目的 研究扶正化瘀胶囊(FZHc)对肝纤维化小鼠肝细胞中核因子相关因子2(Nrf2)表达的影响. 方法 将昆明小鼠70只随机分为对照组和模型组.对照组24只,包括正常组(A1)、矿物油组(A2)、FZHc组(A3),每组8只;模型组46只,腹腔注射CCL4,包括模型6周组(B1) 10只、模型10周组(B2) 12只、FZHc低剂量组(C1) 12只、FZHc高剂量组(C2) 12只.其中A3、C1、C2组在第7周起用FZHc药粉(由双蒸水配制成浓度为0.1 g/ml药液)灌胃,连续4周.模型组第6、10周末分批处死小鼠,收集肝脏标本HE染色观察组织炎症程度变化,Masson染色观察纤维化病变;免疫组织化学检测Nrf2、醌氧化还原酶l(Nqol)、α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)表达;Western blot检测细胞内Nrf2、Nqol总蛋白表达量及Nrf2核转移情况;RT-PCR检测Nrf2 mRNA表达量.采用SPSS13.0软件进行统计学分析,计量资料采用单因素方差分析,两两比较选用LSD检验,计数资料采用Ridit分析.结果 B1组9只小鼠中有7只(77.8％)为肝纤维化Ⅱ期,有2只小鼠为肝纤维化Ⅲ期.B2组8只小鼠中5只(62.5％)为肝纤维化Ⅲ期,3只为可见假小叶形成.FZHc干预可减轻肝脏炎症及纤维化程度,C1组10只小鼠有6只(60％)为肝纤维化Ⅱ期,4只小鼠肝纤维化Ⅲ期,C2组9只小鼠中有2只(22.2％)为肝纤维化Ⅰ期,5只为肝纤维化Ⅱ期,2只为肝纤维化Ⅲ期,多组间的Kruskal-Wallis检验,x2=53.321,P＜0.01.B2、C1、C2组α-SMA阳性表达平均吸光度值分别为0.09±0.01、0.06±0.01、0.04±0.00;FN阳性表达平均吸光度值分别为0.08±0.01、0.07±0.01、0.05±0.01,F=77.421和F=118.262,P值均＜0.05.不同剂量FZHc干预后α-SMA、FN表达较B2组明显减少,并呈剂量依赖性.B2、C1、C2组小鼠肝组织中Nrf2阳性表达平均吸光度值分别为0.07±0.01、0.10±0.01、0.17±0.01;Nqol平均光吸光度分别为0.06±0.01、0.09±0.01、0.14±0.01,F=182.537和F=75.615,P值均＜0.05;Nrf2总蛋白表达的相对灰度值分别为0.79±0.05、0.94±0.02、1.12±0.08,F=45.664,P＜ 0.05.不同剂量FZHc干预可显著增加Nrf2、Nqol表达,并呈剂量依赖性.C1、C2、B2组Nrf2核蛋白表达的相对灰度值分别为0.97±0.09、1.24±0.10和0.67±0.04,F=94.787,P＜0.05,差异有统计学意义.FZHc促进Nrf2 mRNA相对表达量,C1组为4.00±0.12、C2组为4.65±0.08、B2组为2.15±0.11,F=3230.105,P＜0.05,差异有统计学意义,并具有剂量依赖性. 结论 FZHc可能通过促进Nrf2 mRNA、蛋白表达并促进Nrf2胞核转移,促进其下游靶基因Nqol的表达,抑制HSCs活化及FN合成,从而减轻肝脏纤维化.
