The Level Of MMPs And CCL2 In Serum Of IPF Patients With Qi-Yin Deficiency And Blood Stasis Syndrome And The Effect Of Yiqi Yan Gyin Huoxue Method On Src/STAT3 Signaling In IPF Model Rats

Objectives:1.Study on the correlation between MMP-2,MMP-9,CCL2 and TGF-β1 in the serum of IPF patients with deficiency of lung and kidney Qi and Yin and blood s tasis in the development and progression of IPF.2.In vivo experiments were conducted to investigate whether Shenqichongcao For mula（SQCCF）,which is cubed with the method of benefiting Qi,nourishing Yin a nd activating Blood,could inhibit the expression of ASS1 and its downstream src/STAT3 signaling pathway,thereby slowing down BLM-induced lung fibrosis in rat s.Method:1.Clinical studyThirty patients identified as IPF with deficiency of both lung and kidney qi and y in with blood stasis and 30 healthy physical examiners were selected as the contro l group.The serum expression levels of MMP-2,MMP-9,CCL2 and TGF-β1 in the two groups were compared and their correlation with IPF was made.2.Experimental research2.1 One hundred and twenty SPF grade SD male rats were selected and divided i nto five groups using the random number table method:blank group（K group）,m odel group（Model group）,SQCCF group（SF group）,pirfenidone group（BT group）and ADI group.Except for group K,all the other four groups used a disposable endotracheal drip of BLM to induce the preparation of the rat model of IPF.The dosing regimen was 28 days.Six rats in each group were taken at four time point s（days 7,14,21 and 28）of the drug intervention.2.2 HE and Masson staining were performed to observe the histopathology of lung tissue in each group.Giemsa staining was used to analyze different kinds of infla mmatory cells in BALF.2.3 The levels of serum cytokines CCL2 and TGF-β₁in each group were measur ed by ELISA.2.4 Western Blot to determine the protein expression of src,STAT3 in lung tissue s.2.5 The expression levels of ASS1,src,STAT3,MMP-2,MMP-9 in lung tissue of each group were detected by RT-q PCR.Results:1.Clinical trial1.1 Comparison of serum MMP-2 and MMP-9 levels in the two groupsMMP-2 and MMP-9 in the IPF observation group were significantly higher th an those in the control group（t₁=31.038,P₁<0.01;t₂=21.992,P₂<0.01）.1.2 Comparison of serum CCL2 and TGF-β1 levels in the two groupsCCL2 and TGF-β1 were significantly higher in the IPF observation group th an in the control group（t₁=4.157,P₁<0.01;t₂=17.097,P₂<0.01）.2.Animal experiments2.1 HE stainingAfter the BLM Model was established by one-time tracheal instillation,on the7th day in the Model group,the pulmonary septum was slightly enlarged,there was exudation in alveoli,inflammatory cells such as lymphocytes and macrophages were infiltrated,and inflammatory reaction was observed.With the proliferation of fibroblasts,light blue collagen fibers appeared.On the 14th day,alveolar septa wi dened more obviously,alveolar structure was slightly damaged,some alveoli were broken,lung tissue structure was disordered,and a large number of inflammatory cells such as lymphocytes and macrophages infiltrated in alveolar cavity and strom a,and the proliferation of fibroblasts was more obvious than that on the 7th day.On the 21st day,the alveolar structure in the visual field was decomposed,and th e septum disappeared.Compared with the 7th day,the inflammatory lesions showe d a decreasing trend,but there were still some inflammatory cells infiltrating,whic h was slightly less than before,and spindle-shaped fibroblasts appeared.On the 28th day,the inflammatory lesion was obviously relieved compared with that on the7th day.Under the microscope,the alveolar structure was seriously damaged,the a lveolar septum was obviously widened and deformed,and the lung tissue showed obvious fibrosis changes.Compared with Model group,the pathological changes of SF group,BT group and ADI group are basically the same,but the damage degr ee of lung tissue in rats is significantly reduced,the pulmonary septum is thinner,the alveolar structure is relatively complete,the infiltration degree of inflammator y cells is significantly reduced,and the inflammatory reaction is lighter.2.2 Masson stainingIn Model group,a large number of blue collagen fibers were deposited in the lung tissue,and the thickness of alveolar wall was different,and fibroblasts prolif erated.With the increase of time,fibroblasts proliferated gradually,and the blue st aining area gradually expanded and deepened.From the 7th day to the 28th day,t he proliferation of collagen fibers showed an increasing trend.At 28 days,a large number of dense diffuse blue collagen fibers proliferation,a large number of fibr oblasts proliferation and collagen precipitation can be seen in the interstitial lung,f orming a flaky or fascicular distribution of fibrosis lesions.Compared with Model group,the collagen fiber deposition in SF group,BT group and ADI group decrea sed,but the collagen deposition in ADI group was more obvious.2.3 Different types of inflammatory cells in BALFAfter intratracheal instillation of BLM,a large number of inflammatory cells in the lung of rats accumulated and infiltrated,and neutrophils,macrophages