中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
7期
519-523
,共5页
张文雍%李洋%李婷%宁佐伟%李维%李旭
張文雍%李洋%李婷%寧佐偉%李維%李旭
장문옹%리양%리정%저좌위%리유%리욱
肝硬化%大鼠%体外研究
肝硬化%大鼠%體外研究
간경화%대서%체외연구
Liver cirrhosis%Rats%In vivo
目的 探讨醛固酮拮抗剂对肝纤维化大鼠NOX4蛋白表达的抑制作用. 方法 体内实验:雄性Wistar大鼠24只,随机分为3组,每组8只.模型组:用四氯化碳(CCl4)油2.5 ml/kg皮下注射,3次/周;安体舒通组:CCl4油注射的同时予以安体舒通20 mg/kg灌胃,1次/d;对照组:用橄榄油皮下注射,于4周后处死实验动物取材.用HE染色观察肝组织形态结构;Masson染色进行METAVIR肝纤维化评分；免疫组织化学法检测NOX4蛋白的表达.体外实验:将大鼠HSC-T6细胞株与不同剂量的醛固酮(10-9、10-7、10-5 mol/L,观察剂量效应)作用不同时间(6、12、24h)以观察时间效应.给予醛固酮(Ald)1μmol/L、依普利酮1μmol/L预处理60min,再给予Ald刺激大鼠HSC-T6细胞,设立阴性对照组.Western blot方法检测NOX4蛋白的表达.采用Oneway ANOVA方差分析法,首先用Levene方法进行方差齐性检验,确定方差齐且整体比较组间差异有统计学意义后进一步做多重比较,多重比较采用LSD法. 结果 CCl4组纤维化程度较对照组明显增高(相对表达量为16.060±0.300比2.471±0.160),两组比较,差异有统计学意义.CCl4+Sp组纤维化程度较CCl4组低(相对表达量为5.761±0.152比16.060±0.300),两组比较,差异有统计学意义.免疫组织化学结果显示,与对照组相比,CCl4组NOX4蛋白表达明显增高(相对表达量为7.231±0.211比1.350±0.252),两组比较,差异有统计学意义.与CCl4组相比,CCl4+Sp组NOX4蛋白表达下降(相对表达量为4.270±0.242比7.231±0.211),两组比较,差异有统计学意义.Ald刺激HSC-T6细胞后NOX4蛋白表达增强,并呈时间、浓度依赖性,在10-5 mol/L浓度刺激24h达到峰值；与对照组相比,Ald组NOX4表达明显增加(相对表达量为0.710±0.011比0.316±0.015),两组比较,差异有统计学意义.与Ald组相比,Ald+依普利酮组NOX4的表达减少(相对表达量为0.615±0.014比0.710土0.011),两组比较,差异有统计学意义.结论 醛固酮拮抗剂通过抑制Ald诱导的氧化应激效应抑制肝纤维化大鼠NOX4蛋白的表达.
目的 探討醛固酮拮抗劑對肝纖維化大鼠NOX4蛋白錶達的抑製作用. 方法 體內實驗:雄性Wistar大鼠24隻,隨機分為3組,每組8隻.模型組:用四氯化碳(CCl4)油2.5 ml/kg皮下註射,3次/週;安體舒通組:CCl4油註射的同時予以安體舒通20 mg/kg灌胃,1次/d;對照組:用橄欖油皮下註射,于4週後處死實驗動物取材.用HE染色觀察肝組織形態結構;Masson染色進行METAVIR肝纖維化評分；免疫組織化學法檢測NOX4蛋白的錶達.體外實驗:將大鼠HSC-T6細胞株與不同劑量的醛固酮(10-9、10-7、10-5 mol/L,觀察劑量效應)作用不同時間(6、12、24h)以觀察時間效應.給予醛固酮(Ald)1μmol/L、依普利酮1μmol/L預處理60min,再給予Ald刺激大鼠HSC-T6細胞,設立陰性對照組.Western blot方法檢測NOX4蛋白的錶達.採用Oneway ANOVA方差分析法,首先用Levene方法進行方差齊性檢驗,確定方差齊且整體比較組間差異有統計學意義後進一步做多重比較,多重比較採用LSD法. 結果 CCl4組纖維化程度較對照組明顯增高(相對錶達量為16.060±0.300比2.471±0.160),兩組比較,差異有統計學意義.CCl4+Sp組纖維化程度較CCl4組低(相對錶達量為5.761±0.152比16.060±0.300),兩組比較,差異有統計學意義.免疫組織化學結果顯示,與對照組相比,CCl4組NOX4蛋白錶達明顯增高(相對錶達量為7.231±0.211比1.350±0.252),兩組比較,差異有統計學意義.與CCl4組相比,CCl4+Sp組NOX4蛋白錶達下降(相對錶達量為4.270±0.242比7.231±0.211),兩組比較,差異有統計學意義.Ald刺激HSC-T6細胞後NOX4蛋白錶達增彊,併呈時間、濃度依賴性,在10-5 mol/L濃度刺激24h達到峰值；與對照組相比,Ald組NOX4錶達明顯增加(相對錶達量為0.710±0.011比0.316±0.015),兩組比較,差異有統計學意義.與Ald組相比,Ald+依普利酮組NOX4的錶達減少(相對錶達量為0.615±0.014比0.710土0.011),兩組比較,差異有統計學意義.結論 醛固酮拮抗劑通過抑製Ald誘導的氧化應激效應抑製肝纖維化大鼠NOX4蛋白的錶達.
