Vitamin D alleviates knee osteoarthritis in DMM-induced OA mice model.
A DMM-induced OA mice model was established to explore the protective effects of vitamin D on OA. Knee sections of mice from each group were stained with Safranin-O and Fast Green to explore structural changes of articular cartilage, subchondral bone and bone tissue. The findings showed that treatment with vitamin D ameliorated the rough cartilage surface and the severely exposed subchondral bone compared with the vehicle group (Figure 1A) indicating the protective effect of vitamin D. In addition, vitamin D treatment significantly reduced the osteoarthritis research society international (OARSI) score for mice in the vitamin D group compared with the vehicle group (Figure 1B). Moreover, micro-CT was performed to explore formation of osteophyte in each group. Micro-CT results showed that osteophyte volume was significantly higher in the vehicle group compared with the sham group. However, treatment with vitamin D abrogated increase in osteophyte formation in OA model mice (Figure 1A). Osteophyte score analysis showed that the osteophyte score of the vitamin D group was significantly lower compared with that of the vehicle group. This finding indicates that vitamin D inhibits osteophyte production in DMM-induced OA mice model.
Degradation of ECM protein is an important pathological characteristic of OA[41]. Therefore, immunofluorescence analysis was performed to explore expression levels of ECM proteins (COL2A1 and aggrecan) and ECM degradation associated proteases (MMP13 and ADAMTS5). Immunofluorescence results showed that the expression levels of COL2A1 and aggrecan of the vehicle group were significantly lower compared with the levels of the sham group. Meanwhile, the expression of MMP13 and ADAMTS5 were markedly elevated in vehicle group, which didn’t display in the sham group. Notably, treatment with vitamin D significantly alleviated the decreased expression of ECM proteins (COL2A1 and aggrecan), and abrogated the elevated expression of ECM degradation associated proteases (MMP13 and ADAMTS5) (Figure 1A and D).
OA is characterized by inflammatory response [42], thus inflammatory factors may be responsible for the phenomena observed. Therefore, ELISA kits were used to further explore the levels of major inflammatory factors in each group. ELISA results showed that the vitamin D treatment group significantly reduced the increased levels of IL-1β and IL-18 after DMM surgery compared with the levels of the vehicle group. However, although the secretion of TNF-α was significantly elevated in OA model group, the level was not significantly changed after vitamin D treatment (Figure 1E). These finding indicate that vitamin D effectively abrogates development of OA in the DMM-induced OA mice model.
Vitamin D exerts joint protective effects through VDR
VDR knockout mice (VDR−/− mice) were used to explore whether vitamin D exerted its protective effects through VDR. The findings showed that vitamin D treatment did not protect cartilage against degeneration in VDR−/− mice that have undergone DMM surgery (Figure 2A). OARSI scores showed that vitamin D did not exert protective effect on DMM-induced OA VDR −/− mice model (Figure 2B). In addition, formation of osteophytes was evaluated by micro-CT. The findings indicated that vitamin D did not exhibit the inhibitory effect on osteophyte formation in DMM-induced OA VDR −/− mice model (Figure 2A). Findings from osteophyte score analysis were consistent with the micro-CT analysis (Figure 2C).
Further, the expression level of COL2A1 and aggrecan were explored to determine the status of ECM degradation. Notably, expression levels of COL2A1 and aggrecan were not significant increased after treatment with vitamin D in the DMM-induced OA VDR −/− mice compared with the levels in the vehicle group (Figure 2A and D). ECM degradation associated proteases (MMP13 and ADAMTS5) were not significantly downregulated after vitamin D treatment in the DMM-induced OA VDR −/− mice model compared with the vehicle group (Figure 2A and D).
Expression levels of inflammatory cytokines in each group were explored using ELISA kits. Expression of IL-1β and IL-18 was not significantly downregulated in DMM-induced OA VDR −/− mice after treatment with vitamin D compared with the levels in DMM-induced OA mice (Figure 2E). The result was different from the results observed in DMM-induced OA mice treated with vitamin D. These findings indicate that vitamin D exerts antarthritic effects in a VDR dependent manner.
