| Objective:To assay the the inbihitory avtivity of Citrus alkaloid extract (CAE) on the proliferation of human embryonic lung fibroblasts (MRC-5) and explore the preventive effect on bleomycin (BLM)-induced pulmonary fibrosis (PF) in rats and clarify its possible mechanism.Methods:1. Culture system of MRC-5 cells in the logarithmic growth phase and MTT method were used for the evaluation of inhibitory activity of CAE. The cytotoxicity of CAE was analyzed with lactate dehydrogenase (LDH) assay.2. Rats were randomly divided into six groups:normal, control, prednisone (positive control), CAE-8, CAE-16 and CAE-32 groups. Body weight was measured weekly. Pulmonary fibrosis model was established using bleomycin (BLM). CAE at doses of 8,16 and 32 mg/kg/day, prednisone at dose of 5 mg/kg/day and sodium chloride were administered by gavage for 4 weeks. The rats were killed by decapitation on 28th day, serum samples and lung tissues were collected for assays. Pathological changes were analyzed by HE and Masson stainings to affirm the extent of lung alveolitis and fibrosis under optics microscope. Serum and lung tissue hydroxyproline (HYP) concentrations were measured using alkaline hydrolysis and UV spectrophotometric detection method. mRNA and protein expressions of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and tumor necrosis factor-a (TNF-a) in rat lung tissues were determined by polymerase chain reaction (RT-PCR) and Western blotting.Results:1. In vitro:CAE dose-dependently inbhibited the proliferation of MRC-5. LDH assay clearly revealed that the inhibitory activity of CEA was not due to its cytotoxicity.2. Weight change:CAE-8 group (16 mg/kg/day) group on 7th,21st and 28th days, CAE-32 (32 mg/kg/day) group on 28th day showed a significant higher body weight gain (P<0.01) compared with control group; the body weight of CAE-8 (8 mg/kg/day) on day 7 was higher than prednisone (P<0.01).3. Alveolitis and pulmonary fibrosis:the levels alveolitis, lung fibrosis of CAE treated groups (8,16,32 mg/kg/day) were significantly lower than those of control group (P<0.01 or P<0.05).4. HYP assay results:CAE and prednisone treatments (8,16,32 mg/kg/day) were markedly lowered the HYP levels in both serum and lung tissue compared with control group (P<0.05 or P<0.01).5. RT-PCR and Western blotting results:Compared with normal group, mRNA and protein levels of MMP-9 in lung tissues were significantly decrease (P<0.05 or P<0.01) while mRNA and protein levels of TIMP-1 and TNF-αin lung tissues were significantly elevated in control group (P＜0.05). (1) Compared with control group, mRNA expressions of MMP-9 in lung tissues were significantly up-regulated in CAE-8 and CAE-32 groups (P<0.05 or P<0.01), moreover, mRNA expression of MMP-9 in lung tissues of CAE-16 and prednisone groups also increased but did not reach significance; mRNA expressions of TIMP-1 and TNF-αin lung tissues were significantly decreased in prednisone, CAE-16 and CAE-32 groups (P<0.05 or P<0.01), furthermore, mRNA expression of TIMP-1 and TNF-αin lung tissues of CAE-8 group also lowered but did not statistically significantly; (2) Compared with control group, protein levels of MMP-9 in lung tissues were significantly restored in prednisone, while, CAE-16 and CAE-32 groups (P<0.05 or P<0.01), in addition, protein levels of MMP-9 in lung tissues of CAE-8 group were also elevated but failed to reach significant; protein levels of TIMP-1 in lung tissues were significantly reduced in prednisone, CAE-8, CAE-16 and CAE-32 group (P<0.05 or P<0.01); protein levels of TNF-αin lung tissues were significantly recovered in CAE-32 group (P<0.01), in addition, protein levels of TNF-αin lung tissues of prednisone, CAE-8 and CAE-16 groups were also reduced without significance.Conclusion:CAE dose-dependently inhibited the proliferation of MRC-5. CAE reduced HYP contents of serum and lung tissues, elevated the mRNA and protin expressios of MM9-9, while down regulated mRNA and protein expression levels of TIMP-1 and TNF-α. The results revealed that CAE possessed a preventive effect on BLM-induced PF in rats. The preliminary mechanisms of the effcet might be via inhibition of the expression of TNF-α, regulation of MMP-9 and TIMP-1 balance. |
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