# In Vitro Gene Expression Responses of Bovine Rumen Epithelial Cells to Different pH Stresses
## 4 Discussion
### Effect of pH Stresses on BREC Transcription and Metabolism
It is worth noting that the FAS expression of the ARA group was higher than that of the SARA group. FAS is a transmembrane protein and binding FASL can initiate transduction of the apoptosis signal and cause apoptosis [54]. Studies have shown that upregulation of apoptotic genes could lead to disruption of the potential of the mitochondrial transmembrane, resulting in cytochrome c release and leading to apoptosis induction [55]. Acidosis in the extracellular environment can induce cell death through a pathogenic mechanism mediated by the opening of ROS and the mitochondrial permeability transition pore (mPTP) [56]. Similar to the results of our study, an abundance of proapoptotic response-related proteins (e.g., cytochrome c, BAX, and caspase-3) was upregulated in the rumen in a low-pH environment [39]. Data on the induced expression of apoptotic genes at pH = 4.5 suggest that the cellular mechanism may not protect BRECs against pH-induced apoptosis, while the rate of apoptosis decreased at pH = 5.5 due to upregulation of PI3K; the HSP90 mRNA levels likely helped BRECs activate self-protection mechanisms. Therefore, we suggest that BRECs mediate inflammation through the TNF/MAPK pathway and resist external stimulation in a low-pH environment. Our results agree with a previous report in which the TNF/MAPK pathway at low pH could play a crucial role in inflammation [57]. In addition, the results of 94 shared DEGs observed both in the \"control vs. SARA\" and \"control vs. ARA\" groups showed that cytochrome P450 was also affected by the low-pH treatment. In our study, CYP1A1 was significantly upregulated in the control vs. ARA group and tended to increase in the control vs. SARA group, while CYP26A1 was downregulated in both groups. CYP1A1 is a member of the cytochrome P450 subfamily. Previously, it was reported from clinical research that the P450 subfamily was involved in tumor deterioration and related diseases, and it was also found that the P450 enzyme has certain activity changes in the pathogenesis of many inflammatory diseases [58]. CYP1A1 was downregulated in hepatocytes in an LPS model in vitro [59]. Based on this confirmation, we postulate that regulation of CYP1A1 may indeed participate in the inflammatory response. The transcriptional expression profile of the RNA-Seq analysis in the current study was verified by the RT-qPCR analysis. The metabolome data further showed that the control group had significant metabolic differences compared with the SARA and ARA groups. The drug metabolism-cytochrome P450 pathway was enriched simultaneously. Although there are few studies about drug metabolism involving the cytochrome P450 pathway in BRECs, studies on the CYP2 gene upstream of its pathway show that it can participate in the I3K/Akt/mTOR signaling pathway [60], which is consistent with the transcriptome results. Hence, we speculate that the functional involvement of the P450 family may be one of the mechanisms of inflammation induced by low pH, although the detailed mechanism needs to be further studied. In addition, we found that the differential metabolites in the two groups were mainly concentrated in lipid compounds. Fatty acids can regulate lipid metabolism and immune response in the rumen epithelium [61]. The aforementioned findings thus support the crucial function of the rumen epithelium's metabolic adaption to low pH.