Amelioration of Doxorubicin Induced Muscle Dysfunction with Embryoinic stem cells-Derived Exosomes

项目摘要： 多柔比星（DOX）是用于癌症治疗的最有效的化学治疗剂之一，但它也 导致全身炎症和严重的多器官副作用和许多患者的肌肉功能障碍。 肌肉力量的丧失加上持续的疲劳是癌症患者的负担， 化疗然而，目前还不清楚炎症是否会诱导细胞死亡，即所谓的焦亡（pyroptosis）。 与细胞凋亡和坏死相比，细胞死亡的不同形式）发生在DOX诱导的肌肉毒性中， 功能障碍（DIMT）。我们的初步数据显示，DOX治疗可诱导比目鱼肌焦亡 肌肉（SM），Sol 8细胞，其被源自胚胎干细胞的外泌体（ES-exos）抑制。我们 还观察到促炎性M1巨噬细胞、Toll样受体4（TLR 4）、NLRP 3形成 在DIMT后，DOX治疗后SM中的炎性小体和焦萎特异性半胱天冬酶1、IL-1β和IL-18。 DMIT后用ES-Exos处理的小鼠减少了M1巨噬细胞、TLR 4、NLRP 3炎性小体和SM 焦亡此外，M1巨噬细胞发生表型转换，显著增加了抗- 炎性M2巨噬细胞和减少的焦亡以及不利的SM重塑。miRNA阵列数据 显示ES-Exos中miR 200 c的增加。此外，我们的初步数据显示， ES-Exos处理后DIMT中p38和JNK磷酸化，表明p38/JNK参与 通路因此，基于这一令人信服的数据，我们假设ES-Exos改善了DOX诱导的糖尿病。 通过增加抗炎M2巨噬细胞引起的焦亡和相关的不良肌肉重塑 从而改善DIMT后的肌肉功能。我们建议通过以下3个方面来检验这一假设： 明确的目标。目的1：确定增加的M1巨噬细胞水平是否诱导TLR 4并激活 炎性小体（NLRP 3）在短期和长期DIMT后介导的焦亡。目标2：证明 用ES-Exos治疗是否导致M1表型转换为M2巨噬细胞导致 焦亡和不良SM重塑。我们还将确定miR 200 c介导的p38的机制， 在DIMT中ES-Exos处理后焦亡中的JNK细胞信号传导。此外，我们将研究 存在对细胞信号传导、抗癫痫和抗纤维化途径具有特异性的更广泛miRNA ES-Exos治疗。目的3：确定ES-Exos是否对药物的抗肿瘤功效发挥作用。 DOX治疗癌症，同时改善SM功能。拟议的研究是假设驱动的，机械的， 应该有助于确定ES-Exos对M2巨噬细胞表型转换、促炎和抗炎的影响， 细胞因子、细胞凋亡和细胞凋亡在DIMT进展中的作用。此外，我们预计ES-Exos将没有 干扰DOX诱导的癌细胞杀伤和抑制肿瘤生长。我们认为 从这些研究中获得的结果可以容易地转化为临床的无细胞制剂， 外泌体不会形成畸胎瘤，畸胎瘤在使用人和小鼠ES细胞时是常见的。

Project Summary: Doxorubicin (DOX) is one of the most effective chemotherapeutic agents used for cancer treatment, but it also causes systemic inflammation and serious multi-organ side effects and muscle dysfunction in many patients. The loss of muscle strength in combination with constant fatigue is a burden on cancer patients undergoing chemotherapy. However, it is unknown whether inflammation induced cell death, known as pyroptosis (a distinct form of cell death, compared to apoptosis, and necrosis) occurs in DOX induced muscle toxicity and dysfunction (DIMT). Our preliminary data shows that treatment with DOX induces pyroptosis in Soleus muscle (SM), Sol 8 cells which is inhibited by exosomes derived from embryonic stem cells (ES-exos). We also observed increase in pro-inflammatory M1 macrophages, toll-like receptor 4 (TLR4), formation of NLRP3 inflammasome and pyroptotic specific caspase 1, IL-1β, and IL-18 post-DIMT in SM following DOX treatment. Mice treated with ES-Exos post-DMIT decreased M1 macrophages, TLR4, NLRP3 inflammasome and SM pyroptosis. Moreover, phenoswitching of M1 macrophages occurred which significantly increased anti- inflammatory M2 macrophages and reduced pyroptosis as well as adverse SM remodeling. miRNA array data shows increase in miR200c in ES-Exos. Furthermore, our preliminary data shows significantly decrease in phosphorylated p38 and JNK in DIMT following ES-Exos treatment, suggesting involvement of p38/JNK pathway. Therefore, based on this compelling data, we hypothesize that ES-Exos ameliorate DOX-induced pyroptosis and associated adverse muscle remodeling by increasing anti-inflammatory M2 macrophages thereby improving muscle function post-DIMT. We propose to test this hypothesis through the following 3 specific aims. Aim 1: To determine if increased level of M1 macrophages induce TLR4 and activates inflammasome (NLRP3) mediated pyroptosis during short and long-term post-DIMT. Aim 2 To demonstrate whether treatment with ES-Exos cause phenoswitching of M1 into M2 macrophages results in amelioration of pyroptosis and adverse SM remodeling. We will also determine the mechanisms of miR200c mediated p38 and JNK cell signaling in pyroptosis following ES-Exos treatment in DIMT. Additionally, we will examine the presence of broader range of miRNA specific for cell signaling, anti-pyroptotic, and anti-fibrotic pathways following ES-Exos treatment. Aim 3: To determine whether ES-Exos exert effects on anti-tumor efficacy of DOX in cancer while improving SM function. The proposed studies are hypothesis-driven, mechanistic, and should help define the impact of ES-Exos on M2 macrophage phenoswitching, pro- and anti-inflammatory cytokines, pyroptosis and apoptosis in the progression of DIMT. Also, we expect that ES-Exos would have no interference with the DOX-induced killing of cancer cells and inhibition of tumor growth. We believe that results obtained from these studies can easily be translated to the clinic as the cell free preparation of exosomes will not form teratomas, which is common occurrence with the use of human and mouse ES cells.