Taohong Siwu Decoction Combined With the LncRNA H19/miR-675-5p Axis Repairs Limb Ischemia-Reperfusion Injury Through the Regulation of the Wnt3a/Ca2+ Signaling Pathway.

Abstract

Background: Taohong Siwu decoction (THSWT) has shown therapeutic effects on ischemia/reperfusion injury (IRI). This study tended to investigate the role of THSWT combined with the long non-coding RNA (LncRNA) H19 (H19)/miR-675-5p axis in improving limb IRI (LIRI). Methods: Hind LIRI rats and simulated IRI skeletal myoblasts models were constructed to evaluate the therapeutic effects of THSWT. The mechanism of THSWT treatment on LIRI was investigated by the regulation of the H19/miR-675-5p axis and the wingless/integrated (Wnt)/Ca²⁺ signaling pathway. Various assessments, such as H&E staining, TUNEL staining, flow cytometry, cell counting kit-8 (CCK-8) assay, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunohistochemistry (IHC) staining, enzyme-linked immunosorbent assay (ELISA), biochemical assay, and calcium fluorescence imaging, were conducted to observe skeletal muscle injury, cell apoptosis, skeletal myoblast proliferation, gene and protein expressions, cytokine levels, glucose (Glu) uptake, and Ca²⁺ concentration. Results: THSWT intervention effectively improved skeletal muscle injury in LIRI rats, as evidenced by reduced muscle fiber damage and decreased cell apoptosis, accompanied by downregulation of H19, miR-675-5p, cleaved-Caspase3, Bax, PLC, and PKC expressions and upregulation of Bcl2 expression. Furthermore, silencing of H19 inhibited cell apoptosis of skeletal muscle and reduced IL-1β, IL-6, and TNF-α levels in LIRI rats. Notably, THSWT intervention combined with the silencing of H19 synergistically promoted the repair of skeletal muscle injury in LIRI rats. Mechanistically, THSWT intervention combined with regulation of the H19/miR-675-5p axis promoted the proliferation of skeletal myoblasts damaged by IRI through the Wnt3a/Ca²⁺ signaling pathway, increasing the levels of intracellular Bcl2, while decreasing the levels of Ca²⁺, CaMKⅡ, PLC, PKC, cleaved-Caspase3, Bax, TNF-α, IL-1β, IL-6, Wnt3a, and β-catenin. Conclusions: THSWT combined with the regulation of the H19/miR-675-5p axis effectively improved LIRI by modulating the Wnt3a/Ca²⁺ signaling pathway, providing insights for potential therapeutic strategies for LIRI.