Trichostatin A对博莱霉素诱导的大鼠肺纤维化的抑制作用

Inhibition of Trichostatin A on bleomycin induced pulmonary fibrosis in rats

目的观察非选择性组蛋白去乙酰化酶抑制剂Trichostatin A（TSA）对博莱霉素诱导大鼠肺纤维化的影响以及肺组织中α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原含量及转化生长因子岛（TGF-β1）mRNA表达的变化。方法将46只SPF级雄性160～180gSD大鼠随机分为正常对照组、模型组Ⅰ、模型组Ⅱ、治疗组，其中正常对照组10只，模型组Ⅰ14只，模型组Ⅱ12只，治疗组10只。模型组Ⅰ、模型组Ⅱ及治疗组经气管内注入盐酸博莱霉素（5mg／kg），制备肺纤维化大鼠模型。正常对照组气管内注入相同体积生理盐水。治疗组在造模次日起腹腔内注射TSA2mg／kg溶于二甲基亚砜（DMSO）60μl，并稀释于生理盐水1．2m1中，每天1次，连续3d；模型组Ⅱ在造模次日起腹腔内注射DMSO 60μl稀释于生理盐水1．2ml中，每天1次，连续3d；模型组Ⅰ及正常对照组在造模次日起腹腔内注射生理盐水1．2ml，每天1次，连续3d。其中模型组Ⅰ大鼠死亡5只，模型组Ⅱ大鼠死亡4只，治疗组大鼠死亡2只。于造模第21天处死全部存活大鼠。取肺组织进行HE染色及Masson三色染色，评估肺纤维化程度。碱水解法测定肺组织羟脯氨酸（HYP）含量。酶联免疫吸附法（ELISA）测定肺组织匀浆中α-SMA及Ⅰ型胶原的含量。real—time PCR检测肺组织TGF-β1 mRNA表达的变化。结果①HE染色分析：模型组Ⅰ及模型组Ⅱ肺泡炎程度较正常对照组增高（P〈0．05），模型组Ⅰ及模型组Ⅱ之间肺泡炎程度差异无统计学意义（P〉0．05），治疗组肺泡炎程度均较模型组Ⅰ及模型组Ⅱ减轻（P〈0．05），与正常对照组比较肺泡炎程度差异无统计学意义（P〉0．05）。②Masson染色分析：模型组Ⅰ及模型组Ⅱ肺组织纤维化程度较正常对照组增高（P〈0．05），模型组Ⅰ及模型组Ⅱ之间肺纤维化程度差异无统计学意义（P〉0．05），治疗组肺纤维化程度较模型组Ⅰ及模型组Ⅱ减轻（P〈0．05），与正常对照组比较肺纤维化程度差异无统计学意义（P〉0．05）。③肺组织HYP含量：模型组Ⅰ及模型组Ⅱ的HYP含量较正常对照组高（P〈0．05），模型组Ⅰ及模型组Ⅱ的HYP含量无明显变化（P〉0．05）。治疗组的HYP含量较模型组Ⅰ及模型组Ⅱ降低（P〈0．05），与正常对照组比较差异无统计学意义（P〉0．05）。④肺组织匀浆中α-SMA蛋白浓度：模型组Ⅰ及模型组Ⅱ的α-SMA含量较正常对照组高（P〈0．05），模型组Ⅰ及模型组Ⅱ的α-SMA含量差异无统计学意义（P〉0．05），治疗组的α-SMA含量较模型组Ⅰ及模型组Ⅱ降低（P〈0．05），与正常对照组比较差异无统计学意义（P〉0．05）。⑤肺组织匀浆中Ⅰ型胶原蛋白浓度：模型组Ⅰ及模型组Ⅱ的Ⅰ型胶原含量较正常对照组高（P〈0．05），模型组Ⅰ及模型组Ⅱ的Ⅰ型胶原含量差异无统计学意义（P〉0．05），治疗组的Ⅰ型胶原含量较模型组Ⅰ及模型组Ⅱ降低（P〈0．05），与正常对照组比较差异无统计学意义（P〉0．05）。⑥肺组织TGF-β1 mRNA：模型组Ⅰ及模型组Ⅱ的TGF-β1 mRNA表达较正常对照组升高（P〈0．05），模型组Ⅰ及模型组Ⅱ的TGF-β1 mRNA表达差异无统计学意义（P〉0．05），治疗组的TGF—β1 mRNA表达较模型组Ⅰ及模型组Ⅱ降低（P〈0．05），但高于正常对照组（P〈0．05）。结论气管内注入博莱霉素成功构建肺纤维化大鼠模型。非选择性组蛋白去乙酰化酶抑制剂TSA能减轻博莱霉素诱导大鼠肺纤维化，降低肺组织中纤维化相关蛋白α-SMA、Ⅰ型胶原含量以及TGF—β1 mRNA表达。