目的 研究扶正化瘀膠囊(FZHc)對肝纖維化小鼠肝細胞中覈因子相關因子2(Nrf2)錶達的影響. 方法 將昆明小鼠70隻隨機分為對照組和模型組.對照組24隻,包括正常組(A1)、礦物油組(A2)、FZHc組(A3),每組8隻;模型組46隻,腹腔註射CCL4,包括模型6週組(B1) 10隻、模型10週組(B2) 12隻、FZHc低劑量組(C1) 12隻、FZHc高劑量組(C2) 12隻.其中A3、C1、C2組在第7週起用FZHc藥粉(由雙蒸水配製成濃度為0.1 g/ml藥液)灌胃,連續4週.模型組第6、10週末分批處死小鼠,收集肝髒標本HE染色觀察組織炎癥程度變化,Masson染色觀察纖維化病變;免疫組織化學檢測Nrf2、醌氧化還原酶l(Nqol)、α-平滑肌肌動蛋白(α-SMA)、纖維連接蛋白(FN)錶達;Western blot檢測細胞內Nrf2、Nqol總蛋白錶達量及Nrf2覈轉移情況;RT-PCR檢測Nrf2 mRNA錶達量.採用SPSS13.0軟件進行統計學分析,計量資料採用單因素方差分析,兩兩比較選用LSD檢驗,計數資料採用Ridit分析.結果 B1組9隻小鼠中有7隻(77.8％)為肝纖維化Ⅱ期,有2隻小鼠為肝纖維化Ⅲ期.B2組8隻小鼠中5隻(62.5％)為肝纖維化Ⅲ期,3隻為可見假小葉形成.FZHc榦預可減輕肝髒炎癥及纖維化程度,C1組10隻小鼠有6隻(60％)為肝纖維化Ⅱ期,4隻小鼠肝纖維化Ⅲ期,C2組9隻小鼠中有2隻(22.2％)為肝纖維化Ⅰ期,5隻為肝纖維化Ⅱ期,2隻為肝纖維化Ⅲ期,多組間的Kruskal-Wallis檢驗,x2=53.321,P＜0.01.B2、C1、C2組α-SMA暘性錶達平均吸光度值分彆為0.09±0.01、0.06±0.01、0.04±0.00;FN暘性錶達平均吸光度值分彆為0.08±0.01、0.07±0.01、0.05±0.01,F=77.421和F=118.262,P值均＜0.05.不同劑量FZHc榦預後α-SMA、FN錶達較B2組明顯減少,併呈劑量依賴性.B2、C1、C2組小鼠肝組織中Nrf2暘性錶達平均吸光度值分彆為0.07±0.01、0.10±0.01、0.17±0.01;Nqol平均光吸光度分彆為0.06±0.01、0.09±0.01、0.14±0.01,F=182.537和F=75.615,P值均＜0.05;Nrf2總蛋白錶達的相對灰度值分彆為0.79±0.05、0.94±0.02、1.12±0.08,F=45.664,P＜ 0.05.不同劑量FZHc榦預可顯著增加Nrf2、Nqol錶達,併呈劑量依賴性.C1、C2、B2組Nrf2覈蛋白錶達的相對灰度值分彆為0.97±0.09、1.24±0.10和0.67±0.04,F=94.787,P＜0.05,差異有統計學意義.FZHc促進Nrf2 mRNA相對錶達量,C1組為4.00±0.12、C2組為4.65±0.08、B2組為2.15±0.11,F=3230.105,P＜0.05,差異有統計學意義,併具有劑量依賴性. 結論 FZHc可能通過促進Nrf2 mRNA、蛋白錶達併促進Nrf2胞覈轉移,促進其下遊靶基因Nqol的錶達,抑製HSCs活化及FN閤成,從而減輕肝髒纖維化.
목적 연구부정화어효낭(FZHc)대간섬유화소서간세포중핵인자상관인자2(Nrf2)표체적영향. 방법 장곤명소서70지수궤분위대조조화모형조.대조조24지,포괄정상조(A1)、광물유조(A2)、FZHc조(A3),매조8지;모형조46지,복강주사CCL4,포괄모형6주조(B1) 10지、모형10주조(B2) 12지、FZHc저제량조(C1) 12지、FZHc고제량조(C2) 12지.기중A3、C1、C2조재제7주기용FZHc약분(유쌍증수배제성농도위0.1 g/ml약액)관위,련속4주.모형조제6、10주말분비처사소서,수집간장표본HE염색관찰조직염증정도변화,Masson염색관찰섬유화병변;면역조직화학검측Nrf2、곤양화환원매l(Nqol)、α-평활기기동단백(α-SMA)、섬유련접단백(FN)표체;Western blot검측세포내Nrf2、Nqol총단백표체량급Nrf2핵전이정황;RT-PCR검측Nrf2 mRNA표체량.채용SPSS13.0연건진행통계학분석,계량자료채용단인소방차분석,량량비교선용LSD검험,계수자료채용Ridit분석.결과 B1조9지소서중유7지(77.8％)위간섬유화Ⅱ기,유2지소서위간섬유화Ⅲ기.B2조8지소서중5지(62.5％)위간섬유화Ⅲ기,3지위가견가소협형성.FZHc간예가감경간장염증급섬유화정도,C1조10지소서유6지(60％)위간섬유화Ⅱ기,4지소서간섬유화Ⅲ기,C2조9지소서중유2지(22.2％)위간섬유화Ⅰ기,5지위간섬유화Ⅱ기,2지위간섬유화Ⅲ기,다조간적Kruskal-Wallis검험,x2=53.321,P＜0.01.B2、C1、C2조α-SMA양성표체평균흡광도치분별위0.09±0.01、0.06±0.01、0.04±0.00;FN양성표체평균흡광도치분별위0.08±0.01、0.07±0.01、0.05±0.01,F=77.421화F=118.262,P치균＜0.05.불동제량FZHc간예후α-SMA、FN표체교B2조명현감소,병정제량의뢰성.B2、C1、C2조소서간조직중Nrf2양성표체평균흡광도치분별위0.07±0.01、0.10±0.01、0.17±0.01;Nqol평균광흡광도분별위0.06±0.01、0.09±0.01、0.14±0.01,F=182.537화F=75.615,P치균＜0.05;Nrf2총단백표체적상대회도치분별위0.79±0.05、0.94±0.02、1.12±0.08,F=45.664,P＜ 0.05.불동제량FZHc간예가현저증가Nrf2、Nqol표체,병정제량의뢰성.C1、C2、B2조Nrf2핵단백표체적상대회도치분별위0.97±0.09、1.24±0.10화0.67±0.04,F=94.787,P＜0.05,차이유통계학의의.FZHc촉진Nrf2 mRNA상대표체량,C1조위4.00±0.12、C2조위4.65±0.08、B2조위2.15±0.11,F=3230.105,P＜0.05,차이유통계학의의,병구유제량의뢰성. 결론 FZHc가능통과촉진Nrf2 mRNA、단백표체병촉진Nrf2포핵전이,촉진기하유파기인Nqol적표체,억제HSCs활화급FN합성,종이감경간장섬유화.