VDR directly interacts with NLRP3 to increase ubiquitination level of NLRP3
The findings showed increased IL-1β and IL-18 levels after DMM surgery in mice, indicating activation of NLRP3 inflammasomes. Inflammasome signaling molecule NLRP3 is implicated in progression of OA and is a potential novel biomarker for OA[43]. Previously reported liquid chromatography-mass spectrometry analysis showed that VDR interacts with NLRP3 VDR can affect NLRP3 in bone marrow-derived macrophage(BMDM)[44]. In addition, co-immunoprecipitation was performed to further confirm the interaction between NLRP3 and VDR in THP-1 cells. The findings showed interaction of VDR and NLRP3 in the LPS group and LPS & Nig group (Figure 3A). Moreover, consistent findings were obtained through immunofluorescence colocalization of samples from synovial sections of OA patients and normal subjects (Figure 3B).
NLRP3 activates caspase-1 through formation of inflammasome complex, subsequently leading to secretion of pro-inflammatory cytokines. To explore the effects of VDR-NLRP3 interaction on NLRP3 downstream signals, VDR was knocked down in THP-1 cells. The findings showed that VDR knockdown significantly upregulated expression of caspase-1 and IL-1β secretion in SN after LPS & Nig induced NLRP3 activation (Figure 3C). These findings indicate that VDR is implicated in NLRP3 activation.
Previous studies report that ubiquitination of NLRP3 is a key step in repressing NLRP3 activation[45]. Therefore, the role of VDR in regulating NLRP3 ubiquitination and inhibiting its activation was explored. The findings indicated VDR knockdown significantly reduced ubiquitination level of NLRP3 compared with the LPS & Nig induced negative control (NC) group (Figure 3D). This finding indicated increased NLRP3 activation, which is consistent with the changes in NLRP3 downstream signals presented in Figure 3C. In summary, these findings indicate that VDR directly interacts with NLRP3 and promotes NLRP3 ubiquitination level and activation, subsequently reducing caspase-1 activity and IL-1β secretion in macrophages.
Macrophage VDR partly reverses decrease in chondrocyte proliferation rate caused by NLRP3 activation
Although the relationship between VDR and NLRP3 has been elucidated in above studies, this interaction of macrophages and how its affect chondrocytes should be explored. Therefore, a co-culture system of THP-1 and human primary chondrocytes was established for the subsequent experiments (Figure 4). EdU flow cytometry analysis findings showed no significant changes in proliferation rate of chondrocytes cultured alone with or without LPS & Nig stimulation (Figure 5A and C). However, when chondrocytes were co-cultured with THP-1 without knockdown of VDR (MΦ-NCKD), the proliferation rate of chondrocytes in the co-culture system treated with LPS & Nig was significantly lower compared with that in the co-culture system without LPS & Nig (Figure 5A and C). The findings showed that chondrocyte proliferation rate was significantly reduced when the chondrocytes were co-cultured with THP-1 with knockdown VDR (MΦ-VDRKD) compared with that of chondrocytes co-cultured with MΦ-NCKD after addition of LPS & Nig stimulated co-culture system. These findings were consistent with EdU fluorescence results (Figure 5B and D). The findings indicate that VDR in THP-1 promotes proliferation of chondrocytes.
Macrophage VDR partly abrogates increase in chondrocyte apoptosis rate caused by NLRP3 activation
Apoptosis rate of chondrocytes in each group was determined through the Annexin V-FITC/PI Apoptosis assay. The result showed no difference between the apoptosis rate of chondrocytes in the group of chondrocytes cultured alone treated with LPS & Nig treatment and the group without LPS & Nig treatment (Figure 5E and G). Apoptosis rate of chondrocytes in the co-culture system stimulated by LPS & Nig was significantly increased for chondrocytes were co-cultured with MΦ-NCKD or MΦ-VDRKD. The findings showed that apoptosis rate of chondrocytes co-cultured with MΦ-VDRKD was significantly higher compared with that of chondrocytes co-cultured with MΦ-NCKD after LPS & Nig treatment (Figure 5E and G). Consistent findings were observed for the apoptotic rate of chondrocyte using the TUNEL Bright Green Apoptosis Detection Kit (Figure 5F and H). These findings indicate that the interaction between VDR and NLRP3 in THP-1 can inhibit apoptosis of chondrocytes.
