To investigate the anti-inflammatory effects of Ophiopogon japonicus polysaccharides (OJP) on submandibular gland inflammation in a mouse model of Sjögren’s syndrome (SS) and its impact on the tumor necrosis factor-α (TNF-α)/nuclear factor kappa-B (NF-κB)/NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling pathway.

① Eight C57BL/6J mice served as the control group (Control group), while 32 C57BL/6J mice were subjected to immune induction to establish the SS model and then randomly divided into 4 groups (n=8 each): model group (SS group), low-dose OJP group [OJP (10 mg/kg) group], high-dose OJP group [OJP (20 mg/kg) group], and hydroxychloroquine group (Hyd group). The Control and SS groups received intragastric administration of saline, the OJP (10 mg/kg) and OJP (20 mg/kg) groups received 0.2 mL of OJP solution at 10 mg/kg or 20 mg/kg, respectively, and the Hyd group received 0.2 mL of hydroxychloroquine solution at 100 mg/kg, once daily for 4 weeks. Water consumption and salivary flow rate were measured. Serum levels of inflammatory cytokines interleukin-6 (IL-6), interleukin-1β (IL-1β), and TNF-α were detected by ELISA. Pathological changes in submandibular gland tissue were observed by HE staining. Protein expression related to the TNF-α/NF-κB/NLRP3 pathway was assessed by Western blot, and TNF-α and NLRP3 protein expression was detected by immunofluorescence. ② RAW264.7 macrophages were divided into blank control group (Control group), model group (SS group), low-dose OJP group (OJP1 group), medium-dose OJP group (OJP2 group), and high-dose OJP group (OJP4 group). After 24 hours of culture, the medium was removed; the Control group received 2 mL of complete DMEM medium, the SS group received 2 mL of complete DMEM medium containing 16 μg LPS, and the different OJP dose groups received 2 mL of complete DMEM medium containing 16 μg LPS plus OJP at concentrations of 1, 2, or 4 mg/mL, respectively. Inflammatory cytokine levels were measured by ELISA, TNF-α/NF-κB/NLRP3 pathway-related protein expression was detected by Western blot, and TNF-α protein expression was detected by immunofluorescence.

① Compared with the Control group, the SS group showed increased water consumption and decreased salivary flow rate (P<0.05), elevated serum levels of IL-1β, IL-6, and TNF-α (P<0.01), aggravated pathological damage observed by HE staining in submandibular glands, and increased expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cysteinyl aspartate specific proteinase (Caspase-1), IL-1β, TNF-α, TNF receptor type 1 (TNFR1)/glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and phosphorylated NF-κB p65 (p-NF-κB p65)/NF-κB p65 (P<0.01), with immunofluorescence also showing elevated TNF-α and NLRP3 protein expression. Compared with the SS group, the Hyd group and both OJP dose groups exhibited decreased water consumption, increased salivary flow rate (P<0.01), reduced serum levels of IL-1β, IL-6, and TNF-α, alleviated submandibular gland pathological damage, decreased expression of NLRP3, ASC, Caspase-1, IL-1β, TNF-α, TNFR1/GAPDH, and p-NF-κB p65/NF-κB p65 proteins by Western blotting (P<0.01), and reduced TNF-α and NLRP3 protein expression by immunofluorescence. ② Compared with macrophages treated solely with LPS, those treated with OJP intervention showed reduced levels of inflammatory cytokines and decreased expression of NLRP3, ASC, Caspase-1, IL-1β, TNF-α, TNFR1/GAPDH, and p-NF-κB p65/NF-κB p65 (P<0.01).

OJP may exert anti-inflammatory effects by inhibiting the activation of the TNF-α/NF-κB/NLRP3 signaling pathway, thereby alleviating the inflammatory response in SS mice.