目的 探讨小鼠肝脏缺血再灌注(IR)后血红素加氧酶1(HO-1)表达与自噬的相关性以及对肝功能的影响.方法 建立对照组,IR组,HO-1诱导剂Hemin预处理后IR组(Hemin+ IR组)及HO-1抑制剂Znpp预处理后IR组(Znpp+ IR组).按不同灌注时间,IR组分为0、2、6、12、24、48、72 h组,Hemin+IR组和Znpp+ IR组分为0、2、6、12、24 h组.检测各组小鼠血清ALT、AST水平以评估肝功能；HE染色评估肝脏形态学改变,电镜检查观察肝脏组织内自噬小体情况；Western blot法检测HO-1及自噬相关蛋白LC3Ⅱ的蛋白水平.多组间比较采用方差分析,两组比较采用t检验,两两之间比较采用SNK-q检验.结果 IR组、Hemin+ IR组及Znpp+ IR组小鼠各时间点血清ALT、AST值均高于对照组,组间比较,差异有统计学意义(F =96.17,85.53,P＜0.05)；IR组缺血再灌注后12、24 h的血清ALT、AST值均高于Hemin+ IR组相应时间点的值,两者比较,差异有统计学意义(qALT=-14.46,-7.85；qAST=-12.98,-5.26,P＜0.05)；而低于Znpp +IR组相应时间点的值,两者比较,差异有统计学意义(qALT=4.25,4.94；qAST=4.98,3.53,P ＜0.05).HE染色示肝脏IR损伤在Hemin+ IR组较IR组和Znpp+ IR组显著改善,应用Suzuki分级进行半定量分析,对照组、IR 12 h组、Hemin+ IR 12 h组、Znpp+IR 12 h组肝脏IR损伤评分分别为:0.5±0.3、2.6±0.5、1.1±0.3、3.0±0.4,组间比较,差异有统计学意义(F =53.62,P＜0.05).IR 12 h组分别与Hemin +IR 12 h组和Znpp +IR 12 h组比较,差异有统计学意义(q=10.67,14.02,P＜0.05).电镜检查示:IR组小鼠肝脏自噬小体较对照组明显增加,在Hemin+ IR组增加更明显,而Znpp+ IR组较IR组有所减少,对照组、IR 12 h组、Hemin+ IR 12 h组、Znpp +IR 12 h组每10个视野的自噬小体平均数量分别为:(0.4±0.2)个、(1.8±0.6)个、(4.0±1.8)个、(0.7±0.5)个,组间比较,差异有统计学意义(F =21.35,P＜0.05)；IR 12 h组分别与Hemin +IR 12 h组和Znpp+ IR 12 h组比较,差异有统计学意义(q=6.05,-3.03,P＜0.05).HO-1及自噬相关蛋白LC3Ⅱ的表达在IR组及Hemin+ IR组显著增加,且Hemin+ IR 6、12 h组HO-1蛋白相对表达量分别为0.64±0.17、0.51 ±0.12；LC3Ⅱ蛋白相对表达量分别为1.04±0.20、1.20±0.23,高于IR组相应时间点的蛋白相对表达量HO-1:0.45±0.08、0.17 ±0.03；LC3Ⅱ:0.58±0.04、0.95±0.14,两组比较,差异有统计学意义(tHO-1=4.03,4.69；tLC3Ⅱ=4.29,6.69,P＜0.05)；而Znpp+ IR 6、12 h组HO-1蛋白相对表达量分别为0.23 ±0.03、0.14±0.02；LC3Ⅱ蛋白相对表达量分别为0.35 ±0.04,0.49±0.14,低于IR组相应时间点的蛋白相对表达量,两组比较,差异有统计学意义(tHO-1=13.82,7.04；tLC3Ⅱ=7.21,4.03,P＜0.05).结论 HO-1的表达与肝功能改善呈正相关,且其可能是通过诱导体内自噬来实现的.
