Angelica sinensis polysaccharide attenuates constipation-associated colorectal carcinogenesis by inhibiting the NLRP3/Caspase-1/IL-1β inflammasome axis

Figure 1 Angelica sinensis polysaccharide dose-dependently suppresses NOD-like receptor protein 3 expression in deoxycholic acid-stimulated NCM460 cells. A: Representative immunofluorescence images showing CK20 (green), NOD-like receptor protein 3 (red), DAPI (blue), and merged images in control, culture in vehicle adding equimolar sodium chloride to control for osmolality, deoxycholic acid, Angelica sinensis polysaccharide-L, Angelica sinensis polysaccharide-H, and nigericin groups; B: Quantification of NOD-like receptor protein 3 relative fluorescence intensity expressed as fold change vs control. ^aP < 0.05. OC: Culture in vehicle adding equimolar sodium chloride to control for osmolality; DCA: Deoxycholic acid; ASP: Angelica sinensis polysaccharide; CA19-9: Carbohydrate antigen 19-9.

Figure 2 Angelica sinensis polysaccharide dose-dependently reduces ASC speck formation in deoxycholic acid-stimulated NCM460 cells. A: Representative immunofluorescence images showing CK20 (green), ASC (red), DAPI (blue), and merged images; B: Quantification of ASC relative fluorescence intensity. ^aP < 0.05. OC: Culture in vehicle adding equimolar sodium chloride to control for osmolality; DCA: Deoxycholic acid; ASP: Angelica sinensis polysaccharide; CA19-9: Carbohydrate antigen 19-9.

Figure 3 Angelica sinensis polysaccharide dose-dependently inhibits caspase-1 activation in deoxycholic acid-stimulated NCM460 cells. A: Representative immunofluorescence images showing CK20 (green), cleaved caspase-1 p20 (red), DAPI (blue), and merged images; B: Quantification of cleaved caspase-1 relative fluorescence intensity. ^aP < 0.05. OC: Culture in vehicle adding equimolar sodium chloride to control for osmolality; DCA: Deoxycholic acid; ASP: Angelica sinensis polysaccharide; CA19-9: Carbohydrate antigen 19-9.

Figure 4 NOD-like receptor protein 3 inhibitor MCC950 attenuates nigericin-induced caspase-1 activation in Angelica sinensis polysaccharide-treated cells. A: Representative immunofluorescence images showing CK20 (green), cleaved caspase-1 p20 (red), DAPI (blue), and merged images in control, culture in vehicle adding equimolar sodium chloride to control for osmolality, deoxycholic acid, Angelica sinensis polysaccharide (ASP), ASP + nigericin, and ASP + MCC950 groups; B: Quantification of cleaved caspase-1 relative fluorescence intensity. ^aP < 0.05. OC: Culture in vehicle adding equimolar sodium chloride to control for osmolality; DCA: Deoxycholic acid; ASP: Angelica sinensis polysaccharide; CA19-9: Carbohydrate antigen 19-9.

Figure 5 Caspase-1 inhibitor VX-765 blocks interleukin-1β expression while extracellular adenosine 5′-triphosphate disodium salt enhances it. A: Representative immunofluorescence images showing CK20 (green), interleukin-1β (red), DAPI (blue), and merged images in control, culture in vehicle adding equimolar sodium chloride to control for osmolality, deoxycholic acid, Angelica sinensis polysaccharide (ASP), ASP + adenosine 5′-triphosphate disodium salt, and ASP+VX-765 groups; B: Quantification of interleukin-1β relative fluorescence intensity. ^aP < 0.05. OC: Culture in vehicle adding equimolar sodium chloride to control for osmolality; DCA: Deoxycholic acid; ASP: Angelica sinensis polysaccharide; ATP: Adenosine 5′-triphosphate disodium salt; CA19-9: Carbohydrate antigen 19-9.

Figure 6 Combined pathway modulation confirms the NOD-like receptor protein 3/caspase-1/interleukin-1β axis as the target of Angelica sinensis polysaccharide. A: Representative immunofluorescence images showing CK20 (green), cleaved caspase-1 p20 (red), DAPI (blue), and merged images in control, deoxycholic acid, deoxycholic acid + Angelica sinensis polysaccharide (ASP), ASP + nigericin, ASP + nigericin + adenosine 5′-triphosphate disodium salt, and ASP + nigericin + VX-765 groups; B: Quantification of cleaved caspase-1 relative fluorescence intensity. ^aP < 0.05. DCA: Deoxycholic acid; ASP: Angelica sinensis polysaccharide; ATP: Adenosine 5′-triphosphate disodium salt; CA19-9: Carbohydrate antigen 19-9.

Figure 7 Angelica sinensis polysaccharide suppresses inflammatory mediator mRNA expression via the NOD-like receptor protein 3/caspase-1/ interleukin-1β pathway. A: IL-1β; B: IL-18; C: TNF-α; D: IL-6; E: COX-2; F: MMP-9. Reverse-transcription PCR analysis of interleukin (IL)-1β, IL-18, tumor necrosis factor-alpha, IL-6, COX-2, and matrix metalloproteinase-9 mRNA expression in control, deoxycholic acid, deoxycholic acid + Angelica sinensis polysaccharide (ASP), ASP + nigericin, ASP + nigericin + cycplase 09, and ASP + nigericin + VX-765 groups. ^aP < 0.05. DCA: Deoxycholic acid; ASP: Angelica sinensis polysaccharide; CON: Control; CY-09: Cycplase 09; TNF-α: Tumor necrosis factor-alpha; MMP-9: Matrix metalloproteinase-9; IL: Interleukin; COX-2: Cyclooxygenase-2.

Figure 8 Potential mechanistic model of Angelica sinensis polysaccharide inhibiting the constipation-to-colorectal cancer inflammation cascade. Angelica sinensis polysaccharide is proposed to act at or upstream of NOD-like receptor protein 3 (NLRP3) inflammasome assembly, suppressing caspase-1 activation and downstream inflammatory mediators [interleukin (IL)-1β, IL-18, tumor necrosis factor-alpha, and IL-6]. The precise molecular target remains unclear, with candidate mechanisms including transcriptional suppression of NLRP3, interference with nuclear factor kappa-B priming, or blockade of potassium efflux/mitochondrial reactive oxygen species. Validation will require NLRP3-knockout mouse models, reactive oxygen species scavenging assays, and potassium flux assessments. Dashed arrows denote putative regulatory relationships pending experimental confirmation. ASP: Angelica sinensis polysaccharide; CRC: Colorectal cancer; NLRP3: NOD-like receptor protein 3; IL-1β: Interleukin-1 beta; IL-18: Interleukin-18; TNF-α: Tumor necrosis factor-alpha; IL-6: Interleukin-6; NF-κB: Nuclear factor kappa-B; ROS: Reactive oxygen species; KO: Knockout.