| Objective:1 Clinical study:Study the correlation between the content of NF-κB p65,AP-1,VEGF,TGF-β1 in the serum of IPF patients with Yin deficiency and lung dryness syndrome and Qi deficiency and blood stasis syndrome and syndrome types.2 Experimental study:Using molecular biological techniques such as ELISA and Western blot,to study the effect of Shenqi Chongcao recipe on MAPK/ERK signal pathway,to explore the mechanism of its intervention in IPF inflammatory response and delaying pulmonary fibrosis,and to provide scientific basis for the study of traditional Chinese medicine in the prevention and treatment of IPF.Methods:1 Clinical study:In this study,60 patients with idiopathic pulmonary fibrosis whose TCM syndromes were identified as yin deficiency and lung dryness and qi deficiency and blood stasis,and 30 normal healthy subjects were collected.They were divided into Yin deficiency and lung dryness syndrome group,Qi deficiency and blood stasis group and healthy control group,30 cases in each group.The contents of NF-κB p65,AP-1,VEGF,and TGF-β1 in the serum of the three groups were detected,and the correlation between them and syndrome types was analyzed.2 Experimental study:160 SPF SD rats were randomly divided into 5 groups:Blank group(group A),Model group(group B),Shenqi Chongcao group(group C),Pirfenidone group(group D),and ADI group(group E),with 32 rats in each group.Except the Blank group,the other four groups of rats were used to construct IPF models by intratracheal instillation of BLM.On the 7th,14 th,21st and 28 th days after drug intervention,samples were taken in batches,and the pathological changes of lung tissue were observed by HE staining and Masson staining.Detection of serum TGF-β1 and IL-10 expression level by ELISA.Detection the relative expression level of P-MEK,P-ERK,NF-κB p65 and AP-1protein in lung tissue by Western blot.Results:1 Clinical study1.1NF-κB p65,AP-1 content:Compared with healthy control group,the content of NF-κB p65 and AP-1 in IPF patients increased significantly(P<0.01);compared with yin deficiency and lung dryness syndrome group,the content of NF-κB p65 and AP-1 in IPF patients of qi deficiency and blood stasis group decreased(P<0.05).1.2VEGF,TGF-β1 Content:Compared with the healthy control group,the content of NF-κB p65 and AP-1 in IPF patients increased significantly(P<0.01);compared with yin deficiency and lung dryness syndrome group,VEGF and TGF-β1 in IPF patients of qi deficiency and blood stasis group content increased(P<0.05).2 Experimental study2.1 General condition of rats2.1.1 General condition of rats:After model establishment,group A rats were in the best condition and group B rats were in the worst condition at the same time point.Among the three groups C,D and E,the general condition of rats in group C is relatively good.The state of rats in groups C,D and E treated with drug intervention in the 3-4 weeks was improved compared with that in the 1-2 weeks.2.1.2 Weight of rats:There was no significant difference in weight of rats in each group before modeling(P>0.05).On the 7th day after modeling,compared with group A,the weight of rats in the other four groups decreased to different degrees(P<0.05,P<0.01).On the 14 th day after modeling,compared with group A,the weight of rats in group B,D and E decreased(P<0.05,P<0.01).On the 21 th and 28 th days after modeling,compared with group A,the weight of rats in group B,D and E decreased(P<0.05,P<0.01);Compared with group B,the weight of rats in group C,D and E after drug intervention increased(P<0.05,P<0.01).2.2 HE staining:The internal structure of lung tissue of rats in group A is complete,the outline is clear,no inflammatory cell infiltration is found in the alveoli,and no obvious hyperplasia is found in the alveolar wall.In group B rats,on the 7th day,the internal alveolar structure of the lung tissue was destroyed,the alveolar septa were slightly widened,part of the alveolar wall was broken,the alveolar cavity fused,and there were many inflammatory cells in the lung tissue.On the 14 th day,the alveolar structure was significantly damaged,the interval was widened,the inflammatory cells were further gathered,and some fibroblasts proliferation and angiogenesis were seen.On the 21 st day,the number of collapsed and atrophic alveoli increased,the number of inflammatory cells decreased slightly,and the number of fibroblasts increased,and collagen deposition was visible.On the 28 th day,a large number of alveoli were damaged,the alveolar septa were significantly widened,the infiltration of inflammatory cells was slightly improved,the infiltration of fibroblasts and collagen deposition were significantly increased,and the lung