Study On Mechanism Of Yiqi-Huatan-Quyu Recipe On Pulmonary Vasular Relaxation In COPD Via NO-cGMP-KATP Pathway

1 Objective This study investigats the evolution of pathogenesis in the development of chronic obstructive pulmonary disease（COPD） based on the theories of traditional Chinese medicine. Then the effect of Qibai Pingfei Capsule（QBPF） on pulmonary vasoconstriction in vitro, the pulmonary function, hemodynamic phenomena and the pathological alteration of pulmonary vascular were observed in the experimental study. The aim is to explore the regulatory mechanism of QBPF in the prevention and treatment of COPD, and the possible correlation between ATP-sensitive K+ channel（K_ATP channel） and NO-c GMP-dependent pathway.2 Method 2.1 pulmonary artery rings on experiment study in vitro The 3rd-4th level branch of right pulmonary artery was connected to prepare pulmonary artery rings. The vasodilator effect of QBPF capsule on pulmonary artery ring in rats was observed by multi-wire myograph system. The maximum relaxation（Emax） of QBPF（0.125g/Kg, 0.25g/Kg, 0.5g/Kg, 1g/Kg, 2g/Kg）-induced relaxation of under endothelium-integrity,endothelium-denudation, L-NAME, ODQ, GLYB was detected respectively.2.2 animal study in vivoSprague-Dawley（SD） rats were randomly divided into a normal group, a model group, a positive group of Nicorandil, a high dose group（H-QBPF group）, a middle dose group（M-QBPF group） and a low dose group（（L-QBPF group）） of QBPF. The model rats with phlegm and blood stasis syndrome of COPD were established with compound methods of forced swimming, smoking and hypoxia. Then the pulmonary function, blood gas, hemodynamic phenomena and the pathological alteration of pulmonary vascular were observed. Then the content of NO and c GMP were detected using ELISA, e NOS and i NOS protein expression were detected using immunohistochemical method, Furthermore, the expressions of Kir6.1 and SUR2 B in the lung tissues were determined respectively by real time quantitative PCR（RT-q PCR） and Western blot analysis. 2.3 cell culture study in vitro Qibai Pingfei capsule was provided to rats via continuous gavage for 10 days to obtain QBPF. Primary rat PASMCs were cultured using tissue explants adherent method. Methyl thiazolyl tetrazolium（MTT） was used to detect the effect of QBPF on PASMCs proliferation under hypoxia. Laser scanning confocal microscopy was used to authenticate α-smooth muscle actin. RT-q PCR and Western blot were applied to detect the transcription and protein expression levels of Kir6.1 and SUR2 B gene in PASMCs.3 Results 3.1 pulmonary artery rings on experiment study in vitro 3.1.1 Relaxant effects of QBPF on U46619- induced pre-constrictionQBPF relaxed the U46619-preconstricted endothelium-intact pulmonary artery rings via a concentration-dependent manner with a Ec50 of 0.56 g/L and Emax of 84.30 ± 6.27%. Compared with the values in endothelium-intact rings, the vasorelaxation effect was significantly attenuated inendothelium-denuded rings with EC50 of 1.33 g/L and Emax of 47.91 ± 4.09%（P <0.01）.3.1.2 Effects of L-NAME and ODQ on QBPF-induced vasorelaxation The results of endothelium-intact rings indicated that L-NAME or ODQ incubation had the similarly marked inhibition effect on QBPF–induced relaxation with Emax of 45.81 ± 8.72% in L-NAME and 50.42 ± 7.5% in ODQ, while Emax of 84.30 ± 6.27% in QBPF-control（P <0.01）. Meanwhile the above inhibitory effects also had no notable difference compared to the endothelium-denuded pulmonary artery rings（P >0.05）.3.1.3 Effects of GLYB on QBPF-induced vasorelaxation GLYB showed the inhibitory effects on QBPF-induced vasorelaxation in endothelium-intact rings（P <0.01, Emax: 84.30 ± 6.27 in QBPF-control, 51.34 ± 9.58% in GLYB-QBPF）. 