From The Perspective Of Phlegm And Blood Stasis, Explore The Effects Of Feixian Recipe On The Activation And Proliferation Of Lung Fibroblasts Mediated By VEGFR2

Objective:To explore the relationship between phlegm and blood stasis theory of TCM and the microscopic pathological basis of IPF through the combination of literature research,theoretical exploration and experimental research,and to explore the mechanism of Feixian formula against pulmonary fibrosis.Method:1.Combining the understanding of phlegm and blood stasis theory in IPF process and the study of microscopic pathological basis of IPF in modern medicine,from phlegm-heat obstructing lung and inflammatory injury,turbidity and collagen,blood stasis and neovascularization,stasis-phlegm type and lung fibroblasts,pulmonary microvascular endothelial cells to explore the correlation between stasis-phlegm type and the microscopic pathological basis of IPF.2.The lung fibroblasts were isolated and cultured from the lung tissue of suckling mice.VEGFR2(40,20,10,5 mg/L)was used to intervene the lung fibroblasts to construct the activation and proliferation model of lung fibroblasts.Western-Blot method was used to detect the expression of α-SMA protein,which was used to determine the concentration of VEGFR2.The lung fibroblasts were treated with different concentrations of Feixian formula and prednisone.The inhibition rate of cell proliferation was determined by MTT assay,and the maximum non-toxic concentration of Feixian formula and prednisone was determined.Mediated lung fibroblasts were given to the decoction of Feixian formula decoction(100,25,6.25 mg/L)and prednisone solution(50mg/L)as a positive control,the intervention time was 24h,and than MTT assay was used to detect the proliferation of fibroblasts after intervention.The expressions of mRNA and protein of α-SMA and Col Ⅰ were detected by RT-PCR and Western-Blot.The expression of TGF-β1,TβR Ⅰ and Smad3 mRNA was detected by RT-PCR.The expression of protein of PI3K,AKt and pAKt mRNA was detected by Western-Blot.Result:1.There is a correlation between phlegm-heat obstructing lung and inflammatory injury,turbidity and collagen,blood stasis and neovascularization in terms of source,function,nature and characteristics.There is a correlation between stasis-phlegm type and pulmonary fibroblasts and pulmonary microvascular endothelial cells.We believe that the mutual promotion of lung fibroblasts and pulmonary microvascular endothelial cells in function and pathological products is consistent with the interaction of sputum and stasis in Chinese medicine theory.In the process of clinical treatment of IPF,we must pay attention to co-therapy of sputum and blood stasis.2.VEGFR2 can promote the expression of a-SMA protein in lung fibroblasts(P<0.05),and the increase range is positively correlated with the concentration of VEGFR2 in a certain range.20,40mg/L VEGFR2 intervention cells,the cells expressed the highest concentration of a-SMA protein.3.High concentration of Feixian formula and prednisone can significantly inhibit cell proliferation(P<0.01),25,6.25mg/L of lung fiber and 12.5mg/mL of prednisone can promote cell proliferation(P<0.05).100 mg/L Feixian formula had a slight inhibitory effect on cell proliferation(P>0.05).50mg/L prednisone had a slight promoting effect on cell proliferation(P>0.05).4.The model group and each experimental group had strong inhibitory effects on lung fibroblasts.The Feixian formula high,middle dose group and prednisone group significantly inhibited the model cells(P<0.01 or P<0.05).5.Compared with the blank group,the expression of a-SMA gene in the model group was slightly lower than that in the blank group(P>0.05),the expression of Col Ⅰ gene was significantly decreased(P<0.01),and the expression of a-SMA and Col Ⅰ protein was significantly increased(P<0.01).Compared with the model group,the expression of α-SMA gene was up-regulated in the high,medium-dose of Feixian formula and prednisone groups(P<0.01),and the high,medium and low-dose groups could significantly down-regulate the Col I gene.Expression(P<0.01).The expression of a-SMA protein was significantly down-regulated in the high,medium and low doses of Feixianfang(P<0.05 or P<0.01).The expression of Col I protein was significantly down-regulated in each drug-administered group(P<0.01).6.Compared with the blank group,the expression of TβR Ⅰ gene in the model group was significantly increased(P<0.01),and there was no significant difference in the levels of TGF-β1 and Smad3(P>0.05).Compared with the model group,the expression of TGF-β1 was significantly up-regulated in the high-dose and low-dose groups and the prednisone group(P<0.01),and the expression of TβR Ⅰ gene in the high-dose group was significantly increased(P<0.01).The expression of TβR Ⅰ gene in the low-dose group was significantly decreased(P<0.01),and the expression of Smad3 gene was up-regulated in each experimental group(P<0.01 or P<0.05).7.Compared with the blank group,the expression of PI3K and pAKt protein in the model group was significantly increased(P<0.01),and there was no significant difference in the expression of AKt protein(P>0.05).Compared with the model group,the low-dose group of Feixianfang could significantly down-regulate the expression of PI3K protein(P<0.05).There was no significant difference in the expression of AKt protein in the high,middle and low dose groups of pulmonary fibrosis(P>0.05).The middle dose group and the prednisone group could significantly down-regulate the expression of pAKt protein(P<0.05 or P<0.01).Conclusion:1.There is a correlation between phlegm and blood stasis theory of TCM and the microscopic pathological basis of IPF.In the treatment of IPF,attention should be paid to the co-therapy of sputum and blood stasis.2.VEGFR2 promotes the transformation of lung fibroblasts into myofibroblasts.3.Feixian formula can exert anti-fibrotic effect by inhibiting the proliferation and differentiation of lung fibroblasts and reducing collagen deposition.The mechanism may be related to PI3K/AKt signaling pathway.