Curcumin nanoparticles mitigate early inflammation in severe acute pancreatitis by modulating peritoneal macrophage polarization via Nrf2/HO-1 pathway activation

Figure 1 Characterization and safety verification of curcumin nanoparticles. A: Morphological characteristics of curcumin nanoparticles (Cur-NPs); B: Particle size distribution of Cur-NPs; C: Polydispersion index of Cur-NPs; D: Zeta potential of curcumin nanoparticles; E: Cumulative drug release rate of Cur-NPs under different pH (5, 6.5, 7.4) conditions; F: Representative images of rhodamine B fluorescence signal in peritoneal macrophages of each group (Blank, Rhod, Lipo@Rhod); the mean fluorescence intensity in each group was quantitatively analyzed by flow cytometry; G: Encapsulation efficiency and drug loading capacity of Cur-NPs; H: Plasma concentrations of curcumin (Lipo, free curcumin, Cur-NPs) after 6 hours, 12 hours and 24 hours of treatment; I: Cell viability after 6 hours, 12 hours, and 24 hours of treatment in each group; J: Hemolysis rate (%) of Cur-NPs at each concentration; K: Coagulation index (%) of Cur-NPs at each concentration; L: Representative hematoxylin and eosin stained sections of five major organs: Heart, liver, spleen, lung, and kidney after 24 hours and 7 days of Cur-NPs treatment. Cur-NPs: Curcumin nanoparticles.

Figure 2 The therapeutic effect of curcumin nanoparticles in a rat model of severe acute pancreatitis. A: Representative photomicrographs of pancreatic tissue sections (hematoxylin and eosin staining); B: Histopathological scoring of pancreatic tissues for evaluating the severity of edema, inflammation, and necrosis. Orange arrows indicate areas of necrosis, inflammatory cell infiltration, and hemorrhage; C: The expression level of serum amylase; D: The expression level of serum lipase; E: The expression level of serum pro-inflammatory cytokine tumor necrosis factor-α; F: The expression level of serum interleukin-1β. The data were expressed as mean ± SD (n = 6 rats in each group). ^aP < 0.05 vs sham, ^bP < 0.05 vs severe acute pancreatitis. Cur-NPs: Curcumin nanoparticles; SAP: Severe acute pancreatitis.

Figure 3 Curcumin nanoparticles induce polarization of peritoneal macrophages toward the M2 phenotype in vivo. A: Flow cytometric analysis of CD86 and CD163, specific proteins for peritoneal macrophages, showing co-expression of CD68 and CD45 in both M1 and M2 macrophages; B: The ratio of M1/M2 positive cells; C: The expression levels of inducible nitric oxide synthase (iNOS) and CD163 mRNA, with the internal reference gene GAPDH normalized; D: The expression level of pro-inflammatory cytokine IL-1β in cell homogenate; E: The expression level of the anti-inflammatory cytokine IL-10 in the cell homogenate; F: Representative immunoblots of iNOS and CD163 proteins with corresponding quantitative grayscale analysis. The data were expressed as mean ± SD (n = 6 rats in each group). ^aP < 0.05 vs sham, ^bP < 0.05 vs severe acute pancreatitis. Cur-NPs: Curcumin nanoparticles; SAP: Severe acute pancreatitis; iNOS: Inducible nitric oxide synthase.

Figure 4 Curcumin nanoparticles activate nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway in peritoneal macrophages in vivo. A: The expression levels of t nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) mRNA, with the internal reference gene GAPDH normalized; B: Representative immunoblots of Nrf2 and HO-1 proteins with corresponding quantitative grayscale analysis. The data were expressed as mean ± SD (n = 6 rats in each group). ^aP < 0.05 vs sham, ^bP < 0.05 vs severe acute pancreatitis. Cur-NPs: Curcumin nanoparticles; SAP: Severe acute pancreatitis; iNOS: Inducible nitric oxide synthase; HO-1: Heme oxygenase-1; Nrf2: Nuclear factor erythroid 2-related factor 2.