tissue showed obvious fibrosis.The pathological changes of rats in Group C,Group D and Group E were similar to those in Group B.The degree of alveolar destruction and the aggregation of inflammatory cells and fibroblasts in the lung tissue of group C and D were significantly improved compared with that of group B.There was no significant difference in lung histopathology between group C and group D at the same time.In group E,partial alveolar destruction and fibrous formation were observed,but at the same time,it was lighter than group B,and slightly heavier than group C and group D.2.3 Masson staining:The bronchial and vascular wall structures of rats in group A were normal,and thin blue collagen fibers were visible.In group B,on the 7th day,the alveolar tissue space thickened,and there were more light blue collagen fibers around the trachea and blood vessels.On the 14 th day,the number of collagen fibers around trachea,blood vessels and damaged alveoli increased,and the blue degree was significantly deepened.On the 21 st day,the trachea and blood vessels at all levels were surrounded by a large amount of blue collagen,and the number of blue-stained sites increased,and the color was similar to that on the 14 th day.On the 28 th day,the lung interstitium was filled with a large number of blue collagen fibers,and the collagen was dense,showing a thick strip or large distribution,and the lung tissue was diffuse fibrosis.The formation trend of collagen fibers in group C,D and E was similar to that in model group.Blue collagen deposition can be seen in the trachea wall and blood vessel wall of Group C and D,and the blue staining area is smaller than that of the model group,and the blue degree is lighter than that of the model group.There was no significant difference in collagen formation between group C and group D.There were many collagen fibers in the trachea and blood vessel wall of group E,and the blue staining area was smaller than that of model group,and larger than that of group C and D;The degree of blue is similar to that of model group,but darker than that of group C and group D.2.4 ELISA results:Compared with group A,The expression of TGF-β1 of rats in the other four groups at the same time point was significantly increased(P<0.01)and the expression of IL-10 was significantly decreased(P<0.01).Compared with group B,The expression of TGF-β1 of rats in group C,D and E at the same time point was significantly decreased(P<0.01),and the expression of IL-10 was significantly increased(P<0.01).Compared with group C,there was no significant difference in the expression of TGF-β1 and IL-10 at the same time point of rats in group D(P>0.05).The expression of TGF-β1 of rats in group E at the same time points was significantly increased(P<0.01),and the expression of IL-10 was significantly decreased(P<0.01).Compared with group D,the expression of TGF-β1 of group E rats at the same time point was significantly increased(P<0.01),and the expression of IL-10 was significantly decreased(P<0.01).2.5 Western blot test results:Compared with group A,the protein expression of P-MEK,P-ERK,NF-κB p65 and AP-1 in lung tissue of rats in the other four groups increased significantly at the same time points(P<0.01).Compared with group B,the protein expression of P-MEK,P-ERK and NF-κB p65 and AP-1 in lung tissue of rats in group C,D and E decreased significantly at the same time points(P<0.01).Compared with group C,There was no significant difference in protein expression of P-MEK,P-ERK,NF-κB p65 and AP-1 in lung tissue of rats in group D at the same time points(P>0.05).The protein expression of P-MEK,P-ERK,NF-κB p65 and AP-1 in lung tissue of rats in group E increased significantly at the same time points(P<0.01).Compared with group D,The protein expression of P-MEK,P-ERK,NF-κB p65 and AP-1 in lung tissue of rats in group E increased significantly at the same time point(P<0.01).Conclusion:1.Clinical research shows that IPF patients are in a state of high expression of inflammatory mediators and angiogenesis factors.Between the two syndrome types,the syndrome of yin deficiency and lung dryness is characterized by high levels of inflammatory mediators,and the syndrome of qi deficiency and blood stasis is characterized by high levels of angiogenesis factors.2.Experimental study shows that Shenqi Chongcao recipe can down-regulate NF-κB p65,AP-1 protein expression by inhibiting MAPK/ERK signal pathway,increase IL-10 expression level,and reduce TGF-β1 expression level,thus inhibiting BLM-induced excessive inflammatory reaction in the lung,reducing collagen deposition,and delaying the progression of IPF. |
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