陈皮挥发油对大鼠肺纤维化的干预作用

目的：研究陈皮挥发油（essential oil extracted from Citrus reticulata,EOcR）对人胚肺成纤维细胞（humanembryonic lung fibroblast,HELF）增殖的影响,以及对博莱霉素诱导的大鼠肺纤维化模型的干预作用,并探讨其作用机制。方法：将HELF传代培养,待细胞进入对数生长期后进行实验,采用四甲基偶氮唑盐比色法测定不同浓度陈皮挥发油对HELF增殖的影响。大鼠随机分为正常组、模型组、强的松组、陈皮挥发油25mg（EOCR-25）组、陈皮挥发油50mg（EOCR-50）组、陈皮挥发油100mg（EOCR-100）组和陈皮挥发油200mg（EOCR-200）组。除正常组外,其余各组气管内注入博来霉素建立大鼠肺纤维化模型。建模后除正常组、模型组两组大鼠外,其余各组大鼠分别给予相应药物灌胃。各组大鼠于实验第28天处死并留取血清和肺组织。分光光度法测定血清和肺组织超氧化物岐化酶（superoxi dedismutase,SOD）及丙二醛（malondialdehyde,MDA）含量;双抗夹心酶联免疫吸附法测定肺组织I型胶原蛋白（typeⅠcollagen,ColⅠ）含量;根据积分量表对肺组织病理学变化进行半定量分析;免疫组织化学和原位杂交法检测肺组织中结缔组织生长因子（connective tissue growth factor,CTGF）蛋白及mRNA表达,并进行图像半定量分析。结果：不同浓度EOcR均可抑制HELF的增殖。建模后第7、14、21、28天时,EOCR-50、EOCR-100和EOCR-200组大鼠体质量增幅较模型组显著增加（P〈0．05或P〈0．01）;EOCR-100和EOCR-200组肺泡炎、肺纤维化积分显著低于模型组（P〈0．01）;EOCR-50、EOCR-100和EOCR-200组血清及肺组织SOD活性显著高于模型组（P〈0．01）;EOCR-50、EOCR-100和EOCR-200组血清及肺组织MDA含量显著低于模型组（P〈0．05）;EOCR-100和EOCR-200组肺组织ColⅠ含量显著低于模型组（P〈0．01）,肺组织CTGF蛋白及mRNA表达量显著低于模型组（P〈0．01）。结论：陈皮挥发油对博莱霉素诱导所致大鼠肺纤维化具有干预作用,其作用机制可能是通过调节氧化和抗氧化失衡,降低CTGF蛋白及其mRNA表达,减少胶原沉积以减轻纤维化程度。

Preventive effects of Citrus reticulata essential oil on bleomycin-induced pulmonary fibrosis in rats and the mechanism

OBJECTIVE: To investigate the effects of essential oil of Citrus reticulata (EOCR) on proliferation of human embryonic lung fibroblasts (HELFs),and to explore its protective effects on bleomycin (BLM)-induced lung fibrosis in rats.METHODS: Routinely cultured HELFs during the logarithmic phase of growth were divided into control and treated groups,and applied for evaluation of inhibitory activity using methylthiazol tetrazolium (MTT) assay.A rat model of BLM-induced pulmonary fibrosis was used for the evaluation of antifibrotic effect of EOCR.Forty-two Sprague-Dawley rats were randomly divided into normal group,model group,prednisone group and different doses of EOCR groups.BLM was intratracheally instilled into all the rats except those in the normal group,and EOCR was orally given to BLM-treated rats at doses of 25,50,100 and 200 mg/kg once per day for four weeks.The rats in the normal group were intratracheally administered the same volume of saline.On the 28th day,rats were sacrificed under anesthesia,and the serum and lung tissues were collected.Superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents in serum and lung tissues were analyzed with corresponding kits; type Ⅰ collagen (Col Ⅰ ) content in lung tissues was evaluated with enzyme-linked immunosorbent assay; pulmonary fibrosis was assessed by lung histology; protein and mRNA expressions of connective tissue growth factor (CTGF) in lung tissues were measured with immunohistochemical and in situ hybridization semiquantitative image analyses,respectively.RESULTS: The EOCR at different concentrations displayed inhibitory activity on proliferation of HELFs.In in vivo experiment,the weight gain of the rats in groups treated with EOCR at doses of 50,100 and 200 mg/kg per day was significantly higher than those in the model group at the 7th,14th,21st and28th day (P＜0.05 or P＜0.01).The scores of alveolitisand pulmonary fibrosis in the groups treated with EOCR at doses of 100 and 200 mg/kg per day were significantly lower than those in the model group (P＜0.01); the SOD levels in serum and pulmonary tissues of the EOCR (50,100 and 200 mg/kg) groups were markedly increased compared with the model group (P＜0.01 ),while the MDA levels in both serum and pulmonary tissues were markedly reduced (P＜0.05) ; the Col Ⅰ level in pulmonary tissues of the EOCR (100 and 200 mg/kg per day) groups were markedly lower than that of the model group (P＜0.01) ; the protein and mRNA expressions of CTGF in the groups treated with EOCR at doses of 100 and 200 mg/kg per day were down-regulated compared with the model group (P＜0.01).CONCLUSION: The results indicate that EOCR has preventive effects on BLM-induced pulmonary fibrosis in rats.The mechanism may be via adjusting the unbalance of oxidation and antioxidation,down-regulating CTGF protein and mRNA expressions,and reducing collagen deposition and fibrosis.