Toddalolactone protects against osteoarthritis by ameliorating chondrocyte inflammation and suppressing osteoclastogenesis

Abstract

Background: Osteoarthritis (OA) is widely recognized as the most common chronic joint disease accompanied by progressive cartilage and subchondral bone damage. Toddalolactone (TOD), a natural compound extracted from Toddalia asiatica (L.) Lam., has been widely used in the treatment of stroke, rheumatoid arthritis, and oedema. Nevertheless, what TOD acts as in the pathogenesis and progression of OA hasn't been reported. In this investigation, we have aimed to determine how TOD affects OA in vitro and in vivo.

Methods: LPS (10 µg/ml) and IL-1β (10 ng/ml) were employed to induce chondrocyte inflammation or RANKL to induce osteoclast differentiation in bone marrow derived macrophages (BMMs). The effects of TOD on chondrocyte inflammation and osteoclast differentiation were evaluated. Anterior cruciate ligament transection (ACLT) was performed to develop an OA animal model and study the effects of TOD.

Results: We found that TOD inhibited the expression of inflammatory and catabolic mediators (IL-6, IL-8, TNF-α, MMP2, MMP9, and MMP13) in inflammatory chondrocytes in vitro. Furthermore, TOD was proven to inhibit RANKL-induced-osteoclastogenesis and inhibit the expression of osteoclast marker genes. Our data also confirmed that TOD suppressed the destruction of articular cartilage and osteoclastogenesis via inhibiting the activation of NF-κB and MAPK signalling pathways. In the ACLT mouse model, we found that TOD attenuated cartilage erosion and inhibited bone resorption.

Conclusions: These results showed that TOD can be adopted as a potential therapeutic agent for OA.

Keywords: Cartilage; Inflammation; Osteoarthritis; Osteoclastogenesis; Toddalolactone.

Fig. 1

TOD inhibited the expression of inflammatory and catabolic mediators in LPS-induced chondrocytes. A Chemical structure of TOD. B The effect of TOD on the cell viability of SW1353 was evaluated by CCK-8. C Gradient concentrations (0, 0.5, 1, 5, 10, 20, 40 µM/ml) of TOD on the apoptosis level of SW1353 was measured by flow cytometry. D Western blotting performed after SW1353 cells were treated with gradient concentrations (0, 5, 10, and 20 µM/ml) of TOD; β-Tubulin was used as the loading control. E Pro-inflammatory cytokines, including IL-6, IL-8, TNF-α, MMP2, MMP9, and MMP13 were measured by RT-PCR. F ELISA was used to measure the expression level of MMP2, MMP9, MMP13, IL-6, and TNF-α. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^{**, ##} indicates p < 0.01, ^ns indicates not significant

Fig. 2

TOD inhibits RANKL-induced osteoclast differentiation and bone resorption without cytotoxicity in vitro. A The cell viability of BMMs exposed to TOD was measured by CCK-8. B The cell viability of RAW264.7 cells exposed to TOD was assessed by CCK-8. C BMMs were cultured with M-CSF (25 ng/ml), RANKL (50 ng/ml), and the indicated concentrations (0, 5, 10, and 20 µM) of TOD for 5 days. The effect of TOD on BMMs differentiation was detected using TRAP staining. Scale bar: 100 μm. D The area and number of osteoclasts were quantified per well. E BMMs were cultured with M-CSF (25 ng/ml) and RANKL (50 ng/ml) for 5 days, and TOD (20 µM) was added at different stages during osteoclast differentiation. The effect of TOD on BMMs differentiation was detected using TRAP staining. Scale bar: 100 μm. F The area and number of osteoclasts were quantified per well. G BMMs were cultured with M-CSF (25 ng/ml), RANKL (50 ng/ml), and the indicated concentrations (0, 5, 10, and 20 µM) of TOD for 5 days. Scanning electron microscopy was used to observe bone resorption pits. Scale bar: 100 μm. H The resorption area and number of rings were quantified. I BMMs were cultured with M-CSF (25 ng/ml) and RANKL (50 ng/ml) for 5 days, and TOD (20 µM) was added at different stages during osteoclast differentiation. Scanning electron microscopy was used to observe bone resorption pits. Scale bar: 100 μm. J The resorption area and number of rings were quantified. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^**,## indicates p < 0.01, ^ns indicates not significant

