| BackgroundPulmonary fibrosis (PF) is a kind of fiber proliferative disease with no effective treatments.Patients with this kind of disease finally die of progressive dyspnea, concurrent lung cancer or cardiovascular diseases. Idiopathic Pulmonary Fibrosis(IPF), Nonspecific Interstitial Pneumonitis(NSIP) are the most common type in Interstitial Lung Disease(ILD),among them,JPF is a kind of diffuse pulmonary disease which involves the pulmonary alveoli and bronchioles,the function of alveolar capillary membrance gradually disappears,and the lung finally develops into diffuse pulmonary fibrosis and honeycomb lung. IPF has a high fatality rate and poor prognosis,the median survival of it is 2-3 years after diagnosisAlthough we have already had a better understanding of the pathogenesis of ILD especially IPF these years,we are not so clear about its cause and molecular mechanism yet.There is no other effective treatment except lung transplantation and long-term oxygen therapy. Therefore an effectived therapeutic is needed imperiouslyTraditional Chinese medicine and pharmacy is a good complement of western medicine.Due attention has been paid to traditional Chinese medicine and pharmacy in the treatment of pulmonary fibrosis.Many studies show that astragalus membranceus has an antioxidant effect,but research in astragalus membranceus about pulmonary fibrosis is little.The unbalance of oxidation and antioxidant palys an important role in the development of PF. The living organism produces free radicals every moment,but the free radicals has not resulted serious consequences,because the living organism also has a system of antioxidant,oxidant and antioxidant keep relative balance.The lung also has oxidant and antioxidant system,many researches show the unbalance of oxidation and antioxidant in IPF patients.If we correct the imbalance of oxidation and antioxidant in IPF patients,we may relieve PF.We suppose that astragalus membranceus has a function of antioxidant,it may inhibits bleomycin-induced pulmonary fibrosis in mice by maintaining the balance of oxidant-antioxidant.Our experiments adopt the method of intratracheal aerosol inhaled by MicroSprayer atomization devices to optimize the PF mice model,and then the mice were intraperitoneal injected with astragalus membranceus parenteral solution. All mice were sacrificed 14 days after the treatment,the lungs and serum were collected for detection.The lungs were stained with hematoxylin eosin and Masson's trichrome for the pathological and fibrosis examination, examined with RT-PCR for the expression of Superoxide Dismutasel/2/3(SOD1/2/3)?Catalase (CAT), NADPH oxidase2/4 (NOX2/4)? a-SMA mRNA and examined with western blot for the expression ofa-SMA? NOX2/4 protein.The serum was assayed by colorimetric method for concentration of malondialdehyde (MDA) and total antioxidant capacity(T-AOC).This study explored the possible mechanism of astragalus membranceus in reliving pulmonary fibrosis,it may produce some experimental base in the treatment of pulmonary fibrosis for reference.Method1. Astragalus membranceus inhibits bleomycin-induced pulmonary fibrosis in mice8 weeks female mice were randomly divided into three groups,control group, bleomycin group and astragalus membranceus group. Bleomycin group mice received intratracheal aerosol inhaled of BLM (3mg/kg, 100?L),while in control group, mice received the same volume of noamal saline intratracheal aerosol inhaled. Astragalus membranceus group mice received intraperitoneal injected with astragalus membranceus parenteral solution at a dose of (1.7g/kg.,100ul) after intratracheal aerosol inhaled of BLM (3mg/kg, 100?L), bleomycin group mice and control group mice received intraperitoneal injected with same volume of noamal saline. Mice were sacrificed on 14d after the treatment.,the lungs and serum were collected for detection.The lungs were dissected and fixed in 10% buffered formalin.After embedding in paraffin,the sections were evaluated by HE and Masson's trichrome respectively.Lung injury and fibrosis grading were performed using Mikawar and Ashcroft scoring systems.The lungs were also examined with RT-PCR for the expression of a-SMA mRNA and examined with western blotting for the expression of a-SMA protein.2.The influence of astragalus membranceus