3.1.4 QBPF-induced relaxation in modeling rats of pulmonary vascular ringsTo study the vasorelaxation function in the model of phlegm and blood stasis syndrome combined with COPD, QBPF-induced vascular relaxation was used to compare between the control group and the model group. We found that QBPF-induced vascular relaxation was weaken in pulmonary artery rings of the model group（P <0.01）. The rate of vascular relaxation was lower 22.22% than that of control group（Emax:62.63%±10.02 in model, 84.30± 6.27% in control, and EC50:0.72g/L in model, 0.56g/L in control）. 3.2 animal study in vivo 3.2.1 Observation on macroscopic view on modelingThe rats underwent modeling, some respiratory symptoms such as sneezing, coughing, polypnea and secretion of nasal and mouth etc. were observed accompanying with diet and weight growth to reduce or even no weight growth. Simultaneously, lips, nose and Onyx cyanosis, accidie, and fur owing luster were also looked into. While the above symptoms of rats inH-QBPF group, M-QBPF group and Nicorandil group were improved after treatment. And symptoms of rats in the L-QBPF group fell somewhere in the middle.3.2.2 Effect of QBPF on pulmonary function parameters Compared with the control group, the values of FEV0.3, FVC, and FEV0.3/FVC in the model group were significantly decreased（P<0.01）. While the values of pulmonary functions parameters significantly increased in the H-QBPF group, M-QBPF group, L-QBPF group and Nicorandil group compared with the model group（P<0.01）.3.2.3 Effect of QBPF on blood gas analysis Compared with the control group, the values of p H, Pa O₂, and Sa O₂ in the model group were significantly decreased and Pa CO₂ were increased（P<0.01）. Compared with the model group, the H-QBPF, M-QBPF, L-QBPF and Nicorandil up-regulated the values of p H, Pa O₂, and Sa O₂, at the same time, down-regulated the values of Pa CO₂（P < 0.05 or P < 0.01） 3.2.4 Effect of QBPF on RVSP, m PAP and Ventricular fat thickness Compared with the control group, the values of RVSP, m PAP and Ventricular fat thickness in the model group were significantly increased（P<0.01）. While the values of pulmonary functions parameters significantly decreased in the H-QBPF group, M-QBPF group, L-QBPF group and Nicorandil group compared with the model group（P < 0.05 or P < 0.01）. 3.2.5 Effect of QBPF on the morphology of rat pulmonary arterioles The lung tissues of the model group were aggravated with the thickening of pulmonary vascular wall and the formation of emphysema. The above pathological changes were relieved in all the treatment groups.3.2.6 Effect of QBPF on the content of NO and c GMP Compared with the control group, the values of NO and c GMP in the model group were significantly decreased（P<0.01）. While the values of NOsignificantly increased in the H-QBPF group, M-QBPF group and Nicorandil group, the values of NO significantly increased in the H-QBPF group, M-QBPF group,L-QBPF group and Nicorandil group（P < 0.05 or P < 0.01）. 3.2.7 Effect of QBPF on e NOS and i NOS protein expression in rat lung arterioles The e NOS protein showed strong positive expression,While the i NOS protein showed weakly positive expression in the control group. After the intervention of QBPF or Nicorandil, the e NOS protein expression was up-regulated and the i NOS protein expression was down-regulated（P < 0.05 or P < 0.01）.3.2.8 Effect of QBPF on Kir6.1 and SUR2 B m RNA expression in rat lungs Compared with the control group, the values of Kir6.1 and SUR2 B m RNA in the model group were significantly decreased（P<0.01）. While the values of NO significantly increased in the H-QBPF group, M-QBPF group and Nicorandil group, the values of Kir6.1 and SUR2 B m RNA significantly increased in the H-QBPF group, M-QBPF group, L-QBPF group and Nicorandil group（P < 0.05 or P < 0.01）. 