国际呼吸杂志
國際呼吸雜誌
국제호흡잡지
INTERNATIONAL JOURNAL OF RESPIRATION
2012年
22期
1709-1714,1761
,共7页
叶清%李燕芹%熊剑飞%徐佳波%刘斌%邹静%秦慧
葉清%李燕芹%熊劍飛%徐佳波%劉斌%鄒靜%秦慧
협청%리연근%웅검비%서가파%류빈%추정%진혜
肺纤维化%组蛋白去乙酰化酶抑制剂%α-平滑肌肌动蛋白%Ⅰ型胶原%转化生长因子β1
肺纖維化%組蛋白去乙酰化酶抑製劑%α-平滑肌肌動蛋白%Ⅰ型膠原%轉化生長因子β1
폐섬유화%조단백거을선화매억제제%α-평활기기동단백%Ⅰ형효원%전화생장인자β1
Pulmonary fibrosis%Histone deacetylase inhibitors%α-smooth muscle actin%Collagen Ⅰ%Transforming growth factor-β1
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β1(TGF-β1)mRNA表达的变化.方法 将46只SPF级雄性160～180 g SD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Ⅰ 14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TSA 2 mg/kg溶于二甲基亚砜(DMSO) 60μl,并稀释于生理盐水1.2 ml中,每天1次,连续3d;模型组Ⅱ在造模次日起腹腔内注射DMSO 60 μl稀释于生理盐水1.2 ml中,每天1次,连续3 d；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1.2 ml,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-β1mRNA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-β1mRNA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.
目的 觀察非選擇性組蛋白去乙酰化酶抑製劑Trichostatin A(TSA)對博萊黴素誘導大鼠肺纖維化的影響以及肺組織中α-平滑肌肌動蛋白(α-SMA)、Ⅰ型膠原含量及轉化生長因子β1(TGF-β1)mRNA錶達的變化.方法 將46隻SPF級雄性160～180 g SD大鼠隨機分為正常對照組、模型組Ⅰ、模型組Ⅱ、治療組,其中正常對照組10隻,模型組Ⅰ 14隻,模型組Ⅱ12隻,治療組10隻.模型組Ⅰ、模型組Ⅱ及治療組經氣管內註入鹽痠博萊黴素(5mg/kg),製備肺纖維化大鼠模型.正常對照組氣管內註入相同體積生理鹽水.治療組在造模次日起腹腔內註射TSA 2 mg/kg溶于二甲基亞砜(DMSO) 60μl,併稀釋于生理鹽水1.2 ml中,每天1次,連續3d;模型組Ⅱ在造模次日起腹腔內註射DMSO 60 μl稀釋于生理鹽水1.2 ml中,每天1次,連續3 d；模型組Ⅰ及正常對照組在造模次日起腹腔內註射生理鹽水1.2 ml,每天1次,連續3d.其中模型組Ⅰ大鼠死亡5隻,模型組Ⅱ大鼠死亡4隻,治療組大鼠死亡2隻.于造模第21天處死全部存活大鼠.取肺組織進行HE染色及Masson三色染色,評估肺纖維化程度.堿水解法測定肺組織羥脯氨痠(HYP)含量.酶聯免疫吸附法(ELISA)測定肺組織勻漿中α-SMA及Ⅰ型膠原的含量.real-time PCR檢測肺組織TGF-β1mRNA錶達的變化.結果 ①HE染色分析:模型組Ⅰ及模型組Ⅱ肺泡炎程度較正常對照組增高(P＜0.05),模型組Ⅰ及模型組Ⅱ之間肺泡炎程度差異無統計學意義(P＞0.05),治療組肺泡炎程度均較模型組Ⅰ及模型組Ⅱ減輕(P＜0.05),與正常對照組比較肺泡炎程度差異無統計學意義(P＞0.05).②Masson染色分析:模型組Ⅰ及模型組Ⅱ肺組織纖維化程度較正常對照組增高(P＜0.05),模型組Ⅰ及模型組Ⅱ之間肺纖維化程度差異無統計學意義(P＞0.05),治療組肺纖維化程度較模型組Ⅰ及模型組Ⅱ減輕(P＜0.05),與正常對照組比較肺纖維化程度差異無統計學意義(P＞0.05).③肺組織HYP含量:模型組Ⅰ及模型組Ⅱ的HYP含量較正常對照組高(P＜0.05),模型組Ⅰ及模型組Ⅱ的HYP含量無明顯變化(P＞0.05).治療組的HYP含量較模型組Ⅰ及模型組Ⅱ降低(P＜0.05),與正常對照組比較差異無統計學意義(P＞0.05).④肺組織勻漿中α-SMA蛋白濃度:模型組Ⅰ及模型組Ⅱ的α-SMA含量較正常對照組高(P＜0.05),模型組Ⅰ及模型組Ⅱ的α-SMA含量差異無統計學意義(P＞0.05),治療組的α-SMA含量較模型組Ⅰ及模型組Ⅱ降低(P＜0.05),與正常對照組比較差異無統計學意義(P＞0.05).⑤肺組織勻漿中Ⅰ型膠原蛋白濃度:模型組Ⅰ及模型組Ⅱ的Ⅰ型膠原含量較正常對照組高(P＜0.05),模型組Ⅰ及模型組Ⅱ的Ⅰ型膠原含量差異無統計學意義(P＞0.05),治療組的Ⅰ型膠原含量較模型組Ⅰ及模型組Ⅱ降低(P＜0.05),與正常對照組比較差異無統計學意義(P＞0.05).⑥肺組織TGF-β1 mRNA:模型組Ⅰ及模型組Ⅱ的TGF-β1 mRNA錶達較正常對照組升高(P＜0.05),模型組Ⅰ及模型組Ⅱ的TGF-β1 mRNA錶達差異無統計學意義(P＞0.05),治療組的TGF-β1mRNA錶達較模型組Ⅰ及模型組Ⅱ降低(P＜0.05),但高于正常對照組(P＜0.05).結論 氣管內註入博萊黴素成功構建肺纖維化大鼠模型.非選擇性組蛋白去乙酰化酶抑製劑TSA能減輕博萊黴素誘導大鼠肺纖維化,降低肺組織中纖維化相關蛋白α-SMA、Ⅰ型膠原含量以及TGF-β1 mRNA錶達.