to MDA/T-AOC in bleomycin-induced pulmonary fibrosis mice8 weeks female mice were randomly divided into three groups,control group, bleomycin group and astragalus membranceus group.The mice were handling as above. Mice were sacrificed on 14d after the treatment,the serum was collected for detection.The concentration of MDA and T-AOC were detected by colorimetric method in serum following the instruction of MDA kit and T-AOC kit.3.The influence of astragalus membranceus to oxidant associated gene in bleomycin-induced pulmonary fibrosis mice8 weeks female mice were randomly divided into three groups,control group, bleomycin group and astragalus membranceus group.The mice were handling as above. Mice were sacrificed on 14d after the treatment,the lungs were collected for detection. The lungs were then examined with RT-PCR for the expression of NOX2/4 mRNA and examined with western blotting for the expression of NOX2/4 protein.4. The influence of astragalus membranceus to antioxidant associated gene in bleomycin-induced pulmonary fibrosis mice8 weeks female mice were randomly divided into three groups,control group, bleomycin group and astragalus membranceus group.The mice were handling as above. Mice were sacrificed on 14d after the treatment,the lungs were collected for detection. The lungs were then examined with RT-PCR for the expression of SOD1/2/3> CAT mRNA.5.Statistical analysisThe data was statistical analyzed with SPSS19.0,data was presented as mean±SD for at least three separate experiments.All the data was peformed with test of normality and test of homogeneity of variance.ANOVA and LSD test were used for multiple group comparisons when data met test of normality and test of homogeneity of variance. P<0.05 compared with bleomycin group was considered statistically significant.ANOVA and Dunnett T3 test were used for multiple group comparisons when data met test of normality and but do not met test of homogeneity of variance. P <0.05 compared with bleomycin group was considered statistically significant.Results1. Astragalus membranceus inhibits bleomycin-induced pulmonary fibrosis in mice1.1 HE staining of the lungs:the lungs of the control group mice had clear alveolar structure,with thin alveolar walls,less inflammatory cells between the interstitial tissues.While the lungs of the bleomycin group mice had destructive alveolar structure,with thick alveolar walls and more inflammatory cells between the interstitial tissue,the pathological demage of the lungs in astragalus membranceus group was between bleomycin group and control group.The pathological score of the mice in three groups was 1.50±1.73?7.75±0.96? 4.5±0.58, The pathological score of the lungs in bleomycin group was significantly higher than that in control group (P<0.01).After astragalus membranceus treatment, the pathological score was significantly decreased (P?0.01)1.2 Masson's trichrome staining of the lungs:the lungs of the control group mice had complete alveolar structure,with barely any inflammatory cells and collagen deposition.While the lungs in bleomycin group mice had destructive alveolar structure,with lots of inflammatory cells and collagen deposition.The fibrosis demage of the lungs in astragalus membranceus group was between bleomycin group and control group.The fibrosis score of the mice in three groups was 1.50±0.58?7.25±0.50? 3.25±0.50, The pathological score of the lungs in bleomycin group was significantly higher than that in control group (P<0.01).After astragalus membranceus treatment, the pathological score was significantly decreased (P?0.01)1.3?-SMA mRNA expression of the lungs:Thea-SMA mRNA expression of the lungs in bleomycin group was significantly higher than that in control group (1.33±0.07 VS 0.99±0.04, P<0.01).After astragalus membranceus treatment, the a-SMA mRNA expression was significantly decreased (0.99±0.05 VS 1.33±0.07, P <0.01)1.4a-SMA protein expression of the lungs:Thea-SMA protein expression of the lungs in bleomycin group was significantly higher than that in control group (0.55±0.03 VS 0.36±0.02, P<0.01).After astragalus membranceus treatment, the a-SMA protein expression was significantly decreased (0.36±0.01 VS 0.55±0.03, P <0.01)2.The influence of astragalus membranceus to MDA/T-AOC in bleomycin-induced pulmonary fibrosis