3.2.9 Effect of QBPF on Kir6.1 and SUR2 B protein expression in rat lungs The expression of SUR2 B in the model group was down-regulated by comparison to that in the control group（P <0.01）. Compared with the model group, QBPF and Nicorandil treatment increased the SUR2 B expression（P<0.01 or P <0.05）. The expression of KIR6.1 in the model group had a slight decrease, but there were no significant difference in each group（P >0.05）. 3.3 Cell culture study in vitro 3.3.1 Morphological characteristics of rat PASCMsAfter the adherence of tissue explants for 3-5 days, small amount of irregular elongated fusiform cells crawled out, and they showed diffusion later.The cells grew into parallel arrangement after 7-10 days, and a part of them showed multilayer overlap and some showed monolayer with high and low fluctuation, which were densely distributed as smooth muscle cell-distinctive “peak-trough” shape. Using laser scanning confocal microscope, green fluorescence with positive filamentous distribution was observed, which was α-SMA. The cell nucleus showed blue fluorescence with long-rod or oval shape.3.3.2 Effect of QBPF at different concentrations on proliferation of PASCMS at different time points The result indicated that, compared with the normoxia group, the OD of 12 h, 24 h, 48 h, and 72 h under hypoxia were all significantly increased, and PASCMs showed proliferation（P <0.01）. At each time point, the QBPF at a concentration of 5%, 10%, and 20% could inhibit the proliferation of PASCMs induced under hypoxia at different degrees, and OD was significantly decreased（P <0.05 or P <0.01）. The effect of inhibition in the 20% QBPF group under hypoxia for 24 h was the most significant3.3.3 Effect of QBPF at different concentrations on Kir6.1 and SUR2 B protein in PASCMS at different time points The Kir6.1 and SUR2 B protein showed low expression under normoxia. The Kir6.1 and SUR2 B protein were up-regulated under hypoxia, and the peak expression was at 24h（P < 0.05 or P < 0.01）. 3.3.4 Effect of HYXQ on Kir6.1 and SUR2 B protein expression in PASMCs Compared with the normoxia group, the Kir6.1 and SUR2 B protein level in the hypoxia group were significantly increased（P <0.01） under hypoxia for 24 h. Especially, the Kir6.1 and SUR2 B protein level m RNA level of 10%, and 20% QBPF group were significantly increased compared with the hypoxia group（P <0.05 or P <0.01）.3.3.5 Effect of GLYB on Kir6.1 and SUR2 B m RNA expression in PASMCsCompared with the normoxia group, the Kir6.1 and SUR2 B protein expression in the hypoxia group and 20% HYXQ group were significantly increased（P <0.01） under hypoxia for 24 h. The Kir6.1 and SUR2 B protein expression were significantly decreased after the intervention of GLYB（P <0.05 or P <0.01）. 3.3.6 Effect of L-NAME on Kir6.1 and SUR2 B m RNA expression in PASMCsCompared with the normoxia group, the Kir6.1 and SUR2 B protein expression in the hypoxia group and 20% HYXQ group were significantly increased（P <0.01） under hypoxia for 24 h. The Kir6.1 and SUR2 B protein expression were significantly decreased after the intervention of L-NAME（P <0.05 or P <0.01）. 4 ConclusionThe model rats with phlegm and blood stasis syndrome of COPD were established with compound methods based on the analysis of macroscopic view, pulmonary function, blood gas, hemodynamic phenomena and the pathological alteration of pulmonary vascular.The Yiqi-Huatan-Quyu recipe, QBPF, has the role of relaxation on endothelial dependent in vitro pulmonary artery rings, improvement of the pulmonary function, reduction of m PAP, inhibition of hypoxia-induced proliferation of PASCMs and prevention and treatment of COPD to PAH.The possible mechanism is the activation of the K_ATP channel in pulmonary vascular smooth muscle via NO-c GMP-dependent pathway.