The Role Of Connective Tissue Growth Factor Antibody In The Development Of Pulmonary Fibrosis Induced By Silica Particles In Rats

Objectives: Silicosis is a kind of chronic pulmonary diseases which is characterized by inflammation and diffuse fibrosis caused by long-term inhalation of silica particles,accompanied with decreased lung function and quality of life.Although the mechanism of silicosis has not been fully elucidated at present,inflammation processes,epithelial-mesenchymal transition(EMT)and fibrosis play important roles in the development of silicosis.Studies have shown that connective tissue growth factor(CTGF)can not only promote inflammation,but also participate in the development of fibrosis in cooperation with TGF-?.However,the exact role of CTGF in the development of pulmonary inflammation and fibrosis caused by silica paritcles is not well understood.Therefore,in this study we mainly explore the role and possible mechanism of CTGF antibody in the process of lung inflammation,EMT and fibrosis caused by silica particles.Methods: We established a silicosis model by intratracheally instilled silica particles to rats and CTGF antibody was intraperitoneally injected to directly neutralize the silica-induced CTGF in rats.A total of 192 Wistar rats were randomly divided into 6 groups as blank group,normal saline group,silica group,Ig G group,silica+ Ig G group,silica+ anti-CTGF group(n=32 each group).Rats were tracheal instillation of 1 ml saline or 50 mg silica particles(50mg/ml,1ml),intraperitoneal injection of saline,Ig G or CTGF antibody.After instilled treatment,eight rats of each group were sacrificed on days 1,7,28 and 84.The levels of lung inflammation and fibrosis were scored in the middle lobe of the right lung by histological sections which were stained with H&E and Masson.The expression of CTGF,IL-1? and TGF-? were measured by ELISA.And the expression of E-cadherin,p120 ctn and Rho A were measured by Western blotting.Result:(1)The results of HE and Masson staining showed that the structure of lungs was damaged,the alveolar wall became more thicken,the alveolar space was broken,and there were a lot of cellular nodules and severe inflammation when exposure to silica particles for 7 day in the silica groups.And there was no obvious fibrosis when compared with normal saline group from the results of masson.After 7 days of Ig G or CTGF antibody neutralization,the degree of inflammatory infiltration and the size of cellular nodules in lung tissue were significantly decreased when compared with silica group.After exposed to the silica particles for 28 and 84 days,inflammation was gradually reduced in comparsion with silica group on the 7th day,collagen-like changes appeared around the cell nodules and the accumulated collagen fibers in the pulmonary parenchyma gradually increased in silica group.The degree of inflammation infiltration and collagen fiber deposition after intraperitoneal injection of Ig G antibody were similar to the silica group on the 7th day.After intraperitoneal injection of CTGF antibody,the size of cellular nodules and collagen fiber deposition were significantly lower than the silica+ Ig G group.From the results of inflammatory response and fibrosis score,intraperitoneal administration of Ig G antibody can temporarily reduce the inflammatory response on the 7th day(P <0.05).After CTGF antibody administration,the inflammatory response can be significantly reduced on the 7,28,and 84 days when compared with silica group(P <0.05).After CTGF antibody administration,compared with silica group the degree of fibrosis response could be significantly reduced on the 7,28,and 84 days(P <0.05).(2)ELISA results showed that the expression of TGF-? in BALF of the silica group and silica+ Ig G group were significantly increased on days 84(P <0.05).Compared with silica+Ig G group,the expression of TGF-? in BALF could significantly inhibit by CTGF antibody(P <0.05).(3)Western blot results showed that compared with saline group the expression of p120 ctn isoform I and and Rho A were up-regulated in the silica group on day 7(? P <0.05).After exposed to silica particles for 7days,the expression of p120 ctn isoform ? and were significantly reduced? after intraperitoneal injection of CTGF antibody or Ig G,the expression of Rho A were significantly reduced by CTGF antibody(P <0.05).The expression of E-cadherin and p120 ctn isoform ?and ?were significantly down-regulated in the silica group on day 28(P <0.05).The expression of E-cadherin,p120 ctn isoform ? and Rho A were significantly up-regulated by CTGF antibody and returned to the levels of normal saline group on day 28(P <0.05).The expression of p120 ctn isoform ?and ?in silica,silica+Ig G and silica+anti-CTGF group were still significantly lower than the normal saline group on day 84(P <0.05).And there were no significant differences in the levels of p120 ctn isoform ? and ? between silica group and silica+anti-CTGF group on day 84.Conclusion:(1)CTGF antibody plays an active role in the process of silica-induced lung inflammation and fibrosis.Blocking CTGF can significantly inhibit the degree of lung inflammation and fibrosis.And CTGF antibody could attenuate silica-induced pulmonary fibrosis in rats by inhibiting the expression of TGF-? when exposed to silica particles for 84 days.(2)The expression of E-cadherin,p120 ctn isoform ?and ?were decreased significantly by silica particles,indicating that silica particles could lead to pulmonary EMT when exposure to 28 days.CTGF antibody could increase the expression of E-cad,p120 ctn isoform and? Rho A to inhibit the development of EMT induced by silica particles.