The Therapeutic Effect Of Oleanane Triterpenoid CDDO-Me On Radiation Induced Lung Injury In C57BL/6 Mice

Objective To investigate the therapeutic effect of CDDO-Me on radiation induced lung inflammation and radiation induced lung fibrosis in C57BL/6 mice, and the potential mechanism of its action.Methods 88 C57BL/6 female mice of six to eight weeks were divided into radioactive pneumonia group and radioactive pulmonary fibrosis group.Subseqently control group 、simple irradiation group 、 irradiation +CDDO-Me group and CDDO-Me group were established in both pnermonia and fibrosis studies. The right hemithoraxes of C57BL/6 mice were irradiated using varian linear accelerator with 6MV-X ray.The radiation dose in pneumonia group was 12.5 Gy and in fibrosis group was 22.5 Gy. Mice in control group and CDDO-Me group were only anesthetized but not irradiated.In pneumonic study, mice were gavaged in a dose of 600ng(200μl) CDDO-Me in 1 day befor and 1,3,5 day after irradiation respectively. In fibrotic study, mice were gavaged in a dose of 600ng(200μl) CDDO-Me in 1day befor and 1,3,5,7,9 day after irradiation respectively. Mice in control group and irradiation group were gavaged with the same volume normal saline at the same time. After 3weeks of irradiation in pneumonia group, up lobes of right lungs of 6 mice were processed for HE staining, middle lung lobes were used for real time-PCR analysis to investigate α-SMA 、Col1a1、FN m RNA expression levels, and remaining lung lobes were used for ELISA analysis to investigate TGF-β1、IL-6 and IL-10 levels. The remaining 6 mice, right lung alveoli were lavaged and collected the bronchoalveolar lavage fluid(BALF).Cells in the collected BALF were counted and classification, and protein concentration was analyzed using BCA method. After 12 weeks of irradiation in fibrosis group, right lungs from micewere processed for Masson staining. Remaining lung lobes were used for quantitative real time-PCR(q PCR) analysis to investigate α-SMA 、Col1a1、FN m RNA expression levels and examine the lung tissue hydroxyproline(Hyp) content by alkali hydrolysis method.Results(1)Pathological examination showed in R + CDDO-Me groups, lung tissue damages were mitigated remarkably compared with R groups.Pneumonia score and Masson semi-quantitative analysis showed significant differences between two groups, P<0.001;(2)ELISA testing showed, TGFβ1、IL – 6、IL – 10 levels were significantly rised in irradiation group compared with control group, P < 0.001; TGFβ1 and IL – 6 levels were significantly reduced in irradiation+CDDO-Me group compared with irradiation group, P<0.05; IL – 10 levels were significantly increased in irradiation+CDDO-Me group compared with irradiation group, P< 0.01;(3)BALF investigation results showed, total amount of cells、the percentage of inflammatory cells and protein contents in BALF were all distinctly increased in irradiation group compared with control group, P< 0.001; total amount of cells and inflammatory cells percentage were dramatically decreased in irradiation+CDDO-Me group compared with R group, P < 0.001; protein contents were markedly lessened in irradiation+CDDO-Me group compared with irradiation group, P< 0.01;(4)q PCR results showed,in pneumonia group,pro-fibrotic factors of α-SMA 、 Col1a1 and FN m RNA expression levels were all elevated notably in irradiation group compared with control group,P < 0.001;and m RNA expression of these three factors were all decreased distinctly in irradiation+CDDO-Me group compared with irradiation group, P < 0.001; in fibrosis group,pro-fibrotic markers of α-SMA 、Col1a1and FN m RNA expression levels were all rised clearly in irradiation group compared with control group, P < 0.001;α-SMA 、Col1a1and FN m RNA expression were reduced distinctly in irradiation+CDDO-Me group compared with irradiation group, P<0.05;(5) hydroxyproline of lung tissue quatitative analysis showed, Hyp contents were promoted significantly in irradiation group compared with control group,P<0.001;which were reduced distinctly in irradiation+CDDO-Me group compared withirradiation group,P< 0.001.Conclusion(1)This experiment successfully built radiation induced lung injury model in mice;(2)CDDO-Me can improve radiation induced lung inflammation obviously;(3)CDDO-Me can play a strong anti-fibrosis effect in the radiation induced mice lung fibrotic model;(4) CDDO- Me can reduce TGFβ1 level in pneumonia stage,which might be one of the mechanisms of its anti-inflammatory and anti-fibrosis functions,and the therapeutic mechanism of CDDO-Me for RILI needs further study.