目的 评价肾缺血后处理(ischemic postconditioning,IPo)对热休克蛋白(heat shock protein,HSP)70、HSP27和血红素加氧酶-1(heme oxygenase-1,HO-1,即HSP32)表达的影响及在减轻肾缺血/再灌注损伤(ischemia/reperfusion injury,I/RI)中的作用.方法 健康雄性SD大鼠140只,体重250 g～280 g,采用随机数字表法随机分为4组(每组35只):假手术组(S组)仅开腹,游离双侧肾脏,分离双侧肾蒂不夹闭；缺血/再灌注(ischemia/reperfusion,I/R)组,夹闭双侧肾蒂缺血45 min,恢复灌注；IPo组,夹闭双侧肾蒂45 min,再灌注10 s,缺血10s,反复3次,恢复灌注；HSP抑制剂槲皮黄酮+IPo组(Q+IPo组),缺血前lh腹腔注射槲皮黄酮100 mg/kg,余操作同IPo组.于再灌注即刻(T0)、1、3、6、12、24、48 h(T1～6)时取5只大鼠经心脏抽血后迅速处死取肾,采用逆转录-多聚酶链反应(RT-PCR)和免疫组织化学法分别检测各时点肾组织HSP70、HSP27和HO-1的mRNA和蛋白表达,测定T3时血清肌酐(creatinine,Cr)和尿素氮(urea nitrogen,BUN)浓度、肾组织丙二醛(methylene dioxyamphetamine,MDA)含量和超氧化物歧化酶(superoxide dismutase,SOD)活性、肾组织核因子-κB(nuclear factor-kappa B,NF-κB)表达和血清肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)浓度,光镜下观察肾组织病理学结果. 结果 S组HSP70、HSP27和HO-1的mRNA有微量表达,蛋白几乎无表达,其余组在To时开始表达,逐渐升高,T3时达高峰,随后逐渐下降.与S组比较,其余组各时点HSP70、HSP27和HO-1的mRNA和蛋白表达上调(P＜0.05),IPo组较I/R组T2～5时HSP70、HSP27和HO-1的mRNA和蛋白表达上调(P＜0.05),Q+IPo组较IPo组T2～5时HSP70、HSP27和HO-1的mRNA和蛋白表达下调(P＜0.05).T3时血清Cr、BUN和TNF-α浓度I/R组分别为(102±5) μmol/L、(25.7±3.9) mmol/L、(2.29±0.18) μg/L,IPo组分别为(64±5)μmol/L、(11.3±3.0) mmol/L、(1.76±0.13)μg/L,Q+IPo组分别为(101±6)μmol/L、(26.5±4.5) mmol/L、(2.31±0.17) μg/L,均高于S组(46±6) μmol/L、(5.1±1.9) mmol/L和(1.13±0.14) μg/L(P＜0.05),IPo组三者浓度较I/R组降低(P＜0.05),Q+IPo组较IPo组升高(P＜0.05).T3时MDA含量I/R组(2.20±0.23) nmol/mgprot、IPo组(1.35±0.13) nmol/mgprot和Q+IPo组(2.25±0.16) nmol/mgprot较S组(1.02±0.19) nmol/mgprot升高(P＜0.05),SOD活性I/R组(104±6) U/mgprot、IPo组(124±4) U/mgprot和Q+IPo组(106±5) U/mgprot较S组(147±6) U/mgprot 降低(P＜0.05),IPo组与I/R组比较MDA含量降低和SOD活性升高(P＜0.05),Q+Ipo组与IPo组比较MDA含量升高和SOD活性降低(P＜0.05),肾组织NF-κB表达I/R组、IPo组和Q+IPo组较S组增高,IPo组较I/R组表达降低,Q+IPo组较IPo组表达增高(P＜0.05).I/R组与Q+Ipo组相比,各指标差异无统计学意义(P＞0.05).与S组比较,其余3组有程度不等的肾组织病理学损伤,IPo组损伤较I/R组减轻,Q+IPo组损伤程度与I/R组相似. 结论 IPo上调了HSP70、HSP27和HO-1的表达；HSP高表达参与了肾IPo减轻肾I/RI的过程.
