitochondria. Most importantly, we found that this clearance process helps alleviate inflammation. In short, our research demonstrates that the C4BPA gene regulates both mitophagy and inflammation. Leveraging this mechanistic understanding, the present work proposes novel molecular targets for the prediction of bovine mastitis.

Mastitis is a prevalent disease in the dairy cattle industry and has adverse effects on dairy cows’ health and milk quality. Importantly, mastitis is associated with the inflammatory response and mitophagy. As a complement-regulatory factor, C4b-binding protein alpha (C4BPA) has been shown to modulate inflammatory factors. This study further investigates its role and mechanisms in regulating mitophagy and inflammatory responses. Following C4BPA knockout, bovine mammary epithelial cells (BMECs) exhibited reduced expression of TLR4 and key pro-inflammatory cytokines, namely the tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Electron microscopy revealed a marked increase in mitochondrial membrane rupture, as well as cristae disorder and damage and increased reactive oxygen species (ROS) levels. Moreover, Pink1 and Parkin protein levels were increased, as was LC3B lipidation (LC3B-II), whereas p62 protein expression was significantly downregulated. Immunofluorescence indicated substantially increased LC3 colocalization with mitochondria, suggesting that C4BPA gene knockout activated Pink1/Parkin-mediated mitophagy. The fact that C4BPA knockout decreased the levels of p-IκB and p-p65 while increasing those of IκBα and p65 therefore indicates its regulatory role in the NF-κB-mediated inflammatory response. Together, these findings reveal that C4BPA deficiency in BMECs not only activates Pink1/Parkin-mediated mitophagy but also suppresses the NF-κB-mediated inflammatory response. This study provides novel potential molecular targets for predicting mastitis in dairy cattle.