Abstract: The aim of this study was to investigate the effect of L-Arginine (L-Arg) on the proliferation and apoptosis of bovine intestinal epithelial cells injured by heat stress. The bovine primary intestinal epithelial cells cultured in vitro were divided into control group (Con group, 37℃) and experimental groups (after being cultured at 42℃ for 6 h, the medium without L-Arg (HS group) or the medium with different concentrations (2, 4, 6, 8, 10 mmol·L^-1) of L-Arg were changed to culture at 37℃ for 12 h). The cell viability was detected by CCK-8 and the best healing concentration of L-Arginine was screened. Then the third generation bovine intestinal epithelial cells with good condition were divided into control group (Con group, cultured at 37℃), heat stress group (HS group, cultured at 42℃ for 6 h and then cultured at 37℃ for 12 h) and 6 mmol·L^-1 L-Arg group (42℃ for 6 h and then replaced with 6 mmol·L^-1 L-Arg medium at 37℃ for 12 h). The morphological changes of cells were observed by DAPI staining, the leakage of lactate dehydrogenase (LDH) was detected by micro method, and the cell cycle was detected by flow cytometry. The relative expression levels of cysteine proteolytic enzyme-3 (Caspase-3), cysteine aspartate proteolytic enzyme-9 (Caspase-9), Bcl-2-associated X protein (Bax) and B lymphocyte tumor-2 (Bcl-2) mRNA and protein were detected by real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA) to evaluate the effect of L-Arg on the proliferation and apoptosis of bovine intestinal epithelial cells injured by heat stress. The results showed that in the range of 2~6 mmol·L^-1, compared with HS group, the activity of intestinal epithelial cells was increased by adding L-Arg under heat stress and the activity was the highest when the concentration of L-Arg was 6 mmol·L^-1(P<0.05), which could be used as the experimental concentration of L-Arg group in the follow-up experiment. Compared with Con group, the nuclear morphology of HS group was irregular, chromatin was condensed, the leakage of LDH was significantly increased (P<0.05), the ratio of cells at S stage was significantly decreased (P<0.05), the relative expression levels of Bax, Caspase-9, Caspase-3 mRNA and protein were significantly increased(P<0.05), while the protein content of Bcl-2 was significantly decreased in HS group (P<0.05). These results indicated that heat stress promoted the apoptosis of intestinal epithelial cells in dairy cows. Compared with HS group, the nuclear morphology was regular and the leakage of LDH in intestinal epithelial cells of dairy cows treated with 6 mmol·L^-1 L-Arg were significantly decreased (P<0.05), the S stage cell ratio increased (P<0.05), and the relative expression levels of Bax, Caspase-3, Caspase-9 mRNA and protein were significantly decreased(P<0.05), while the relative expression levels of Bcl-2 mRNA and prfotein were significantly increased (P<0.05). It is concluded that L-Arg can promote the proliferation of small intestinal epithelial cells injured by heat stress, and reduce cell apoptosis thus alleviate the damage of heat stress on intestinal epithelial cells of dairy cows in vitro.

Key words: L-Arginine, dairy cows, heat stress, intestinal epithelial cell, apoptosis