Mechanical Stretching Of Pulmonary Vein Stimulates Expression Of Inflammatory Factor In Pulmonary Hypertension Due To Left Heart Disease In Rats

Objective:In this study,the activation of Mitogen-activation protein kinase(MAPKs)pathway and expressions of inflammatory factors ilterluekin-6(IL-6)and tumor necrosis factor-α(TNF-α)were observed in pulmonary hypertension due to left heart disease(PH-LHD)rat model,to explore the the changes of pulmonary vein in the early stage of PH-LHD and mechanism of pulmonary vascular remodeling caused by pulmonary vein mechanical stretch.At the same time,primary cells of pulmonary vein smooth muscle(PVSMCs)were cultured with modified pulmonary vein extraction to establish a basis for subsequent cell level studies.Methods:A total of 66 SD rats(3-4 weeks old,weight 80-100 g)were randomly divided into the sham group(S1-S3,n=6),PH-LHD group(including M1-M7 group,n=6)and PVSMCs group(M8 group,n=6).The S1-S3 group only exposed the ascending aorta without banding procedure,while M1-M8 group received Banding precedure to build the PH-LHD model.On day 25 or 35 after surgery,pulmonary vein,pulmonary artery and lung tissue samples were prepared.1.The pulmonary vein of S1,S2 were given a mechanical strength of 0 g or 2.0 g for 60 minutes.2.The pulmonary vein of M1,M2 were given a mechanical strength of 0 g or 2.0 g for60 minutes.3.The pulmonary vein of M3-M6 were pretreated with MAPKs inhibitors for 60minutes before given a mechanical strength of 2.0 g for 60 minutes.4.The pulmonary veins of M8 were isolated guided by intravenous puncture needle trocar.Primary pulmonary vein smooth muscle cells(PVSMCs)were cultured by tissue block adherent method,and the cells were purified by differential adherent method for20 minutes.Activation of MAPKs pathway and expressions of IL-6 and TNF-α in serum or pulmonary vein were detected by ELISA,Western blot,HE staining and immunohistochemistry(IHC).The purity of PVSMCs was determined by IHC.Results:1.Establishment of PH-LHD rat model(1)Twenty-five days after operation,the rats in the PH-LHD group showed different degrees of left heart failure by echocardiography.(2)Compared with sham group,rats in the PH-LHD group had increased left heart weight,right ventricular pressure and left atrial pressure.(3)The results of lung tissue HE staining showed no significant difference between the sham and the PH-LHD group.2.Comparison of IL-6 and TNF-α expressions in pulmonary vein,pulmonary artery and lung tissues of PH-LHD rats in the early stage of disease(1)Twenty-five days after operation,ELISA results showed that IL-6 and TNF-α expressions in serum of the PH-LHD group was increased than sham group.(2)On day 25 after operation,the results of lung tissue IHC showed that IL-6 and TNF-α expressions around pulmonary vein was significantly higher than that around pulmonary artery and lung tissue,but there were no such results in the sham group.The results of pulmonary vein IHC showed that the expressions of IL-6 and TNF-α in the PH-LHD group were significantly higher than sham group.On day 35 after operation,the results of lung tissue IHC showed that expressions of IL-6 and TNF-α were increased in pulmonary vein,pulmonary tissue and pulmonary artery.(3)On day 25 after operation,the results of pulmonary veins Western blot showed that the expressions of IL-6 and TNF-α in the PH-LHD group were higher than pulmonary arteries and lung tissues.On day 35 after operation,Western blot results showed expressions of IL-6 and TNF-α were increased in pulmonary vein,pulmonary tissue and pulmonary artery.3.In the early stage of PH-LHD rats,the expressions of IL-6 and TNF-α in pulmonary veins were increased and was antagonized by SAC/MAPKs signaling pathway inhibitors.(1)The results of pulmonary veins Western blot showed no significant difference in the expressions of IL-6 and TNF-α in S1 and S2.On day 25 after operation,the expressions of IL-6 and TNF-α in M1 were significantly higher than S1 and S2,which was lower than M2.(2)The results of pulmonary veins Western blot showed that the expressions of IL-6 and TNF-α in M3-M6 were decreased than M2.4.Mechanical stretch induced increased expression of MAPKs signaling pathway protein in pulmonary vein of PH-LHD rats,which was antagonized by SAC/MAPKs pathway inhibitors.(1)The results of pulmonary veins Western blot showed no significant difference in the expressions of p-p38,p-JNK1 and p-ERK1/2 in S1 and S2.The expressions of p-p38,p-JNK1 and p-ERK1/2 in M1 were significantly higher than S1 and S2,which was lower than M2.(2)The results of pulmonary veins Western blot showed expressions of p-p38,p-JNK1 and p-ERK1/2 in M3,M4,M5 and M6 were significantly decreased than M2.5.Primary extraction of pulmonary vein smooth muscle cells.PVSMCs were cultured by tissue block adherent method.PVSMCs appeared in one or two days.IHC results showed that the third generation of PVSMCs had complete cell morphology and clearly visible cell nuclei.The α-SMA positive rate of smooth muscle cells was more than 90%,which showed cell purity was relatively high.Conclusion:1.In the early stage of PH-LHD,increasing of IL-6 and TNF-α expressions were occurred in pulmonary vein,which were earlier than pulmonary artery and lung tissues,can be antagonized by SAC/MAPKs signaling pathway inhibitors.At the same time,the activation of MAPKs pathway was occurred in pulmonary vein.In the PH-LHD rat model,mechanical stretch of pulmonary vein mediates the increased expressions of inflammatory cytokines IL-6 and TNF-α through SAC/MAPKs pathway,which is involved in the formation of pulmonary hypertension.2.When culture Primary PVSMCs,the difficulty of pulmonary vein isolated can be reduced by using intravenous needle trocar as a guidance,and PVSMCs with strong proliferation ability and high purity can be obtained with differential adhesion method.