Fig. 3

TOD inhibits the formation of F-actin ring. A BMMs were cultured with M-CSF (25 ng/ml), RANKL (50 ng/ml), and the indicated concentrations (0, 5, 10, and 20 µM) of TOD for 5 days. After differentiation, BMMs were fixed and stained for F-actin ring. Scale bar: 100 μm. B The size per F-actin ring and area of osteoclasts were quantified. C BMMs were cultured with M-CSF (25 ng/ml) and RANKL (50 ng/ml) for 5 days, and TOD (20 µM) was added at different stages during osteoclast differentiation. After differentiation, BMMs were fixed and stained for F-actin ring. Scale bar: 100 μm. D The size per F-actin ring and area of osteoclasts were quantified. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^{**, ##} indicates p < 0.01, ^ns indicates not significant

Fig. 4

TOD suppresses RANKL-induced gene expression during osteoclastogenesis in vitro. A mRNA expression of osteoclast marker genes (NFATc1, Trap, Ctr, Ctsk, DC-STAMP, and VATPase D2) were detected by RT-PCR. B Western blotting performed that TOD suppressed the activation of osteoclast related protein (Trap, Ctsk, c-Fos, and NFATc1); GAPDH was used as the loading control. C ImageJ software was used to quantify the density of the western blot bands shown in B. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^{**, ##} indicates p < 0.01, ^ns indicates not significant

Fig. 5

TOD inhibits the activation of the NF-κB and MAPK signalling pathways in SW1353 cells. A Western blotting performed the activity of NF-κB and MAPK signalling pathways in SW1353 cells; GAPDH was used as the loading control. B ImageJ software was used to quantify the density of the western blot bands (P-P65, IκBα, P-P38, and P-JNK) shown in A. C Western blots performed the protein expression of P65 in the nucleus and cytoplasm in SW1353 cells. D ImageJ software was used to quantify the density of the western blot bands in C. E Immunofluorescence was used to determine nuclear translocation of p65 in SW1353 cells. Scale bar: 100 μm. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^{**, ##} indicates p < 0.01, ^ns indicates not significant

Fig. 6

TOD attenuates the activation of the NF-κB and MAPK signalling pathways in BMMs. A Western blotting performed the activity of NF-κB and MAPK signalling pathways in RAW264.7 cells; GAPDH was used as the loading control. B ImageJ software was used to quantify the density of the western blot bands (P-P65, IκBα, P-P38, and P-JNK) shown in A. C Western blots performed the protein expression of P65 in the nucleus and cytoplasm in RAW264.7 cells. D ImageJ software was used to quantify the density of the western blot bands in C. E Immunofluorescence was used to determine nuclear translocation of p65 in RAW264.7 cells. Scale bar: 100 μm. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^{**, ##} indicates p < 0.01, ^ns indicates not significant

Fig. 7

TOD protects against ACLT-induced cartilage destruction and inhibits osteoclastogenesis in vivo. A H&E staining, Safranin O/Fast Green staining, Toluidine Blue, and TUNEL DAPI/FITC staining were performed respectively. Scale bar: 100 μm. B Immunohistochemical analysis of Collagen II. Scale bar: 100 μm (upper); 10 μm (lower). C Immunohistochemical analysis of P65. Scale bar: 100 μm (upper); 10 μm (lower). D TRAP staining of the osteoclasts in the subchondral plate. Scale bar: 200 μm. E The OARSI scores of different groups were assessed. F The percentage of apoptotic chondrocytes in cartilage was calculated. G Quantitative analysis of the immunohistochemical staining of Collagen II. H Quantitative analysis of the immunohistochemical staining of P65. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^{**, ##} indicates p < 0.01, ^ns indicates not significant

Fig. 8

TOD protects against ACLT-induced bone loss in vivo. A 3D reconstruction micro-CT images were performed for each group. Scale bar: 2 mm. B Quantification of SMI, BV/TV, Tb.Th, Tb.Sp, Tb.N, and BMD. C H&E staining of important organs. Scale bar: 200 μm; Body weights were recorded once per week. Data represent means ± SD of triplicate independent experiments. ^{*, #} indicates p < 0.05, ^**,## indicates p < 0.01, ^ns indicates not significant

Fig. 9

The mechanism of that TOD protects against osteoarthritis by ameliorating chondrocyte inflammation and suppressing osteoclastogenesis