mice.2.1 The concentration of MDA in the serium:The concentration of MDA in the serium in bleomycin group was significantly higher than that in control group (282.54±6.93 VS 9.88±0.83, P<0.01).After astragalus membranceus treatment, the concentration of MDA in the serium was significantly decreased (9.02±2.89 VS 282.54±6.93, P<0.01)2.2 The concentration of T-AOC in the serium:The concentration of T-AOC in the serium in bleomycin group was significantly higher than that in control group (15.01±1.06 VS 9.44±1.26, P<0.01).After astragalus membranceus treatment, the concentration of T-AOC in the serium was no significantly difference with that in bleomycin group (13.01±1.70 VS 15.01±.06 P=0.068)2.3 MDA/T-AOC in the serium:MDA/T-AOC in the serium in bleomycin group was significantly higher than that in control group (18.90±1.52 VS 1.06±0.17, P <0.01).After astragalus membranceus treatment, MDA/T-AOC in the serium was significantly decreased (0.69±0.20 VS 18.90±1.52, P<0.01)3.The influence of astragalus membranceus to oxidant associated gene in bleomycin-induced pulmonary fibrosis mice.3.1NOX2 mRNA expression of the lungs:The NOX2 mRNA expression of the lungs in bleomycin group was significantly higher than that in control group (1.31±0.14 VS 0.88±0.09, P<0.01).After astragalus membranceus treatment, the NOX2 mRNA expression was significantly decreased (0.56±0.05 VS 1.31±0.14, P <0.01)3.2NOX4 mRNA expression of the lungs:The NOX4 mRNA expression of the lungs in bleomycin group was significantly higher than that in control group (1.26±0.01 VS 1.16±0.02, P<0.05).After astragalus membranceus treatment, the NOX4 mRNA expression was significantly decreased (0.71±0.08 VS 1.26±0.01, P <0.01)3.3NOX2 protein expression of the lungs:The NOX2 protein expression of the lungs in bleomycin group was significantly higher than that in control group (0.41±0.01 VS 0.17±0.01, P<0.01).After astragalus membranceus treatment, the NOX2 protein expression was significantly decreased (0.17±0.03 VS 0.41±0.01, P <0.01).3.4NOX4 protein expression of the lungs:The NOX4 protein expression of the lungs in bleomycin group was significantly lower than that in control group (0.11±0.02 VS 0.84±0.05, P<0.01).After astragalus membranceus treatment, the NOX4 protein expression was significantly increased (0.23±0.02 VS 0.11±0.02, P <0.05)4. The influence of astragalus membranceus to antioxidant associated gene in bleomycin-induced pulmonary fibrosis mice.4.1 SOD1 mRNA expression of the lungs:The SOD1 mRNA expression of the lungs in bleomycin group was significantly lower than that in control group (0.55±0.21 VS 1.09±0.22, P<0.01)).After astragalus membranceus treatment, the SOD1 mRNA expression was no significantly difference with that in bleomycin group (0.47±0.15 VS 0.55±0.21, P=0.552),but still significantly lower than that in control group (0.47±0.15 VS 1.09±0.22, P?0.01)4.2 SOD2 mRNA expression of the lungs:There were no significantly differences of the SOD2 mRNA expression of the lungs among the mice in three groups (1.03±0.03 VS 1.04±0.01 VS 1.05±0.04 P=0.580)4.3 SOD3 mRNA expression of the lungs:The SOD3 mRNA expression of the lungs in bleomycin group was significantly higher than that in control group (1.32±0.04 VS 1.10±0.04, P<0.01).After astragalus membranceus treatment, the SOD3 mRNA expression was significantly decreased (0.39±0.10 VS 1.32±0.04, P <0.01)4.4 CAT mRNA expression of the lungs:The CAT mRNA expression of the lungs in bleomycin group was significantly lower than that in control group (0.99±0.09 VS 1.26±0.06, P<0.01).After astragalus membranceus treatment, the CAT mRNA expression was significantly lower that in bleomycin group and control group (0.43±0.02 VS 0.99±0.09, P?0.01; 0.43±0.02 VS 1.26±0.06, P?0.01)Conclusionastragalus membranceus has the function of antioxidant,it can relieve bleomycin-induced pulmonary fibrosis.The mechanism may be that astragalus membranceus can maintain the balance of oxidant-antioxidant in bleomycin-induced pulmonary fibrosis mice and inhibit the injury of the architecture cell of the lung.The research of antioxidant and anti-fibrosis of astragalus membranceus may produce some experimental base of astragalus membranceus in the treatment of pulmonary fibrosis for reference. |
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