Icariin alleviates osteoarthritis by inhibiting NLRP3-mediated pyroptosis

Abstract

Background: Osteoarthritis (OA) is the common chronic degenerative joint bone disease that is mainly featured by joint stiffness and cartilage degradation. Icariin (ICA), an extract from Epimedium, has been preliminarily proven to show anti-osteoporotic and anti-inflammatory effects in OA. However, the underlying mechanisms of ICA on chondrocytes need to be elucidated.

Methods: LPS-treated chondrocytes and monosodium iodoacetate (MIA)-treated Wistar rats were used as models of OA in vitro and in vivo, respectively. LDH and MTT assays were performed to detect cytotoxicity and cell viability. The expression levels of NLRP3, IL-1β, IL-18, MMP-1, MMP-13, and collagen II were detected by qRT-PCR and Western blotting. The release levels of IL-1β and IL-18 were detected by ELISA assay. Caspase-1 activity was assessed by flow cytometry. Immunofluorescence and immunohistochemistry were used to examine the level of NLRP3 in chondrocytes and rat cartilage, respectively. The progression of OA was monitored with hematoxylin-eosin (H&E) staining and safranin O/fast green staining.

Results: ICA could suppress LPS-induced inflammation and reduction of collagen formation in chondrocytes. Furthermore, ICA could inhibit NLRP3 inflammasome-mediated caspase-1 signaling pathway to alleviate pyroptosis induced by LPS. Overexpression of NLRP3 reversed the above changes caused by ICA. It was further confirmed in the rat OA model that ICA alleviated OA by inhibiting NLRP3-mediated pyroptosis.

Conclusion: ICA inhibited OA via repressing NLRP3/caspase-1 signaling-mediated pyroptosis in models of OA in vitro and in vivo, suggesting that ICA might be a promising compound in the treatment of OA.

Keywords: Caspase-1 signaling; Icariin; NLRP3 inflammasome; Osteoarthritis; Pyroptosis.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1

Effects of LPS on chondrocytes inflammation and collagen formation. a The mRNA levels of NRLP3, IL-1β, and IL-18 were analyzed by qRT-PCR in rat chondrocytes treated with LPS. b The release levels of IL-1β and IL-18 in the cell supernatant of LPS treated rat chondrocytes was checked by ELISA assay. c The expression level of collagen formation and pyroptosis-related protein was analyzed by Western blotting in rat chondrocytes treated with LPS. d Quantitative analysis of protein band gray in c. GAPDH was used as internal control. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 2

Effects of ICA on chondrocytes injury and pyroptosis. a The cell activity was detected by MTT assay in chondrocytes treated with various concentrations of ICA for 24 h. b Leakage of LDH was assessed by ELISA assay in chondrocytes treated with LPS or ICA. The mRNA levels of IL-1β (c) and IL-18 (d) were analyzed by qRT-PCR in chondrocytes treated with LPS or ICA. The release level of IL-1β (e) and IL-18 (f) were analyzed by ELISA assay in chondrocytes treated with LPS or ICA. g The protein level of collagen formation and pyroptosis-related protein was analyzed by Western blotting in rat chondrocytes treated with LPS or ICA. h Quantitative analysis of protein band gray in g. GAPDH was used as internal control. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 3

ICA exerted negative effects on LPS-induced NLRP3 expression and pyroptosis. a Immunofluorescent was used to detect NLRP3 in chondrocytes treated with LPS or ICA. b Flow cytometry analysis was used to measure caspase-1 activity in chondrocytes treated with LPS or ICA. c Quantitative statistics of doublepositive for caspase-1 and PI stain. d The protein level of pyroptosis-related protein was analyzed by Western blotting in chondrocytes treated with LPS or ICA. e Quantitative analysis of protein band gray in d. GAPDH was used as internal control. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 4

ICA suppressed chondrocytes injury and pyroptosis through NLRP3. a The mRNA level of NLRP3 was validated by qRT-PCR in chondrocytes. b The protein level was assessed by Western blotting in chondrocytes. GAPDH was used as internal control. c Leakage of LDH was assessed by ELISA assay in chondrocytes. The release level of IL-1β (d) and IL-18 (e) were analyzed by ELISA assay in chondrocytes. f The protein level of pyroptosis-related protein was analyzed by Western blotting in rat chondrocytes. g Quantitative analysis of protein band gray in e.GAPDH was used as internal control. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 5

ICA protected rat against osteoarthritis. a H&E staining and safranin O/fast green staining were used to evaluate cartilage histopathology in OA rats, and immunohitochemical analysis was used to detect NLRP3 in OA rat cartilage tissue. b Statistical analysis of NLRP3 positive rate. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 6

ICA attenuated pyroptosis and promoted collagen formation in OA rats. The mRNA levels of NLRP3 (a), IL-1β (b), and IL-18 (c) were examined with qRT-PCR in OA rat cartilage tissue. d The expression level of collagen formation and pyroptosis-related protein was analyzed by Western blotting in OA rat cartilage tissue. e Quantitative analysis of protein band gray in d. GAPDH was used as internal control. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *p < 0.05 and **p < 0.01