SZC-6 Promotes Diabetic Wound Healing in Mice by Modulating the M1/M2 Macrophage Ratio and Inhibiting the MyD88/NF-χB Pathway

Abstract

Background/Objectives: The prolonged M1-like pro-inflammatory polarization of macrophages is a key factor in the delayed healing of diabetic ulcers (DU). SIRT3, a primary mitochondrial deacetylase, has been identified as a regulator of inflammation and represents a promising new therapeutic target for DU treatment. Nonetheless, the efficacy of existing SIRT3 agonists remains suboptimal. Methods: Here, we introduce a novel compound, SZC-6, demonstrating promising activity levels. Results: SZC-6 treatment down-regulated the expression of inflammatory factors in LPS-treated RAW264.7 cells and reduced the proportion of M1 macrophages. Mitosox, IF, and JC-1 staining revealed that SZC-6 preserved cellular mitochondrial homeostasis and reduced the accumulation of reactive oxygen species. In vivo experiments demonstrated that SZC-6 treatment accelerated wound healing in diabetic mice. Furthermore, HE and Masson staining revealed increased neovascularization at the wound site with SZC-6 treatment. Tissue immunofluorescence results indicated that SZC-6 effectively decreased the proportion of M1-like cells and increased the proportion of M2-like cells at the wound site. We also found that SZC-6 significantly reduced MyD88, p-IκBα, and NF-χB p65 protein levels and inhibited the nuclear translocation of P65 in LPS-treated cells. Conclusions: The study concluded that SZC-6 inhibited the activation of the NF-χB pathway, thereby reducing the inflammatory response and promoting skin healing in diabetic ulcers. SZC-6 shows promise as a small-molecule compound for promoting diabetic wound healing.

Keywords: SZC-6; diabetic; macrophages; wound healing.

Figure 1

A selective SIRT3 activator, SZC-6, enhanced deacetylation activity. (A) Three-dimensional view of SIRT3 protein structure. (B) Chemical structure of SZC-6 and C12. (C,D) RAW264.7 cells were treated with various doses of SZC-6. Total cellular deacetylation was probed with an anti-acetyl lysine antibody to evaluatelysine acetylation. (E) The predicted co-crystal structure of SZC-6 and SIRT3. Left, overall structure with SIRT3 depicted in grey. Right, enlarged view of the isomeric site of SZC-6. SIRT3 is represented as a grey cartoon, while SZC-6 is illustrated as a rod. Dotted lines represent hydrogen bonds. Data are presented as mean ± SD (n = 3). ** p < 0.01.

Figure 2

SZC-6 reduces ROS accumulation and increases mitochondrial potential in vitro. (A) Mitochondrial membrane potential (Δψm) of RAW264.7 was assessed using Image-iT™ TMRM dye after treatment with HG + LPS and HG + LPS + SZC-6. Scale bar: 50 μm. (B) Intracellular reactive oxygen species (ROS) content was measured using a ROS detection kit. Scale bar: 50 μm. (C) SZC-6 reduces intracellular mitochondrial ROS accumulation. Scale bar: 50 μm. (D) Mitochondrial membrane potential (Δψm) of RAW264.7 was measured with the JC-1 probe after treatment with HG + LPS and HG + LPS + SZC-6. Red and green fluorescence corresponded to JC-1 aggregates and monomers, respectively. Scale bar: 50 μm. (E,F) Statistical plots showing mitochondrial membrane potential (Δψm) and ROS fluorescence intensity. (G) Quantification of the red/green fluorescence ratio in (D). (H) Total intracellular ATP levels. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. NC refers to negative control, LPS refers to the high-glucose model group, SZC-6-L denotes 5 μM SZC-6, and SZC-6-H denotes 10 μM SZC-6.