目的 評價腎缺血後處理(ischemic postconditioning,IPo)對熱休剋蛋白(heat shock protein,HSP)70、HSP27和血紅素加氧酶-1(heme oxygenase-1,HO-1,即HSP32)錶達的影響及在減輕腎缺血/再灌註損傷(ischemia/reperfusion injury,I/RI)中的作用.方法 健康雄性SD大鼠140隻,體重250 g～280 g,採用隨機數字錶法隨機分為4組(每組35隻):假手術組(S組)僅開腹,遊離雙側腎髒,分離雙側腎蒂不夾閉；缺血/再灌註(ischemia/reperfusion,I/R)組,夾閉雙側腎蒂缺血45 min,恢複灌註；IPo組,夾閉雙側腎蒂45 min,再灌註10 s,缺血10s,反複3次,恢複灌註；HSP抑製劑槲皮黃酮+IPo組(Q+IPo組),缺血前lh腹腔註射槲皮黃酮100 mg/kg,餘操作同IPo組.于再灌註即刻(T0)、1、3、6、12、24、48 h(T1～6)時取5隻大鼠經心髒抽血後迅速處死取腎,採用逆轉錄-多聚酶鏈反應(RT-PCR)和免疫組織化學法分彆檢測各時點腎組織HSP70、HSP27和HO-1的mRNA和蛋白錶達,測定T3時血清肌酐(creatinine,Cr)和尿素氮(urea nitrogen,BUN)濃度、腎組織丙二醛(methylene dioxyamphetamine,MDA)含量和超氧化物歧化酶(superoxide dismutase,SOD)活性、腎組織覈因子-κB(nuclear factor-kappa B,NF-κB)錶達和血清腫瘤壞死因子-α(tumor necrosis factorα,TNF-α)濃度,光鏡下觀察腎組織病理學結果. 結果 S組HSP70、HSP27和HO-1的mRNA有微量錶達,蛋白幾乎無錶達,其餘組在To時開始錶達,逐漸升高,T3時達高峰,隨後逐漸下降.與S組比較,其餘組各時點HSP70、HSP27和HO-1的mRNA和蛋白錶達上調(P＜0.05),IPo組較I/R組T2～5時HSP70、HSP27和HO-1的mRNA和蛋白錶達上調(P＜0.05),Q+IPo組較IPo組T2～5時HSP70、HSP27和HO-1的mRNA和蛋白錶達下調(P＜0.05).T3時血清Cr、BUN和TNF-α濃度I/R組分彆為(102±5) μmol/L、(25.7±3.9) mmol/L、(2.29±0.18) μg/L,IPo組分彆為(64±5)μmol/L、(11.3±3.0) mmol/L、(1.76±0.13)μg/L,Q+IPo組分彆為(101±6)μmol/L、(26.5±4.5) mmol/L、(2.31±0.17) μg/L,均高于S組(46±6) μmol/L、(5.1±1.9) mmol/L和(1.13±0.14) μg/L(P＜0.05),IPo組三者濃度較I/R組降低(P＜0.05),Q+IPo組較IPo組升高(P＜0.05).T3時MDA含量I/R組(2.20±0.23) nmol/mgprot、IPo組(1.35±0.13) nmol/mgprot和Q+IPo組(2.25±0.16) nmol/mgprot較S組(1.02±0.19) nmol/mgprot升高(P＜0.05),SOD活性I/R組(104±6) U/mgprot、IPo組(124±4) U/mgprot和Q+IPo組(106±5) U/mgprot較S組(147±6) U/mgprot 降低(P＜0.05),IPo組與I/R組比較MDA含量降低和SOD活性升高(P＜0.05),Q+Ipo組與IPo組比較MDA含量升高和SOD活性降低(P＜0.05),腎組織NF-κB錶達I/R組、IPo組和Q+IPo組較S組增高,IPo組較I/R組錶達降低,Q+IPo組較IPo組錶達增高(P＜0.05).I/R組與Q+Ipo組相比,各指標差異無統計學意義(P＞0.05).與S組比較,其餘3組有程度不等的腎組織病理學損傷,IPo組損傷較I/R組減輕,Q+IPo組損傷程度與I/R組相似. 結論 IPo上調瞭HSP70、HSP27和HO-1的錶達；HSP高錶達參與瞭腎IPo減輕腎I/RI的過程.
