Study On The Mechanism Of Intervention Of IPF By The Method Of Replenishing Qi, Nourishing Yin, Activating Blood And Dredging Collaterals Based On Oxidative Stress

Purpose:To demonstrate the mechanism of benefiting qi&yin and regulating blood flow and meridians（Yiqi Yangyin Huoxue Tongluo）treatment to idiopathic pulmonary fibrosis（IPF）multiple-targeted in regulating oxidative stress in vivo and vitro study and investigate its effect on NO,H₂O₂,SOD,CAT,GSH-px,Keap1/Nrf2 signal transduction pathway and its downstream target HO-1,NQO-1 andγ-GCS protein and mRNA expression.This study is to gain more evidences and supports in guiding the clinical treating for idiopathic pulmonary fibrosis.Material and method:144 SPF male Wistar rats were randomly divided into six groups,namely control group,model control group,positive control group,high dosage group,medium dosage group and low dosage group,through the general conditions of rats,lung pathological changes,HE staining,alveolar inflammation pathological score results,pathological score of pulmonary fibrosis of rat model of pulmonary fibrosis was evaluated,and determination of experimental materials and index for seventh days,fourteenth days,twenty-eighth days three time points.By colorimetry and UV Vis spectrophotometric detection of free radicals（NO,H₂O₂）and antioxidant enzymes（SOD,CAT,GSH-px）expression,to elucidate the effect of Yiqi Yangyin Huoxue Tongluo method on antioxidant ability of pulmonary fibrosis in rats;Western-blot and RT-PCR methods were used to reveal the dynamic expression of Keap1（cytosolic protein）,Nrf2（nuclear protein）,p-Nrf2（nuclear protein）and mRNA in lung tissue at different time points,to clarify the mechanism of Yiqi Yangyin Huoxue Tongluo control rat model of pulmonary fibrosis keap1/Nrf2 signal transduction network;Western-blot and RT-PCR were used to reveal the dynamic expression of HO-1,NQO-1,γ-GCS protein and mRNA in lung tissue at different time points at different levels and angles,further study of the mechanism of Yiqi Yangyin Huoxue Tongluo regulation of IPF rat model of oxidative stress.Results:1.Yiqi Yangyin Huoxue Tongluo can improve the general condition of the rats and effectively inhibit the early alveolitis,pulmonary fibrosis in the late deposition and improve the pathological morphology,improve the lung tissue of rats.2.Yiqi Yangyin Huoxue Tongluo can increase SOD,CAT,GSH-px content,reduced NO and H₂O₂ antioxidant effect,delaying the process of pulmonary fibrosis,and the effect of high dose of Chinese medicine is the best.Compared with the blank control group,the NO and H₂O₂ contents in the other groups were significantly increased,and the oxidative stress reaction and the free radical generation were observed;Compared with the model control group,the content of NO in the western medicine group,the high,middle and low dose group of Chinese medicine were significantly reduced,and the western medicine and the traditional Chinese medicine could play an important role in antioxidation;Compared with the western medicine group,the content of NO and H₂O₂ in the high dose group was not statistically significant,whitch shows that Chinese medicine was the best curative effect;Compared with the control group,the other groups in the lung tissues of SOD,CAT,GSH-px content decreased obviously,it can be seen that oxidative stress reaction in lung tissue of rats with pulmonary fibrosis leads to the decrease of SOD,CAT,GSH-px activity and content;Compared with the model group,the SOD,CAT,GSH-px content in the western medicine group,the high,middle and low dose group of Chinese medicine were significantly increased,which showed that the western medicine and traditional Chinese medicine could play an important role in improving SOD,CAT and GSH-px;Compared with the western medicine group,the content of NO and H₂O₂ were not significantly different between the high dose group and the western medicine group,High dose of traditional Chinese medicine and Western medicine have similar antioxidant effect,and the antioxidant effect of traditional Chinese medicine increases with the increase of drug concentration.3.Yiqi Yangyin Huoxue Tongluo can regulate the Keap1/Nrf2 signal transduction pathway of antioxidant,slow lung fibrosis.The Keap1 protein and mRNA in the blank control group showed high expression in senen days,fourteenth days and twenty-eighth days,that is the expression of Keap1 protein and mRNA in normal lung tissue were higher than those in the control group;The Nrf2 protein and mRNA in the blank control group had only a small amount of expression in seven days,fourteenth days and twenty-eighth days,that is the expression of Nrf2 protein and mRNA in normal lung tissue was low;Blank control group p-Nrf2 protein in seventh days,fourteenth days,twenty-eighth days,only a small amount of expression in the three time points.After bleomycin after modeling,rats in the model control group Keap1 protein and mRNA compared with the blank control group was significantly reduced in varying degrees,and with the advance of time gradually decreased,that of IPF in lung tissue during oxidative stress in the expression of Keap1 protein and mRNA showed reduced state;The rats in the model control group Nrf2 protein and mRNA compared with the blank control group was significantly increased in varying degrees;The model control group rats p-Nrf2 protein compared with the blank control group was significantly increased in varying degrees.After drug treatment,with a slowdown in the degree of pulmonary fibrosis,compared with the model group,by western medicine group,high-dose group,middle dose group,low dose of Chinese medicine group of Keap1 protein and mRNA in varying degrees;Compared with the model control group,western medicine group,Chinese medicine high dose group,middle dose group,low dose group of Chinese medicine Nrf2 protein and mRNA were increased in varying degrees;Compared with the model group,western medicine group,high dose group