原肌球蛋白3对缺氧/复氧诱导的大鼠心肌细胞焦亡及成纤维细胞活化的影响

Effects of tropomyosin 3 on pyroptosis of cardiomyocytes and fibroblast activation induced by hypoxia/reoxygenation in rats

目的:探讨原肌球蛋白3(TPM3)在缺氧/复氧(H/R)刺激的心肌细胞焦亡和成纤维细胞活化中的作用。方法:利用H/R方法处理大鼠心肌细胞(H9c2细胞),体外模拟心肌缺血/再灌注(I/R)损伤,用细胞计数试剂盒8(CCK-8)评估细胞增殖活性,实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质免疫印迹试验(Western blotting)检测TPM3表达,确定最佳缺氧时间。构建TPM3-shRNA慢病毒稳定表达的H9c2细胞株,并给予H/R处理(缺氧3 h、复氧4 h),RT-qPCR检测TPM3表达,Western blotting检测TPM3、天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)、NOD样受体蛋白3(NLRP3)、Gasdermin家族蛋白D的N末端产物(GSDMD-N)表达,免疫荧光检测caspase-1表达,酶联免疫吸附试验(ELISA)检测细胞上清液中白细胞介素(IL-1β、IL-18)含量,阐明干扰TPM3对心肌细胞焦亡的影响。使用上述细胞上清液孵育大鼠心肌成纤维细胞,Western Blotting检测Ⅰ型和Ⅲ型胶原蛋白、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶抑制因子2(TIMP2)的表达,明确H/R条件下TPM3干扰的心肌细胞对成纤维细胞活化的影响。结果:与对照组比较,H/R处理4 h可显著降低H9c2细胞存活率〔(25.81±1.90)%比(99.40±5.54)%,P<0.01〕,促进TPM3 mRNA和蛋白表达〔TPM3/GAPDH(2-ΔΔCt):3.87±0.50比1,TPM3/β-Tubulin:0.45±0.05比0.14±0.01,均P<0.01〕,并同时促进焦亡相关蛋白caspase-1、NLRP3、GSDMD-N的表达以及细胞因子IL-1β、IL-18的释放〔cleaved caspase-1/caspase-1:0.89±0.04比0.42±0.03,NLRP3/β-Tubulin:0.39±0.03比0.13±0.02,GSDMD-N/β-Tubulin:0.69±0.05比0.21±0.02,IL-1β(μg/L):13.84±1.89比4.31±0.33,IL-18(μg/L):17.56±1.94比5.36±0.63,均P<0.01〕。然而,与H/R组比较,干扰TPM3则明显减弱了H/R对这些蛋白和细胞因子的促进效应〔cleaved caspase-1/caspase-1:0.57±0.05比0.89±0.04,NLRP3/β-Tubulin:0.25±0.04比0.39±0.03,GSDMD-N/β-Tubulin:0.27±0.03比0.69±0.05,IL-1β(μg/L):8.56±1.22比13.84±1.89,IL-18(μg/L):9.34±1.04比17.56±1.94,均P<0.01〕。此外,H/R处理的心肌细胞上清液可显著增加大鼠心肌成纤维细胞Ⅰ型和Ⅲ型胶原蛋白、TIMP2及MMP-2的表达(Ⅰ型胶原蛋白/β-Tubulin:0.62±0.05比0.09±0.01,Ⅲ型胶原蛋白/β-Tubulin:0.44±0.03比0.08±0.00,TIMP2/β-Tubulin:0.73±0.04比0.20±0.03,TIMP2/β-Tubulin:0.74±0.04比0.17±0.01,均P<0.01)。然而,与H/R组比较,干扰TPM3则削弱了这些促进效应(Ⅰ型胶原蛋白/β-Tubulin:0.18±0.01比0.62±0.05,Ⅲ型胶原蛋白/β-Tubulin:0.21±0.03比0.44±0.03,TIMP2/β-Tubulin:0.37±0.03比0.73±0.04,TIMP2/β-Tubulin:0.45±0.03比0.74±0.04,均P<0.01)。结论:靶向干扰TPM3可以减弱H/R诱导的心肌细胞焦亡以及心肌细胞对成纤维细胞的活化,提示TPM3可作为心肌I/R损伤的潜在靶点。

