Effect Of Ang-(1-7) On Hypoxia-induced PASMCs Proliferation And The Expression Of PKCα/ERK1/2 Signaling Molecules

Objective:Proficient in primary mouse PASMCs acquisition methods.To explore the effects of Ang-(1-7),on cobalt chloride-induced PASMCs in mice,and the effects of PKCα,ERK1/2 signaling molecules.Methods:1.C57 BL / 6J mice were selected,infused iron powder agarose through the right ventricle,harvested lung lobes,minced and digested,and obtained iron-containing pulmonary arteries using a magnetic separator for primary and passage cell culture.Cells were identified by immunofluorescence with smooth muscle cell-specific antibodies.2.At 24 h,48 h,and 72 h,PASMCs were treated with different concentrations of cobalt chloride(0,50,100,200,400 μmol / L),and the OD value of each experimental group was determined by CCK-8 to determine the optimal concentration of cobalt chloride on PASMCs.3.PASMCs were deled with cobalt chloride,Ang-(1-7)and its Mas receptor antagonist A779,grouped into groups: normoxia group,normoxia + Ang-(1-7)group,cobalt chloride group,cobalt chloride + Ang-(1-7)group,cobalt chloride + Ang-(1-7)+A779 group and cobalt chloride + A779 group.4.The proliferation of PASMCs in each treatment group was determined by CCK-8and flow cytometry.The expression of PKC α and ERK 1 / 2 signaling molecules was determined by qRT-PCR and Western-blot.Results:1.We were proficient in mouse primary PASMCs culture and passage techniques.Immunofluorescence using the PASMCs-specific α-SMA antibody suggested that more than 90% of the cells showed positive expression.2.CCK-8 method detected the proliferation of PASMCs after 24 h,48 h and 72 h:after 24 h,the OD value in the 50 μmol/L cobalt chloride and 100 μmol/L cobalt chloride group compared with the normoxia group(all P< 0.05);the OD value in the 50 μmol/L cobalt chloride group(P< 0.05).After 48 h and 72 h of treatment,the OD value of cobalt chloride was less than that at the same time point(all P< 0.05).When the 50 μmol/L cobalt chloride group in 24 h group was compared with the 50 μmol/L cobalt chloride group in 48 h group and 72 h group respectively,the OD value of 72 h group was less than the 24 h group(both P< 0.05),and there was no significant difference between the24 h group and 48 h group(P> 0.05).3.The proliferation of PASMCs after Ang-(1-7)and its antagonist A779 by CCK-8 assay: after 24 h of different drug groups,OD values increased in group 50,50 +Ang-(1-7),50 + Ang-(1-7)+ A779 group and 50 + A779 group compared with the normoxia group(all P< 0.05).Compared with the 50 groups,the OD increased in group50 + Ang-(1-7)+ A779,50 + A779(both P< 0.05),and there was no significant difference in the OD values of group 50 + Ang-(1-7)(P> 0.05).4.Flow cytometry of Ang-(1-7)and its antagonist A779: the proportion of S+G2cells in 50,50 + Ang-(10-7)+ A779 + A779 and 50 group compared with the normoxia group(P< 0.05),but no significant difference in normoxia + 1-7 group(P> 0.05).5.qRT-PCR for mRNA expression of PKCα after Ang-(1-7)and its antagonist:compared with the normoxia group,50,50 + Ang-(1-7)group,50 + Ang-(1-7)+ A779 +A779 group,and 50 + A 779 group(P< 0.05),but no significant difference in the normoxia + Ang-(1-7)group(P> 0.05).Compared with the 50 group,both the 50 +Ang-(1-7)+ A779 and 50 + A779 groups had increased expression(all P> 0.05),while there was no significant difference in the 50 + Ang-(1-7)group.6.Westen blot detected the molecular expression of PKCα and ERK1/2 with Ang-(1-7)and its antagonist A779:50,50,50 + Ang-(1-7)+ A779 and 50 + A779 groups(all P< 0.05);50 + A779 group compared to 50 group(P< 0.05).Compared with the normoxia group,ERK1/2 protein expression was increased among 50,50 + Ang-(1-7)and 50 +A779 groups(P< 0.05),but no significant difference in C + Ang-(1-7)group(P> 0.05).Conclusion:1.Using the magnetic separation method,the high purity of primary mouse PASMCs can be obtained,and can obtain the distal pulmonary artery,highly reproducible and economical.2.We successfully completed the chemically induced-hypoxic cell model,cobalt chloride promoted the proliferation of primary PASMCs in mice,the most suitable conditions were 24 h,50 μmol / L,and found that with the increase of action and concentration of cobalt chloride,the apoptosis of PASMCs was greater than proliferation.3.The Mas receptor antagonist A779 exacerbates the proliferation of mouse PASMCs induced by cobalt chloride,while stimulating the expression of PK Cα and ERK1/2 signaling molecules.However,no significant effect of Ang-(1-7)was found on PASMCs proliferation and the expression of PKCα and ERK1/2 signaling molecules.