Effects Of Dietary Energy Intake On Growth Of Rumen Epithelium And Its Underlying Mechanism In Goats

It is widely accepted that energy-rich diets promote the growth of rumen epithelium, however the mechanisms underlying are not clear. The study systemically studied the effects of energy-rich diets on the growth of rumen epithelium. The underlying cellular and molecular mechanisms were also discussed in this paper.1Effect of dietary energy level on growth of rumen epithelium in goatsIn order to study the effect of dietary energy level on rumen epithelium growth of goats, eighteen young crossbred goats (Boer×Yangtze River Delta White,120d, BW14.27±0.69kg) were used in this study. Goats were randomly allocated to two, high energy level (HL, n=9, ME:1.00MJ/(kg0.75·d)) and low energy level (LL, n=9, ME:0.60MJ/(kg0.75·d)). The empty weights of goats were determined on each Monday morning. After42d feeding period, goats were slaughtered and rumen epithelium samples were collected. Surface area of rumen papilla was calculated by sliding caliper; Section of rumen papillae was stained with hematoxylin-eosin (HE) for morphological determination; Protein, DNA, and RNA content of rumen epithelium was detected by biochemical analytical technique; Cell cycle was analyzed by flow cytometry; Gene expression was determined by Real-time Q-PCR; Protein expression was determined by western blot.The results showed that:(1) The average daily gain (ADG)(P<0.05), empty weight of rumen (P<0.05), length and surface of rumen papillae (P<0.05) were significantly increased in the HL group than in the LL group.(2) The number of substratum spinosum (SS) and stratum granulosum (SG) in HL group was significantly higher than in LL group (P<0.05), while the cell size of rumen epithelium was decreased in response of high energy level of diet (P<0.05), demonstrating that energy-rich diet promoting growth of rumen epithelium through cell proliferation rather than hypertrophy.(3) The results of cell cycle measured by flow cytometry showed that compared with LL group the percentage of cell number in G1-phase was lower (P<0.05), while the S-phase (the phase of DNA synthesis)(P<0.05) and the G2/M-phase (the phase of cell mitosis)(P<0.05) was higher in HL group, further indicating that energy-rich diet accelerated cell cycle progression and promoted rumen epithelial cells proliferation.(4) The mRNA abundance of cyclin D1was about32%higher in HL group than that in LL group (P<0.05). Both the protein expression of cyclin D1(P<0.05) and CDK4(P<0.05) was significant higher in HL than those in LL group, suggesting that energy-rich diet promoted rumen epithelial cell proliferation through accelerating cell cycle progression associated with increment of cyclin D1and CDK4expression.In summary, enegy-rich diet increased the expression of cyclin D1of rumen epithelium, accelerated cell cycle and the growth of rumen, and promoted cell proliferation.2Mechanisms study of diet energy level on growth of rumen epithelium of goatsThe study was taken on both in vivo and in vitro to study the mechanisms of diet energy level on growth of rumen epithelium of goats. Animal experiments and sample collection methods were the same with series one. Before the experiment, on14d and35d, blood samples were collected via jugular venipuncture respectively. Serum IGF-1concentration was measured by radioimmunoassay. Protein expression of ERK, pERK, AKT and pAKT was analyzed by western blot.The results in vivo showed that:(1) Serum IGF-1concentration was significant higher in the HL group than that in the LL group at each time point (P<0.05), showed that energy-rich diet promoting growth of rumen epithelium was associated with the increase of serum IGF-1concentration.(2) In vivo the pERK/ERK ratio in HL group was significantly higher than that in LL group (P<0.05), indicating that the energy-rich diet promoted phosphorylation of ERK. In vivo pERK protein was not detected.Rumen papillae were taken from the ventral blind sac of goat of healthy young goats (n=4) after slaughter, and were digested by fractional trypsinization and incubated at37℃癈with5%CO2for24h to allow attachment. The attached cells were treated by IGF-1, IGF-1R inhibitor, ERK inhibitor Ⅱ, and AKT inhibitor Ⅳ for8h in24-well plate and for24h in25ml cell culture flasks. Proliferation of ruminal epithelial cell in culture was assessed using [3H] thymidine (3H-TdR) incorporation. Gene expression was determined by Real-time Q-PCR; Protein expression was determined by western blot.The results in vitro showed that: (1) In vitro the IGF-1administration (25μg/L) markedly enhanced the3H-TdR incorporating into the DNA of rumen epithelial cells. And the effect was inhibited by IGF-1R inhibitor, indicating that IGF-1promoted rumen epithelial cell proliferation due to its binding with IGF-1R.(2) In vitro IGF-1significantly increased cyclin D1(P<0.05) but not CDK4(P>0.05) gene and protein expression of rumen epithelial cells. Additionally, the enhanced cyclin D1protein expression was inhibited by IGF-1R inhibitor. It was suggested that IGF-1up-regulated cyclin D1-CDK4assembly by enhancing cyclin D1expression, which induced acceleration of cell cycle progression, resulting in cell proliferation of rumen epithelium.(3) In vitro the IGF-1administration markedly swelled the pERK/ERK ratio of rumen epithelial cells (P<0.05). Additionally, the enhanced cyclin D1protein expression was inhibited by ERK inhibitor (P<0.05). However, there was no effect of either IGF-1or ERK inhibitor on CDK protein expression. It indicated that in response to energy-rich diet, the up-regulated IGF-1enhanced cyclin D1protein expression through Ras/Raf/MEK/ERK signaling pathway.(4) There was no pAKT protein expression detected in rumen epithelial cells in vitro. But the enhacned cyclin D1protein expression was inhibited by AKT inhibitor (P<0.05). It was suggested that PI3K/AKT signaling pathway might concerned with up-regulated cyclin D1protein expression of rumen epithelial cells by IGF-1. But the mechanism was not clear.In summary, enegy-rich diet increased the expression of cyclin D1of rumen epithelium, accelerated cell cycle and the growth of rumen, and promoted cell proliferation. Furthermore, the study also demonstrated the effect of energy-rich diet on growth of rumen epithelium was mediate by IGF-1throuth Ras/Raf/MEK/ERK signaling pathway.