Objective To explore the role of tropomyosin 3(TPM3)in hypoxia/reoxygenation(H/R)-induced cardiomyocyte pyroptosis and fibroblast activation.Methods Rat cardiomyocytes(H9c2 cells)were treated with H/R method to simulate myocardial ischemia/reperfusion(I/R)injury,and cell proliferation activity was evaluated with cell counting kit-8(CCK8).The expression of TPM3 mRNA and protein was detected by quantitative real-time polymerase chain reaction(RT-qPCR)and Western blotting.H9c2 cells with stable TPM3-short hairpin RNA(shRNA)expression were constructed and treated with H/R(hypoxia for 3 hours,and reoxygenation for 4 hours).The expression of TPM3 was measured by RT-qPCR.The expressions of TPM3,pyroptosis-related proteins including caspase-1,NOD-like receptor protein 3(NLRP3)and Gasdermin family proteins-N(GSDMD-N)were measured by Western blotting.The expression of caspase-1 was also observed by immunofluorescence assay.The levels of human interleukins(IL-1β,IL-18)in the supernatant were determined by enzyme-linked immunosorbent assay(ELISA)to elucidate the effect of sh-TPM3 on pyroptosis of cardiomyocytes.Rat myocardial fibroblasts were incubated with the above cell supernatant,and the expressions of human collagenⅠ,collagenⅢ,matrix metalloproteinase-2(MMP-2),and matrix metalloproteinase inhibitor 2(TIMP2)were detected by Western blotting to determine the effect of TPM3-interfered cardiomyocytes on the activation of fibroblasts under H/R conditions.Results Compared with the control group,H/R treatment for 4 hours significantly decreased the survival rate of H9c2 cells[(25.81±1.90)%vs.(99.40±5.54)%,P<0.01],promoted the expression of TPM3 mRNA and protein[TPM3/GAPDH(2-ΔΔCt):3.87±0.50 vs.1,TPM3/β-Tubulin:0.45±0.05 vs.0.14±0.01,both P<0.01],and promoted the expressions of caspase-1,NLRP3,GSDMD-N,and the enhanced release of cytokines IL-1βand IL-18[cleaved caspase-1/caspase-1:0.89±0.04 vs.0.42±0.03,NLRP3/β-Tubulin:0.39±0.03 vs.0.13±0.02,GSDMD-N/β-Tubulin:0.69±0.05 vs.0.21±0.02,IL-1β(μg/L):13.84±1.89 vs.4.31±0.33,IL-18(μg/L):17.56±1.94 vs.5.36±0.63,all P<0.01].However,compared with the H/R group,sh-TPM3 significantly weakened the promoting effects of H/R on these proteins and cytokines[cleaved caspase-1/caspase-1:0.57±0.05 vs.0.89±0.04,NLRP3/β-Tubulin:0.25±0.04 vs.0.39±0.03,GSDMD-N/β-Tubulin:0.27±0.03 vs.0.69±0.05,IL-1β(μg/L):8.56±1.22 vs.13.84±1.89,IL-18(μg/L):9.34±1.04 vs.17.56±1.94,all P<0.01].In addition,the expressions of collagenⅠ,collagenⅢ,TIMP2,and MMP-2 in myocardial fibroblasts were significantly increased by the cultured supernatants from the H/R group(collagenⅠ/β-Tubulin:0.62±0.05 vs.0.09±0.01,collagenⅢ/β-tubulin:0.44±0.03 vs.0.08±0.00,TIMP2/β-tubulin:0.73±0.04 vs.0.20±0.03,TIMP2/β-Tubulin:0.74±0.04 vs.0.17±0.01,all P<0.01).However,these boosting effects were weakened by sh-TPM3(collagenⅠ/β-Tubulin:0.18±0.01 vs.0.62±0.05,collagenⅢ/β-Tubulin:0.21±0.03 vs.0.44±0.03,TIMP2/β-Tubulin:0.37±0.03 vs.0.73±0.04,TIMP2/β-Tubulin:0.45±0.03 vs.0.74±0.04,all P<0.01).Conclusion Interference with TPM3 can alleviate H/R-induced cardiomyocyte pyroptosis and fibroblast activation,suggesting that TPM3 may be a potential target of myocardial I/R injury.