研究脂肪幹細胞條件培養基對暴露於懸浮微粒與高脂肪飲食的心肌細胞之影響及相關機轉

Abstract

空氣污染與肥胖為心血管疾病(cardiovascular diseases, CVDs)的重要危險因子。流行病學研究指出，空氣污染可加劇肥胖所誘導的心臟損傷。脂肪間質幹細胞(adipose-derived stem cells, ADSCs)的旁分泌作用已被證明可以減輕心臟損傷，然而，其對空氣污染與肥胖共同誘導之心肌細胞凋亡的影響及其潛在機制仍未明確。本研究利用棕櫚酸(palmitic acid, PA)與高脂飲食(high-fat diet, HFD)誘導肥胖模型，並以懸浮微粒(particulate matter, PM)模擬空氣污染，探討ADSC條件培養液(ADSC-conditioned medium, ADSC-CM)對PA+PM處理後的H9c2細胞及HFD+PM處理小鼠心肌細胞之凋亡效應及其相關分子機轉。 透過Western blot分析檢測凋亡相關蛋白表現，研究結果顯示，在PA+PM處理之H9c2細胞與HFD+PM處理之小鼠心肌細胞中，促凋亡蛋白PUMA與cleaved Caspase-3表現顯著上升，抗凋亡蛋白Bcl-2表現則下降。然而，ADSC-CM處理可有效降低PUMA與cleaved Caspase-3的表現，同時增加Bcl-2表現。另外，TUNEL與Annexin V/PI染色結果進一步證實，ADSC-CM顯著減少PA+PM與HFD+PM誘導之心肌細胞凋亡。RT-qPCR結果顯示，PA+PM與HFD+PM處理顯著降低miR221/222的表現，而ADSC-CM處理則可回復其表現。進一步使用miR221/222基因剔除(knockout, KO)與轉基因(transgenic, TG)小鼠模型，證實ADSC-CM中的miR221/222可緩解HFD+PM誘導之心肌細胞凋亡。此外，本研究也發現PA+PM處理會增加細胞內活性氧(reactive oxygen species, ROS)生成，進而誘發粒線體分裂，促進細胞凋亡；然而，ADSC-CM可有效抑制ROS產生並調控粒線體分裂，進而減緩細胞凋亡現象。綜合以上結果，ADSC-CM可透過正向調控miR221/222表現與降低ROS產生，進一步調控粒線體功能，從而減輕PA+PM誘導之心肌細胞凋亡，展現其潛在的心肌保護效果。

Air pollution and obesity are critical risk factors for cardiovascular diseases (CVDs), and epidemiological studies have shown that air pollution exacerbates obesity-induced cardiac damage. Adipose-derived stem cells (ADSCs) exert paracrine effects that have been reported to attenuate cardiac injury; however, the effects of ADSCs in preventing cardiomyocyte apoptosis induced by obesity and air pollution, as well as the underlying mechanisms, remain unclear. In this study, palmitic acid (PA) and high-fat diet (HFD) were used to induce obesity, and particulate matter (PM) was applied to simulate air pollution exposure. We studied the effect of adipose-derived stem cell-conditioned medium (ADSC-CM) on apoptosis of PA+PM-treated H9c2 cells and HFD+PM-treated mouse cardiomyocytes and its related mechanisms. Western blot analysis was conducted to assess the expression of apoptosis-related proteins. The results showed that the apoptosis-related proteins PUMA and cleaved Caspase-3 were significantly upregulated in PA+PM-treated H9c2 cells and HFD+PM-treated mouse cardiomyocytes, while the anti-apoptosis-related protein Bcl-2 was markedly decreased. Notably, treatment with ADSC-CM effectively reduced the protein expression of PUMA and cleaved Caspase-3 expression, while increasing that of Bcl-2. TUNEL assay and Annexin V/PI staining further confirmed that ADSC-CM significantly attenuated PA+PM- and HFD+PM-induced cardiomyocyte apoptosis. RT-qPCR detection found that PA+PM and HFD+PM significantly reduced miR221/222 levels, while ADSC-CM treatment increased miR221/222 levels. Furthermore, knockout (KO) and transgenic (TG) mice were used to demonstrate that miR221/222 in ADSC-CM ameliorated cardiac apoptosis induced by HFD+PM treatment. In addition, PA+PM treatment increased the production of reactive oxygen species (ROS), which triggered mitochondrial fission and contributed to apoptosis. However, ADSC-CM could effectively reduce ROS and regulate mitochondrial fission, alleviating cellular apoptosis. In conclusion, these findings demonstrated that ADSC-CM attenuates PA+PM-induced cardiomyocyte apoptosis through the modulation of miR221/222 and the suppression of ROS.