Angiotensin-converting Enzyme 2(ACE2) In LPS-induced Intestinal Inflammatory Injury In Pigs And Its Molecular Mechanism

Angiotensin-converting enzyme 2(ACE2)is a key regulator of the renin angiotensin system(RAS),which has been shown to have extensive anti-inflammatory and anti-injury effects in various tissues such as heart,liver,and kidney.But in pigs,the role about intestinal inflammation is unclear.In this study,we studied the protective effect of ACE2 on inflammatory injury of porcine intestinal tract in diarrhea-preserved porcine jejunal tissue and porcine intestinal epithelial cells(IPEC-J2),and explored its related mechanisms.The research includes the following three aspects:1.Expression and correlation of Renin-Angiotensin System pathways in jejunal inflammatory injury in diarrhea-preserved pigsIn this chapter we collected jejunum tissues of some diarrhea conservation pigs The levels of inflammatory factors,AngⅡ and Ang(1-7)in jejunum were detected by ELISA.Histopathological changes were observed by pathological section.The expression levels of ACE2 and Mas gene were analyzed by RT-PCR.The method of Western-blotting was performed to detect protein expression levels of ACE2,Mas,ACE and AT1 from jejunum tissues.The results showed:1)The contents of proinflammatory cytokines including TNF-α and IL-1β in jejunal tissues of diarrhea conservation pigs were significantly higher than those in the normal group,but anti-inflammatory factor such as IL-10 had no significant different;2)The histopathology changes showed that the jejunum of diarrhea pigs had flank hair loss,thickened glands,and a large number of bleeding points.3)Compared with the normal group,the expression levels of ACE2 and Mas mRNA and protein in jejunum of diarrhea pigs decreased,and the expression levels of ACE and AT1 protein increased.The contents of AngⅡ increased and the Ang(1-7)decreased.4)The ratio of ACE2/ACE protein was lower than that of the normal group.Conclusion:The jejunum of pigs with diarrhea was proliferative enteritis.At this time,the local RAS of jejunum tissue was activated,and the expression of ACE-AngⅡ-AT1 axis was higher than that of ACE2-Ang(1-7)-Mas axis,the anti-inflammatory damage effect of ACE2 was at a disadvantage.2 Expression of angiotensin-converting enzyme 2(ACE2)in porcine intestinal epithelial cells(IPEC-J2)and its relationship with cellular inflammatory injuryIn this chapter,the relationship between ACE2 and inflammatory injury was explored by LPS-induced inflammatory injury model in porcine intestinal epithelial cells(IPEC-J2).The study included:1)The methods of western-blotting and immunofluorescence were used to determine the cytological localization and distribution of ACE2 in IPEC-J2 cells;2)Different concentrations of LPS(0.001 μg/mL,0.01 μg/mL,0.1 μg/mL,1 μg/mL,10μg/mL)was used for establishing inflammation model in IPEC-J2 cell.3)To detect the content of TNF-α,IL-1β,IL-8,IL-10,AngII and Ang(1-7)in the supernatant by LPS-induced and the expression level of other members of RAS in the cells,and to explore the relationship between RAS and LPS-induced inflammatory injury.3)The potential about ACE2 to resist inflammatory damage was verified by the AngII receptor inhibitor telmisartan and the addition of exogenous active ACE2 protein.RESULTS:1)The expression of ACE2 protein in IPEC-J2 cells was distributed in the membrane and cytoplasm.2)The inflammatory injury model of IPEC-J2 cells induced by different concentrations of LPS was successfully established,and the LPS stimulation time was determined to be 6h.It was embodied in:cell morphology changes,cell gap was not obvious,ROS in cells increased,proinflammatory factors TNF-α,IL-1(3 and IL-8 were up-regulated or significantly up-regulated,anti-inflammatory factor IL-10 was significantly down-regulated;3)ACE2,Ang(1-7)and Mas decreased in cells,ACE,AngII and AT1 increased with the increasing of LPS concentration;4)Correlation analysis found that ACE2-Ang(1-7)-Mas axis and LPS-induced pro-inflammatory factors TNF-α,IL-1β and IL-8 was negatively correlated,and had a positive correlation with the anti-inflammatory factor IL-10.The ACE-AngII-AT1 axis had an inverse correlation;5)Exogenously added ACE2 can significantly reduce the release of anti-inflammatory factors under LPS stimulation.Addition of telmisartan(AT1R inhibitor)can inhibit LPS-induced activation of the AT1 pathway.Relieves cellular inflammatory responses.The results suggest that ACE2 has the effect of resisting LPS-induced inflammatory injury of IPEC-J2 cells in a concentration-dependent manner.Its resistance may be related to the degradation of AngII and the production of Ang(1-7),which inhibits the activation of LPS-induced ACE-AngII-AT1 pathway.3 Molecular mechanism of angiotensin-converting enzyme 2 against LPS-induced inflammatory injury in porcine intestinal epithelial cellsIn the previous experiment we found that ACE2 was negatively correlated with the degree of inflammatory injury induced by LPS and had a certain anti-inflammatory effect.In order to further clarify the anti-inflammatory mechanism of ACE2,in this chapter we intended to use the CRISPR/Cas 9 gene editing technology to construct the ace2 gene targeted knockout system of porcine intestinal epithelial cells(IPEC-J2),and used ace2 knockout cells to reversely explore protective effect of ACE2 on inflammation in porcine intestinal epithelial cell,in addition,Using exogenous active ACE2 protein and its Mas receptor inhibitor(A779)to analyze changes in key protein factors of inflammatory signaling pathways such as TLR4,NF-κB and MAPK.Revealing the molecular mechanism of ACE2,which effect on anti-inflammatory damage.Results:1)The ace2 gene in IPEC-J2 cells was knocked out by the CRISPR-Cas9 system,and the ace2 deleted IPEC-J2 cell line was established.The methods of western-blotting and immunofluorescence showed that the expression of ace2 in the cells was significantly decreased after knockout,the inflammatory response in the knockout cells was aggravated,the expression of ACE2 and Mas were significantly down-regulated,TLR4 was activated,and the release of inflammatory factors increased;2)The addition of active ACE2 protein can alleviate the increase of inflammatory factor,and A779 can reverse the anti-inflammatory effect of ACE2.3)ACE2 can inhibit the phosphorylation of p65 and ERK1/2 in the NF-κB and MAPK pathways induced by LPS.This effect can also be blocked by A779.Conclusion:ACE2 degraded AngⅡ and mediated Ang(1-7)-Mas to protect inflammatory injury by LPS,which through regulating the p65 of NF-κB and ERK1/2 of MAPK pathways to decreased the release of inflammatory cytokines.In summary,all members of RAS exist in the intestine of the porcine,the two axes antagonizing each other have damage and anti-injury effects when there is external stimuli causing intestinal or cellular inflammatory damage,ACE2 mediated Ang(1-7)-Mas axis exerts an anti-injury effect and has a certain protective and repairing effect on inflammatory damage of jejunal tissue or intestinal epithelial cells.