Figure 3

SZC-6 maintains mitochondrial functional homeostasis. (A) Immunofluorescence (IF) showing Drp1 and Mfn2 protein expression levels after LPS and LPS + SZC-6 treatment. Scale bar: 75 μm. (B,C) Statistical plots of Mfn2 and Drp1 fluorescence intensity. (D) Western blot (WB) detection of Mfn2 protein levels in each cell group (Figure S2). (E) Statistical plots of the grey value of Mfn2 protein bands for each group. Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. NC refers to the negative control, LPS refers to the high-glucose model group, SZC-6-L denotes 5 μM SZC-6, and SZC-6-H denotes 10 μM SZC-6.

Figure 4

SZC-6 inhibits M1 polarization and reduces the M1/M2 ratio in vitro. (A) Flow cytometry was employed to determine the ratio of F4/80+/CD86+ in each group. (B–E) RT-qPCR was used to measure mRNA levels of iNos, TNF-α, Arg-1, and Retnla. (F,G) Representative immunofluorescence images of CD206 and CD86 after 24-h treatment with HG + LPS and HG + LPS + SZC-6 (L, H) (n = 3). Scale bar: 100 μm. (H,I) Quantitative statistical plots of immunofluorescence intensity of CD206 and CD86 proteins in each group (n = 3). (J) Western blot analysis (n = 3) showing iNos and Arg-1 protein expression levels after 24-h treatment with HG + LPS and HG + LPS + SZC-6 (L, H) (Figures S3 and S4). (K,L) Quantitative analysis of iNos and Arg-1 protein expression. Data are presented as means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. NC refers to negative control, LPS refers to the high-glucose model group, SZC-6-L denotes 5 μM SZC-6, and SZC-6-H denotes 10 μM SZC-6.

Figure 5

Treatment with SZC-6 improves wound healing in STZ-induced diabetic mice. (A) Schematic illustration of SZC-6 administration for diabetic wounds. (B) Representative images of diabetic scald wounds treated with the compound SZC-6 at each time point (the diameter of the ring inside the red circle is 1.5 cm). (C) Statistics of wound healing rates in diabetic scald wounds at each time point in each group (n = 6). (D,E) Representative images of diabetic scald wounds on day 9 with HE staining. (F,G) Representative images of Masson staining of diabetic scald wounds on day 9. Data are presented as means ± SD (n = 6). *** p < 0.001. NC refers to negative control.

Figure 6

SZC-6 reduces the M1/M2 ratio in diabetic mice. (A) Representative images of CD86 immunofluorescence in diabetic scald wounds on day nine. (B) Representative images of CD206 immunofluorescence in diabetic scald wounds on day nine. Scale bar of Merge = 100 μm, scale bar of Zoom = 50 μm. (C–E) We quantified the proportion of M1 and M2 macrophages by counting CD86- and CD206-positive nuclei, expressed as a percentage of total F4/80-positive. Data are presented as means ± SD (n = 6). *** p < 0.001. NC refers to negative control.

Figure 7

SZC-6 Modulation of the NF-χB pathway in LPS-treated RAW264.7 cells. (A) Venn diagram showing overlapping potential compound targets of SZC-6 and therapeutic targets for DU treatment. (B) Bubble plot of GO function analysis of potential compound targets of SZC-6 in DU treatment. (C) Bubble plot of KEGG enrichment of potential compound targets of SZC-6 in DU treatment. (D,E) Effects of SZC-6 at different doses on MyD88, p-IκBα, and p65 expression in LPS-treated RAW264.7 cells (Figure S6). (F) Effects of SZC-6 at different doses on the nuclear translocation of p65 in LPS-treated RAW264.7 cells. Scale bar = 25 μm. Data are presented as Mean ± SEM, n = 3, ** p < 0.01, and *** p < 0.001 indicate significant differences compared to the NC group, while ^# p < 0.05 and ^## p < 0.01 indicate significant differences compared to the HG + LPS group. NC refers to negative control, HG + LPS refers to the high-glucose model group, SZC-6-L denotes 5 μM SZC-6, and SZC-6-H denotes 10 μM SZC-6.