and middle dose group,low dose of Chinese medicine group p-Nrf2 protein had increased in different degrees,which suggested that expression of Yiqi Yangyin Huoxue Tongluo method can inhibit Keap1 protein and mRNA,promote the expression of Nrf2protein and mRNA,increase the expression of p-Nrf2 protein to enhance antioxidant activity in vivo,inhibiting excessive oxidation,and compared with western medicine group,lung tissue in rats of high dose of Chinese medicine in the expression level of Keap1 protein and mRNA protein,Nrf2 and mRNA,p-Nrf2 protein had no significant difference,suggesting that the high dose of traditional Chinese medicine and Western medicine medicine intervention effect of prednisone equivalent.It is speculated that the Yiqi Yangyin Huoxue Tongluo intervention by Keap1/Nrf2 signal transduction pathway,thereby inhibiting lung tissue damage and excessive repair.4.Yiqi Yangyin Huoxue Tongluo can upregulate pulmonary fibrosis model rats HO-1,NQO-1,γ-GCS protein and mRNA expression,antioxidant effect.The results showed that the HO-1protein and mRNA in the blank control group had only a small amount of expression at seventh,14 and 28 days in the three groups.After bleomycin after the model rats of HO-1compared with the blank control group was significantly increased in varying degrees,showed increased expression of HO-1 in lung tissue and pulmonary fibrosis was closely related to the formation of the guide play an important role in lung inflammation and lung injury in medium.After drug treatment,compared with model control group,the expression of HO-1 and mRNA in lung tissue of rats in each group were up-regulated.Compared with the western medicine group,Chinese medicine high dose group expression was significantly upregulated at protein level,the expression level of protein was significantly lower in the low-dose group of Chinese medicine,Chinese medicine dose group protein expression level was not statistically significant,suggesting that the best curative effect of high dose of Chinese medicine group.Compared with the blank control group,the expression of NQO-1protein and mRNA in the lung tissue of model control group was significantly lower at seventh,14,and 28.After bleomycin after the model rats of NQO-1 compared with the blank control group was significantly reduced in varying degrees,presumably play an important role in inflammation and lung injury in the alveoli.After drug treatment,compared with model control group,the expression of NQO-1 protein and mRNA in lung tissue of rats in each group were up-regulated,It is shown that the effect of drugs on the antioxidant activity in vivo.Compared with the western medicine group,the expression level of protein in Chinese medicine group was significantly lowered,and the protein expression level was significantly lower in the low dose group,there was no significant difference in the expression of high dose group of Chinese medicine.Compared with the traditional Chinese medicine high dose group,the middle dose group and low dose group of Chinese medicine were significantly lower.It can be seen that the traditional Chinese medicine high dose group.In the blank control group,the expression ofγ-GCS protein and mRNA at seventh,14 and 28days was only a small amount of expression,that is,the expression ofγ-GCS protein and mRNA in normal lung tissue was lower than that in the control group.After bleomycin after model rats compared withγ-GCS than control group significantly increased in varying degrees,showed that the expression ofγ-GCS increased in lung tissue and pulmonary fibrosis was closely related to the formation of the guide play an important role in lung inflammation and lung injury in medium.After drug treatment,compared with model control group,the expression ofγ-GCS protein and mRNA in lung tissue of rats in each group were up-regulated.Compared with the western medicine group,Chinese medicine high dose group expression was significantly upregulated at protein level,the expression level of protein was significantly lower in the low-dose group of Chinese medicine,Chinese medicine dose group protein expression level was not statistically significant,suggesting that the best curative effect of high dose of Chinese medicine group.By increasing HO-1,NQO-1 andγ-GCS levels indicate the effect of Yiqi Yangyin Huoxue Tongluo,antioxidant effect,achieve the purpose of treating pulmonary fibrosis.Conclusion:1.Based on the theory of collateral disease and the theory of collateral effect,it is proposed that the core pathogenesis of IPF is"deficiency of lung and collateral stasis",Lung deficiency of lung qi and yin deficiency two,"Blood stasis"that is blood stasis veins,put forward the"Yiqi Yangyin Huoxue Tongluo"and using ShenLong decoction treatment.2.Yiqi Yangyin Huoxue Tongluo can reduce the content through down-regulation of NO in lung tissue and oxygen free radicals such as H₂O₂ content and increase SOD,CAT,GSH-px and other antioxidant enzymes of Yiqi Yangyin Huoxue Tongluo,inhibit pulmonary fibrosis and oxidative stress,thus delaying the progress of IPF.3.Yiqi Yangyin Huoxue Tongluo can act on method through down-regulation of Keap1protein and mRNA content,mRNA content and expression of Nrf2 and upregulation of p-Nrf2 protein content in the regulation of Keap1/Nrf2 signal transduction pathway,inhibit pulmonary fibrosis and oxidative stress,thus delaying the progress of IPF.4.Yiqi Yangyin Huoxue Tongluo can improve the expression HO-1,γ-GCS,NQO-1 protein and mRNA,promote the downstream target gene and Keap1/Nrf2 signal transduction pathway,inhibition of oxidative stress in pulmonary fibrosis,and the progression of IPF.5.Yiqi Yangyin Huoxue Tongluo regulation of oxidative stress related to oxygen free radical and antioxidant enzymes,may be achieved through the downstream target protein and gene regulation of Keap1/Nrf2 signal transduction pathway and promotes Keap1/Nrf2 signaling pathway regulation,and delay the development of IPF.