Agnuside Alleviates Synovitis and Fibrosis in Knee Osteoarthritis through the Inhibition of HIF-1 α and NLRP3 Inflammasome

Abstract

Increasing evidence has shown that NLRP3 inflammasome activation participates in chronic aseptic inflammation and is related to tissue fibrosis. Our last study also revealed the vital role of NLRP3 inflammasome, highly associated with tissue hypoxia, in the onset and development of knee osteoarthritis (KOA). In this study, we tried to find a possible benign intervention for that pathological process. Agnuside (AGN), a nontoxic, natural small molecule isolated from the extract of Vitex negundo L. (Verbenaceae), has been demonstrated to have antioxidation, anti-inflammatory, analgesia, and many other properties as an iridoid glycoside, although its specific target is still unclear. Therefore, we established MIA-induced KOA model rats and investigated the effects of AGN oral gavage on oxygen-containing state, NLRP3 inflammasome, synovitis, and fibrosis in KOA. Pimonidazole staining and HIF-1α immunohistochemical assay both showed that AGN at the oral dose of 6.25 mg/kg can effectively relieve local hypoxia in synovial tissue. Besides, we observed a decrease of HIF-1α, caspase-1, ASC, and NLRP3 after AGN intervention, both in the mRNA and protein levels. In addition, rats treated with the AGN showed less inflammatory reaction and fibrosis, not only in the expression of NLRP3, inflammasome downstream factors IL-1β and IL-18, and fibrosis markers TGF-β, TIMP1, and VEGF but also in the observation of HE staining, anatomical characteristics, Sirius Red staining, and type I collagen immunohistochemistry. Subsequently, we established LPS-induced models of fibroblast-like synoviocytes (FLSs) mimicking the inflammatory environment of KOA and activating NLRP3 inflammasome. FLSs treated with AGN (3 μM) resulted in a downregulation of HIF-1α and the components required for NLRP3 inflammasome activation. Meanwhile, the content of proinflammatory factors IL-1β and IL-18 in FLS supernatant was also reduced by AGN. In addition, both mRNA and protein levels of the fibrotic markers were significantly decreased after AGN management. To conclude, this study demonstrates that AGN alleviates synovitis and fibrosis in experimental KOA through the inhibition of HIF-1α accumulation and NLRP3 inflammasome activation. Additionally, not only does it reveal some novel targets for anti-inflammatory and antioxidant effects of AGN but also announces its potential value in treating KOA in humans.

Figure 1

Agnuside relieves the state of hypoxia in KOA rats. (a) The chemical structure of AGN. (b) Representative synovial tissues stained with pimonidazole, 200x, scale bar = 100 μm. (c) Representative HIF-1α immunohistochemical sections of synovial tissues in each group, 200x, scale bar = 100 μm. (d) The percentage of HIF-1α-positive areas in each group. Data were analyzed by ImageJ. ^∗P < 0.05, compared with the normal group. ^#P < 0.05, compared with the KOA group. (e) mRNA level of HIF-1α between groups. ^∗P < 0.05 compared with the normal group. ^#P < 0.05, compared with the KOA group.

Figure 2

Agnuside may inhibit NLRP3 inflammasome and alleviate synovitis in KOA Rats. (a) mRNA levels of caspase-1, ASC, and NLRP3 in each group. ^∗P < 0.05, in comparison with the normal group. ^#P < 0.05, in comparison with the KOA group. (b) Typical protein bands for each group. (c) Protein level comparison of pro-caspase-1, caspase-1, p10, ASC, and NLRP3 between groups. ^∗P < 0.05, ^∗∗P < 0.01, compared with the normal group. ^##P < 0.01, compared with the KOA group. (d) Serum levels of IL-1β and IL-18 between groups. ^∗P < 0.05, ^∗∗P < 0.01, compared with the normal group. ^#P < 0.05, compared with the KOA group. (e) Representative synovial tissues of each group stained with stained with HE staining, 200x, scale bar = 100 μm. (f) Synovitis score according to Kenn's criteria. ^∗∗P < 0.01, compared with the normal group. ^##P < 0.01, compared with the KOA group.

Figure 3

Agnuside alleviates synovial fibrosis in KOA rats. (a) Anatomical changes of synovial tissues (red box area) in each group. (b) Collagen deposition was revealed through Sirius Red staining, 200x, scale bar = 100 μm. (c) Representative type I collagen immunohistochemical sections of synovial tissues in each group, 200x, scale bar = 100 μm. (d) Semiquantification of immunohistochemical sections was evaluated by calculating the percentage of collagen I-positive areas. ^∗∗P < 0.01, in comparison with the normal group. ^#P < 0.05, in comparison with the KOA group. Data were analyzed by ImageJ. (e) Comparison of TGF-β, TIMP1, and VEGF mRNA levels between groups. ^∗P < 0.05, compared with the normal group. ^#P < 0.05, compared with the KOA group. (f) Typical protein bands for each group. (g) Comparison of TGF-β, TIMP1, and VEGF protein expressions between groups. ^∗∗P < 0.01, compared with the normal group. ^#P < 0.05, ^##P < 0.01, compared with the KOA group.

Figure 4

Agnuside inhibits HIF-1α accumulation and NLRP3 inflammasome activation in LPS-treated FLSs. (a) Relative caspase-1 activity for each group. ^∗P < 0.05, in comparison with the normal group. ^#P < 0.05, in comparison with the LPS group. (b) mRNA level of HIF-1α, caspase-1, ASC, and NLRP3 in each group. ^∗P < 0.05, ^∗∗P < 0.01, in comparison with the normal group. ^#P < 0.05, ^##P < 0.01, in comparison with the KOA group. (c) Typical protein bands for each group. (d) Protein level comparison of HIF-1α, pro-caspase-1, caspase-1 p10, ASC, and NLRP3 between groups. ^∗∗P < 0.01, compared with the normal group. ^##P < 0.01, compared with the KOA group. (e) FLS supernatant contents of IL-1β and IL-18 between groups. ^∗∗P < 0.01, compared with the normal group. ^##P < 0.01, compared with the KOA group.

Figure 5

Agnuside downregulates fibrosis marker expression in LPS-treated FLSs. (a) Relative mRNA levels of TGF-β, TIMP1, and VEGF in FLSs in each group. ^∗P < 0.05, ^∗∗P < 0.01, in comparison with the normal group. ^#P < 0.05, in comparison with the KOA group. (b) Typical protein bands for each group. (c) Comparison of TGF-β, TIMP1, and VEGF protein expressions between groups. ^∗∗P < 0.01, compared with the normal group. ^#P < 0.05, ^##P < 0.01, compared with the KOA group.