Objective To investigate the effect of Fuzhenghuayu compound (FZHc) on expression of nuclear factor E2-related factor 2 (Nrf2) in hepatocytes under conditions of hepatic fibrosis using a mouse model.Methods Mice were randomly assigned to a control group and a hepatic fibrosis model group.The control group was further divided into three subgroups for use as normal controls (A1),mineral oil-treated controls (A2),and FZHc-treated controls (A3); the hepatic fibrosis model group was administered carbon tetrachloride (CC14 dissolved in mineral oil and injected intraperitoneally) and further divided into four subgroups for use as 6-weeks models (B1),10-weeks models (B2),low-dose (L)-FZHc models (C1),and high-dose (H)-FZHc models (C2).The FZHc (capsule powder diluted with double-distilled water to 0.1 g/mL) was administered via gastric perfusion to groups A3,C1,and C2 starting at week 7 of the experiment.At the end of week 6 and 10,hepatic specimens were collected and evaluated for degree of hepatic fibrosis and inflammation using routine haematoxylin-eosin staining and Masson staining.Immunohistochemical analysis was performed to measure the hepatocyte expression of Nrf2,NAD(P)H quinine oxidoreductase 1 (Nqol),α-smooth muscle actin (α-SMA) and fibronectin (FN).Real-time fluorescence quantitative PCR was used to measure Nrf2 mRNA expression.western blotting was used to detect Nrf2 and Nqol total protein expression and Nrf2 nuclear translocation.F test,LSD test and ridit test were used for statistical analyses.Results Compared with the B2 group (ridit value:0.09),the model groups treated with FZHc showed significantly lower degrees of hepatic inflammation and fibrosis for both the low (C1 group,ridit value:0.32) and high doses (C2 group,ridit value:0.40) (F =82.927,P ＜ 0.05).In addition,compared with the B2 group,the model groups treated with FZHc showed significantly decreased expression of α-SMA and FN proteins,with a dose-dependent trend (by immunohistochemistry:C 1 group at the end of 10 weeks,F =77.421,118.262,P ＜ 0.05; C2 group,P =0.002,0.013) and significantly increased expression of Nrf2 and Nqol proteins (by immunohistochemistty:C1 and C2 groups at the end of 10 weeks,F =182.537,75.615,P ＜ 0.05 and by westem blotting:F =45.664,127.673,P ＜ 0.05),which also showed a dose-dependent trend (C2 group,P =0.000,0.014; 0.005,0.014).Western blotting also indicated that the amount of nuclear transported Nrf2 was higher in the C1 and C2 groups at the end of 10 weeks (vs.B2 group,F =94.787,P ＜ 0.05),and the amount of nuclear transported Nrf2 was significantly higher in the C2 group (vs.C1 group,P =0.044).Nrf2 mRNA expression was significantly higher in the C1 group than in the B2 group (F =3230.105,P ＜ 0.05),and the C2 group had more substantially increased expression (P =0.001); there was no statistical difference found between groups B1 and B2 (P =0.094).Conclusion Fuzhenghuayu compound increased the expression of Nrf2 mRNA and protein under conditions of hepatic fibrosis in mice and stimulated Nrf2 nuclear transport,as well as increased expression of the Nrf2 target gene Nqol that is known to suppress activation of hepatic stellate cells and decrease the deposition of FN.Therefore,Fuzhenghuayu compound may ameliorate hepatocyte injury in hepatic fibrosis in mice by exerting an antihepatic fibrosis effect.
