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

项目总结： 阿霉素(DOX)是治疗癌症最有效的化疗药物之一，但它也 在许多患者中导致全身炎症和严重的多器官副作用和肌肉功能障碍。 肌肉力量的丧失和持续的疲劳是癌症患者接受 化疗。然而，目前尚不清楚炎症是否会导致细胞死亡，即所谓的上睑下垂(a 不同形式的细胞死亡，与细胞凋亡和坏死相比，在DOX诱导的肌肉毒性和 功能障碍(DIMT)。我们的初步数据显示，DOX治疗可导致比目鱼肌下垂 肌肉(SM)，由胚胎干细胞(ES-exos)衍生的外切体(ES-exos)抑制的Sol8细胞。我们 还观察到促炎症的M1巨噬细胞，Toll样受体4(TLR4)，NLRP3的形成 多柔比星治疗后SM患者DIMT后炎症体和嗜酸性半胱氨酸特异性caspase1、IL-1β和IL-18的表达。 Dmit后ES-Exos处理的小鼠减少了M1巨噬细胞、TLR4、NLRP3炎症体和SM 上睑下垂。此外，M1巨噬细胞发生了表型转换，显著增强了抗 炎症的M2巨噬细胞和减轻的下垂以及不利的SM重塑。MiRNA阵列数据 显示ES-Exos中miR200c的增加。此外，我们的初步数据显示， ES-Exos治疗后DIMT中p38和JNK的磷酸化，提示p38/JNK参与 路径。因此，基于这些令人信服的数据，我们假设ES-Exos改善了DOX诱导的 通过增加抗炎M2巨噬细胞引起的上睑下垂和相关的不良肌肉重塑 从而改善DIMT术后的肌肉功能。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M1巨噬细胞水平升高是否诱导TLR4并激活 炎性小体(NLRP3)介导DIMT术后短期和长期的下垂。目标2：演示 ES-Exos治疗是否导致M1向M2巨噬细胞的表型切换导致改善 上睑下垂和SM不良重塑。我们还将确定miR200c介导的p38和p38的机制。 JNK细胞在DIMT患者ES-Exos治疗后下垂中的信号转导。此外，我们还将研究 存在更广泛的针对细胞信号转导、抗焦链和抗纤维化通路的miRNA 在接受ES-Exos治疗后。目的3：确定ES-Exos是否影响其抗肿瘤作用 在改善SM功能的同时，在癌症中应用DOX。建议的研究是假设驱动的、机械化的和 应有助于确定ES-Exos对M2巨噬细胞表型转换、促炎和抗炎的影响 细胞因子、上睑下垂和细胞凋亡在DIMT进展中的作用此外，我们预计ES-Exos将没有 干扰DOX诱导的肿瘤细胞杀伤和抑制肿瘤生长。我们相信 这些研究的结果可以很容易地作为无细胞制剂转移到临床上。 外切体不会形成畸胎瘤，这是使用人和小鼠ES细胞时常见的情况。

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.