| The modern nutrition,cell line models, western blot, real-time quantitative PCR(RT-PCR) and other technical means are applied in this project. Aimed to research the interrelation between Ala-Gln and the intestinal epithelia autophagy in piglets,and clarify the mediating role of mTOR/p38 MAPK signaling pathway in the intestinal epithelial cells in the process of autophagy. The results can provide a necessary theory evidence for revealing the mechanism of regulatory effect of Ala-Gln on autophagy in intestinal epithelial cells and provide a theoretical basis for the applications of Ala-Gln in the piglet feed.Experiment ?: The effect of Ala-Gln on the cell viability and autophagy of the intestinal porcine epithelial cellsUsing single factor to design experiment, and a total of six treatments were designed in this test. Culture intestinal porcine epithelial cells(IPEC-1) in vitro, adding final concentration of 0, 0.2, 0.4, 0.8, 1.6, 3.2mM of Ala-Glu, and four repeats for each treatment. MTT was used to establish IPEC-1 cell concentration standard curve, growth curve, and to detect the cell viability. When the cell confluence comes to 80%-90%, the cell samples were collected for Western blot. Results show that:(1) All groups of adding Ala-Gln, the cell survival rate were significantly greater than no adding group one(P<0.01), in addition, when adding up to 3.2mM, the cell survival rate was 51.08% higher than the control group(P<0.01), and extremely significantly higher than 0.2mM and 0.4mM addition groups(P<0.01), significantly higher than 0.8mM one.(2) The expression of LC3-? protein increased firstly and then decreased with the increasing of Ala-Gln. when the concentration of Ala-Gln was 1.6mM, the expression of LC3-? reached the maximum and significantly higher than other treatments. On the other hand, with the increasing of Ala-Gln, the expression of p62 shows a change trend from high to low, and present extremely significant different between groups(P <0.01). It can be seen that along with the increase of Ala-Gln, the aotophagy of IPEC-1 cells was gradually enhanced. To sum up all results above, Ala-Gln can improve the cell survival rate of IPEC-1 and enhance autophagy of IPEC-1 cells.Experiment ?: mTOR and p38 MAPK signaling pathway mediate the affection of Ala-Gln on piglets intestinal epithelial cells autophagyIn this test, intestinal porcine epithelial cells(IPEC-1) were cultivated, which was set as cell model in vitro. cell viability of IPEC-1 were determined by MTT,to detect the influence of different concentration of rapamycin and SB203580, which were mTOR and p38 MAPK signaling pathway specific inhibitors. Then to detect the affection of mTOR and p38 MAPK signaling pathway related proteins and autophagy related proteins by Western blot method after adding specific inhibitors, to research the affection of mTOR and p38 MAPK signaling pathway mediated Ala-Gln on the autophagy of piglets intestinal epithelial cells. Results showed that:(1) there was no difference(P >0.05) on cell vitality between the group of adding 0.11 uM Rapamycin and no adding one. With the increase the addition of Rapamycin, the cell viability declined gradually and present extremely significant different between groups(P <0.01); with the increase addition of SB203580, the cell vitality of IPEC-1 gradually reduced and present extremely significant different between groups(P<0.01).(2) After adding 1uM Rapamycin, the protein expression of mTOR(P =0.024),p-mTOR(P=0.016), p62 decreased significantly(P<0.01), and could increased the LC3-? protein expression significantly(P<0.01); With the increase of Ala-Gln concentration, protein expression of mTOR and LC3-? improves significantly(P <0.01), while protein expression of p-mTOR and p62 decreased obviously(P<0.01); Ala-Gln and Rapamycin has extremely significant interaction to protein expression of LC3-? and p62(P<0.01), and significant interaction to p-Mtor(P=0.012).(3) After adding 40 uM SB203580, there was no regularity function to p38 MAPK and LC3-? protein, while significantly reduction on p-p38MAPK(P<0.01) and significantly promotion on p62(P <0.01); With the increase of Ala-Gln concentration, the protein expression of p-p38 MAPK and LC3-? increased significantly(P<0.01), protein expression of p62 significantly decreased(P <0.01). Ala-Gln and SB203580 has extremely significant interaction to protein expression of LC3-?(P <0.01), and significant interaction to p62(P =0.034). Based on the above, mTOR signaling pathway could inhibited cell autophagy of IPEC-1, and p38 MAPK signaling pathway might promote IPEC-1 cell autophagy.Experiment ?: The impact of Ala-Gln on mTOR signaling pathways of piglets intestinal porcine epithelial cellsUsing single factor to design experiment, and a total of 6 treatments were designed in this test. To cultivate IPEC-1(piglet intestinal porcine epithelial cells) in vitro, adding final concentration of 0, 0.2, 0.4, 0.8, 1.6, 3.2mM of Ala-Gln, and three repeats for each treatment. Using Western blot, fluorescence quantitative PCR technique to detect the effect of different concentration of Ala-Gln on mTOR signaling pathways. Results show that:(1) in the mTOR signaling pathways, after adding Ala-Gln, the amount of p-mTOR protein expression significantly reduced(P<0.01).(2) After adding Ala-Gln, the amount of mRNA expression on ATG13 and ULK1 were increased significantly(P<0.01), in addition, all presented a trend of first increased and then decreased with the increase of Ala-Gln concentration. When the concentration of Ala-Gln was 1.6mM, mRNA expression was maximum. The mRNA expression on p53 and Bax improved significantly(P<0.01)with the increase of Ala-Gln concentration. In conclusion: adding Ala-Gln could inhibite the phosphorylation of mTOR. The addition of Ala-Gln could rasie the gene expression of ULK1 and ATG13 which are downstream signaling molecules of mTOR, at the same time increased the expression of gene p53 and Bax which related to mTOR pathway. In conclution,Ala-Gln affecte the signaling molecules above, and then promote autophagy of IPEC-1. |
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