CAJ | 학술논문

目的观察瘦素对大鼠气道平滑肌细胞(ASMC)蛋白激酶-B(Akt)、磷酸化蛋白激酶-B(Pho-Akt)、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关x蛋白(Bax)、半胱氨酸天冬氨酰蛋白酶-3(caspase-3)的表达及细胞凋亡的影响,探讨瘦素影响ASMC凋亡的可能机制.方法体外培养大鼠ASMC,按随机数字表法分为空白对照组、瘦素50 μg/L组(Lep50组)、瘦素100 μg/L组(Lep100组)、瘦素200 μg/L组(Lep200组)及瘦素200μg/L±LY294002组[1-磷脂酰肌醇3-激酶(PI3K)抑制剂组].各组均孵育24h后用Annexin-V/PI双标记流式细胞仪分析法检测细胞凋亡率,Western blot测定Akt、Pho-Akt、Bcl-2、Bax和caspase-3的蛋白表达情况.结果 Lep50组、Lep100组、Lep200组的细胞凋亡率分别为(3.97±0.39)％、(1.88 ±0.72)％和(0.77±0.1 1)％,均低于空白对照组的(7.38±0.49)％(F =89.57,P＜0.05),且各组细胞凋亡率与瘦素浓度呈负相关(r=-0.711,P＜0.05)；PI3K抑制剂组凋亡率为(3.29±0.36)％,高于Lep200组(F =89.57,P＜0.01).Western blot结果显示随着瘦素浓度的升高Bcl-2表达增加,且与浓度呈正相关(r=0.939,P＜0.05),Bax和caspase-3表达下降,与瘦素浓度成负相关(r值分别为-0.908和-0.961,均P＜0.05),Bcl-2/Bax比值增加(F=20.56,P ＜0.05),Pho-Akt表达上调,且与瘦素浓度呈正相关(r=0.958,P ＜0.05)；PI3K抑制剂组较Lep200组Pho-Akt、Bcl-2表达下降(F值分别为32.93和19.48,均P＜0.05),Bax和caspase-3表达升高(F值分别为10.10和29.86,均P＜0.05),Bcl-2/Bax比值下降(F =20.56,P＜0.05).结论瘦素可以抑制ASMC的凋亡,其机制可能与激活P13K/Akt通路有关.
목적 관찰수소대대서기도평활기세포(ASMC)단백격매-B(Akt)、린산화단백격매-B(Pho-Akt)、B세포림파류/백혈병-2(Bcl-2)、Bcl-2상관x단백(Bax)、반광안산천동안선단백매-3(caspase-3)적표체급세포조망적영향,탐토수소영향ASMC조망적가능궤제.방법 체외배양대서ASMC,안수궤수자표법분위공백대조조、수소50 μg/L조(Lep50조)、수소100 μg/L조(Lep100조)、수소200 μg/L조(Lep200조)급수소200μg/L±LY294002조[1-린지선기순3-격매(PI3K)억제제조].각조균부육24h후용Annexin-V/PI쌍표기류식세포의분석법검측세포조망솔,Western blot측정Akt、Pho-Akt、Bcl-2、Bax화caspase-3적단백표체정황.결과 Lep50조、Lep100조、Lep200조적세포조망솔분별위(3.97±0.39)％、(1.88 ±0.72)％화(0.77±0.1 1)％,균저우공백대조조적(7.38±0.49)％(F =89.57,P＜0.05),차각조세포조망솔여수소농도정부상관(r=-0.711,P＜0.05)；PI3K억제제조조망솔위(3.29±0.36)％,고우Lep200조(F =89.57,P＜0.01).Western blot결과현시수착수소농도적승고Bcl-2표체증가,차여농도정정상관(r=0.939,P＜0.05),Bax화caspase-3표체하강,여수소농도성부상관(r치분별위-0.908화-0.961,균P＜0.05),Bcl-2/Bax비치증가(F=20.56,P ＜0.05),Pho-Akt표체상조,차여수소농도정정상관(r=0.958,P ＜0.05)；PI3K억제제조교Lep200조Pho-Akt、Bcl-2표체하강(F치분별위32.93화19.48,균P＜0.05),Bax화caspase-3표체승고(F치분별위10.10화29.86,균P＜0.05),Bcl-2/Bax비치하강(F =20.56,P＜0.05).결론 수소가이억제ASMC적조망,기궤제가능여격활P13K/Akt통로유관.
Objective To observe the effects of leptin on the expression of Akt,Pho-Akt,Bcl-2,Bax,caspase-3 and the apoptosis of airway smooth muscle cells (ASMCs),and to explore the possible mechanisms.Methods ASMCs were derived from rat airway tissue and cultured in vitro.The cells were randomly divided into 5 groups including a control group,leptin at concentrations of 50,100,200 μg/L groups (group Lep50,Lep100,Lep200),and PI3K specific antagonist with Lep200 group.Then the cells of different groups were incubated for 24 h.An apoptosis detection kit was used for annexin V and PI staining.The expression of Akt,phosphorylation Akt,Bcl-2,Bax,caspase-3 were measured by Western blot.Results The apoptosis rates of ASMCs in group Lep50,Lep100 and Lep200 were (3.97 ±0.39)％,(1.88 ± 0.72) ％ and (0.77 ±0.11) ％,respectively,all significantly lower than that in the control group (7.38 ±0.49)％ (F =89.57,P ＜ 0.05).Furthermore,the concentration of leptin was negatively related to the apoptosis rate (r =-0.711,P ＜ 0.05).The apoptosis rates of PI3 K specific antagonist with Lep200 group (3.29 ± 0.36) ％ was higher than that of group Lep200 (0.77 ± 0.1 1) ％ (F =89.57,P ＜ 0.01).After the intervention of leptin,the expression of Bcl-2 was upregulated and positively correlated with leptin concentration (r =0.939,P ＜ 0.05); Bax was downregulated and negatively related to the leptin concentration (r =-0.908,P ＜ 0.05) ; while the Bcl-2/Bax ratio was raised after leptin treatment (F =20.56,P ＜0.05).Leptin inhibited the activation of caspase-3 in the negative way.(r =-0.961,P ＜0.05).The results also showed that leptin significantly increased phosphorylation of Akt that positively related to leptin concentration (r =0.958,P ＜ 0.05).Compared with group Lep200,the expression of Pho-Akt and Bcl-2 in PI3K specific antagonist with Lep200 group were downregulated (F =32.93,19.48,respectively,P ＜ 0.05),while the expression of Bax and caspase-3 was increased (F =10.10,29.86,respectively,P ＜0.05) ; the Bcl-2/Bax ratio was lower in group Lep200 as compared to the PI3K specific antagonist with Lep200 group (F =20.56,P ＜ 0.05).Conclusion Leptin can significantly inhibit ASMC apoptosis partially via the PI3K/Akt signaling pathway.