and lymp hocytes in Model group increased significantly compared with those in K group at the same time point（P<0.01）.The neutrophils,macrophages and lymphocytes in the Intervention group were lower than those in the Model group at the same time po int（P<0.05）.SF+BLM neutrophils were lower than those in the Model group only on the 7th,21st and 28th day,and there was no significant difference between them on the 14th day（P>0.05）.There was no significant difference in macrophages and l ymphocytes between ADI group and Model group on 28th day（P>0.05）.2.4 Expression of cytokines in serum of IPF ratsCCL2 and TGF-β1 in the serum of rats in the early stage of Model group（7d and 14d）increased sharply,and the levels continued to increase with the progress of time,which were significantly higher than those in K group（P<0.05）.At the same time,the levels of serum CCL2 and TGF-β1 in SF group,BT group and ADI gro up were lower than those in Model group（P<0.05）.The levels of serum CCL2 and TGF-β1 in SF group,BT group and ADI group increased rapidly in the first 14 d ays,then increased slowly on the 21st and 28th days,and reached the peak on the28th day.2.5 Protein expression of src and STAT3 in lung tissueCompared with K group,p-src and p-STAT3 levels were highly expressed in Model group at different time points（P<0.05）.P-src and p-STAT3 levels were sig nificantly increased at 7d and 14d,and decreased at 21d and 28d,but still signific antly higher than K group.SF group,BT group and ADI group were lower than Model group at the same time points.2.6 Expression levels of ASS1,src,STAT3,MMP-2 and MMP-9 in lung tissues of each group2.6.1 Levels of ASS1 in lung tissues of rats in each groupCompared with the K group,the level of ASS1 in the lung tissue of rats in t he BLM group increased significantly at 7d and 14d,and decreased at 21d and 28d,but the level increased at the 28th day compared with the previous one.The le vel of ASS1 in the lung tissue of the remaining Intervention groups was significan tly lower than that of the Model group at all time points（P<0.05）;the level of th e SF and BT groups was not significantly different at all time points,but was lo wer than that of the ADI group.2.6.2 Levels of src in lung tissue of rats in each groupCompared with group K,the level of src in lung tissue of rats in Model gro up increased significantly at all time points,with an increasing trend on the 7th a nd 14th days,with the highest level on the 14th day and a decrease on the 21st and 28th days.The level of src in lung tissue in the remaining Intervention group was significantly lower than that in the Model group at all time points（P<0.05）.There was no significant difference in content between SF group and BT group at each time point.But they were all lower than those in ADI group（P<0.05）.2.6.3 STAT3 levels in lung tissues of rats in each groupCompared with group K,STAT3 in lung tissue of rats in Model group increased s ignificantly at all time points（P<0.01）,with the fastest increase from 7th to 14th day,and it still showed an upward trend on 21st and 28th day,but the increase r ate slowed down.STAT3 in lung tissue of rats in Yugan pre-treatment group was significantly lower than that in Model group at each time point（P<0.05）.There w as no significant difference between SF group and BT group at each time point,b ut they were lower than ADI group.2.6.4 MMP-2 levels in lung tissues of rats in each groupCompared with the K group,the level of MMP-2 in the lung tissue of rats i n the Model group was significantly higher at all time points（P<0.01）;the level o f MMP-2 in the lung tissue of rats in the remaining Intervention groups was signi ficantly lower than that in the Model group at all time points（P<0.05）;the level of MMP-2 in the SF group and BT group at all time points was not significantly different,but they were all lower than that in the ADI group.2.6.5 MMP-9 levels in lung tissues of rats in each groupCompared with the K group,the level of MMP-9 in the lung tissue of rats i n the Model group was significantly higher at all time points（P<0.01）;the level o f MMP-9 in the lung tissue of rats in the remaining Intervention group was signif icantly lower than that in the Model group at all time points（P<0.05）;the level o f MMP-9 in the SF group and BT group at all time points was not significantly different,but was lower than that in the ADI group.Conclusion:1.High expression expression of various cytokines,chemokines and proteases existed in patients with IPF lung-kidney qi-yin deficiency with blood stasis evidenc e.2.After BLM was injected into the trachea of rats,inflammatory cells such a s macrophages,neutrophils and lymphocytes accumulated and infiltrated heavily in the lung tissue of rats.Meanwhile,BLM can induce the activation of ASS1 in inf lammatory cells,upregulate src/STAT3 expression,promote the secretion of various cytokines such as CCL2 and TGF-β₁,promote the proliferation of FB and collag en deposition,and finally lead to the development of interstitial lung fibrosis.3.SQCCF may ameliorate BLM-induced lung fibrosis in rats by inhibiting the ASS1-mediated src/STAT3 signaling pathway in inflammatory cells.