ABSTRACT
The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.
Subject(s)
Animals , Humans , Chlorocebus aethiops , Interleukin-6/genetics , Janus Kinase 2/pharmacology , Infectious bronchitis virus/metabolism , STAT3 Transcription Factor/metabolism , Angiotensin-Converting Enzyme 2/pharmacology , Cytokine Receptor gp130/metabolism , Vero Cells , Signal Transduction , Inflammation , RNA, MessengerABSTRACT
This article aimed at exploring the effects and protective mechanism of β-CM7 on renin angiotensin system (RAS) in diabetic rats myocardial tissue. We divided 32 male SD rats into 4 groups: control group, diabetic model control group, insulin (3.7x10(-8) mol/d) treatment group and β-CM7 (7.5x10(-8) mol/d) treatment group. After 30 days, all rats were decapitated and myocardical tissues were collected immediately. After injection, β-CM7 could decrease the content of Ang II, increase the content of Angl-7. And β-CM7 could improve the mRNA of AT1 receptor and Mas receptor. β-CM7 also could improve the mRNA of ACE and ACE2, enhance the activity of ACE and ACE2. These data confirmed tli β-CM7 could activate ACE2-Angl-7-Mas axis, negative passage in RAS, to inhibit the expression ACE mnRiJA and protein in rat myocardium, alleviate the myocardial tissue damage induced by Ang II. The effect of β-CM7 on inhibiting myocardium damage might be related to ACE/ACE2 passageway.
Subject(s)
Animals , Male , Rats , Angiotensin II , Metabolism , Diabetes Mellitus, Experimental , Drug Therapy , Diabetic Cardiomyopathies , Drug Therapy , Endorphins , Pharmacology , Myocardium , Metabolism , Pathology , Peptide Fragments , Pharmacology , Peptidyl-Dipeptidase A , Metabolism , RNA, Messenger , Rats, Sprague-Dawley , Receptor, Angiotensin, Type 1 , Metabolism , Receptors, G-Protein-Coupled , Metabolism , Renin-Angiotensin SystemABSTRACT
New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.
Subject(s)
Animals , Rabbits , Blotting, Western , DNA Primers , Ducks , Virology , Electrophoresis, Polyacrylamide Gel , Immune Sera , Parvovirus , Polymerase Chain Reaction , Recombinant Proteins , Genetics , Viral Proteins , GeneticsABSTRACT
Objective: The aim of the present study was to evaluate the stability of native bovine ? casomorphin 7 in duodenum of adult rats, and the local effect on expression of cholecystokinin in intestinal mucosa. Method: (1) According to the variety of infused ? casomorphin 7 concentrations between absorptive and un-absorptive groups in vivo, its characteristic absorption in duodenum was observed. (2) To characterize the stability of the ? casomorphin 7, rat intestinal mucosa homogenate was digested and the enzyme activities were determined in vitro. (3) The effect on cholecystokinin mRNA expression of ? casomorphin 7 after luminal administration was analyzed by RT-PCR. Results:? casomorphin 7 was unable to be absorbed by intestinal epithelia and quickly degraded in the intestinal tract. It contributed to elevate cholecystokinin mRNA expression in intestinal mucosa and this effect was partly blocked by naloxone. This peptide also significantly inhibited the somatostatin mRNA expression. Conclusion: The present data demonstrated that the local effect on expression of cholecystokinin of ? casomorphin 7 might be related to opioid system and directly or indirectly affected by somatostatin.