Effects of resveratrol-derived carbonized polymer dots on macrophage polarization and osteogenic differentiation of human periodontal ligament stem cells under inflammatory conditions

Objective To investigate the effect and mechanism of resveratrol-derived carbonized polymer dots (RSV-CPDs) on macrophage polarization and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions, and to provide an experimental basis for the treatment of periodontitis with RSV-CPDs. Methods RSV-CPDs were prepared by high-temperature pyrolysis of resveratrol (RSV) in the presence of ammonia as a catalyst, and RSV-CPDs were characterized by transmission electron microscope (TEM), Fourier transform infrared spectrometer (FTIR), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). CCK8 was used to detect the cytotoxicity of RSV-CPDs. The effects of RSV-CPDs on the apoptosis and cell polarization of macrophages stimulated by<i> Porphyromonas gingivalis</i>-lipopolysaccharide (<i>P.g</i>-LPS) were detected by flow cytometry: ① For the apoptosis detection experiment, the macrophages (RAW264.7) were divided into the control group (no treatment), <i>P.g</i>-LPS group [treated with <i>P.g</i>-LPS (2 μg/mL) for 24 h], RSV group [treated with <i>P.g</i>-LPS (2 μg/mL) + RSV (10 μg/mL) for 24 h], and RSV-CPDs group [treated with <i>P.g</i>-LPS (2 μg/mL) + RSV-CPDs (50 μg/mL) for 24 h]. ② For the cell polarization experiment, the macrophages (RAW264.7) were divided into four groups. They were the control group (no treatment), <i>P.g</i>-LPS + IFN-γ group [<i>P.g</i>-LPS (200 ng/mL) + IFN-γ (20 ng/mL) treated cells for 24 h], RSV group [<i>P.g</i>-LPS (200 ng/mL) + IFN-γ (20 ng/mL) + RSV (10 μg / mL) treated cells for 24 h], RSV-CPDs group [<i>P.g</i>-LPS (200 ng / mL) + IFN-γ (20 ng / mL) + RSV-CPD (50 μg / mL) treated cells for 24 h]. The supernatant of macrophages in the above four groups of cell polarization experiments was collected and mixed with osteogenic induction medium at a 1:1 ratio to culture hPDLSCs. The hPDLSCs were divided into the control group, <i>P.g</i>-LPS + IFN-γ group, RSV group, and RSV-CPDs group. The osteogenic trend of hPDLSCs was detected by alkaline phosphatase (ALP) staining and alizarin red staining (ARS). Real-time quantitative PCR (RT-qPCR) was used to detect the expression of osteogenesis-related genes. Western blot was used to detect the expression of osteogenesis-related proteins in hPDLSCs. Finally, transcriptome tests were used to explore the mechanism of the effect of RSV-CPDs on the phenotype of macrophages (THP-1) stimulated by inflammation. Results TEM results showed that RSV-CPDs exhibited a uniform spherical structure. FTIR results showed the O-C=O peak of RSV-CPDs. XRD results confirmed that the newly synthesized RSV-CPDs exhibited an amorphous structure. XPS results showed that RSV-CPDs formed a hydrophilic carboxyl group. CCK-8 results showed that RSV had specific toxicity to RAW264.7 when the concentration exceeded 10 μg/mL (<i>P</i> = 0.011), while RSV-CPDs still had good biosafety to cells when the concentration reached 50 μg/mL (<i>P</i> &gt; 0.05). Therefore, the concentration of RSV was 10 μg/mL and RSV-CPDs was 50 μg/mL. The results of flow cytometry showed that RSV-CPDs inhibited the apoptosis of macrophages under inflammatory stimulation (<i>P</i> = 0.008), and the inhibitory effect was better than that of its precursor RSV (<i>P</i> = 0.009). Compared with the <i>P.g</i>-LPS + IFN-γ group, CD86⁺ cells in the RSV group and RSV-CPDs group decreased by varying degrees (<i>P</i> &lt; 0.001, <i>P</i> = 0.004), while CD206⁺ cells increased by varying degrees (<i>P</i> = 0.006, <i>P</i> = 0.008), and the proportion of CD206⁺ cells in the RSV-CPDs group was higher than that in the RSV group (<i>P</i> = 0.010). Compared with the <i>P.g</i>-LPS + IFN-γ group, the supernatant of macrophages treated with RSV-CPDs significantly increased the ALP expression (<i>P</i> = 0.005) and ARS level (<i>P</i> = 0.006) of hPDLSCs. The mRNA expression of osteogenic-related genes RUNX-2, OCN, and COL-1 significantly increased (<i>P </i>&lt; 0.05), and the level of RUNX-2 protein also significantly increased (<i>P</i> = 0.001). Transcriptome results showed that compared with the <i>P.g</i>-LPS + IFN-γ group, the nuclear factor kappa-B (NF-κB) signaling pathway and tumor necrosis factor (TNF) signaling pathway in the RSV-CPDs group showed downward trends. Conclusion RSV-CPDs can inhibit the apoptosis of macrophages in the inflammatory state, promote M2 polarization, and bolster the osteogenic differentiation of hPDLSCs. The mechanism involved may be related to the inhibition of NF-κB and TNF signaling pathways.