Xanthohumol alleviates palmitate-induced inflammation and prevents osteoarthritis progression by attenuating mitochondria dysfunction/NLRP3 inflammasome axis

Abstract

Osteoarthritis (OA) is a prevalent chronic degenerative joint disease worldwide. Obesity has been linked to OA, and increased free fatty acid levels (e.g., palmitate) contribute to inflammatory responses and cartilage degradation. Xanthohumol (Xn), a bioactive prenylated chalcone, was shown to exhibit antioxidative, anti-inflammatory, and anti-obesity capacities in multiple diseases. However, a clear description of the preventive effects of Xn on obesity-associated OA is unavailable. This study aimed to assess the chondroprotective function of Xn on obesity-related OA. The in vitro levels of inflammatory and ECM matrix markers in human chondrocytes were assessed after the chondrocytes were treated with PA and Xn. Additionally, in vivo cartilage degeneration was assessed following oral administration of HFD and Xn. This study found that Xn treatment completely reduces the inflammation and extracellular matrix degradation caused by PA. The proposed mechanism involves AMPK signaling pathway activation by Xn, which increases mitochondrial biogenesis, attenuates mitochondrial dysfunction, and inhibits NLRP3 inflammasome and the NF-κB signaling pathway induced by PA. In summary, this study highlights that Xn could decrease inflammation reactions and the degradation of the cartilage matrix induced by PA by inhibiting the NLRP3 inflammasome and attenuating mitochondria dysfunction in human chondrocytes.

Keywords: AMPK/NF-κB signaling pathway; Mitochondria dysfunction; NLRP3 inflammasome; Osteoarthritis; Palmitate; Xanthohumol.

Fig. 1

The cytotoxicity of Xn on human chondrocytes. A. Chemical structure of Xn. B. Human primary chondrocytes examined by toluidine blue staining. C. Expression of collagen II, MMP3, and MMP13 detected by qPCR in normal human and OA chondrocytes. D and E. Cytotoxic effects of Xn on human chondrocytes determined at 24h and 48h by CCK8 assay. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2

Blocking the levels of PA-induced inflammatory mediators by Xn in human chondrocytes. A. mRNA expression of IL-1β and TNF-α detected using qPCR. B. Production of IL-1β and TNF-α detected using ELISA. C. mRNA expression of iNOS and COX-2 detected using qPCR. D. Production of NO and PGE2 detected using ELISA. E. Protein expression of COX2 and iNOS detected using western blotting and quantified. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3

Attenuation of PA-induced ECM degradation by Xn in human chondrocytes. A. mRNA expression of collagen II, MMP1, MMP3, MMP13, and Adamts5 detected using qPCR. Protein expression of collagen II, MMP1, MMP3, MMP13, and Adamts5 detected using western blotting (B) and quantification results (C). D. Expression of collagen II and MMP13 detected by immunofluorescence. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 4

Prevention of PA-induced NF-κB activation by Xn in human chondrocytes. Protein expression of p-IkBα, IkBα, p-p65, and p65 detected using western blotting (A) and quantified (B). C. Nuclear translocation of p65 observed by immunofluorescence. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5

Inhibition of PA-NLRP3 inflammasome axis-induced inflammation and ECM degradation by Xn. A. mRNA expression of NLRP3, ASC, and caspase 1 detected using qPCR. Protein expression of NLRP3, ASC, caspase-1, pro-caspase-1, IL-1β, and pro-IL-1β detected using western blotting (B) and quantification results (C). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 6

Increased mitochondrial biogenesis and prevention of mitochondrial dysfunction by Xn. A. mRNA expression of PGC-1α and TFAM detected using qPCR. Protein expression of p-AMPKα, AMPKα, PGC-1α, and TFAM detected using western blotting (B) and quantification results (C). D. Protein expression of p-AMPKα, AMPKα, TFAM, NLRP3, Collagen II, and MMP3 detected using western blotting with or without compound C treatment. E. Modular structure of Xn. F. The modular structure of AMPK. G. Interaction between Xn and AMPK. H. Content of ROS measured by a detection kit. I. Content of MDA measured by a detection kit. J. Content of ATP measured by detection kit. K. Expression of mtDNA detected using qPCR. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 7

Alleviation of OA progression by Xn in the DMM rat model. A. Degeneration of articular cartilage observed in S–O staining. B. Immunohistochemistry of MMP3 and collagen II employed to assess the effect of Xn on the cartilage in the DMM models. C. Schematic illustration of the prospective protection conferred by Xn and the underlying mechanism in PA-induced OA development.