Effects Of Ang-(1-7) On The Proliferation And Expression Of PKCα/Akt Signaling Molecule In Hypoxia-induced Primary Mouse Pulmonary Artery Smooth Muscle Cells

Objective:To investigate the effect of Ang-（1-7）on hypoxia-induced mouse pulmonary artery smooth muscle cells and the expression of PKCα/Akt signaling molecule in hypoxia-induced mouse pulmonary artery smooth muscle cells.Methods:1.Choose healthy adult mice C57BL/6J,from the right ventricle to the pulmonary artery perfusion agarose solution containing iron powder,after in vitro chopped lung tissue,in combination with using the enzyme digestion method and magnetic separation process for iron pulmonary vessels in mice,put in CO₂incubator for the generation and subculture with smooth muscle cells specificity of alpha-SMA antibodies immunofluorescence staining identification of the cells.2.Mouse PASMCs were placed in a hypoxic incubator and treated with hypoxic treatment.Ang-（1-7）and MAS receptor antagonist-A779 were used for drug intervention.The experiment was divided into six groups:Normoxia group,Normoxia+Ang-（1-7）group,Hypoxia group,Hypoxia+Ang-（1-7）group,Hypoxia+Ang-（1-7）+A779 group and Hypoxia+A779 group.3.The proliferation of PASMCs was detected by CCK-8.The molecular expression of PKCα/Akt in each group was detected by immunofluorescence and Western-blot.Results:1.The primary culture of mouse pulmonary artery smooth muscle cells was successfully cultured,and the cells were successfully passed to the third generation.The immunofluorescence staining of smooth muscle cell specificα-SMA antibody confirmed that about 90%of the cells were positive.2.CCK-8 method was used to detect the proliferation of PASMCs in each group after 24h,48h and 72h hypoxia culture.The results showed that after 24 hours of hypoxia,compared with the Normoxia group,the viability of cells in Hypoxia+Ang-（1-7）+A779group and Hypoxia+A779 group was significantly improved.Compared with Hypoxia group,survival of Hypoxia+Ang-（1-7）+A779 group and Hypoxia+A779 group increased significantly;There was no statistical difference between the Hypoxia+Ang-（1-7）+A779 group and the Hypoxia+A779 group.After 48h and 72h of hypoxia,compared with Normoxia group,all hypoxia groups showed decreased cell viability.3.The protein molecular expression of PKCαand Akt in each experimental group was qualitatively analyzed by immunofluorescence.The results showed that compared with the Normoxia group,the expression of PKCαprotein molecule in the Hypoxia group and the Hypoxia+Ang-（1-7）group was decreased,and the expression of the Hypoxia+Ang-（1-7）+A779 group and the Hypoxia+A779 group was increased.However,there was no significant difference in the expression of Akt protein molecule in the Normoxia group,Normoxia+Ang-（1-7）group and Hypoxia group,while the expression of Akt protein molecule increased in the Hypoxia+Ang-（1-7）group,Hypoxia+Ang-（1-7）+A779 group and Hypoxia+A779 group.4.The molecular expression of PKCαand Akt in each experimental group was quantitatively analyzed by western blot.Compared with the Normoxia group,the expression of PKCαin Normoxia+Ang-（1-7）group,Nypoxia group and Hypoxia+Ang-（1-7）group was decreased,and the expression of PKCαin Hypoxia+Ang-（1-7）+A779 group and Hypoxia+A779 group was increased.Compared with the Hypoxia group,the expression levels of the Hypoxia+Ang-（1-7）+A779 group and the Hypoxia+A779 group were significantly increased,while the expression levels of the Hypoxia+Ang-（1-7）group were not statistically different.There was no statistical difference between Hypoxia+Ang-（1-7）+A779 group and Hypoxia+A779 group.There was no significant difference in the expression of Akt protein between the Normoxia group,the Normoxia+Ang-（1-7）group and the Hypoxia group.Compared with the Hypoxia group,the expression of Akt protein was increased in the Hypoxia+Ang-（1-7）group,the Hypoxia+Ang-（1-7）+A779 group and the Hypoxia+A779 group.Compared with the Hypoxia+Ang-（1-7）+A779 group,the expression of Akt protein in the Hypoxia+Ang-（1-7）+A779 group increased,and the expression of Akt protein in the Hypoxia+Ang-（1-7）+A779 group increased compared with the Hypoxia+Ang-（1-7）+A779group.Conclusion:1.The combination of enzyme digestion method and magnetic separation method can stably obtain high purity mouse pulmonary artery smooth muscle cells.This method is beneficial to obtain mouse distal pulmonary arteriole,easy to operate,high repeatability.2.Hypoxia may have no significant effect on the proliferation of primary PASMCs in vitro,and apoptosis may occur with the prolongation of hypoxia.Inhibition of Ang-（1-7）specific Mas receptor can promote the proliferation of PASMCs in mice under hypoxic environment,which may be realized by increasing the expression of PKCαand Akt signaling molecules.Ang-（1-7）can inhibit the expression of Akt signaling molecules during this process,but has not been found to affect the expression of PKCαsignaling molecules and cell proliferation.