CAJ | 학술논문

目的：探讨瞬时感受器电位V1型（transient receptor potential vanilloid type 1，TRPV1）在精子氧化应激中对精子凋亡的调控作用及其分子机制。方法手淫法收集健康生育男性精液，Percoll 梯度离心法分离精液精子，采用次黄嘌呤、黄嘌呤氧化酶制备精子氧化应激体外实验模型。实验分5组：（1）A 组为精子悬液；（2） B组为精子悬液+次黄嘌呤（终浓度为1 mmol/L）+黄嘌呤氧化酶（终浓度为50 mU/ml）；（3）C 组为精子悬液+capsazepine（终浓度为1μmol/L）+次黄嘌呤（终浓度为1 mmol/L）+黄嘌呤氧化酶（终浓度为50 mU/ml）；（4） D 组为精子悬液+ CsA（终浓度为0.2μmol/L）+次黄嘌呤（终浓度为1 mmol/L）+黄嘌呤氧化酶（终浓度为50 mU/ml）；（5）E 组为精子悬液+ CsA（终浓度为0.2μmol/L）+capsazepine（终浓度为1μmol/L）+次黄嘌呤（终浓度为1 mmol/L）+黄嘌呤氧化酶（终浓度为50 mU/ ml）。采用JC-1探针法检测精子线粒体膜电位的变化，FITC-Annexin V探针法检测精子凋亡，蛋白印迹法检测细胞色素c表达，分光光度法检测Caspase3、9的活性。结果各组精子线粒体膜电位检测发现，与A组比较，B~E 组的荧光强度变化明显（P ＜0.01）；与C组比较，D组和E组的荧光强度变化较小（P＜0.01）；D组与E组比较无统计学差异（P＞0.05）。精子凋亡率及凋亡指数检测发现，与A组比较，B~E 组精子凋亡率及凋亡指数明显增加（P＜0.01）；与C组比较，D组和E组精子凋亡率及凋亡指数降低（P ＜0.01）；D组与E组比较无统计学差异（P ＞0.05）。精子蛋白印迹检测显示，与B组比较， A、C~E 组精子线粒体细胞色素C明显降低（P ＜0.01）；与C组比较，D组和E组精子线粒体细胞色素C表达降低（P＜0.01）；D组与E组比较差异无统计学意义（P ＞0.05）。分光光度法检测精子线粒体Caspase3/9的活性显示，与B组比较，A、C~E 组精子线粒体Caspase3/9的活性明显降低（P＜0.01）；与C组比较，D组和E组精子线粒体Caspase3/9的活性降低（P＜0.01）；D组与E组比较差异无统计学意义（P＞0.05）。结论在精子氧化应激反应中， TRPV1可能通过调控细胞内钙离子浓度，诱发线粒体通透性转换孔开放，导致细胞色素C释放于胞浆，激活caspase-9和3，从而参与诱导经线粒体途径的精子凋亡。阻断TRPV1的作用有助于减轻氧化应激反应所致精子凋亡。目的：探討瞬時感受器電位V1型（transient receptor potential vanilloid type 1，TRPV1）在精子氧化應激中對精子凋亡的調控作用及其分子機製。方法手淫法收集健康生育男性精液，Percoll 梯度離心法分離精液精子，採用次黃嘌呤、黃嘌呤氧化酶製備精子氧化應激體外實驗模型。實驗分5組：（1）A 組為精子懸液；（2） B組為精子懸液+次黃嘌呤（終濃度為1 mmol/L）+黃嘌呤氧化酶（終濃度為50 mU/ml）；（3）C 組為精子懸液+capsazepine（終濃度為1μmol/L）+次黃嘌呤（終濃度為1 mmol/L）+黃嘌呤氧化酶（終濃度為50 mU/ml）；（4） D 組為精子懸液+ CsA（終濃度為0.2μmol/L）+次黃嘌呤（終濃度為1 mmol/L）+黃嘌呤氧化酶（終濃度為50 mU/ml）；（5）E 組為精子懸液+ CsA（終濃度為0.2μmol/L）+capsazepine（終濃度為1μmol/L）+次黃嘌呤（終濃度為1 mmol/L）+黃嘌呤氧化酶（終濃度為50 mU/ ml）。採用JC-1探針法檢測精子線粒體膜電位的變化，FITC-Annexin V探針法檢測精子凋亡，蛋白印跡法檢測細胞色素c錶達，分光光度法檢測Caspase3、9的活性。結果各組精子線粒體膜電位檢測髮現，與A組比較，B~E 組的熒光彊度變化明顯（P ＜0.01）；與C組比較，D組和E組的熒光彊度變化較小（P＜0.01）；D組與E組比較無統計學差異（P＞0.05）。精子凋亡率及凋亡指數檢測髮現，與A組比較，B~E 組精子凋亡率及凋亡指數明顯增加（P＜0.01）；與C組比較，D組和E組精子凋亡率及凋亡指數降低（P ＜0.01）；D組與E組比較無統計學差異（P ＞0.05）。精子蛋白印跡檢測顯示，與B組比較， A、C~E 組精子線粒體細胞色素C明顯降低（P ＜0.01）；與C組比較，D組和E組精子線粒體細胞色素C錶達降低（P＜0.01）；D組與E組比較差異無統計學意義（P ＞0.05）。分光光度法檢測精子線粒體Caspase3/9的活性顯示，與B組比較，A、C~E 組精子線粒體Caspase3/9的活性明顯降低（P＜0.01）；與C組比較，D組和E組精子線粒體Caspase3/9的活性降低（P＜0.01）；D組與E組比較差異無統計學意義（P＞0.05）。結論在精子氧化應激反應中， TRPV1可能通過調控細胞內鈣離子濃度，誘髮線粒體通透性轉換孔開放，導緻細胞色素C釋放于胞漿，激活caspase-9和3，從而參與誘導經線粒體途徑的精子凋亡。阻斷TRPV1的作用有助于減輕氧化應激反應所緻精子凋亡。
목적：탐토순시감수기전위V1형（transient receptor potential vanilloid type 1，TRPV1）재정자양화응격중대정자조망적조공작용급기분자궤제。방법수음법수집건강생육남성정액，Percoll 제도리심법분리정액정자，채용차황표령、황표령양화매제비정자양화응격체외실험모형。실험분5조：（1）A 조위정자현액；（2） B조위정자현액+차황표령（종농도위1 mmol/L）+황표령양화매（종농도위50 mU/ml）；（3）C 조위정자현액+capsazepine（종농도위1μmol/L）+차황표령（종농도위1 mmol/L）+황표령양화매（종농도위50 mU/ml）；（4） D 조위정자현액+ CsA（종농도위0.2μmol/L）+차황표령（종농도위1 mmol/L）+황표령양화매（종농도위50 mU/ml）；（5）E 조위정자현액+ CsA（종농도위0.2μmol/L）+capsazepine（종농도위1μmol/L）+차황표령（종농도위1 mmol/L）+황표령양화매（종농도위50 mU/ ml）。채용JC-1탐침법검측정자선립체막전위적변화，FITC-Annexin V탐침법검측정자조망，단백인적법검측세포색소c표체，분광광도법검측Caspase3、9적활성。결과각조정자선립체막전위검측발현，여A조비교，B~E 조적형광강도변화명현（P ＜0.01）；여C조비교，D조화E조적형광강도변화교소（P＜0.01）；D조여E조비교무통계학차이（P＞0.05）。정자조망솔급조망지수검측발현，여A조비교，B~E 조정자조망솔급조망지수명현증가（P＜0.01）；여C조비교，D조화E조정자조망솔급조망지수강저（P ＜0.01）；D조여E조비교무통계학차이（P ＞0.05）。정자단백인적검측현시，여B조비교， A、C~E 조정자선립체세포색소C명현강저（P ＜0.01）；여C조비교，D조화E조정자선립체세포색소C표체강저（P＜0.01）；D조여E조비교차이무통계학의의（P ＞0.05）。분광광도법검측정자선립체Caspase3/9적활성현시，여B조비교，A、C~E 조정자선립체Caspase3/9적활성명현강저（P＜0.01）；여C조비교，D조화E조정자선립체Caspase3/9적활성강저（P＜0.01）；D조여E조비교차이무통계학의의（P＞0.05）。결론재정자양화응격반응중， TRPV1가능통과조공세포내개리자농도，유발선립체통투성전환공개방，도치세포색소C석방우포장，격활caspase-9화3，종이삼여유도경선립체도경적정자조망。조단TRPV1적작용유조우감경양화응격반응소치정자조망。
