# 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  
In the present study, the preliminary finding is that low pH reduces the viability of BRECs in the two treatment groups and causes apoptosis in ARA groups. This could be explained by endoplasmic reticulum stress and altered mitochondrial membrane potential, which induces apoptotic signaling [38]. Of the several hundred DEGs induced or repressed during pH stress in vitro, the transcription analysis showed that they were primarily associated with inflammation, cell regulation, and apoptosis, including elevated protein abundance of acidified NF-kappa B p65, IL-6, and tumor necrosis factor (TNF), which were consistent with previous studies [39]. It is worth noting that the expressions of DEGs connected with inflammation were higher at SARA pH levels than at the pH of the ARA group; this may be due to significant apoptosis in the ARA group of cells. In the SARA group, the KEGG analysis showed that pathways associated with inflammation included the MAPK signaling pathway and TNF signaling pathway. Our results showed an increased significance (\(p<0.05\)) of inducible TNF in SARA groups at the mRNA level [17]. Likewise, several inflammation-related genes, such as JUN, PKA, and MAPK3K, were also significantly upregulated; NF-kB was downregulated under pH 5.5 stress, which signals the involvement of the inflammatory signaling pathway [40; 41; 42; 43]. TNF is a proinflammatory cytokine. The goal of inflammation is to wipe out the irritating agent and stimulate tissue regeneration. An inadequate inflammatory reaction can lead to tissue damage and in severe cases, even to organ failure and death [44]. TNF receptors (TNFRs) are a subset of the TNF receptor superfamily (TNFRSF), which mostly consists of TNFR1 and TNFR2. Responses to TNFR1 stimulation may be inflammatory or apoptotic, while TNFR2 is particularly important for cell activation, migration, and proliferation [45]. TNF can activate NF-kB via the 1 kB kinase (IKK: an inhibitory protein of NF-kB kinase) complex [46]. Similarly, TNF can activate JNK via either caspase-dependent or caspase-independent pathways [47]. An essential branch of the MAPK pathway is the JNK signal transduction pathway. The JNK signal pathway is crucial for numerous physiological and pathological processes, including cell stress, apoptosis, reproduction, and the cell cycle [48]. In particular, JNK regulates the production of TNF and can regulate the inflammatory response through the expression of e-selectin [49; 50]. This shows that under in vitro conditions, BRECs can regulate inflammation through the TNF/MAPK pathway at pH 5.5. In addition, we noted that the PI3K gene was upregulated and TLR4 was downregulated in the PI3K-AKT signaling pathway. The PI3K-PKB signaling pathway can promote cell survival; activated PKB can directly phosphorylate precursor apoptotic proteins and can, in the short term, prevent activation of the apoptotic pathway, leading to cell death [51]. CCL20 was also upregulated in the SARA group compared with the control and ARA groups. CCL20, as a chemokine, can participate in the NF-kappa B and Akt signaling pathways to regulate cellular inflammation [52; 53]. However, it was significantly downregulated in the ARA group compared to the control group. This may be the result of a general breakdown of cellular homeostasis in the ARA group. Many results show that BRECs regulate their inflammatory response through other pathways in the pH 5.5 environment to maintain their survival and physiological functions. The PI3K-Akt signaling pathway and TNF signaling pathway were also enriched in the ARA group. Our results showed that the ARA group significantly induced apoptosis. In combination with the transcriptional results, data on the induced expression of genesat pH = 4.5 suggest that the cellular mechanism may not protect BRECs. To study the differences in apoptosis between the SARA and ARA groups, we examined the expressions of apoptotic genes.