The Research Of Interferon-γ And Curcumin On Bleomycin-induced Pulmonary Fibrosis And Fibroblasts Apoptosis In Rats

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive, highlylethal disease of unknown etiology that is characterized by rapid fibroticdestruction of the lung. On surgical biopsy specimens, IPF is associatedwith the histologic pattern referred to as usual interstitial pneumonia(UIP). The prognosis for IPF patients is extremely poor, with a mediansurvival generally reported as 2 to 3 years from the time of diagnosis. Theconventional hypothesis that IPF was an inflammatory alveolitis led to anemphasis on anti-inflammatory therapy, but it did not improve diseaseoutcome. Now evolving hypothesis is that fibrosis results from epithelialinjure and abnormal wound healing, so future therapies should aim atpreventing or inhibiting the fibroproliferative response and enhancingnormal alveolar reepithelialization. However, few agents have beenadequately studied in humans. This study is to observe the effect andmechanism of interferon-γand curcumin on pulmonary fibrosis inducedby bleomycin in rats.Partâ… : The effect of interferon-γon bleomycin-induced pulmonaryfibrosis in ratsMethods: 80 male SD rats were randomly divided into 4 groups: normal control group, model control group, hydrocortisone treated group andIFN-γtreated group. The rats were killed on the 3, 7, 14 and 28th day ofthe study. The concentration of IFN-γ, and transforming growth factor-β₁(TGF-β₁) in bronchoalveolar lavage fluid (BALF) were measured, theseverity of alveolitis and fibrosis, the content of hydroxyproline, theexpression of transforming growth factor-β₁ (TGF-β₁) mRNA andinterferon-γ(IFN-γ) mRNA in lung tissues were analyzed.Results: (1)The severity of alveolitis: The scores of alveolitis in modelcontrol group, hydrocortisone group and IFN-γgroup at day 3,7,14,28 allwere higher than that in normal control group. The scores in IFN-γ, groupat day 14 was higher than that in the model control group at day 3,7,14than that in the hydrocortisone group (P＜0.05). (2)The scores ofpulmonary fibrosis: The scores in model control group, hydrocortisonegroup and IFN-γ, group at day 14,28 were higher than that in normalcontrol group, but no difference among these three groups. (3)Thecontents of hydroxyproline of lung tissue: The contents in model controlgroup, hydrocortisone group and IFN-γgroup at day 14,28 were higherthan that in normal control group, but no difference between modelcontrol group and IFN-γgroup, The content in IFN-γ, group at day 28 washigher than that in the hydrocortisone group (P＜0.05). (4)Theconcentrations of IFN-γin BALF: The concentrations in model controlgroup at day 3,7 were higher than that in normal control group. The concentrations in IFN-γtreated group were higher than that in modelcontrol group at day 3,7, 14,28 and higher than that in the hydrocortisonegroup at day 3,7,14 (P＜0.05). (5) The expressions of IFN-γmRNA oflung tissue: The expressions in model control group, hydrocortisonegroup at day 3,7,14 were higher than that in normal control group, Theexpressions in IFN-γtreated group were higher than that in normalcontrol group at day 3,7,14,28 and higher than that in the hydrocortisonegroup at day 3,7 (P＜0.05). (6)The concentrations of TGF-β₁ in BALF:The concentrations in model control group, hydrocortisone group andIFN-γgroup at day 3,7,14,28 all were higher than that in normal controlgroup, the concentrations in IFN-γtreated group was higher than that inmodel control group at day 3 and higher than that in the hydrocortisonegroup at day 3,7,14 (P＜0.05). (7) The expressions of TGF-2₁ mRNA inlung tissues: The expressions in model control group, hydrocortisonegroup and IFN-γgroup at day 3,7,14,28 all were higher than that innormal control group, the expressions in IFN-γtreated group was higherthan that in model control group at day 7 and higher than that in thehydrocortisone group at day 3,7 (P＜0.05).Partâ…¡: The effect of curcumin on bleomycin-induced pulmonaryfibrosis in ratsMethods: 54 SD rats were randomly divided into 3 groups, each groupcontained 18 rats. The control group, which received a single intratracheal dose of 2 ml/kg of sterile physiological saline at day 0,andfrom 14d, 0.5ml/kg of sterile physiological saline was injectedintraperitoneally every day; The bleomycin group, which received asingle intratracheal dose of 5mg/kg of bleomycin A₅, and from 14d, 0.5ml/kg of suspension of 6% alcohol and 