Targeting the sirtuin 6–NF-κB p65 axis by 6-hydroxyhyoscyamine hydrobromide: a deacetylation-driven new therapy for diabetic wounds

Abstract Background Diabetic wounds are a debilitating complication of diabetes mellitus with a high rate of amputation and mortality. 6-Hydroxyhyoscyamine hydrobromide (6-HH) is a belladonna alkaloid with smooth-muscle-relaxing and microcirculation-enhancing properties, yet its role in diabetic wounds remains unknown. Methods In vivo, diabetic wounds were established in streptozotocin (STZ)-induced type 1 diabetic mice, high fat diet (HFD)/STZ-induced type 2 diabetic mice, and db/db diabetic mice to investigate the therapeutic effects of 6-HH, and skin samples were collected for hematoxylin and eosin (H&E) and Masson’s trichrome staining, immunofluorescence, RNA-seq analysis, and western blotting. In vitro, the mechanism of action of 6-HH on cytokines, nuclear factor kappa B (NF-κB) signals, and the interaction between sirtuin 6 (SIRT6) and p65 in lipopolysaccharide (LPS)-induced macrophages were detected by using enzyme-linked immunosorbent assay (ELISA), western blotting, reverse-transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and immunoprecipitation, while molecular docking was used to evaluate the binding energy between 6-HH and SIRT6 protein. Coculture systems of THP-1 cells–human umbilical vein endothelial cells (HUVECs)/immortalized human keratinocytes (HaCaTs) were established to investigate the effects of 6-HH on cross-talk between macrophages and endothelial cells/keratinocytes. SIRT6-specific inhibitor Oss_128167 was applied in vitro and in vivo to verify the mechanism of 6-HH in diabetic wound healing. Results 6-HH exhibited excellent pro-healing effect in the three types of diabetic mouse model. RNA-seq analysis found that 6-HH recovered diabetic-induced aberrant expression changes of genes in the local wounds, especially those related to M1 macrophage polarization with downregulation of Toll-like receptor (TLR) signals and nicotinamide adenine dinucleotide phosphate (NADP+) nucleosidase activity. Molecular docking analysis found that 6-HH could effectively bind to the active site of the SIRT6 protein. Remarkably, decline of SIRT6 in M1 macrophages resulted in lysine hyperacetylation, while activation and stabilization of SIRT6 by 6-HH suppressed M1 macrophage polarization and hyperacetylation through inhibiting p65 transcription with deacetylation of p65Lys310 and H3K9, contributing to improve angiogenesis and re-epithelization through interaction between macrophages and endothelial cells/keratinocytes. However, pharmacological inhibition of SIRT6 reversed the action of 6-HH in macrophages and diabetic wounds. Conclusions Collectively, deacetylase SIRT6 might be a direct pharmacological target of 6-HH that downregulates the hyperacetylated state of macrophages, thus contributing to diabetic wound healing. Graphical Abstract