| In this study, three experiments are included:1. Effects of concentrate diet on apoptosis and autophagy in rumen epithelial of goat; 2. Effects of starvation on goat rumen epithelium apoptosis and autophagy; 3. Effects of pH and SCFA on primitive cultured goat rumen epithelial cells apoptosis and autophagy in vitro.1 Effects of dietary concentrate level on apoptosis and autophagy in rumen epithelial of goatFifteen young crossbred goats (Boer x Yangtze River Delta White)with permanent rumen fistula were divided into high concentrate group (HC group, n= 9) and high forage group (HF group, n= 6), all of the goats were fed in the single sheepfold, water were avaliable at any time, the goats in two groups were fed at 8:00 and 17:00 every day respectively. After 94 days feeding, all the goats were slaughtered for sampling.After the goats were all slautered, it was found that the final body weight, empty weight of rumen and 5×5cm2 weight of rumen in HC group were significantly higher than those in HF group (P<0.05); The pH of rumen fluid in HC group was significantly lower than that in HF group (P< 0.05);The SCFA in the rumen fluid were detected by gas chromatography, it was found that the concentration of TSCFA, acetate, propionate, butyrate, isobutyrate, isovalerate and valerate of rumen fluid in HC group were significantly higher than those in HF group (P<0.01);Using Real-time Q-PCR method to detect mRNA expression levels of genes related to apoptosis such as p53,Bcl-2,Bax,Caspase3 and Caspase9, it was found that the p53、Bcl-2、Bax、Caspase3、and Caspase9 mRNA expression levels of rumen epithelium in HC group were significantly higher than those in HF group (P< 0.05), the value of Bcl-2/Bax had no significant difference between the two groups (P>0.05); Using Real-time Q-PCR method to detect mRNA expression levels of genes related to autophagy such as Beclinl and Bcl-2, it was found that the Bcl-2 mRNA expression level of rumen epithelium in HC group were significantly higher than that in HF group (P< 0.05), the Beclinl mRNA expression level of rumen epithelial had no significant difference between the two groups (P>0.05); Using TUNEL apoptosis detection, it was found that the apoptotic index of rumen epithelium in HC group were significantly higher than those in HF group (P< 0.05).In summary, Increasing dietary concentrate levels induced apoptosis in rumen epithelial cells of goats,and at the same time increasing dietary concentrate level inhibited autophagy in rumen epithelium cells.2 Effects of starvation on autophagy and apoptosis in rumen epithelial of goatThirteen young crossbred goats (Boer × Yangtze River Delta White)with permanent rumen fistula were divided into no starvation group (NS group,n= 4),short starvation group (SS group,n= 4) and long starvation group (LS group,n= 5),all of the goats were fed in the single sheepfold, peanut stems and water were avaliable at any time.The goats in three groups were fed with 100 grams concentrate at 8:00,11:00,14:00 and 17:00 every day respectively, after 42 days feeding, all of the goats were slaughtered for sampling.The pH of rumen fluid in SS group and LS group was significantly higher than that in NS group (P<0.05), the pH of rumen fluid in LS group was significantly higher than that in SS group (P< 0.05);The SCFA in the rumen fluid were detected by gas chromatography, it was found that the concentration of TSCFA,acetate, propionate and butyrate of rumen fluid in SS group and LS group were significantly lower than those in NS group (P<0.05), the concentration of TSCFA and butyrate of rumen fluid in LS group were significantly lower than those in SS group (P<0.05), the concentration of acetate and propionate of rumen fluid had no significant difference between LS group and SS group (P>0.05);Using transmission electron microscope, it was found that under the condition of starvation some phenomenon happened in rumen epithelial cells,such as the nuclear morphological abnormalities, the mitochondria mild swelling, scattered autophagic vacuoles in the cytoplasm,and so on.Using Real-time Q-PCR method to detect mRNA expression levels of genes related to autophagy such as