목적 평개신결혈후처리(ischemic postconditioning,IPo)대열휴극단백(heat shock protein,HSP)70、HSP27화혈홍소가양매-1(heme oxygenase-1,HO-1,즉HSP32)표체적영향급재감경신결혈/재관주손상(ischemia/reperfusion injury,I/RI)중적작용.방법 건강웅성SD대서140지,체중250 g～280 g,채용수궤수자표법수궤분위4조(매조35지):가수술조(S조)부개복,유리쌍측신장,분리쌍측신체불협폐；결혈/재관주(ischemia/reperfusion,I/R)조,협폐쌍측신체결혈45 min,회복관주；IPo조,협폐쌍측신체45 min,재관주10 s,결혈10s,반복3차,회복관주；HSP억제제곡피황동+IPo조(Q+IPo조),결혈전lh복강주사곡피황동100 mg/kg,여조작동IPo조.우재관주즉각(T0)、1、3、6、12、24、48 h(T1～6)시취5지대서경심장추혈후신속처사취신,채용역전록-다취매련반응(RT-PCR)화면역조직화학법분별검측각시점신조직HSP70、HSP27화HO-1적mRNA화단백표체,측정T3시혈청기항(creatinine,Cr)화뇨소담(urea nitrogen,BUN)농도、신조직병이철(methylene dioxyamphetamine,MDA)함량화초양화물기화매(superoxide dismutase,SOD)활성、신조직핵인자-κB(nuclear factor-kappa B,NF-κB)표체화혈청종류배사인자-α(tumor necrosis factorα,TNF-α)농도,광경하관찰신조직병이학결과. 결과 S조HSP70、HSP27화HO-1적mRNA유미량표체,단백궤호무표체,기여조재To시개시표체,축점승고,T3시체고봉,수후축점하강.여S조비교,기여조각시점HSP70、HSP27화HO-1적mRNA화단백표체상조(P＜0.05),IPo조교I/R조T2～5시HSP70、HSP27화HO-1적mRNA화단백표체상조(P＜0.05),Q+IPo조교IPo조T2～5시HSP70、HSP27화HO-1적mRNA화단백표체하조(P＜0.05).T3시혈청Cr、BUN화TNF-α농도I/R조분별위(102±5) μmol/L、(25.7±3.9) mmol/L、(2.29±0.18) μg/L,IPo조분별위(64±5)μmol/L、(11.3±3.0) mmol/L、(1.76±0.13)μg/L,Q+IPo조분별위(101±6)μmol/L、(26.5±4.5) mmol/L、(2.31±0.17) μg/L,균고우S조(46±6) μmol/L、(5.1±1.9) mmol/L화(1.13±0.14) μg/L(P＜0.05),IPo조삼자농도교I/R조강저(P＜0.05),Q+IPo조교IPo조승고(P＜0.05).T3시MDA함량I/R조(2.20±0.23) nmol/mgprot、IPo조(1.35±0.13) nmol/mgprot화Q+IPo조(2.25±0.16) nmol/mgprot교S조(1.02±0.19) nmol/mgprot승고(P＜0.05),SOD활성I/R조(104±6) U/mgprot、IPo조(124±4) U/mgprot화Q+IPo조(106±5) U/mgprot교S조(147±6) U/mgprot 강저(P＜0.05),IPo조여I/R조비교MDA함량강저화SOD활성승고(P＜0.05),Q+Ipo조여IPo조비교MDA함량승고화SOD활성강저(P＜0.05),신조직NF-κB표체I/R조、IPo조화Q+IPo조교S조증고,IPo조교I/R조표체강저,Q+IPo조교IPo조표체증고(P＜0.05).I/R조여Q+Ipo조상비,각지표차이무통계학의의(P＞0.05).여S조비교,기여3조유정도불등적신조직병이학손상,IPo조손상교I/R조감경,Q+IPo조손상정도여I/R조상사. 결론 IPo상조료HSP70、HSP27화HO-1적표체；HSP고표체삼여료신IPo감경신I/RI적과정.
