The present study aimed to extract and purify the glycoprotein from Cirsii Herba (CHPs), and investigate its immunomodulatory activity and molecular mechanism in RAW264.7 macrophages. The results showed that CHPs contained 14.8% carbohydrates and 80.4% proteins. CHPs were identified as glycoprotein around 70 kDa and contained 17 different amino acids, in which the Glu and Asp were predominant. The carbohydrate chain in CHPs was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose with the molecular ratio of 6.387: 24.358: 5.766: 8.877: 12.098: 20.427: 7.090: 14.997. CHPs significantly boosted pinocytic and phagocytic activities, increased the secretions of inflammatory factors (NO, TNF-α and IL-6) and chemokines (CXCL2 and CXCL10), and promoted the expressions of accessory and costimulatory molecules (CD40, CD80, CD86, MHC I and MHC II). RNA-seq analysis identified 721 DEGs, 1575 GO terms and 69 KEGG pathways. The pathway inhibition assay presented that MAPK and NF-κB pathways were essential to macrophage activation by CHPs. TLR4 was revealed as a functional receptor and involved in the early recognition of CHPs. These results indicated that CHPs as a glycoprotein promoted macrophage polarization to M1 phenotype mainly via TLR4-dependent MAPK and NF-κB pathways.

01 Feb 2025·Cytotechnology

Triptolide attenuates LPS-induced chondrocyte inflammation by inhibiting inflammasome activation via the Wnt/β-catenin and NF-κB signaling pathways

Article

Author: Zou, Yang ; Liu, Qiming ; Shi, Hangchu ; He, Wang ; Ma, Xuming ; Shen, Xiaoqiang

Osteoarthritis (OA) is a common form of arthritis characterized by subchondral bone proliferation and articular cartilage degeneration. Recently, the Nod-like receptor pyrin domain 3 (NLRP3) inflammasome has gained attention due to its association with synovial inflammation in OA. Triptolide (TP), known for its immunosuppressive and anti-inflammatory effects, has been studied in various diseases. However, the specific impact of TP on OA and its underlying mechanism remains largely unexplored. In this study, chondrocytes were treated with a specific concentration of TP, and subsequent analysis through Western blotting and immunofluorescence staining revealed decreased expression levels of MMP-13, NLRP3, Caspase-1, ASC, β-catenin, p-p65, and IκB compared to the model group. ELISA results demonstrated significantly lower levels of IL-1β, IL-18, and TNF-α in the TP treatment group compared to the model group. In addition, triptolide ameliorates the degradation of the extracellular matrix (ECM) by enhancing the expression of collagen-II. In conclusion, our findings suggest that TP exhibits anti-inflammatory effects on chondrocytes in the presence of LPS-induced inflammation by inhibiting the activation of the NLRP3 inflammasome via the Wnt/β-catenin and NF-κB pathway. These results contribute to a better understanding of TP's potential therapeutic benefits in managing OA.

01 Feb 2025·Current Molecular Medicine

Polyphyllin I Mitigated IL-1β-Induced Chondrocytes Damage through Downregulating TWIST1 Expression

Article

Author: Yan, Liangliang ; Ji, Xiaopeng ; Han, Chaoyong ; Liu, Feng ; Yang, Deshun ; Han, Bowen

Background:Osteoarthritis (OA) is a chronic joint disease characterized by the degradation of articular cartilage. Polyphyllin I (PPI) has anti-inflammatory effects in many diseases. However, the mechanism of PPI in OA remains unclear.Methods:HC-a cells treated with IL-1β were identified by immunofluorescence staining and microscopic observation. The expression of collagen II and DAPI in HC-a cells was detected by immunofluorescence. The effects of gradient concentration of PPI on IL-1β-induced cell viability, apoptosis, senescence, and inflammatory factor release were detected by MTT, flow cytometry, SA-β-Gal assay and ELISA, respectively. Expressions of apoptosis-related genes, extracellular matrix (ECM)- related genes, and TWIST1 were determined by qRT-PCR and western blot as needed. The above-mentioned experiments were conducted again after TWIST1 overexpression in IL-1β-induced chondrocytes.Results:IL-1β reduced the number of chondrocytes and the density of collagen II. PPI (0.25, 0.5, 1 μmol/L) had no effect on cell viability, but it dose-dependently elevated the inhibition of cell viability regulated by IL-1β. The elevation of cell apoptosis, senescence and expression of IL-6 and TNF-α were suppressed by PPI in a dosedependent manner. Additionally, PPI reduced the expression of cleaved caspase-3, bax, MMP-3, and MMP-13 and promoted the expression of collagen II. TWIST1 expression was diminished by PPI. TWIST1 overexpression reversed the abovementioned effects of PPI on chondrocytes.Conclusion:PPI suppressed apoptosis, senescence, inflammation, and ECM degradation of OA chondrocytes by downregulating the expression of TWIST1.

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