Objective To investigate the effects of non selective histone deacetylase inhibitors Trichostatin A （TSA） on bleomyein-induced pulmonary fibrosis. To investigate the effects of non selective histone deacetylase inhibitors TSA on transforming growth factor-β1 （TGF-β1） mRNA, α-smooth muscle actin （α-SMA）,collagen Ⅰ in pulmonary fibrosis rats. Methods SPF level male SD rats weight 160-180 g were selected. 46 rats were randomly divided into four groups： ten for normal control group, fourteen for model control group Ⅰ , twelve for model control group Ⅱ and ten for treatment group. Rat puhnonary fibrosis was induced by bleomycin （5 mg/kg） via single intratracheal perfusion in the two model control groups and treatment group. Normal control mice were instilled with a corresponding volume of 0.9% saline intratracheally. Treatment group was treated by the dilution of TSA 2 mg/kg, DMSO 60 μl and 0.9% saline 1.2 ml intraperitoneal injection from the next day, once a day for three days. Model control group Ⅱ was treated by the dilution of DMSO 60 μl and 0.9% saline 1.2 ml intraperitoneal injection from the next day,once a day for three days. Model control group Ⅰ and normal control group were treated by 0.9% saline 1.2 ml intraperitoneal injection from the next day,once a day for three days. The death were no for normal control group, five for model control group Ⅰ , four for model control group Ⅱ and two for treatment group. All the animals were sacrificed on the 21st day after modeling. The pathological changes were observed by hematoxylin and eosin stain and masson trichrome stain. The lung hydroxyproline （HYP） was measured by alkaline hydrolysis. The expression of TGF-β1 mRNA was measured by real-time PCR. The protein level of α-SMA and collage Ⅰ in lung tissue homogenate was measured by ELISA. Results ① Alveolitis grades of the two model control groups increased compared with the normal control group and treatment group （P 〈 0.05）. There was no significant difference in alveolitis grades between the two model control groups （ P 〉0.05）. There was no significant difference in alveolitis grades between treatment group and normal control group （ P 〉0.05）. ②Pathological semi-quantitative analysis showed that pulmonary fibrosis degree of the two model control groups increased compared with normal control group and treatment group（ P 〈0.05）. There was no significant difference in the pulmonary fibrosis degree between the two model control groups （ P 〉0.05）. There was no significant difference in the pulmonary fibrosis degree between treatment group and normal control group （ P 〉0.05）. ③The HYP level in lung tissue of the two model control groups was higher than normal control group and treatment group （ P 〈0.05）. There was no significant difference in lung tissue HYP level between the two model control groups （ P 〉0.05）. There was no significant difference in lung tissue HYP level between treatment group and normal control group （ P 〉0.05）. ④The protein level of α-SMA increased in the two model control groups compared with normal control group and treatment group （ P 〈0.05）. There was no significant difference in the protein level of α-SMA of lung tissue homogenate between the two model control groups by ELISA （P 〉 0.05）. There was no significant difference in the protein level of α-SMA between treatment group and normal control group （ P 〉 0.05）. ⑤The protein level of collagen Ⅰ increased in the two model control groups compared with normal control group and treatment group （ P 〈0.05）. There was no significant difference in the protein level of collagen Ⅰ of lung tissue homogenate between the two model control groups by ELISA （P 〉0.05）. There was no significant difference in the protein level of collagen Ⅰ between treatment group and normal control group （ P 〉0.05）. ⑥The expression of lung tissue TGF-β1 mRNA increased in the two model control groups compared with normal control group （ P 〈0.05）. There was no significant difference in the expression of lung tissue TGF-β1 mRNA between the two model control groups by real-time PCR （ P 〉 0.05）. The expression of lung tissue TGF-β1 mRNA decreased in the treatment group compared with the two model control groups （ P 〈 0.05）. The expression of lung tissue TGF-β1 mRNA increased in treatment group compared with normal control group （ P 〈 0.05）. Conclusions Experimental results show that TSA can alleviate pulmonary fibrosis. Non selective histone deacetylase inhibitors TSA can reduce the bleomycin induced pulmonary fibrosis in rats. It can reduce the protein α-SMA, collagen Ⅰ content and TGF-β1 mRNA expression associated with lung tissue fibrosis.