| Objective1 To study the correlation between the levels of VEGF,PI3K,AKT,HIF-1?,D-D and T cell subsets and the syndrome types in the blood of IPF patients with qi-deficiency and blood-stasis and qi-yin deficiency.2 To study the effect of Shenqi cordyceps sinensis containing serum on PMVECs migration and PI3K/AKT/HIF-1?signaling pathway.Methods1Clinical study:30 IPF patients with qi-yin deficiency syndrome and qi-yin deficiency and blood-stasis syndrome were collected,30 healthy subjects were taken as the normal group,and the expression levels of VEGF,PI3K,AKT,HIF-1?,D-D and T cell subgroup in the blood of the three groups were collected and analyzed to compare the correlation between the groups.2 In vitro cell experiments:(1)Twenty SD male rats were randomly divided into two groups,and the model was made by one-time intratracheal infusion of bleomycin.After twenty eight days,lung tissue was taken and isolated and cultured into PMVECs.Normal saline was used instead of normal saline in the blank group.(2)Forty SD male rats were randomly divided into two groups.The experimental group was given Shenqi Cordyceps Sinensis Recipe every day,twice a day for three consecutive days.Blood was collected from the abdominal aorta and serum was separated under sterileconditions one hour after the last gavage.The blank group was replaced with normal saline.(3)MTT method was used to detect the effects of different concentrations of drug-containing serum on the proliferation of PMVECs,and the optimal concentration was selected for the experiment.(4)Transwell method was used to detect the migration ability of PMVECs,and Western blot method was used to detect the expression levels of PI3K,AKT,HIF-1?and VEGF proteins in PMVECs at the optimal concentration ofdrug-containing serum.Results1 Clinical research1.1D-D content in IPF patients is significantly higher than normal people(P<0.01).The content of D-D in qi-deficiency and blood-stasis group was higher than that in qi-yin deficiency group(P<0.05).1.2 The expression levels of CD3~+T,CD4~+T and CD4~+/CD8~+T in the two groups of IPF decreased significantly compared with the normal group(P<0.01),while the expression levels of CD8~+T in the two groups of IPF increased significantly compared with the normal group(P<0.01).There was no difference in the expression levels of CD3~+T,CD4~+T,CD8~+T,and CD4~+/CD8~+T in the qi-deficient blood-stasis group and the qi-yin deficiency group(P>0.05).1.3 The serum levels of PI3K,AKT,HIF-1?and VEGF in IPF patients were higher than those in normal patients(P<0.01).The content of PI3K in qi-deficiency and blood-stasis group was higher than that in qi-yin deficiency group(P<0.05).Compared with the qi-yin deficiency group,the levels of AKT,HIF-1?and VEGF in the qi-deficiency and blood-stasis group were significantly increased(P<0.01).2.1 MTT results:The cell activity of the model group was significantly enhanced compared with that of the blank group(P<0.01).Compared with the model group,there was no difference in cell viability in the 5%drug-containing serum group(P>0.05),and the cell viability decreased in the 10%and 20%drug-containing serum groups(P<0.05).Compared with the 5%serum group,the cell viability of the 10%and 20%drug-containing serum groups decreased(P<0.05).The cell activity of 10%drug-containing serum group was lower than that of 20%drug-containing serum group(P<0.05).2.2 Migration ability:Compared with the blank group,the number of migrated cells in the model group increased significantly(P<0.01).Compared with the model group,the number of migrating cells in the 10%drug-containing serum group,LY294002 group and LY294002+10%drug-containing serum group significantly decreased(P<0.01).The number of migrated cells in LY294002 group and LY294002+10%drug-containing serum group was significantly reduced compared with the 10%drug-containing serum group(P<0.01).There was no significant difference between LY294002 group and LY294002+10%drug-containing serum group(P>0.05).2.3Expression of VEGF protein in PMVECs:There was a certain amount of basic expression of VEGF in PMVECs of rats in the blank group.Compared with the blank group,the expression level of VEGF in the model group was significantly increased(P<0.01).Compared with the model group,the expression of VEGF in the 10%drug-containing serum group and the LY294002+10%drug-containing serum group decreased significantly(P<0.01).There was no significant difference in LY294002group(P>0.05).Compared with the 10%drug-containing serum group,the expression level of VEGF in the LY294002 group increased significantly(P<0.01),and there was no difference in the expression level of VEGF in the LY294002+10%drug-containing serum group(P>0.05).Compared with the LY294002 group,the expression level of VEGF in the LY294002+10%drug-containing serum group decreased significantly(P<0.01).2.4Expression of PI3K protein in PMVECs:There was a certain amount of basic expression of PI3K in PMVECs of rats in the blank group,and the expression of PI3K in the model group was increased compared with that in the blank group(P<0.01).Compared with the model group,the expression level of PI3K in the 10%drug-containing serum group,LY294002 group and LY294002+10%drug-containing serum group all decreased(P<0.01).Compared with the 10%drug-containing serum group,the expression level of PI3K in LY294002 group and LY294002+10%drug-containing serum group both decreased(P<0.05).2.5Expression of AKT in PMVECs:There was a certain amount of basic expression of AKT in PMVECs of rats in the blank group,and the expression of AKT in the model group was significantly increased compared with that in the blank group(P<0.01).Compared with the model group,the expression of AKT in the 10%drug-containing serum group,LY294002 group and LY294002+10%drug-containing serum group all decreased significantly(P<0.01).The expression level of AKT in LY294002 group and LY294002+10%serum group decreased compared with the 10%serum group(P<0.05).2.6Expression of HIF-1?protein in PMVECs:There was a certain amount of basal expression of HIF-1?in PMVECs in the blank group.Compared with the blank group,HIF-1?expression in the model group was significantly increased(P<0.01).Compared with the model group,HIF-1?expression was significantly decreased in the 10%drug-containing serum group,LY294002 group and LY294002+10%drug-containing serum group(P<0.01).Compared with the 10%drug-containing serum group,the expression level of HIF-1?in LY294002 group and LY294002+10%drug-containing serum group decreased significantly(P<0.01).Conclusion1 IPF patients have a hypercoagulable state,and the hypercoagulable state of patients with qi-deficiency and blood-stasis syndrome was more obvious than that of patients with qi-yin deficiency syndrome.2 There is no significant difference in the degree of immune dysfunction between patients with IPF and those with qi-deficiency and blood-stasis syndrome and qi-yin deficiency syndrome.3 The expression levels of VEGF,PI3K,AKT and HIF-1?were increased in IPF patients,and the expression levels of qi-deficiency and blood-stasis syndrome were higher than those of qi-yin deficiency syndrome.4 The inhibition of proliferation of PMVECs was the strongest in 10%drug-containing serum.5 Angiogenesis is involved in the pathogenesis of IPF,and Shenqi Cordyceps Sinensis Recipe can inhibit the formation of pulmonary fibrosis by inhibiting the migration of PMVECs.6 Shenqi Cordyceps Sinensis Recipe can inhibit the expression of VEGF protein inPMVECs and inhibit the PI3K/AKT/HIF-1?signaling pathway,which to some extent interferes with the formation of pulmonary fibrosis. |
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