163. In Vitro Analysis of Menthol-Mediated Responses in Bovine Ruminal Epithelial Cells with Heat Stress

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Abstract This study aimed to determine the concentration and temporal dependence of menthol on cell viability and transcriptome changes in bovine rumen epithelial cells (BREC1) under heat stress conditions. Heat stress disrupts cell cycle regulation, prompting a multifaceted cellular response to mitigate damage and maintain tissue integrity. Menthol, a cooling essential oil, is recognized for its anti-inflammatory and antioxidant properties. Cell viability and proliferation was determined in BREC1 cultured with DMEM (Con), 40 µM menthol (Low), or 1,000 µM (High) menthol for 8 and 24 hr. Similarly, differentially expressed genes (DEGs) through transcriptomic analysis was performed on Con, Low, or High treated cells cultured for 8 and 24 hr at 37 °C (thermal neutral) and 41 °C (heat stress). Cell viability was increased (P < 0.05) at Low_8 hr vs. High_8 hr. Interestingly, a significant interaction between menthol concentration, treatment period, and temperature was observed in cell proliferation (P < 0.05). After 8 hr, High_41 °C showed increased cell proliferation compared with Low_37 °C and Con_37 °C (P < 0.05). Additionally, at 41 °C, Low_8 hr showed significantly higher proliferation than Con_8 hr (P < 0.05). Heat stress had a significant impact on BREC1 DEGs across cellular senescence pathways (cell cycle arrest in response to stress), resulting in TP53 upregulation (padj < 0.05) in Con_41 °C vs. Con_37 °C after both 8 and 24 hr. Heath shock proteins were increased in Con_41 °C compared with Con_37 °C at both 8 and 24 hr (padj < 0.05). A larger number of DEGs were observed in heat stressed cells treated with menthol for 8 hr versus 24 hr. After 8 hr of treatment, SLC7A11 (cellular antioxidant defense), and CLCC1 (protects cells from stress) were increased in the High_41 °C vs. Con_41 °C (padj < 0.05). Neither gene was differentially expressed between Low_41 °C and Con_41 °C. Moreover, expression of TRPC1 (store-dependent and store-independent Ca2+ entry), two fatty acid transporters (SLC27A5, SLC27A3), and TCAF1 (positively regulates cold sensing receptor TRPM8) was increased (padj < 0.05) in High_41 °C vs. High_37 °C. Similarly, expression of TRPC2 and SLC27A5 and TCAF1 was increased (padj < 0.05) in Low_41 °C vs. Low_37 °C. In contrast, after 24 hr, GPX genes (antioxidants) were increased (padj < 0.05) in Low_41 °C versus Con_41 °C. Interestingly, TRPV4 (Ca2+ influx), TCAF1, and SLC27A4 genes were significantly upregulated (padj < 0.05) in High_41 °C and Low_41 °C vs. High_37 °C and Low_37 °C, respectively. In conclusion, menthol is a cost-effective essential oil that can enhance the viability and proliferation of ruminal cells under heat stress through modulating stress responses, antioxidant activity, and Ca2+ influx signaling pathways. These characteristics could prove favorable for the cattle industry by increasing production and profitability.

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