Obiective To investigate the effect of ischemic postconditioning on expression of heat shock protein (HSP)70,HSP27 and heme oxygenase-1 (HO-1) and the role of which in reduction renal ischemia/reperfusion injury (I/RI) in rats.Methods One hundred forty healthy male SD rats weighing 250 g-280 g were randomly divided into 4 groups (n=35):group Ⅰ sham operation group (group S) ; group ]Ⅱ I/R; group Ⅲ IPo; group Ⅳ quercetin+IPo group (group Q +IPo).The rats were anesthetized with intraperitoneal (IP) chloral hydrate 300 mg/kg.Bilateral kidneys were exposed and their pedicels were clamped for 45 min followed by reperfusion in group Ⅱ -Ⅳ.In group Ⅲ and Ⅳ 3 cycles of 10 s reperfusion followed by 10 s ischemia were applied immediately after 45 min kidney ischemia.In group Ⅳ quercetin (a inhibitor of HSP) 100 mg/kg was given IP at 30 min before ischemia.Five rats were sacrificed at 0, 1,3,6, 12,24,and 48 h (T0-6) of reperfusion and the kidneys were immediately removed for determination the expression of HSP70 mRNA,HSP27 mRNA,HO-1 mRNA,HSP70,HSP27 and HO-1.At T3 before the rats were sacrificed blood samples were obtained for determination of serum creatinine (Cr),urea nitrogen (BUN) and tumor necrosis factor α (TNF-α) concentrations and kidneys were removed for determination of the expression of nuclear factor-kappa B (NF-κB),the methylene dioxyamphetamine (MDA) content and superoxide dismutase (SOD) activity,and observation of histopathology with light microscope. Results The expression of HSP70 mRNA,HSP27 mRNA,HO-1 mRNA,HSP70,HSP27 and HO-1 was traced in group S,and in the other three groups the expression of HSP70 mRNA,HO-1 mRNA and HSP70,HO-1 increased at T1 and reached peakpoint at T3,then began to decrease.Compared with group S,the expression of HSP70 mRNA,HSP27 mRNA,HO-1 mRNA,HSP70,HSP27 and HO-1 was up-regulated at T0-6 in other 3 groups (P＜0.05),.Compared with group I/R,the expression of HSP70 mRNA,HSP27 mRNA,HO-1mRNA,HSP70,HSP27 and HO-1 was up-regulated at T2-5 in group IPo (P＜0.05),Compared with group IPo,the expression of HSP70m RNA,HSP27m RNA,HO-1m RNA,HSP70,HSP27 and HO-1 was down-regulated at T2-5 in group Q+IPo (P＜0.05).There were no significant difference between group I/R and group Q+IPo (P＞0.05).Serum Cr,BUN and TNF-αconcentrations at T3 in group I/R (102±5) μmol/L,(25.7±3.9) mmol/L,(2.29±0.18) μg/L,in group IPo (64±5) μmol/L,(11.3±3.0) mmo]/L,(1.76±0.13) μg/L,and in grou Q+IPo (101±6) μ mol/L,(26.5±4.5) mmol/L,(2.31±0.17) μg/L were significantly increased than those in group S (46±6) μmol/L, (5.1±1.9) mmol/L and (1.13±0.14) μg/L (P＜0.05),and the serum Cr,BUN and TNF-αconcentrations in group IPo were decreased than those in group I/R,and those in group Q+IPo were increased than those in group IPo (P＜0.05).There were no significant difference between group I/R and group Q+IPo (P＞0.05).The MDA content (2.20±0.23) nmol/mgprot in group I/R,(1.35±0.13) nmol/mgprot in group IPo and (2.25±0.16) nmol/mgprot in group Q+IPo was increased than that in group S (P＜0.05),the activity of SOD (104±6) U/mgprot in group I/R,(124±4) U/mgprot in group IPo and (106±5) U/mgprot in group Q+IPo was decreased than that in group S (P＜0.05); the MDA content was decreased and SOD activity was increased in group IPo compared with group I/R,and the MDA content was increased and SOD activity was decreased in group Q+IPo compared with group IPo (P＜0.05).The expression of NF-KB in group was up-regulated at T3 in group I/R,IPo and Q+IPo compared with group S,that in group IPo was down-regulated compared with group I/R,and that in Q+IPo was up-regulated compared with group IPo (P＜0.05).There were no significant difference between group I/R and group Q+IPo (P＞0.05).Microscope examination showed that the renal injury was attenuated in group IPo compared with group I/R while there was no significant difference between group I/R and group Q+IPo. Conclusions IPo increased the expression of HSP70,HSP27,and HO-1 and the expression of HSP is involved in the reduction of renal I/R injury by IPo in rats.
