CAJ | 학술논문

目的评价α7烟碱型乙酰胆碱(α7nACh)受体激动剂对缺氧复氧大鼠心肌细胞糖原合成酶激酶3β(GSK-3β)活性的影响.方法大鼠心肌细胞培养72 h后,按照随机数字表法分为3组(n=18):对照组(C组)、缺氧复氧组(AR组)和α7nACh受体激动剂组(PNU282987组).C组心肌细胞正常培养;AR组心肌细胞缺氧6h后复氧;PNU282987组心肌细胞缺氧6h后复氧,在复氧培养基中加入用DMSO溶解的PNU282987,终浓度30 μmol/L,C组和AR组加入等浓度DMSO.于复氧6h时采用比色法检测心肌细胞乳酸脱氢酶(LDH)漏出率,Annexin V/PI双染流式细胞术检测凋亡情况,Westernblot法检测GSK-3β与磷酸化GSK-3β(p-GSK-3β Ser9)、NF-κcB p65与磷酸化NF-κB p65 (p-NF-κB p65Ser536)蛋白的表达,ELISA法检测心肌细胞培养上清液IL-6与TNF-α的浓度.结果与C组比较,AR组和PNU282987组心肌细胞LDH漏出率升高,正常细胞百分比降低,早期凋亡细胞百分比和晚期凋亡细胞百分比均升高,心肌细胞p-GSK-3β Ser9、NF-κB p65和p-NF-κB p65 Ser536蛋白表达上调,心肌细胞培养上清液IL-6和TNF-α浓度升高(P＜0.01),心肌细胞GSK-3β蛋白表达差异无统计学意义(P＞0.05);与AR组比较,PNU282987组LDH漏出率降低,正常细胞百分比升高,早期凋亡细胞百分比降低(P＜O.01),晚期凋亡细胞百分比差异无统计学意义(P＞0.05),心肌细胞p-GSK-3β Ser9蛋白表达上调,心肌细胞p-NF-κB p65 Ser536蛋白表达下调,心肌细胞培养上清液IL-6和TNF-α浓度均降低(P＜0.01),NF-κB p65蛋白表达差异无统计学意义(P＞0.05).结论 α7nACh受体激动剂可通过降低GSK-3β活性抑制炎性反应,从而减轻大鼠心肌细胞缺氧复氧损伤.
목적 평개α7연감형을선담감(α7nACh)수체격동제대결양복양대서심기세포당원합성매격매3β(GSK-3β)활성적영향.방법 대서심기세포배양72 h후,안조수궤수자표법분위3조(n=18):대조조(C조)、결양복양조(AR조)화α7nACh수체격동제조(PNU282987조).C조심기세포정상배양;AR조심기세포결양6h후복양;PNU282987조심기세포결양6h후복양,재복양배양기중가입용DMSO용해적PNU282987,종농도30 μmol/L,C조화AR조가입등농도DMSO.우복양6h시채용비색법검측심기세포유산탈경매(LDH)루출솔,Annexin V/PI쌍염류식세포술검측조망정황,Westernblot법검측GSK-3β여린산화GSK-3β(p-GSK-3β Ser9)、NF-κcB p65여린산화NF-κB p65 (p-NF-κB p65Ser536)단백적표체,ELISA법검측심기세포배양상청액IL-6여TNF-α적농도.결과 여C조비교,AR조화PNU282987조심기세포LDH루출솔승고,정상세포백분비강저,조기조망세포백분비화만기조망세포백분비균승고,심기세포p-GSK-3β Ser9、NF-κB p65화p-NF-κB p65 Ser536단백표체상조,심기세포배양상청액IL-6화TNF-α농도승고(P＜0.01),심기세포GSK-3β단백표체차이무통계학의의(P＞0.05);여AR조비교,PNU282987조LDH루출솔강저,정상세포백분비승고,조기조망세포백분비강저(P＜O.01),만기조망세포백분비차이무통계학의의(P＞0.05),심기세포p-GSK-3β Ser9단백표체상조,심기세포p-NF-κB p65 Ser536단백표체하조,심기세포배양상청액IL-6화TNF-α농도균강저(P＜0.01),NF-κB p65단백표체차이무통계학의의(P＞0.05).결론 α7nACh수체격동제가통과강저GSK-3β활성억제염성반응,종이감경대서심기세포결양복양손상.
Objective To evaluate the effect of α7 nicotinic acetylcholine (α7nACh) receptor agonist on the activity of glycogen synthase kinase-3β (GSK-β) in rat cardiomyocytes subjected to anoxia/reoxygenation (A/R).Methods After being cultured for 72 h,the cardiomyocytes were randomly divided into 3 groups (n =18 each) using a random number table:control group (group C),A/R group and α7nACh receptor agonist PNU282987 group (PNU282987 group).A/R and PNU282987 groups were exposed to 6 h anoxia followed by 6 h reoxygenation.In addition,PNU282987 with the final concentration of 30 μmol/L (in dimethyl sulfoxide) was added to the culture media for reoxygenation in PNU282987 group,while the equal concentration of dimethyl sulfoxide was added to the culture media for reoxygenation in C and AR groups.At 6 h of reoxygenation,the rate of lactate dehydrogenase (LDH) released was detected using colorimetric method,apoptosis in cardiomyocytes was detected u sing annexin V/PI double-staining assay,the expression of GSK-3β,phosphorylated GSK-3β Ser9 (pGSK-3β Ser9),NF-κB p65 and phosphorylated NF-κB p65 Ser536 (p-NF-κB p65 Ser536) was detected by Western blot,and the concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant were measured using ELISA.Results Compared with C group,the rate of LDH released was significantly increased,the percentage of normal cells was decreased,the percentage of apoptotic cells in the early and late stages was increased,the expression of p-GSK-3β Ser9,NF-κB p65 and p-NF-κB p65 Ser536 was upregulated,the concentrations of TNF-α and IL-6 were increased,and no significant change was found in the expression of GSK-3β in AR and PNU282987 groups.Compared with AR group,the rate of LDH released was significantly decreased,the percentage of normal cells was increased,the percentage of apoptotic cells in the early stage was decreased,no significant change was found in the percentage of apoptotic cells in the late stage,the expression of p-GSK-3β Ser9 was up-regulated,the expression of p-NF-κB p65 Ser536 was down-regulated,the concentrations of TNF-α and IL-6 were decreased,and no significant change was found in the expression of NF-κB p65 in PNU282987 group.Conclusion α7nACh receptor agonist can attenuate A/R injury to rat cardiomyocytes through decreasing GSK-3β activity and inhibiting inflammatory responses.