The Expression Of Porcine CXCR1/2 In Pneumonia And The Preparation Of Swine Antagonistic Protein

The previous medical research has shown that CXCR1 and CXCR2(CXCR1/2)plays an important role in many diseases.In dorder to analyze porcine CXCR1/2 expression in pneumonia,and develop new anti-inflammatory drugs for animals,we expressed porcine CXCR1/2 protein,prepared polyclonal antibody,and analyzed the expression of CXCR1/2 in bronchopneumonia,interstitial pneumonia,serous pneumonia,fibrinous pneumonia and suppurative pneumonia through Real-time quantitative PCR and immunohistochemistry.In addition,we expressed CXCR1/2 antagonistic proteins,CXCL8N11R/G31P(p N11R)and CXCL8G31P(p G31P),and evaluated the anti-inflammatory effect through cell chemotactic experiment and animal experiment.We hope our study can provide a scientific gist for the development of new veterinary anti-inflammatory drugs.The main contents as follows:1 sample collectionThere are total 136 samples of pig pneumonia tissues were collected from March 2017 to December 2018,including 17 cases of bronchopneumonia,81 cases of interstitial pneumonia,8 cases of serous pneumonia,9 cases of fibrinous pneumonia and 21 cases of suppurative pneumonia.2 The expression of CXCR1/2 m RNA in different types of pneumoniaThe expression of CXCR1/2 m RNA in pneumonia tissues was detected by QPCR,and the results showed that the expression of CXCR1/2 m RNA in 5 types of pneumonia samples was significantly up-regulated compared with normal lung tissues,and the expression of CXCR1 m RNA was higher than that of CXCR2 m RNA.Besides,the CXCR1/2 m RNA expression in the tissues of bronchopneumonia,fibrinous pneumonia and suppurative pneumonia was significantly higher than that in interstitial pneumonia and serous pneumonia.3 Recombinant plasmid construction,protein expression and purification of porcine CXCR1/2CXCR1/2 were amplified by RT-PCR,cloned into p GEX-6p-1,and formed PCXCR1 and PCXCR2.The PCXCR1/2 were transformed into E.coli Rosetta(DE3)cells,and a large number of fusion proteins with GST-tag were obtained after optimizing the expression conditions.The recombinant CXCR1/2 porcine were purified by AKTA prime 10,and the results of SDS-PAGE and Western-blot showed that recombinant CXCR1/2 porcine were relatively pure.4 Expression and distribution of pig CXCR1/2 in different types of pneumoniaThe recombinant CXCR1/2 protein were immunized in mice,and then polyclonal antibodies were preparation.Immunohistochemistry(IHC)was used to detect the distribution and expression of CXCR1/2 in pneumonia.The results showed that CXCR1/2 were positively expressed in the cytoplasm and cytomembrane of bronchial epithelial cells and neutrophils,the expression of CXCR1/2 in 5 types of pneumonia was significantly upregulated compared with control group.Furthermore,except of serous pneumonia,the expression of CXCR1 in the other types of pneumonia was significantly higher than CXCR2,it was consistent with the result of QPCR.In summary,the expression of CXCR1/2 was positively correlated with the degree of neutrophil infiltration in tissues,and CXCR1 may play a major role in inducing neutrophils to participate in the regulation of inflammatory response.5 Effect of porcine CXCR1/2 antagonistic proteins on neutrophil chemotaxisThe porcine CXCR1/2 antagonistic proteins p N11 R and p G31 P were prepared.And the effect of p N11 R and p G31 P on neutrophil chemotaxis was detected by the agarose chemotactic model through comparing the migration distance of neutrophil.It was showed that the ability of p N11 R and p G31 P induce neutrophil migration was significantly lower than that of CXCL8.6 Evaluation of anti-inflammatory effects of porcine CXCR1/2 antagonistic proteins on LPS-induced mouse pneumonia model16-18 g female BALB/c mice were randomly divided into 4 groups,one group was inoculated intranasally with 20μL normal saline as control group,and the other four groups were intranasal inoculated with 10μL lipopolysaccharide(LPS)to induce pneumonia.At the same time,10μL normal saline(LPS group),10μL p CXCL8(CXCL8 group),10μL p N11R(N11R group)and 10μL p G31P(G31P group)were subcutaneous injected into the back of mice.After 24 h of treatment,the mice were sacrificed,the examination of histopathology of lung,myeloperoxidase(MPO)activity,neutrophil count of bronchiolar alveolar lavage(BALF),inflammatory factors expression in lung were carried out to evaluate the therapeutic effect of porcine CXCR1/2 antagonistic proteins.The results showed that lung inflammation,MPO activity,CXCL1,TNF-α,CXCL8 expression,neutrophil count of BALF of mice in N11 R group and G31 P group was significantly attenuate compared with CXCL8 group and LPS group(P<0.05).And the MPO activity,neutrophil count of BALF and TNF-α expression in G31 P group were significantly lower than those in N11 R group(P<0.05).The study indicated that CXCR1/2 were positively expressed in the cytoplasm and cytomembrane of bronchial epithelial cells and neutrophils,and the expression of CXCR1/2 was positively correlated with the degree of neutrophil infiltration in tissues.CXCR1 may play a major role in inducing neutrophils to participate in the regulation of inflammatory response.Porcine CXCR1/2 antagonistic protein CXCL8(3-72)N11R/G31 P and CXCL8(3-72)G31 can significantly attenuate pneumonia of mice by decreasing CXCL8 expression and inhibiting neutrophil migration,and the anti-inflammatory effect of CXCL8(3-72)G31P may be better.