목적 관찰비선택성조단백거을선화매억제제Trichostatin A(TSA)대박래매소유도대서폐섬유화적영향이급폐조직중α-평활기기동단백(α-SMA)、Ⅰ형효원함량급전화생장인자β1(TGF-β1)mRNA표체적변화.방법 장46지SPF급웅성160～180 g SD대서수궤분위정상대조조、모형조Ⅰ、모형조Ⅱ、치료조,기중정상대조조10지,모형조Ⅰ 14지,모형조Ⅱ12지,치료조10지.모형조Ⅰ、모형조Ⅱ급치료조경기관내주입염산박래매소(5mg/kg),제비폐섬유화대서모형.정상대조조기관내주입상동체적생리염수.치료조재조모차일기복강내주사TSA 2 mg/kg용우이갑기아풍(DMSO) 60μl,병희석우생리염수1.2 ml중,매천1차,련속3d;모형조Ⅱ재조모차일기복강내주사DMSO 60 μl희석우생리염수1.2 ml중,매천1차,련속3 d；모형조Ⅰ급정상대조조재조모차일기복강내주사생리염수1.2 ml,매천1차,련속3d.기중모형조Ⅰ대서사망5지,모형조Ⅱ대서사망4지,치료조대서사망2지.우조모제21천처사전부존활대서.취폐조직진행HE염색급Masson삼색염색,평고폐섬유화정도.감수해법측정폐조직간포안산(HYP)함량.매련면역흡부법(ELISA)측정폐조직균장중α-SMA급Ⅰ형효원적함량.real-time PCR검측폐조직TGF-β1mRNA표체적변화.결과 ①HE염색분석:모형조Ⅰ급모형조Ⅱ폐포염정도교정상대조조증고(P＜0.05),모형조Ⅰ급모형조Ⅱ지간폐포염정도차이무통계학의의(P＞0.05),치료조폐포염정도균교모형조Ⅰ급모형조Ⅱ감경(P＜0.05),여정상대조조비교폐포염정도차이무통계학의의(P＞0.05).②Masson염색분석:모형조Ⅰ급모형조Ⅱ폐조직섬유화정도교정상대조조증고(P＜0.05),모형조Ⅰ급모형조Ⅱ지간폐섬유화정도차이무통계학의의(P＞0.05),치료조폐섬유화정도교모형조Ⅰ급모형조Ⅱ감경(P＜0.05),여정상대조조비교폐섬유화정도차이무통계학의의(P＞0.05).③폐조직HYP함량:모형조Ⅰ급모형조Ⅱ적HYP함량교정상대조조고(P＜0.05),모형조Ⅰ급모형조Ⅱ적HYP함량무명현변화(P＞0.05).치료조적HYP함량교모형조Ⅰ급모형조Ⅱ강저(P＜0.05),여정상대조조비교차이무통계학의의(P＞0.05).④폐조직균장중α-SMA단백농도:모형조Ⅰ급모형조Ⅱ적α-SMA함량교정상대조조고(P＜0.05),모형조Ⅰ급모형조Ⅱ적α-SMA함량차이무통계학의의(P＞0.05),치료조적α-SMA함량교모형조Ⅰ급모형조Ⅱ강저(P＜0.05),여정상대조조비교차이무통계학의의(P＞0.05).⑤폐조직균장중Ⅰ형효원단백농도:모형조Ⅰ급모형조Ⅱ적Ⅰ형효원함량교정상대조조고(P＜0.05),모형조Ⅰ급모형조Ⅱ적Ⅰ형효원함량차이무통계학의의(P＞0.05),치료조적Ⅰ형효원함량교모형조Ⅰ급모형조Ⅱ강저(P＜0.05),여정상대조조비교차이무통계학의의(P＞0.05).⑥폐조직TGF-β1 mRNA:모형조Ⅰ급모형조Ⅱ적TGF-β1 mRNA표체교정상대조조승고(P＜0.05),모형조Ⅰ급모형조Ⅱ적TGF-β1 mRNA표체차이무통계학의의(P＞0.05),치료조적TGF-β1mRNA표체교모형조Ⅰ급모형조Ⅱ강저(P＜0.05),단고우정상대조조(P＜0.05).결론 기관내주입박래매소성공구건폐섬유화대서모형.비선택성조단백거을선화매억제제TSA능감경박래매소유도대서폐섬유화,강저폐조직중섬유화상관단백α-SMA、Ⅰ형효원함량이급TGF-β1 mRNA표체.
Objective To investigate the effects of non selective histone deacetylase inhibitors Trichostatin A (TSA) on bleomycin-induced pulmonary fibrosis.To investigate the effects of non selective histone deacetylase inhibitors TSA on transforming growth factor-β (TGF-β1) mRNA,a-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 pulmonary 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.
