| This study focused on the effects of ACE2 in the gastrointestinal of weaned piglets.The research consists of three parts:1 The Gene clone of Angiotensin-converting enzyme 2 (ACE2) and its gene expression in weaned pigletsThis study tried to figure out the distribution of ACE2 gene in healthy weaned piglets by cloning the ACE2 gene so as to understand the cellular localization of ACE2 and to lay a theoretical foundation for further study. In this study, all tissue samples collected from the heart, liver, spleen, lung, kidney gastrointestinal tract and brain of 35-day-old weaned piglets and performed the following tests:1) Determining the sequence of ACE2 gene segment by cloning and amplification and doing phylogenetic analysis; 2) Using RT-PCR to determine the expression of ACE2 gene in different kinds of tissues and organs of the weaned piglets; 3) Using immunohistochemical methods to determine the cellular localization of ACE2 protein in these tissues. Results:1) The ACE2 gene segment was successfully cloned. By comparing this ACE2 gene sequence with others on GenBank, we found that the ACE2 gene in weaned piglets had a 98% homology with that in Sus scrofa, a 87.8% homology with that in Bos Taurus, a 78.8% homology with that in human and a low homology with that in Gallus gallus. Phylogenetic analysis showed that piglets had the shortest genetic distance to Bos Taurus and was in a totally different branch with Gallus gallus. These two analyses are consistent.2) RT-PCR showed that the mRNA of ACE2 widely existed in piglets and had a higher level of expression in kidney, stomach, ileum, duodenum, jejunum.3) Gastrointestinal immunohistochemical results showed that ACE2 protein was expressed in the whole gastrointestinal tract, which is especially notable at the bottom of the stomach and in the brush edge parts of intestinal epithelial cells.2 The comparison of the expression of ACE2 in the gastrointestinal tracts of pigletsThe previous research showed that ACE2 expressed in various tissues in piglets and had a higher expression especially in kidney and gastrointestinal. To further investigate the possible roles of ACE2 in piglets’gastrointestinal system, our study selected healthy newborn Sujiang piglets and randomly divided them into two equal groups. Both groups were weaned off on the 21st day after their birth. The group I were executed immediately on that day; the group II were fed for another two weeks and then executed on the 35th day. After execution both groups’gastrointestinal(gastric body, fundic, jejunum, ileum, duodenum, colon) tissue samples were collected and then we performed the following tests:1) Determining of the expression of ACE2 gene in all the tissues above using real-time PCR; 2) Analyzing the differences between the expression of ACE2 protein in gastric fundic and that in jejunum tissues using western blot methods; 3) Determining the RNA/DNA values in gastric fundic and jejunum tissues so as to analyze the relationship between the ratio and the age of piglets; 4) Correlation analysis between ACE2 protein expression and the RNA/DNA ratio in order to analyze the varying trend of ACE2 protein as piglets grow. Results:1) Real-time PCR showed that the mRNA of ACE2 widely existed in piglets gastric fundic, gastric body, jejunum, ileum, duodenum and colon. With the growth of piglets, the expression of ACE2 gene significantly increased (0.31±0.12 VS 0.87±0.15, P<0.05) in the gastric fundic; it has no difference in gastric body(0.31±0.07 VS 0.30±0.11, P> 0.05); in intestinal it increased significantly (0.73±0.15 VS 1.63±0.21, P<0.05) 2) Western blot results showed that ACE2 protein expressed both in gastric fundic and jejunum and with the growth of piglets, its expression increased significantly (P<0.05); 3) The ratio of RNA/DNA in the gastric fundic and jejunum increased with the growth of piglets (1.47±0.21 VS 1.61±0.18,2.17±0.26 VS 3.02±0.15); 4) Correlation analysis showed that:ACE2 protein and the ratio of RNA/DNA were positively correlated in the gastric fundic and jejunum (P<0.05). In other words, the RNA/DNA ratio and ACE2 protein both increase as piglets grow. These results suggested that ACE2 may play an important role in the development of physiological and pathological conditions in piglet gastrointestinal.3 The effect of ACE2 on the secretion of gastric acid in piglets and its possible mechanismIn the previous studies, we found that ACE2 expressed in the gastrointestinal tracts of piglets at different ages and with the growth of the piglets, the expression of ACE2 in gastric fundic and jejunum significantly increased. In order to study the role and possible mechanism of ACE2 in the secretion of gastric acid, we selected healthy newborn Sujiang piglets and randomly divided them into two equal groups. Both groups were weaned off on the 21st day after their birth. The group I were executed immediately on that day; the group II were fed for another two weeks and then executed on the 35th day. After execution both groups’gastrointestinal fundic and blood tissue samples were collected and then we performed the following tests:1) Determining the pH of gastric chime, the serum gastrin content and the enzyme activities of various enzyme including H+-K+-ATP, Na+-K+-ATP and pepsin; 2) Using real-time PCR and Western blot methods to analyzing quantitatively the expression of ACE2 mRNA and protein in the gastric fundic tissues; 3) analyzing quantitatively the expression level of ACE, ACE2, AT1, AT2 and Mas mRNA and determining the AngⅡ content in the gastric fundic tissues. Results:1) With the growth of piglets, the pH of gastric chyme decreased from 4.42±0.07 to 3.65±0.06. The serum gastrin content increased significantly (P<0.01). The enzyme activity of H+-K+-ATP in group II was significantly higher than that in Group I (P<0.05)and the enzyme activity of pepsin and Na+-K+-ATP had no significant differences between the two groups (P> 0.05); 2) The ACE2 mRNA and protein were expressed in gastric fundic and with the growth of the piglets, the expression level significantly increased (P<0.05). Correlation analysis found that ACE2 and gastrin content and H+-K+-ATP activity were positively correlated with each other. Among them, the correlation between ACE2 and H+-K+-ATP was very significant (P<0.05);3) All components of local RAS in gastrin fundic were found. With the growth of the piglets:the expression of ACE, AT1 and AT2 mRNAs did not vary significantly (P> 0.05); AngⅡ content decreased slightly; The mRNA expression of Mas, the receptor of ACE2, increased; the ratios of ACE/ACE2 and ATI/Mas significantly decreased (P<0.05). Conclusion:All members of the local RAS were expressed in gastric fundic. The ACE2-Mas pathway predominated in the secretion of gastric acid. The mechanism may involve increasing the enzyme activity of H-K+-ATP enzyme and promoting the release of gastrin. |
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