# In Vitro Gene Expression Responses of Bovine Rumen Epithelial Cells to Different pH Stresses
## 5 Conclusions  
In the present study, we demonstrated a method to characterize the cellular, transcriptomic, and metabolomic adaptations of BRECs to different pH stress intensities (pH = 5.5, 4.5) in vitro. The study results suggested that the pH = 4.5 treatment caused comparatively more apoptosis of BRECs, and both pH treatments (pH = 5.5, 4.5) changed the transcriptional regulation of BRECs. It was suggested that HASPA1A, TNF, and PIK3 CG regulate the inflammatory response of BRECs through the MAPK/TNF signaling pathway under pH stress. Meanwhile, differential metabolite analysis showed that 2-propyl-2,4-pentadieneoic acid and poly-g-D-glutamate were involved in the changing metabolic process. In general, the change in pH caused an inflammatory reaction of rumen epithelial cells, and the carbohydrate metabolism and amino acid metabolism also changed. The model of the BREC response to pH stress described in the current study provides a theoretical strategy to understand the inflammatory mechanisms of low-pH stress on BRECs when experiencing rumen acidosis.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/ani12192621/s1](https://www.mdpi.com/article/10.3390/ani12192621/s1)]([https://www.mdpi.com/article/10.3390/ani12192621/s1](https://www.mdpi.com/article/10.3390/ani12192621/s1)), Figure S1: Validation of RNA-Seq results by RT-qPCR; Figure S2: Protein-protein interaction (PPI) networks of DEGs significantly enriched pathways associated with BRECs function at different pH; Figure S3: Integrated analysis of the expressions of differentially expressed genes (DEGs) and metabolites in control vs. ARA; Table S1: Primers used in quantitative real-time PCR; Table S2: The significant DEGs of control vs. SARA, control vs. ARA, and SARA vs. ARA ([log2 (FC)] > 1, \(p\) < 0.05); Table S3: The significant DEGs of control vs. SARA, control vs. ARA, and SARA vs. ARA ([log2 (FC)] > 2, \(p\) < 0.01); Table S4: The significant GO terms for control vs. SARA, control vs. ARA, and SARA vs. ARA (_p_ < 0.01); Table S5: The significant KEGG pathways of control vs. SARA, control vs. ARA, and SARA vs. ARA (_p_ < 0.05); Table S6: The GO terms and KEGG analysis of 94 shared DEGs in BRECs between \"control vs. SARA\" and \"control vs. ARA\"; Table S7: The significant different metabolites of control vs. SARA, control vs. ARA, and SARA vs. ARA ([log2 (FC)] > 2, \(p\) < 0.05).  
**Author Contributions:** Conceptualization, H.L., C.Z., Y.L., and L.Z.; methodology, H.L., C.Z., Y.L., and W.L.; formal analysis, H.L., C.Z., Y.L., and L.Z.; investigation, H.L., C.Z., Y.L., and W.L.; resources, L.Z.; writing--original draft preparation, H.L., and L.Z.; writing--review and editing, H.L., T.F., and T.G.; supervision, T.F., and T.G.; funding acquisition, L.Z. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This study was financially supported by the National Natural Science Foundation of China [Grant number 31702138] and the earmarked fund for CARS36.  
**Institutional Review Board Statement:** All procedures involving animals were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agriculture University (Zhengzhou, China) (Permit Number: 12-1328; Date: 05-2021).  
**Informed Consent Statement:** Not applicable, as this research did not involve humans.  
**Data Availability Statement:** The data used in this study are available from the corresponding author on request.  
**Acknowledgments:** We acknowledge [PERSON] from the Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, for guidance and suggestions on this experiment and manuscript.  
**Conflicts of Interest:** The authors declare no conflict of interest.