Figure 5 Curcumin nanoparticles mediate the suppression of nuclear factor kappa-B activation through a nuclear factor erythroid 2-related factor 2-dependent mechanism in vitro. A: The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) mRNA, with the internal reference gene GAPDH normalized; B: Quantitative Nrf2 nuclear translocation based on the ratio of nuclear/cytoplasmic fluorescence intensity; C: Representative immunoblots of Nrf2 and HO-1 proteins with corresponding quantitative grayscale analysis; D: Subcellular localization of Nrf2 (red), nuclei stained by DAPI (blue), with arrows highlighting nuclear translocation; E: Representative immunoblots of pp65 and pIκBα proteins with corresponding quantitative grayscale analysis. The data were expressed as mean ± SD (n = 6 rats in each group). ^aP < 0.05 vs sham, ^bP < 0.05 vs severe acute pancreatitis. Cur-NPs: Curcumin nanoparticles; SAP: Severe acute pancreatitis; iNOS: Inducible nitric oxide synthase; HO-1: Heme oxygenase-1; Nrf2: Nuclear factor erythroid 2-related factor 2.

Figure 6 Curcumin nanoparticles regulate macrophage polarization via nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 signaling pathway in vitro, thereby alleviating inflammation and oxidative stress. A: Flow cytometric analysis of cluster of differentiation 86 (CD86) and CD163, specific proteins for peritoneal macrophages, showing co-expression of CD68 and CD45 in both M1 and M2 macrophages; B: Quantitative results of CD86 and CD163 positive cell counts; C: The ratio of M1/M2 positive cells; D: Representative immunoblots of CD86 and CD163 proteins with corresponding quantitative grayscale analysis; E: Expression and immunofluorescence localization of CD86 (green) and CD163 (red) proteins, with nuclei stained by DAPI (blue); F: Quantitative fluorescence ratio analysis of CD86 and CD163; G: The expression levels of inducible nitric oxide synthase and CD163 mRNA, with the internal reference gene GAPDH normalized; H: Levels of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β; I: Oxidative stress indicators: Malondialdehyde and superoxide dismutase levels. The data were expressed as mean ± SD (n = 6 rats in each group). ^aP < 0.05 vs sham, ^bP < 0.05 vs severe acute pancreatitis. Cur-NPs: Curcumin nanoparticles; SAP: Severe acute pancreatitis; iNOS: Inducible nitric oxide synthase; HO-1: Heme oxygenase-1; Nrf2: Nuclear factor erythroid 2-related factor 2; TBHQ: Tert-butylhydroquinone; SAP-AF: Severe acute pancreatitis ascitic.

Figure 7 During the pathogenesis of severe acute pancreatitis, inflammatory signaling activates the IKK complex (composed of IKKα, IKKβ, and IKKγ). This complex phosphorylates IκB proteins, leading to the release of the nuclear factor kappa-B (NF-κB) p50-p65 heterodimer, which rapidly translocates into the nucleus. It binds to specific sequences within the promoter regions of target genes, initiating the transcription of a wide array of pro-inflammatory factors and promoting macrophage polarization toward the M1 phenotype. Concurrently, in the cytoplasm, Keap1 undergoes conformational changes in response to inflammatory signals, facilitating the ubiquitination and subsequent degradation of nuclear factor erythroid 2-related factor 2 (Nrf2). As a result, the nuclear translocation of Nrf2 is suppressed. Following intervention with curcumin nanoparticles, nuclear translocation of Nrf2 is markedly enhanced. Within the nucleus, abundant Nrf2 forms heterodimers with small Maf proteins. These dimers recognize and bind to the antioxidant response element in the promoter regions of target genes, thereby promoting the expression of heme oxygenase-1 (HO-1). HO-1 subsequently inhibits the phosphorylation of IκB, which in turn prevents the nuclear translocation and transcriptional activity of the NF-κB p50-p65 dimer. This cascade ultimately suppresses the expression of pro-inflammatory cytokines and promotes macrophage polarization toward the M2 phenotype. Cur-NPs: Curcumin nanoparticles; SAP: Severe acute pancreatitis; iNOS: Inducible nitric oxide synthase; HO-1: Heme oxygenase-1; Nrf2: Nuclear factor erythroid 2-related factor 2; ARE: Antioxidant response element; NF-κB: Nuclear factor kappa-B; TNF-α: Tumor necrosis factor-α; IL: Interleukin.