目的 探討小鼠肝髒缺血再灌註(IR)後血紅素加氧酶1(HO-1)錶達與自噬的相關性以及對肝功能的影響.方法 建立對照組,IR組,HO-1誘導劑Hemin預處理後IR組(Hemin+ IR組)及HO-1抑製劑Znpp預處理後IR組(Znpp+ IR組).按不同灌註時間,IR組分為0、2、6、12、24、48、72 h組,Hemin+IR組和Znpp+ IR組分為0、2、6、12、24 h組.檢測各組小鼠血清ALT、AST水平以評估肝功能；HE染色評估肝髒形態學改變,電鏡檢查觀察肝髒組織內自噬小體情況；Western blot法檢測HO-1及自噬相關蛋白LC3Ⅱ的蛋白水平.多組間比較採用方差分析,兩組比較採用t檢驗,兩兩之間比較採用SNK-q檢驗.結果 IR組、Hemin+ IR組及Znpp+ IR組小鼠各時間點血清ALT、AST值均高于對照組,組間比較,差異有統計學意義(F =96.17,85.53,P＜0.05)；IR組缺血再灌註後12、24 h的血清ALT、AST值均高于Hemin+ IR組相應時間點的值,兩者比較,差異有統計學意義(qALT=-14.46,-7.85；qAST=-12.98,-5.26,P＜0.05)；而低于Znpp +IR組相應時間點的值,兩者比較,差異有統計學意義(qALT=4.25,4.94；qAST=4.98,3.53,P ＜0.05).HE染色示肝髒IR損傷在Hemin+ IR組較IR組和Znpp+ IR組顯著改善,應用Suzuki分級進行半定量分析,對照組、IR 12 h組、Hemin+ IR 12 h組、Znpp+IR 12 h組肝髒IR損傷評分分彆為:0.5±0.3、2.6±0.5、1.1±0.3、3.0±0.4,組間比較,差異有統計學意義(F =53.62,P＜0.05).IR 12 h組分彆與Hemin +IR 12 h組和Znpp +IR 12 h組比較,差異有統計學意義(q=10.67,14.02,P＜0.05).電鏡檢查示:IR組小鼠肝髒自噬小體較對照組明顯增加,在Hemin+ IR組增加更明顯,而Znpp+ IR組較IR組有所減少,對照組、IR 12 h組、Hemin+ IR 12 h組、Znpp +IR 12 h組每10箇視野的自噬小體平均數量分彆為:(0.4±0.2)箇、(1.8±0.6)箇、(4.0±1.8)箇、(0.7±0.5)箇,組間比較,差異有統計學意義(F =21.35,P＜0.05)；IR 12 h組分彆與Hemin +IR 12 h組和Znpp+ IR 12 h組比較,差異有統計學意義(q=6.05,-3.03,P＜0.05).HO-1及自噬相關蛋白LC3Ⅱ的錶達在IR組及Hemin+ IR組顯著增加,且Hemin+ IR 6、12 h組HO-1蛋白相對錶達量分彆為0.64±0.17、0.51 ±0.12；LC3Ⅱ蛋白相對錶達量分彆為1.04±0.20、1.20±0.23,高于IR組相應時間點的蛋白相對錶達量HO-1:0.45±0.08、0.17 ±0.03；LC3Ⅱ:0.58±0.04、0.95±0.14,兩組比較,差異有統計學意義(tHO-1=4.03,4.69；tLC3Ⅱ=4.29,6.69,P＜0.05)；而Znpp+ IR 6、12 h組HO-1蛋白相對錶達量分彆為0.23 ±0.03、0.14±0.02；LC3Ⅱ蛋白相對錶達量分彆為0.35 ±0.04,0.49±0.14,低于IR組相應時間點的蛋白相對錶達量,兩組比較,差異有統計學意義(tHO-1=13.82,7.04；tLC3Ⅱ=7.21,4.03,P＜0.05).結論 HO-1的錶達與肝功能改善呈正相關,且其可能是通過誘導體內自噬來實現的.
