<p style="text-align: center;">CHEN Hui ¹, JIN Xin-Yue ¹, SHEN Yu-Jun ¹, WANG Tian-Lin¹, OU Chen ¹, LU Sheng-Feng ¹, CAI Yun ¹, LI Qian ¹, DING Ya-Juan¹ and ZHU Bing-Mei^1,2</p><p style="text-align: center;">(1 Key Laboratory of Acupuncture and Medicine Research of Ministry of Education, Nanjing University of Chinese Medicine, Nanjing 210023, China, 2 Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu, Sichuan, China 610041)</p><p style="text-align: justify;"><strong>Abstract Background</strong>: To investigate whether Stat5 is a true player for cardioprotective effect on remote ischemic preconditioning (RIPC), and explored the role of Stat5 in electro-acupuncture (EA) pretreatment against myocardial I/R, and determine its molecular mechanisms. Methods: Stat5 fl/fl mice and Stat5-cKO mice were randomly divided into 6 groups: Stat5fl/fl+I/R and Stat5-cKO+I/R groups (30/180 min left coronary artery occlusion/reperfusion); Stat5fl/fl+RIPC+I/R and Stat5-cKO+RIPC+I/R groups (applied RIPC with three 5-min cycles of femoral artery occlusion and reperfusion before I/R); Stat5fl/fl+EA+I/R and Stat5-cKO+EA+I/R groups (subjected to 20 min/day of EA treatment for 1 week before I/R). The heart tissue and blood were harvested at the end point of I/R, and the parameters were measured. Results: RIPC activated STAT5 in the heart subjected to I/R. Decreased expressions of Cyt, cleaved caspase-3, and increased Bcl-xL and Bcl-2 were detected only in the presence of STAT5.RIPC increased cardiac HIF-1α, IL10, p-PI3K, and VEGF proteins only in the Stat5fl/fl mice with RIPC. EA reduced myocardial infarct size and TUNEL-positive cells in both presence and absence of STAT5, suggesting its Stat5-independency. RNA-seq data further provided evidence that both RIPC and EA pretreatment activated multiple pathways related their protective role against myocardial I/R injury. Conclusion: RIPC-induced protection against myocardial I/R injury was Stat5 dependent, and was through anti-apoptotic signaling and survival signaling. The cardioprotection induced by EA was Stat5-independent, but Stat5 does participate in the event through anti-apoptotic and survival signaling at certain extent.</p><p><strong>Key words</strong>: Acupuncture, RIPC, Electro-acupuncture pretreatment, Stat5</p><p style="text-align: center;"><br/></p>