Macrophage VDR partly alleviates the decrease in chondrocyte migration caused by NLRP3 activation
In addition, transwell assay and cell scratch area healing assay were conducted to detect the migration ability of chondrocytes in each group. The results of transwell assay showed no significant difference in the number of migrating chondrocytes cultured alone with and without LPS & Nig treatment. However, cell migration rate decreased significantly when chondrocytes were co-cultured with MΦ-NCKD or MΦ-VDRKD after addition of LPS & Nig to the co-culture system. Moreover, the migration rate of chondrocytes co-cultured with MΦ-VDRKD was significantly lower compared with that of MΦ-NCKD group induced by LPS & Nig (Figure 6A and C). Consistent findings were obtained from the cell scratch healing assay (Figure 6B and D). These findings indicate that the interaction between VDR and NLRP3 in THP-1 promotes migration of chondrocytes.
Macrophage VDR inhibited degradation of ECM proteins (COL2A1 and aggrecan) and increase in levels of ECM degradation associated proteases (MMP13 and ADAMTS5) caused by NLRP3 activation in chondrocytes
Expression levels of ECM proteins (COL2A1 and aggrecan) and ECM degradation associated proteases (MMP13 and ADAMTS5) were explore by western blot. The findings showed that addition of LPS & Nig to chondrocytes cultured alone did not cause significant differences in the expression of COL2A1, aggrecan, MMP13 and ADAMTS5 proteins (Figure 6E). However, expression of ECM proteins (COL2A1 and aggrecan) was significantly downregulated whereas expression of ECM degradation associated proteases (MMP13 and ADAMTS5) was significantly upregulated after treatment of the co-culture system with LPS & Nig. Expression of ECM proteins (COL2A1 and aggrecan) in MΦ-VDRKD group with LPS and Nig treatment was significantly downregulated compared with the levels in chondrocytes co-cultured with MΦ-NCKD, whereas expression of ECM degradation associated proteases (MMP13 and ADAMTS5) was significantly upregulated (Figure 6E-F). These findings indicate that the interaction between VDR and NLRP3 in THP-1 inhibits degradation of ECM proteins (COL2A1 and aggrecan) and abrogates increase of ECM degradation associated increased expression of proteases (MMP13 and ADAMTS5) induced by NLRP3 activation in chondrocytes.
VDR inhibits secretion of inflammatory factors by macrophages
The effects reported above may be associated with inflammatory factors secreted by THP-1. Therefore, ELISA kits were used to determine levels of major inflammatory factors in each group. The findings showed that the levels of IL-1β and IL-18 in MΦ-VDRKD group were significantly increased after the LPS & Nig treatment compared with the levels in the MΦ-NCKD group (Figure S1 A-B), whereas the level of TNF-α was not significantly different (Figure S1 C). These findings indicate that VDR inhibits NLRP3 activity thus reducing secretion of IL-1β and IL-18.
1,25VD enhances ubiquitination level of NLRP3 by modulating interaction between VDR and NLPR3
Further analysis was conducted to explore whether 1,25 VD exerted its protective effects on chondrocytes by acting on VDR and by modulation of NLRP3 activation. The findings showed that 1,25 VD inhibited NLRP3 activation and expression of downstream caspase-1 and IL-1β after treatment of THP-1 with LPS & Nig in a dose-dependent manner (Figure 7A). Further analysis was performed to verify that the 1,25 VD modulated interaction between VDR and NLRP3 ubiquitination. The findings showed that 1,25 VD increased ubiquitination level of NLRP3 after LPS & Nig treatment. However, this effect was abrogated by VDR knockdown (Figure 7B). These findings indicate that 1,25VD enhances NLRP3 ubiquitination through VDR and inhibits NLRP3 mediated macrophage inflammation response.