6% polyethylene glycol wasinjected intraperitoneally every day; The curcumin group, whichreceived a single intratracheal dose of 5mg/kg of bleomycin A₅, and from14d, 50mg/kg of curcumin (suspended in 6% alcohol and 6%polyethylene glycol) was injected intraperitoneally every day. 6 rats ineach group were killed at day 17, 21, 28 after modeling in batches. Thesections of lungs were stained with hematoxylin-eosin (HE) andMasson's trichrome to evaluate the severity of alveolitis and pulmonaryfibrosis. The content of hydroxyproline and the expression oftransforming growth factor-β₁ (TGF-β₁) mRNA, interferon-y (IFN-γ)mRNA in lung tissues were analyzed. The concentrations of TGF-β₁ andIFN-γin bronchoalveolar lavage fluid (BALF) were measured.Results: (1) The scores of alveolitis in the curcumin group and thebleomycin group at day 28 were 1.3±0.5, 2.0±0.9, there was asignificant difference between two groups (P＜0.05). (2) The scores ofpulmonary fibrosis in the curcumin group and the bleomycin group were1.3±0.5, 1.8±0.4 at day 21, and 1.2±0.4, 2.2±1.0 at day 28, twogroups were statistically different (P＜0.05). (3) The contents of hydroxyproline in lung tissues in the curcumin group and the bleomycingroup were 1.75±40.36, 2.47±0.24μg/g at day 28, the difference wasstatistically different between two groups (P＜0.01). (4)Theconcentrations of TGF-β₁ in BALF in the curcumin group and thebleomycin group were 20±3, 39±7 ng/L at day 21, and 24±4, 40±7ng/L at day 28, all were statistically different between two groups(P＜0.05). (5) The expression of TGF-β₁ mRNA in lung tissues in thecurcumin group and the bleomycin group were 0.51±0.11, 0.59±0.13 atday 21, and 0.50±0.07, 0.64±0.11 at day 28, all were not statisticallydifferent between two groups (P＞0.05). (6)The concentrations of IFN-γin BALF in the curcumin group and the bleomycin group were 0.49₀.17, 0.50±0.08 at day 21, and 0.52±0.15, 0.52±0.11 at day 28, allwere not statistically different between two groups (P＞0.05). (7) Theexpression of IFN-γmRNA in the curcumin group and the bleomycingroup were 28±5, 35±13 at day 21, and 30±411, 39±13 at day 28,there were no significant differences between two groups (all P＞0.05).Partâ…¢: The effect of curcumin on proliferation and apoptosis offibroblasts from rats with bleomycin-induced pulmonary fibrosisMethods: 10 SD rats were randomly divided into two groups: the controlgroup, which received a single intratracheal dose of 2 ml/kg of sterilephysiological saline, and the bleomycin group, which received a singleintratracheal dose of 5 mg/kg of bleomycin A₅ All rats were killed at day 14. Lungs samples were removed and cut. Lung fibroblasts were isolatedby trypsin dispersion and cultured. Medium with curcumin 5,10,20,30,40μmol/l or without curcumin were supplemented and incubated for 6hours. Fibroblasts proliferation was evaluated MTT (methyl thiazolyltetrazolium), fibroblasts apoptosis and expression of Caspase-3 wereevaluated by flow cytometry analysis.Results: (1) Fibroblasts proliferation: the OD value of proliferation in thecontrol group under the concentration of curcumin 0,5,10,20,40umol/Lwere not statistically differences among five groups,but the OD value inthe bleomycin group were 0.209±0.044,0.161±0.030,0.132±0.031,0.084±0.070 and 0.013±0.006 respectively, there weresignificant differences among five groups (p＜0.05). (2) Fibroblastsapoptosis: the apoptosis in the control group under the concentration ofcurcumin 0,10,20,30umol/L were not statistically different, but theapoptosis in the bleomycin group were 3.58±1.37,20.13±4.97,34.30±7.02 and 42.64±10.40 respectively, five groups were statisticallydifferent (p＜0.05). (3)Fibroblasts expression of Caspase-3: the expressionin the control group under the concentration of curcumin0,10,20,30umol/L were not statistically different among five groups, butthe expression in the bleomycin group were 5.28±2.65,48.38±36.01,60.14±30.35 and 70.01±29.93 respectively, there weresignificant differences among five groups (p＜0.05). Conclusion: (1)Early given IFN-γcan't inhibit composition of TGF-β₁and hydroxyproline and can't prevent the development of alveolitis andpulmonary fibrosis in bleomycin-induced pulmonary fibrosis rats.(2)Curcumin could alleviate alveolitis and pulmonary fibrosis induced bybleomycin in rats, the mechanism was perhaps related to its inhibition ofTGF-β₁. (3)Curcumin could inhibit fibroblasts proliferation and promotefibroblasts apoptosis and expression of Caspase-3.