Beclinl and Bcl-2, it was found that the Beclinl mRNA expression level of rumen epithelium in SS group was significantly higher than that in NS group (P< 0.05), the Bcl-2 mRNA expression level of rumen epithelium had no significant difference between SS group and NS group (P>0.05); The Beclinl mRNA expression level of rumen epithelium in LS group were significantly higher than that in NS group (P< 0.05), but the Bcl-2 mRNA expression level of rumen epithelium was significantly lower than that in NS group (P< 0.05); The Bcl-2 mRNA expression level of rumen epithelium in LS group were significantly lower than that in SS group (P< 0.05), the Beclinl mRNA expression level of rumen epithelium had no significant difference between LS group and SS group (P>0.05); Using Real-time Q-PCR method to detect mRNA expression levels of genes related to apoptosis such as p53, Bcl-2, Bax, Caspase3 and Caspase9, it was found that the Bax mRNA expression levels of rumen epithelium in SS group was significantly higher than that in NS group (P< 0.05), but the value of Bcl-2/Bax was significantly lower than that in NS group (P< 0.05),at the same time, the p53、Bcl-2、Caspase3 and Caspase9 mRNA expression levels of rumen epithelium had no significant difference between SS group and NS group (P>0.05); The p53 mRNA expression levels of rumen epithelium in LS group was significantly higher than that in NS group (P< 0.05), but the Bcl-2 mRNA expression levels of rumen epithelium was significantly lower than that in NS group (P< 0.05),at the same time, the Bax、Caspase3 and Caspase9 mRNA expression levels of rumen epithelium and the value of Bcl-2/Bax had no significant difference between LS group and NS group (P>0.05); The Bcl-2 and Bax mRNA expression levels of rumen epithelium in LS group were significantly lower than those in SS group (P< 0.05), but the value of Bcl-2/Bax was significantly higher than that in SS group (P< 0.05),at the same time, the p53、Caspase3 and Caspase9 mRNA expression levels of rumen epithelium had no significant difference between LS group and SS group (P>0.05);Protein expression of LC3,ERK, pERK, AKT and pAKT was analyzed by western blot, it was foud that two clear stripes LC3-Ⅰ and LC3-Ⅱ appeared in LS group, while one clear stripe LC3-Ⅰ and one fuzzy stripe LC3-Ⅱ appeared in SS group, at the same time, only one stripe LC3-Ⅰ appeared in NS group; The ratio of pERK/ERK in SS group and LS group was significantly higher than that in NS group (P< 0.05),but the ratio of pERK/ERK had no significant difference between LS group and SS group (P>0.05); The ratio of pAKT/AKT in SS group and LS group was significantly lower than that in NS group (P< 0.05),but the ratio of pAKT/AKT had no significant difference between LS group and SS group (P>0.05); In summary, starvation promoted apoptosis in rumen epithelial cells of goats,and at the same time starvation also induced autophagy in rumen epithelium cells.3 Effects of pH and SCFA on apoptosis and autophagy in primitive cultured goat rumen epithelial cells in vitroPrimitive cultured goat rumen epithelial cells were divided into 8 groups:The first group was added in pH 7.4 DMEM (n=4),the second group was added in pH 7.4+20 mM SCFA DMEM (n=4), the third group was added in pH 7.4+40 mM SCFA DMEM (n= 2), the fourth group was added in pH 6.8 DMEM (n=4), the fifth group was added in pH 6.8+20 mM SCFA DMEM (n=4), the sixth group was added in pH 6.8+40 mM SCFA DMEM (n=2), the seventh group was added in pH 6.4 DMEM (n= 4), the eighth group was added in pH 6.4+20 mMSCFA DMEM (n=4).The cells were cultured for 24 hours after that treat, and then collected.Using Real-time Q-PCR method to detect mRNA expression levels of genes related to apoptosis such as p53,Bcl-2,Bax,Caspase3 and Caspase9, it was found that low pH induced apoptosis in rumen epithelial cells, while high SCFA inhibited apoptosis in rumen epithelial cells; Using Real-time Q-PCR method to detect mRNA expression levels of genes related to autophagy such as Beclinl and Bcl-2, it was found that low pH inhibited autophagy in rumen epithelial cells, and high SCFA inhibited autophagy in rumen epithelial cells. |
PDF Full Text Request