The Function Of Autophagy And Synthetic Secretion Of Pulmonary Artery Smooth Muscle Cells In Pulmonary Vascular Remodeling In COPD

Objective: 1.Establish COPD rat model,use autophagy enhancer as a tool medicine,detect the expression of autophagy related parameters(LC3-??LC3-?and Beclin-1)in COPD rat pulmonary artery smooth muscle cells and the parameters of pulmonary vascular remodeling,analysis the relationship between COPD rats PASMCs autophagy and pulmonary vascular remodeling;2.Separat the primitive PASMCs,use autophagy enhancer and autophagy inhibitors as tools,detect the expression of PASMCs autophagy related gene and the change of the synthesis of secretion(IL-6?PDGF-AB),analyse the relationship between PASMCs autophagy and its synthetic secretion.Methods :The first part: The relationship between PASMCs autophagy and pulmonary vascular remodeling in COPD rats.(1)60 SD rats were randomly included in the control group,COPD group,COPD+Rapamycin(autophagy enhancer)intervention group,COPD+ 3-Methyladenine(autophagy inhibitor)intervention group,COPD+Rapamycin solution control group,COPD+3-Methyladenine solution control group,10 rats in each group.The COPD rat model was established according to the traditional cigarette smoke exposure and the injection of lipopolysaccharide in the airway.(2)Extract the rat lung tissue of each group above on the ultra clean bench,pulmonary vascular remodeling index is calculated after sanlian dyeing: calculate the ratio between muscle change type lung small artery blood wall thickness and the vascular diameter(WT %),the ratio between vascular wall area and total area(WA%);use immunofluorescence method to detect the expression quantity of LC3-?,LC3-? and Beclin 1 in PASMCs,correlation analysis to analyze the relationship between autophagy marker gene expression and pulmonary vascular remodeling.The second part: The relationship between PASMCs autophagy and its synthetic secretion in COPD rats induced by LPS.1.The model of COPD rats was established by injecting LPS into the airway and cigarette smoke exposure,then isolate and cultivate the original PASMCs in COPD rats,and the fourth and fifth generation PASMCs were used for follow-up study.2.The experiment was divided into six groups:(1)the control group: no intervention agents were added;(2)LPS group: culture after adding LPS(final concentration is 1 ug/ml)(3)3-MA + LPS groups: 3-MA pretreatment for 30 minutes(final concentration is 5mmol/l)and then culture after adding LPS(final concentration of 1ug/ml)(4)Rapamycin + LPS group: Rapamycin pretreatment for 30 minutes(final concentration of 5ug/L)and then culture after adding LPS(final concentration of 1ug/ml)(5)3-MA individual treatment groups: culture after adding 3-MA(final concentration is 5 mmol/l)(6)Rapamycin individual treatment groups: culture after adding Rapamycin(final concentration is 5?g/L),each group above do 4 holes,repeat the experiment for 2 times.After culturing for 48 hours,collect cells or cell culture supernatant of each group to detect the following indicators:(1)Western Blot was used to detec PASMCs autophagy marker protein LC3-?,LC3-? and protein expression of Beclin1;The enzyme linked immunosorbent assay(ELISA)method was used to detect the changes of IL-6,TGF-?1and PDGF in each group.Correlation analysis was used to analyze the correlation between autophagy and synthetic secretory cytokines.Results: The first part: 1,the expression of LC3-?,LC3-? and Beclin1 in COPD rat pulmonary artery smooth muscle: COPD group,COPD+ Rapamycin intervention group,COPD+3-MA intervention group compared with normal control group the expression of LC3-and Beclin 1 and ? the ratio of LC3-?/LC3-?increased(P < 0.05);COPD+Rapamycin intervention group compared with COPD group the expression of LC3-? and Beclin 1 and the ratio of LC3-?/LC3-?increased(P < 0.05),COPD+ 3-MA intervention group compared with COPD group the expression of LC3-? and Beclin 1 and the ratio of LC3-?/LC3-?decreased(P < 0.05)2.The thickness of the arterial wall of the muscular pulmonary arterial artery was compared with that of the blood vessel(WT%),the wall area of the vessel and the total area of blood vessels(WA%): COPD group,COPD+Rapamycin intervention group,COPD+3-MA intervention group were increased compared with normal control group.COPD+Rapamycin intervention group increased compared with COPD group,and the COPD+3-MA intervention group group decreased compared with LPS group;3.Correlation analysis suggested that the correlation between PASMCs autophagy and pulmonary vascular remodeling in COPD rats is exist(All have R value >0.8,P < 0.05);The second part: 1.The expression quantity of PASMCs autophagy related genes LC3-?,LC3-?and Beclin 1 in rats induced by LPS: LPS group,Rapamycin+LPS group and LPS+3-MA group compared with normal control group the expression of LC3-? and Beclin 1 and the ratio of LC3-?/LC3-?increased(P < 0.05);.Rapamycin+LPS group increased compared with LPS group,and the LPS+ 3-MA group decreased compared with LPS group;2.Changes of synthesis secretion in rats PASMCs induced by LPS: LPS group,Rapamycin+LPS group and LPS+3-MA were increased compared with normal control group.The LPS+ 3-MA group decreased compared with LPS group.3.Correlation analysis suggested that there was a correlation between PASMCs autophagy and its synthetic secretion function in rats induced by LPS(All have R value >0.8,P < 0.05).Conclusions: 1.The correlation between autophagy and pulmonary vascular remodeling of pulmonary arterial smooth muscle cells in COPD rats was confirmed by using autophagy regulators as a tool in integral animal experiment.2.Using autophagy regulators as a tool,through cell experiments,it was proved that lps-induced PASMCs autophagy was related to its synthetic secretion function.3.PASMCs may regulate its synthetic secretion function to participate in pulmonary vascular remodeling by changing the autophagy level.