丹红注射液上调血管内皮细胞外泌体 miR-125b 表达并减轻心肌梗死后心肌细胞凋亡。
Danhong Injection Up-regulates miR-125b in Endothelial Exosomes and Attenuates Apoptosis in Post-Infarction Myocardium.
机构信息
Department of Cardiology, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing, 100010, China.
Beijing Institute of Chinese Medicine, Beijing, 100010, China.
出版信息
Chin J Integr Med. 2023 Dec;29(12):1099-1110. doi: 10.1007/s11655-023-3647-7. Epub 2023 Aug 18.
OBJECTIVE
To investigate the involvement of endothelial cells (ECs)-derived exosomes in the anti-apoptotic effect of Danhong Injection (DHI) and the mechanism of DHI-induced exosomal protection against postinfarction myocardial apoptosis.
METHODS
A mouse permanent myocardial infarction (MI) model was established, followed by a 14-day daily treatment with DHI, DHI plus GW4869 (an exosomal inhibitor), or saline. Phosphate-buffered saline (PBS)-induced ECs-derived exosomes were isolated, analyzed by miRNA microarray and validated by droplet digital polymerase chain reaction (ddPCR). The exosomes induced by DHI (DHI-exo), PBS (PBS-exo), or DHI+GW4869 (GW-exo) were isolated and injected into the peri-infarct zone following MI. The protective effects of DHI and DHI-exo on MI hearts were measured by echocardiography, Masson's trichrome staining, and TUNEL apoptosis assay. The Western blotting and quantitative reverse transcription PCR (qRT-PCR) were used to evaluate the expression levels of miR-125b/p53-mediated pathway components, including miR-125b, p53, Bak, Bax, and caspase-3 activities.
RESULTS
DHI significantly improved cardiac function and reduced infarct size in MI mice (P<0.01), which was abolished by the GW4869 intervention. DHI promoted the exosomal secretion in ECs (P<0.01). According to the results of exosomal miRNA microarray assay, 30 differentially expressed miRNAs in the DHI-exo were identified (28 up-regulated miRNAs and 2 down-regulated miRNAs). Among them, DHI significantly elevated miR-125b level in DHI-exo and DHI-treated ECs, a recognized apoptotic inhibitor impeding p53 signaling (P<0.05). Remarkably, treatment with DHI and DHI-exo attenuated apoptosis, elevated miR-125b expression level, inhibited capsase-3 activity, and down-regulated the expression levels of proapoptotic effectors (p53, Bak, and Bax) in post-MI hearts, whereas these effects were blocked by GW4869 (P<0.05 or P<0.01).
CONCLUSION
DHI and DHI-induced exosomes inhibited apoptosis, promoted the miR-125b expression level, and regulated the p53 apoptotic pathway in post-infarction myocardium.
目的
探讨内皮细胞（ECs）衍生的外泌体在丹红注射液（DHI）抗细胞凋亡作用中的作用机制，以及 DHI 诱导的外泌体对心肌梗死后细胞凋亡的保护作用机制。
方法
建立小鼠永久性心肌梗死（MI）模型，随后给予 DHI 每日治疗 14 天，DHI+GW4869（外泌体抑制剂）或生理盐水。分离磷酸盐缓冲液（PBS）诱导的 ECs 衍生的外泌体，通过 miRNA 微阵列分析，并通过液滴数字聚合酶链反应（ddPCR）进行验证。分离由 DHI（DHI-exo）、PBS（PBS-exo）或 DHI+GW4869（GW-exo）诱导的外泌体，并在 MI 后注入梗死周边区。通过超声心动图、Masson 三色染色和 TUNEL 凋亡检测评估 DHI 和 DHI-exo 对 MI 心脏的保护作用。Western blot 和定量逆转录聚合酶链反应（qRT-PCR）用于评估 miR-125b/p53 介导的途径成分的表达水平，包括 miR-125b、p53、Bak、Bax 和 caspase-3 活性。
结果
DHI 显著改善 MI 小鼠的心脏功能并减少梗死面积（P<0.01），而 GW4869 干预则消除了这种作用。DHI 促进了 ECs 中外泌体的分泌（P<0.01）。根据外泌体 miRNA 微阵列检测结果，在 DHI-exo 中鉴定出 30 个差异表达的 miRNA（28 个上调 miRNA 和 2 个下调 miRNA）。其中，DHI 显著提高了 DHI-exo 和 DHI 处理的 ECs 中 miR-125b 的水平，这是一种公认的抑制 p53 信号的凋亡抑制剂（P<0.05）。值得注意的是，DHI 和 DHI-exo 的治疗可减轻凋亡，提高 miR-125b 的表达水平，抑制 capsase-3 活性，并下调 MI 后心脏中的促凋亡效应物（p53、Bak 和 Bax）的表达水平，而 GW4869 则阻断了这些作用（P<0.05 或 P<0.01）。
结论
DHI 和 DHI 诱导的外泌体抑制凋亡，提高 miR-125b 表达水平，并调节梗死后心肌中的 p53 凋亡途径。
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