Study Of Hydroxysafflow Yellow A On Inhibiting Injury In Different Times Of Chronic Obstructive Pulmonary Disease

The rising prevalence of chronic obstructive pulmonary disease(COPD) hasbecome a serious public health problem, the main mechanism of this disease ischronic inflammation and there is no ideal medicine to treat it. Hydroxysaffloryellow A (HSYA) is the main active ingredient of Carthamus tinctorius L.. We foundthat HSYA was effective to attenute chronic inflammatory injury in COPD rat andcan attenuate pulmonary fibrosis with TGF-β1upregulation in mice. HSYA waseffective to inhibit cellular inflammatory factor expression. In this project we plan toobserve the effect of HSYA to inhibit lung inflammatory signal transduction,attenuate abnormal cytokine expression, pulmonary inflammatory injury and tissueremodeling in different times of COPD rat. In this project we want to study themechanism of chronic inflammatory attenuating effect of HSYA and to provide afoundation for the development of new drug to treat COPD.Method1.Animal experiment: We used passive cigarette smoking and intratrachealinstillation of lipopolysaccharide to establish the COPD rat model. Male wistar ratswere stratified randomly divided into12groups according to body weight(N=10),including two weeks normal control group, two weeks COPD group, two weeksCOPD+35mg/kg HSYA group, two weeks COPD+70mg/kg HSYA group, threeweeks normal control group, three weeks COPD group, three weeksCOPD+35mg/kg HSYA group, three weeks COPD+70mg/kg HSYA group, fourweeks normal control group, four weeks COPD group, four weeks COPD+35mg/kgHSYA group, four weeks COPD+70mg/kg HSYA group. HE staining of lung tissue slice was used to observe the pathological changes, Masson staining of lung tissueslice was used to observe the small airway morphology change and collagendeposition. TNF-α, IL-1β, IL-6and TGF-β1in bronchoalveolar lavage fluid(BALF)were measured by ELISA, the level of p65andα-SMA protein in lung tissue wasdetected by immunohistochemical technology.2.Cell experiment：The model of TGF-β1-induced activation of NIH/3T3cell wasused and NIH/3T3fibroblasts were divided into8groups: normal group, normalHSYA group, TGF-β1group, TGF-β1+0.5μmol/L HSYA group, TGF-β1+1μmol/LHSYA group, TGF-β1+2μmol/L HSYA group, TGF-β1+2mg/L SB-431542group,TGF-β1+2mg/L SB431542+2μmol/L HSYA group. The proliferation of the NIH/3T3cell was observed with MTT assay. The migration of the NIH/3T3cell wasobserved with wound healing assay.Results1.Animal experiment: In COPD model groups, obvious inflammatory cellsinfiltration, bronchioles effusion, and alveolar wall thickening in lung tissue wereobserved. In2weeks COPD group inflammatory cells infiltrating was more seriousthan that in COPD groups of3or4weeks. The thickening of alveolar wallsaggravated gradually as smoking continued and the injuries in4weeks COPD groupwere the most serious; Compared with the COPD groups in same time point HSYAwere found to attenuate the damage especially high dose HSYA. Masson stainingresults: In rats of COPD model groups collagen deposition in small airwaysincreased significantly compared with the normal control groups at the same timepoints. These small airway collagen deposition increased gradually as the smokingcontinued and HSYA can attenuate these injuries especially in high dose; ELISAresults: Compared with the normal control group at the same time points, IL-6, IL-1β,TNF-αand TGF-β1levels in BALF of the COPD groups were significantly increased (all p <0.001) and these elevated expression were inhibited by in HSYA dosedependently at different time points. Immunohistochemical assay results: Comparedwith the normal control group at the same time points, the levels of p65andα-SMA in lung tissue of the COPD groups were significantly increased (all p<0.001),the levels of p65and α-SMA in lung tissue increased time dependently. Comparedthe COPD group at the same time points, the high expression of p65and α-SMAwere inhibited by HSYA dose dependently.2.Cell experiment: From the data of the same time points of MTT test it can beobserved that HSYA (0.5μmol/L,1μmol/L or2μmol/L)attenuated the3T3cellproliferation induced by TGF-β1(TGF-β1group vs. normal group（all p <0.01,TGF-β1+HSYA groups vs. TGF-β1group, all p＜0.05). The cell migrationinduced by TGF-β1can be attenuated by HSYA(TGF-β1group vs. normal group, allp＜0.001; TGF-β1+HSYA groups vs. TGF-β1group, all p＜0.001). The effectHSYA to inhibit TGF-β1induced3T3cell proliferation and migration is time anddose dependent.Conclusion1. HSYA can alleviate inflammatory injuries and COPD pulmonary fibrosis inCOPD rats.2. HSYA inhibits inflammatory cytokines expression, small airway collagendeposition, p65and α-SMA level increase in lung of COPD rats.3. HSYA is active to inhibit TGF-β1-induced activation of NIH/3T3cell.