异佛司可林抑制炎症和纤维化细胞因子减轻小鼠肺纤维化作用研究

Isoforskolin alleviates pulmonary fibrosis in mice by inhibiting inflammatory and fibrotic cytokines

本文旨在探讨异佛司可林(isoforskolin, ISOF)对博莱霉素(bleomycin, BLM)诱导小鼠肺纤维化(pulmonary fibrosis, PF)的影响,并初步探索其作用机制。本研究中动物福利和实验过程均遵循昆明医科大学动物伦理委员会的规定。实验分为假手术组、博莱霉素组(气管滴入BLM 5 mg·kg^(-1))、阳性药物对照组(气管滴入BLM 5 mg·kg^(-1)+腹腔注射地塞米松磷酸钠2.5 mg·kg^(-1))、异佛司可林2.5和10 mg·kg^(-1)组(气管滴入BLM 5 mg·kg^(-1)+灌胃给予异佛司可林2.5和10 mg·kg^(-1))。给药方式为每日1次,连续给药28天。分别在实验第7天和28天处死小鼠,收集肺组织和支气管肺泡灌洗液标本。苏木精-伊红(hematoxylin-eosin, H&E)染色和Masson染色检测肺组织病理变化,碱水解法检测肺组织羟脯氨酸含量, ELISA法(enzyme-linked immunosorbent assay)检测支气管肺泡灌洗液中肿瘤坏死因子α(tumor necrosis factorα, TNF-α)和白细胞介素1β(interleukin 1β, IL-1β)水平。Western blot法检测肺组织转化生长因子β1 (transforming growth factorβ1, TGF-β1)和结缔组织生长因子(connective tissue growth factor, CTGF)蛋白表达。实验结果显示,与博莱霉素组比较,异佛司可林10 mg·kg^(-1)组小鼠肺组织炎症和纤维化程度明显减轻,肺组织羟脯氨酸含量降低,气管灌洗收集肺泡灌洗液(bronchoalveolar lavage fluid, BALF)中TNF-α和IL-1β水平下降,肺组织TGF-β1和CTGF蛋白表达下调,差异有统计学意义(P <0.05)。结果表明,异佛司可林可通过抑制炎症细胞因子TNF-α和IL-1β释放,下调促纤维化因子TGF-β1和CTGF蛋白表达,通过抗炎与抗纤维化双重作用发挥抗肺纤维化作用。

We investigated the therapeutic effect and mechanism of isoforskolin on bleomycin-induced pulmonary fibrosis in mice. The animal welfare and experimental process were in accordance with the Regulations of Animal Ethics Committee of Kunming Medical University. Male Kunming mice were randomly divided into five groups:sham operation group(intratracheal instillation of normal saline), bleomycin group(intratracheal instillation of bleomycin 5 mg·kg^(-1)), positive drug control group(intratracheal instillation of bleomycin 5 mg·kg^(-1)+ intraperitoneal injection of dexamethasone sodium phosphate 2.5 mg·kg^(-1)), isoforskolin(2.5 and 10 mg·kg^(-1)) groups(intratracheal instillation of bleomycin 5 mg·kg^(-1)+ intragastric administration of isoforskolin 2.5 and 10 mg·kg^(-1)). The method of drug administration was once a day for 28 days. On the 7 thand 28 thday of the experiment, the mice were killed, and the lung tissue and bronchoalveolar lavage fluid samples were collected. Hematoxylin eosin(H&E) staining and Masson staining were used to detect the pathological changes of lung tissue. The content of hydroxyproline in lung tissue was detected by alkaline hydrolysis method. The levels of tumor necrosis factor α(TNF-α) and interleukin1β(IL-1β) in bronchoalveolar lavage fluid(BALF) were detected by enzyme linked immunosorbent assay(ELISA).The expressions of transforming growth factor β1(TGF-β1) and connective tissue growth factor(CTGF) in lung tissue were detected by Western blot. The experimental results showed that compared with the bleomycin group,the degree of inflammation and fibrosis in the lung tissue of mice in 10 mg·kg^(-1)isoforskolin group was significantly reduced, the content of hydroxyproline in the lung tissue was decreased, the levels of TNF-α and IL-1β in BALF were decreased, and the protein expressions of TGF-β1 and CTGF in the lung tissue were downregulated, the differences were statistically significant(P < 0.05). The results suggest that isoforskolin can inhibit the release of inflammatory cytokines TNF-α and IL-1β, downregulate the protein expression of profibrotic factors TGF-β1 and CTGF, and exert anti pulmonary fibrosis effect through both anti-inflammatory and anti fibrotic effect.