목적 탐토철고동길항제대간섬유화대서NOX4단백표체적억제작용. 방법 체내실험:웅성Wistar대서24지,수궤분위3조,매조8지.모형조:용사록화탄(CCl4)유2.5 ml/kg피하주사,3차/주;안체서통조:CCl4유주사적동시여이안체서통20 mg/kg관위,1차/d;대조조:용감람유피하주사,우4주후처사실험동물취재.용HE염색관찰간조직형태결구;Masson염색진행METAVIR간섬유화평분；면역조직화학법검측NOX4단백적표체.체외실험:장대서HSC-T6세포주여불동제량적철고동(10-9、10-7、10-5 mol/L,관찰제량효응)작용불동시간(6、12、24h)이관찰시간효응.급여철고동(Ald)1μmol/L、의보리동1μmol/L예처리60min,재급여Ald자격대서HSC-T6세포,설립음성대조조.Western blot방법검측NOX4단백적표체.채용Oneway ANOVA방차분석법,수선용Levene방법진행방차제성검험,학정방차제차정체비교조간차이유통계학의의후진일보주다중비교,다중비교채용LSD법. 결과 CCl4조섬유화정도교대조조명현증고(상대표체량위16.060±0.300비2.471±0.160),량조비교,차이유통계학의의.CCl4+Sp조섬유화정도교CCl4조저(상대표체량위5.761±0.152비16.060±0.300),량조비교,차이유통계학의의.면역조직화학결과현시,여대조조상비,CCl4조NOX4단백표체명현증고(상대표체량위7.231±0.211비1.350±0.252),량조비교,차이유통계학의의.여CCl4조상비,CCl4+Sp조NOX4단백표체하강(상대표체량위4.270±0.242비7.231±0.211),량조비교,차이유통계학의의.Ald자격HSC-T6세포후NOX4단백표체증강,병정시간、농도의뢰성,재10-5 mol/L농도자격24h체도봉치；여대조조상비,Ald조NOX4표체명현증가(상대표체량위0.710±0.011비0.316±0.015),량조비교,차이유통계학의의.여Ald조상비,Ald+의보리동조NOX4적표체감소(상대표체량위0.615±0.014비0.710토0.011),량조비교,차이유통계학의의.결론 철고동길항제통과억제Ald유도적양화응격효응억제간섬유화대서NOX4단백적표체.
Objective To investigate the inhibitory potential of aldosterone antagonist on NOX4 protein expression in hepatic fibrosis by using a rat model of carbon tetracholoride (CCl4)induced hepatotoxicity.Methods Twenty-four male Wistar rats were randomly divided into three equal groups:fibrosis model group (receiving three subcutaneous injections per week of 2.5 ml/kg 40％CCl4); spironolactone (Sp)-treated fibrosis model group (receiving CCl4 regimen plus three injections per day of 20 mg/kg Sp in olive oil); negative-treatment fibrosis model group (receiving CCl4 regimen plus three injections per day of olive oil alone).Unmanipulated rats (receiving no CC14 and no supplemental treatments) served as normal controls.After 4 weeks,liver histology was carried out to assess cytotoxicity (by hematoxylin-eosin staining),fibrosis (by Masson staining and METAVIR scoring),and NOX4 protein expression (by immunohistochemistry).In addition,in vitro analyses of immortalized rat hepatic stellate cells,HSC-T6,were performed to evaluate dose-response (10-9,10-7 and 10-5 mol/L) and time-response (6,12 and 24 h) of aldosterone agonist (Ald) and an aldosterone antagonist,eplerenone (EPLE).Effects on NOX4 protein expression were evaluated by western blotting.Results The fibrosis model group showed significantly more fibrosis than the normal control group (16.060 ± 0.300 vs.2.471 ± 0.160,P =0.000]; however,the Sp-treated fibrosis model group showed significantly less CCl4-induced fibrosis (5.761 ± 0.152 vs.model:16.060 ± 0.300,P =0.000).The fibrosis model group also showed significantly higher NOX4 protein expression in liver tissues than the normal control group (7.231 ± 0.211 vs.1.350 ± 0.252,P =0.000),and the Sp-treated fibrosis model tissues showed significantly less CCl4-induced up-regulated NOX4 protein expression (4.270 ± 0.242 vs.model:7.231 ± 0.211,P =0.000].Ald induced up-regulated NOX4 protein expression in HSC-T6 cells in dose-and concentration-dependent manners,with the peak expression being induced by the 10-5 mol/L concentration and 24 h exposure.The Ald-treated cells expressed significantly more NOX4 protein than the untreated control cells (0.710 ± 0.011 vs.0.316 ± 0.015,P =0.000].and the EPLE-treated cells showed significantly less Ald-induced up-regulated NOX4 expression (0.615 ± 0.014 vs.0.710 ± 0.011,P =0.000].Conclusion Aldosterone antagonists inhibit the firbosis-induced NOX4 protein expression in rat hepatic cells.