목적 탐토소서간장결혈재관주(IR)후혈홍소가양매1(HO-1)표체여자서적상관성이급대간공능적영향.방법 건립대조조,IR조,HO-1유도제Hemin예처리후IR조(Hemin+ IR조)급HO-1억제제Znpp예처리후IR조(Znpp+ IR조).안불동관주시간,IR조분위0、2、6、12、24、48、72 h조,Hemin+IR조화Znpp+ IR조분위0、2、6、12、24 h조.검측각조소서혈청ALT、AST수평이평고간공능；HE염색평고간장형태학개변,전경검사관찰간장조직내자서소체정황；Western blot법검측HO-1급자서상관단백LC3Ⅱ적단백수평.다조간비교채용방차분석,량조비교채용t검험,량량지간비교채용SNK-q검험.결과 IR조、Hemin+ IR조급Znpp+ IR조소서각시간점혈청ALT、AST치균고우대조조,조간비교,차이유통계학의의(F =96.17,85.53,P＜0.05)；IR조결혈재관주후12、24 h적혈청ALT、AST치균고우Hemin+ IR조상응시간점적치,량자비교,차이유통계학의의(qALT=-14.46,-7.85；qAST=-12.98,-5.26,P＜0.05)；이저우Znpp +IR조상응시간점적치,량자비교,차이유통계학의의(qALT=4.25,4.94；qAST=4.98,3.53,P ＜0.05).HE염색시간장IR손상재Hemin+ IR조교IR조화Znpp+ IR조현저개선,응용Suzuki분급진행반정량분석,대조조、IR 12 h조、Hemin+ IR 12 h조、Znpp+IR 12 h조간장IR손상평분분별위:0.5±0.3、2.6±0.5、1.1±0.3、3.0±0.4,조간비교,차이유통계학의의(F =53.62,P＜0.05).IR 12 h조분별여Hemin +IR 12 h조화Znpp +IR 12 h조비교,차이유통계학의의(q=10.67,14.02,P＜0.05).전경검사시:IR조소서간장자서소체교대조조명현증가,재Hemin+ IR조증가경명현,이Znpp+ IR조교IR조유소감소,대조조、IR 12 h조、Hemin+ IR 12 h조、Znpp +IR 12 h조매10개시야적자서소체평균수량분별위:(0.4±0.2)개、(1.8±0.6)개、(4.0±1.8)개、(0.7±0.5)개,조간비교,차이유통계학의의(F =21.35,P＜0.05)；IR 12 h조분별여Hemin +IR 12 h조화Znpp+ IR 12 h조비교,차이유통계학의의(q=6.05,-3.03,P＜0.05).HO-1급자서상관단백LC3Ⅱ적표체재IR조급Hemin+ IR조현저증가,차Hemin+ IR 6、12 h조HO-1단백상대표체량분별위0.64±0.17、0.51 ±0.12；LC3Ⅱ단백상대표체량분별위1.04±0.20、1.20±0.23,고우IR조상응시간점적단백상대표체량HO-1:0.45±0.08、0.17 ±0.03；LC3Ⅱ:0.58±0.04、0.95±0.14,량조비교,차이유통계학의의(tHO-1=4.03,4.69；tLC3Ⅱ=4.29,6.69,P＜0.05)；이Znpp+ IR 6、12 h조HO-1단백상대표체량분별위0.23 ±0.03、0.14±0.02；LC3Ⅱ단백상대표체량분별위0.35 ±0.04,0.49±0.14,저우IR조상응시간점적단백상대표체량,량조비교,차이유통계학의의(tHO-1=13.82,7.04；tLC3Ⅱ=7.21,4.03,P＜0.05).결론 HO-1적표체여간공능개선정정상관,차기가능시통과유도체내자서래실현적.