1,25 VD enhances the inhibitory effect of VDR on inhibition of chondrocyte proliferation caused by NLRP3 activation
The THP-1 and chondrocytes co-culture system was established to explore the effects of 1,25 VD on proliferation of chondrocytes. Flow cytometry results showed that addition of 1,25 VD significantly increased the proliferation rate of chondrocytes when chondrocytes were co-cultured with MΦ-NCKD (Figure 7C-D). However, addition of 1,25 VD did not increase proliferation rate of chondrocytes when chondrocytes were co-cultured with MΦ-VDRKD (Figure 7C-D). These findings were consistent with EdU fluorescence results (Figure 7E-F). The findings indicate that 1,25 VD plays an important role in enhancing chondrocyte proliferation through VDR.
1,25VD enhances inhibitory effect of VDR of chondrocyte apoptosis caused by NLRP3 activation
Annexin V-FITC/PI Apoptosis Detection Kit and the TUNEL Bright Green Apoptosis Detection Kit was used to determine the apoptosis rate of chondrocytes in each group. Flow cytometry results showed that treatment with 1,25VD significantly decreased apoptosis rate of chondrocytes when chondrocytes were co-cultured with MΦ-NCKD (Figure 7G-H). However, addition of 1,25VD did not reduce apoptosis rate of chondrocytes when chondrocytes were co-cultured with MΦ-VDRKD (Figure 7G-H). TUNEL fluorescence results were consistent with flow cytometry results (Figure 7I-J). These findings indicate that 1,25VD plays a vital role in inhibiting chondrocyte apoptosis through VDR.
1,25VD enhances the inhibitory effect of VDR on the decrease of chondrocyte migration caused by NLRP3 activation
Transwell assay and cell scratch area healing assay were performed to determine migration ability of each group of chondrocytes. The number of migrating chondrocytes was significantly increased after treatment with 1,25VD when chondrocytes were co-cultured with MΦ-NCKD (Figure 8A-B). The effect of 1,25VD was abrogated by VDR knockdown in THP-1 cells (Figure 8A-B). This finding indicates that 1,25VD promotes chondrocyte migration through VDR, and similar results were obtained from the cell scratch area healing assay (Figure 8C-D).
1,25VD enhances inhibitory effect of VDR on decrease of ECM proteins (COL2A1 and aggrecan) and increase in ECM degradation associated proteases (MMP13 and ADAMTS5) caused by NLRP3 activation
Western blotting was performed to explore the expression levels of related proteins in each group. The findings showed that the expression levels of ECM proteins (COL2A1 and aggrecan) increased significantly and that of ECM degradation associated proteases (MMP13 and ADAMTS5) decreased significantly in chondrocytes co-cultured with MΦ-NCKD and treated with LPS & Nig after treatment with 1,25 VD (Figure 8E-F). Notably, this effect was abrogated by knockdown of VDR in THP-1 cells (Figure 8E-F). These findings indicated that 1,25VD upregulated expression of ECM proteins (COL2A1 and aggrecan) and downregulated expression of ECM degradation associated proteases (MMP13 and ADAMTS5) through VDR.
1,25VD enhanced ability of VDR to inhibit secretion of inflammatory cytokines by macrophages
The above findings implied that 1,25VD may improve ubiquitination level of NLRP3 by modulating interaction between VDR and NLRP3, thus inhibiting secretion of inflammatory factors by macrophage ultimately playing a protective role on chondrocytes. Therefore, ELISA kits were used to determine the levels of major inflammatory factors in each group of cells. ELISA results showed that 1,25VD had no regulatory effect on TNF-α level (Figure S2 A). However, 1,25VD significantly reduced the levels of IL-1β and IL-18 in the MΦ-NCKD group (Figure S2 B-C). But the effect of 1,25VD on levels of inflammatory factors were abrogated knockdown of VDR in THP-1 cells (Figure S2 B-C).