Impact of Circadian Clock PER2 Gene Overexpression on Rumen Epithelial Cell Dynamics and VFA Transport Protein Expression

Abstract

The circadian gene PER2 is recognized for its regulatory effects on cell proliferation and lipid metabolism across various non-ruminant cells. This study investigates the influence of PER2 gene overexpression on goat rumen epithelial cells using a constructed pcDNA3.1-PER2 plasmid, assessing its impact on circadian gene expression, cell proliferation, and mRNA levels of short-chain fatty acid (SCFA) transporters, alongside genes related to lipid metabolism, cell proliferation, and apoptosis. Rumen epithelial cells were obtained every four hours from healthy dairy goats (n = 3; aged 1.5 years; average weight 45.34 ± 4.28 kg), cultured for 48 h in vitro, and segregated into control (pcDNA3.1) and overexpressed (pcDNA3.1-PER2) groups, each with four biological replicates. The study examined the potential connection between circadian rhythms and nutrient assimilation in ruminant, including cell proliferation, apoptosis, cell cycle dynamics, and antioxidant activity and the expression of circadian-related genes, VFA transporter genes and regulatory factors. The introduction of the pcDNA3.1-PER2 plasmid drastically elevated PER2 expression levels by 3471.48-fold compared to controls (p < 0.01), confirming effective overexpression. PER2 overexpression resulted in a significant increase in apoptosis rates (p < 0.05) and a notable reduction in cell proliferation at 24 and 48 h post-transfection (p < 0.05), illustrating an inhibitory effect on rumen epithelial cell growth. PER2 elevation significantly boosted the expression of CCND1, WEE1, p21, and p16 (p < 0.05) while diminishing CDK4 expression (p < 0.05). While the general expression of intracellular inflammation genes remained stable, TNF-α expression notably increased. Antioxidant marker levels (SOD, MDA, GSH-Px, CAT, and T-AOC) exhibited no significant change, suggesting no oxidative damage due to PER2 overexpression. Furthermore, PER2 overexpression significantly downregulated AE2, NHE1, MCT1, and MCT4 mRNA expressions while upregulating PAT1 and VH+ ATPase. These results suggest that PER2 overexpression impairs cell proliferation, enhances apoptosis, and modulates VFA transporter-related factors in the rumen epithelium. This study implies that the PER2 gene may regulate VFA absorption through modulation of VFA transporters in rumen epithelial cells, necessitating further research into its specific regulatory mechanisms.

Keywords: PER2 gene; circadian rhythm; rumen epithelium; the circadian clock; volatile fatty acid transporter protein.

Figure 1

Relative expression of PER2 gene in goat rumen epithelial cells. The data of each group were mean ± SEM (n = 3), ** showed significant difference (p ≤ 0.01).

Figure 2

Influence of PER2 overexpression on goat rumen epithelial cell proliferation and apoptosis. (A,B) Consequences of PER2 overexpression on goat rumen epithelial cell apoptosis; (C) consequences of PER2 overexpression on goat rumen epithelial cell proliferation. Each group’s data are represented as mean ± SEM (n = 3), ** indicates a significant difference (p ≤ 0.01), * indicates a significant difference (p ≤ 0.05).

Figure 3

Effect of PER2 overexpression on membrane damage in goat rumen epithelial cells.

Figure 4

Effect of PER2 overexpression on ROS levels in goat rumen epithelial cells.

Figure 5

Effect of PER2 overexpression on antioxidant markers in goat rumen epithelial cells.

Figure 6

(a) Construction model of overexpression vector, (b) PER2 CDS amplification results, and (c) pcDNA3.1 double enzyme cut results. M; 5 kb.