Functional validation of miR-99a in vitro and in vivo. (A–E) LIPUS-EVs were added to CD4⁺ T cells in the presence or absence of inhibitor miR-99a. (A) Levels of RORγt mRNA and Foxp3 mRNA expression in vitro by qPCR (RORγt: P = 0.0079 vs. inhibitor NC, P = 0.0377 vs. inhibitor NC + Control-EVs, P = 0.0054 vs. inhibitor NC + LIPUS-EVs; Foxp3: P = 0.0004 vs. inhibitor NC, P = 0.0433 vs. inhibitor NC + Control-EVs, P = 0.0121 vs. inhibitor NC + LIPUS-EVs). (B) The percentages of Th17 cell and Treg cell were assessed by flow cytometry in vitro (CD4⁺ IL-17A⁺: P = 0.0076 vs. inhibitor NC, P = 0.0006 vs. inhibitor NC + Control-EVs, P = 0.0473 vs. inhibitor NC + LIPUS-EVs; CD4⁺ Foxp3⁺: P = 0.0008 vs. inhibitor NC, P = 0.0026 vs. inhibitor NC + Control-EVs, P = 0.0434 vs. inhibitor NC + LIPUS-EVs). (C) The levels of cytokines, including IL-17A and IL-10 in CD4⁺ T cells, were measured by ELISA (IL-17A: P = 0.0001 vs. inhibitor NC, P < 0.0001 vs. inhibitor NC + Control-EVs, P = 0.0266 vs. inhibtor NC + LIPUS-EVs; IL-10: P < 0.0001 vs. inhibitor NC, P = 0.0016 vs. inhibitor NC + Control-EVs, P = 0.0106 vs. inhibitor NC + LIPUS-EVs). (D) The ECAR was measured over time from basal levels and followed by glucose, oligomycin, and 2-DG addition. (E) Cellular glucose uptake and lactate production were measured after EVs addition (glucose uptake: P = 0.0017 P < 0.0001 P = 0.0283; lactate production: P = 0.0007 P < 0.0001 P = 0.0064). (F–P) LIPUS-EVs were injected to iDCM mice in the presence or absence of miR-99a inhibitor. (F, G) Results of H&E staining (scale bars = 100 μm; P = 0.0080 vs. inhibitor NC, P = 0.0167 vs. inhibitor NC + Control-EVs, P = 0.0167 vs. inhibitor NC + LIPUS-EVs). (F, H) Results of masson staining (scale bars = 100 μm; P = 0.0013 vs. inhibitor NC, P = 0.0077 vs. inhibitor NC + Control-EVs, P = 0.0006 vs. inhibitor NC + LIPUS-EVs). (I, J) EF was measured using echocardiography. (P = 0.0054 vs. inhibitor NC, P = 0.0162 vs. inhibitor NC + Control-EVs, P = 0.0444 vs. inhibitor NC + LIPUS-EVs). (K) Levels of BNP in plasma of mice by ELISA (P = 0.0020 vs. inhibitor NC, P = 0.0027 vs. inhibitor NC + Control-EVs, P = 0.0304 vs. inhibitor NC + LIPUS-EVs). Results are expressed as mean ± SD; one-way ANOVA with Bonferroni post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.