The Effects Of IL-17A Antagonists On Fibroblast Proliferation And Secretion Of Caveolin-1

Objective: To clarify IL-17A antagonists’ the molecularmechanisms of intervention in pulmonary fibrosis and provide theoretical andexperimental basis for clinical use of IL-17A antagonist in treatment of IPF bydiscussesing the effect of IL-17A antagonists of hydroxyproline and fibroblastproliferation and secretion of cytokines.Methods:10healthy female Wistar ratswere randomly divided into2groups on average, They were normal saline (NS)group and bleomycin bleomycin (BLM) group. Intratracheal instillation ofBLM was given to BLM group and intratracheal instillation of physiologicalsaline was given to NS group. The rats of two group were killed respectively atday7after intratracheal instillation by right ventricle of heart exsanguinationsunder general anesthesia and bronchoalveolar lavage(BLF) of the right lungwas performed to obtain alveolar macrophages (AMs). The left lung was usedfor pathological HE staining and cultured medium was collected when thegroups’AMs cultured after24hours. Rats’ fibroblasts (208F) were divided into5groups: controlled group (cultured medium of NS group’s AMs) and modelgroup (cultured medium of BLM group AMs), the antibody group (group A andgroup B resistance, resistance to group C)(cultured medium of BLM groupAMs+IL-17A antibody of different concentrations,0.5g/L、1g/L、2g/L).208Fcells proliferation respectively was detected at24hours,48hours and72hoursafter cultivation by using CCK8. At24hours after cultivation,enzyme-linkedimmunosorbnent assay (ELISA) was used to test hydroxyproline in the culturedmedium of208F, immunocytochemisty was used to detecte the expression ofcaveolin-1of208F, real-time Polymerase Chain Reaction(RT-PCR) wasperformed to detecte collagen Ⅰ mRNA and Caveolin-1mRNA expression in208F. Results:(1) pathological HE of rats lung tissue:in NS group, the alveolar structure is complete, edema and inflammatory cells infiltration wasn’t found inthe alveolar interval and inflammatory cells exudation was invisible in thealveolar cavity;in BLM group, hyperemia, edema and inflammatory cellsinfiltration were found in alveolar septa and a large of inflammatory cells wasvisible in alveolar space obviously.(2) the CCK-8results showed thatcompared with the control group, in model group and all antibody groups208Fcell proliferation level at all time points were significantly increased, thedifference was statistically significant (P <0.05); Group compared with modelgroup, the level of208F cell proliferation in all antibody groups at all timepoints were lower, the difference had statistical significance (P <0.05);Between antibody groups, in group A208F cell proliferation resistance level atall time points were higher than other two groups, the difference had statisticalsignificance (P <0.05), and208F cell proliferation levels between group B andgroup C, at all time points, have no obvious difference (P>0.05)(3)ELISAresults suggest: compared with control group, hydroxyproline levels in208Fcell culture supernatant of model group and each antibody group increasedsignificantly, the difference was statistically significant (P <0.05); Groupcompared with model group, levels of hydroxyproline in the culture supernatantof all antibody groups were lower, the difference had statistical significance (P<0.05); in Antibody groups, level of hydroxyproline in the cell culturesupernatant of group A208f were higher than other two groups, the differencehad statistical significance (P <0.05), and in the cell culture supernatant ofgroup B and group C,208f hydroxyproline level had no obvious difference (P>0.05).(4) Immunocytoche-mistry results indicate: compared with controlgroup, in model group and each antibody group208F cells’ Caveolin-1levelswere lower, the difference had statistical significance (P <0.05); Comparedwith model group, the208F cells’ caveolin-1in all antibody groups wereincreased, the difference had statistical significance (P <0.05); in Antibodygroups,208F cell’ caveolin-1level in group A is lower than the other two groups, difference have statistical significance (P <0.05), and208F cells’caveolin-1level of group B and group C had no obvious difference (P>0.05).(5)RT-PCR results showed that:208F cells’ caveolin-1mRNA of Groups,compared with the control group,208F cells’ caveolin-1mRNA levels in modelgroup and each antibody group were lower, difference have statisticalsignificance (P <0.05); Compared with model group,208F cells’ caveolin-1mRNA in each group antibody were increased, the difference had statisticalsignificance (P <0.05); in Antibody groups,208F resistance cells’ caveolin-1mRNA level group A is lower than the other two groups, the difference hadstatistical significance (P <0.05), and208F cells caveolin-1mRNA level ingroup B and group C had no obvious difference (P>0.05).208F cells’Ⅰ typecollagen mRNA of groups, compared with the control group,208F cells’ Ⅰcollagen mRNA in model group and the group A levels increased, differencehave statistical significance (P <0.05),208F type cells’ Ⅰ collagen mRNA ingroup B and group C, had no obvious difference (P>0.05); Compared withmodel group, the208F cells’ Ⅰ collagen mRNA all antibody groups werelower, the difference had statistical significance (P <0.05); in the Antibodygroups,208F cells’ Ⅰ collagen mRNA levels in group A higher than the othertwo groups, difference have statistical significance (P <0.05), and208F cells’collagen mRNA level in group B and group C,had no obvious difference (P>0.05).Conclusions: L-17A antibodies as IL-17A antagonists can reducefacilitative action of alveolar macrophages’ cultured supernatant fromexperimental pulmonary fibrosis rats to the cell proliferation and collagen typeⅠ expression of fibroblast in vitro; Also it can reduce inhibition of caveolin-1expression of fibroblasts by cultured supernatant of alveolar macrophage fromexperimental pulmonary fibrosis rat.