Objective To investigate the role of TRPV1 in apoptosis of sperm in oxidative stress and the possible molecular mechanisms. Methods Semen samples of healthy males were collected by the way of masturbate, and sperms were isolated from semen samples by Percoll gradient centrifugation. in vitro model of sperm oxidative stress was induced by hypoxanthine and xanthine oxidase. The experiment was divided into five groups, groupA( the sperm suspension), groupB(the sperm suspension + hypoxanthine withfinal concentration of 1 mmol/L + xanthine oxidase with final concentration of 50 mU/ml; groupC( the sperm suspension + capsazepine withfinal concentration of 1μmol/L +hypoxanthine with final concentration of 1 mmol/L + xanthine oxidase withfinal concentration of 50 mU/ml), groupD (the sperm suspension+CsA with final concentration of 0.2μmol/L + hypoxanthine with final concentration of 1 mmol/L+xanthine oxidase with final concentration of 50 mU/ml); groupE( the sperm suspension + CsA withfinal concentration of 0.2 μmol/L + capsazepine withfinal concentration of 1μmol/L) + hypoxanthine with final concentration of 1 mmol/L+ xanthine oxidase with final concentration of 50 mU/ml). The sperm viability and motion parameters were detected by computer-aided semen analysis (CASA), the changes of the sperm mitochondrial membrane was measured by JC-1 probe, sperm apoptosis was analyzed usingFITC-Annexin V probe, the expression of cytochrome c was detected by western blot, and the activity of Caspase3, 9 were examined by spectrophotometry. Results Sperm mitochondrial membrane potential of groupB~E was changed significantly compared with that of groupA (P<0.01), the ones of D and E group was slightly changed compared with that of groupC (P<0.01), there is no significant difference between group D and group E (P>0.05). Sperm apoptosis rate and apoptosis index in B ~ E group wereincreased significantly compared with that of groupA (P<0.01), the ones of groupD and group E group were decreased compared with that of groupC(P<0.01) ,there was no significant difference between D and E group (P >0.05). The expression of sperm mitochondrial cytochrome C was decreased significantly in group A, C ~ E compared with that of groupB (P<0.01), it in group D and E was decreased compared with that of groupC(P<0.01) ; there was no significant difference between group D and group E (P> 0.05). Sperm mitochondria Caspase3/9 activity in A, C ~ E Group were significantly decreased compared with that of groupB(P<0.01); it in group D, E was decreased compared with that of groupC(P<0.01); there is no significant difference between group D and group E (P>0.05). Conclusion TRPV1 induced mitochondrial permeability transition pore opening by regulating intracellular calcium concentration in sperm response to oxidative stress, and resulted in releasing cytochrome C into cytoplasm, activating caspase-9 and 3, thereby involving in sperm apoptosis viathe mitochondrial pathway. Blocking the effect of TRPV1 may help to reduce sperm apoptosis induced by oxidative stress.