Objective To investigate the correlation of heme oxygenase-1 (HO-1) expression and autophagy in mice with liver ischemia reperfusion (IR) injury,and to study the influence of HO-1 on the hepatic function.Methods Control group,IR group,Hemin + IR group and Znpp + IR group were constructed.Mice in the Hemin + IR group and the Znpp + IR group were pretreated by Hemin (HO-1 inducer) and Znpp (HO-1 inhibitor) before IR.According to different reperfusion time,the IR group was divided into the 0,2,6,12,24,48 and 72 hours groups,and the Hemin + IR group and the Znpp + IR group were divided into the 0,2,6,12,24 hours groups.The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected to evaluate the hepatic function.The autophagosome and pathological changes of liver were observed under electron microscope and hematoxylin and eosin (HE) staining,respectively.The expressions of HO-1 and autophagyassociated protein (LC3Ⅱ) in the protein level were detected by Western blot.All data were analyzed using the analysis of variance,t test and q test,respectively.Results The levels of ALT and AST of the IR group,Hemin + IR group and Znpp + IR group were significantly higher than those of the control group (F =96.17,85.53,P ＜0.05).The levels of ALT and AST of the IR 12 hours group and 24 hours group were significantly higher than those of the Hemin + IR 12 hours group and 24 hours group (qALT=--14.46,-7.85 ; qAST=-12.98,-5.26,P ＜0.05),but significantly lower than those of the Znpp + IR 12 hours group and 24 hours group (qALT=4.25,4.94;qAST=4.98,3.53,P ＜ 0.05).The results of HE staining showed that hepatic IR injury was significalty alleviated in the Hemin + IR group when compared with the IR group and the Znpp + IR group.The Suzuki degrees of hepatic IR injury in the control group,IR 12 hours group,Hemin + IR 12 hours group and the Znpp + IR 12 hours group were 0.5 ± 0.3,2.6 ± 0.5,1.1 ± 0.3,3.0 ± 0.4,respectively,with significant differences among the 4 groups (F =53.62,P ＜0.05).There were significant difference in the Suzuki degrees of hepatic IR injury between the IR 12 hours group and the Hemin + IR 12 hours group,and between the IR 12 hours group and the Znpp + IR 12 hours group (q =10.67,14.02,P ＜ 0.05).The results of electron microscopy showed that the number of autophagosome in the IR group was greater than the control group and Znpp + IR group,but lesser than the Hemin + IR group.The numbers of autophagosome in every 10 high power fields of the control group,IR 12 hours group,Hemin + IR 12 hours group and the Znpp + IR 12 hours group were 0.4 ±0.2,1.8 ±0.6,4.0 ± 1.8,0.7 ±0.5,respectively,with significant difference among the 4 groups (F =21.35,P ＜ 0.05).There were significant differences in the number of autophagosome between the IR 12 hours group and the Hemin + IR 12 hours group,and between the IR 12 hours group and the Znpp + IR 12 hours group (q =6.05,-3.03,P ＜ 0.05).The expressions of HO-1 and LC3Ⅱ in the IR group and the Hemin + IR group were significantly increased.The relative expressions of HO-1 protein in the Hemin + IR 6 hours group and the 12 hours group were 0.64 ±0.17 and 0.51 ±0.12,which were significantly higher than 0.45 ± 0.08 and 0.17 ± 0.03 of the IR 6 hours group and 12 hours group (t =4.03,4.69,P ＜ 0.05).The relative expressions of LC3Ⅱ protein in the Hemin + IR 6 hours group and the 12 hours group were 1.04 ± 0.20 and 1.20 ± 0.23,which were significantly higher than 0.58 ± 0.04 and 0.95 ±0.14 of the IR 6 hours group and 12 hours group (t =4.29,6.69,P＜0.05).The relative expressions of HO-1 protein in the Znpp + IR 6 hours group and 12 hours group were 0.23 ± 0.03,0.14 ± 0.02,respectively,and the relative expressions of LC3Ⅱ protein were 0.35 ± 0.04 and 0.49 ± 0.14,which were significantly lower than the HO-1 and LC3Ⅱ protein expressions in the IR 6 hours group and 12 hours group (tHO-1 =13.82,7.04; tLC3Ⅱ =7.21,4.03,P ＜0.05).Conclusion HO-1 is positively related to the amelioration of hepatic function,and it may be achieved by induction of autophagy.
