The cytotoxic effects of FST on viability rat chondrocytes
The chemical structure of FST was presented in Fig. 1A. To determine the cytoprotective effects of FST on chondrocytes and the optimal concentration of FST used in this study, chondrocytes were incubated with varying concentrations of FST (5, 10, 20, 40, and 80 µM) for 24, 48, and 72 h for performing CCK-8 assay. Based on the results of cell viability, the concentrations of FST ≤ 40 µM did not produce significant cytotoxicity to chondrocytes at different setting time points. There exhibited a pronounced decline in viability as the concentration of FST was increased from 40 to 80 µM (Fig. 1B-D). Moreover, it was observed that FST restored decreased viability of IL−1β treated chondrocytes in a dose-dependent manner at concentrations of 10 and 20 µM (Fig. 1E). Thus, 10 and 20 µM of FST were employed in the succeeding in vitro trials.
From toluidine blue and safranin O staining, the number and dyeing intensity of chondrocytes were decreased after IL−1β treatment, which were rescued by administration of FST (Fig. 1F). It revealed that FST could prevent IL−1β-induced chondrocytes from cell viability decline and loss of glycosaminoglycans and proteoglycans. Evaluation of cell proliferation showed that EDU-positive cells were reduced following IL−1β exposure, while incubation with FST partly restored proliferation capacity of chondrocytes relative to IL−1β group (Fig. 1G and H). Collectively, these findings suggested that an appropriate dose of FST exerted a cytoprotective effect on chondrocytes followed by IL−1β exposure.
SIRT6 is reduced and senescence phenotype is enhanced in IL-1β-stimulated chondrocytes and injury-induced OA articular cartilage
Recent studies have discovered that SIRT6 was implicated in the occurrence and progression of OA [48–50]. We assessed the expression level of SIRT6 in articular cartilage and cultured chondrocytes between the OA model and control. Firstly, the OA model of rats was successfully established by destabilization of the medial meniscus (Fig. 2A and B). Immunohistochemical staining indicated that the level of SIRT6 was significantly reduced in articular cartilage from OA rats to sham counterparts (Fig. 2C and D). The western blot analysis revealed that cultured rat chondrocytes treated with IL−1β exhibited a decreased level of SIRT6 in a concentration-dependent manner relative to the control (Fig. 2E and F). The results of cellular immunofluorescence further confirmed the reduction of SIRT6 in the in vitro OA model (Fig. 2H and I). In addition, cellular senescence biomarker P16 was markedly upregulated in cartilage tissues of DMM-induced OA rats (Fig. 2J and K) and in chondrocytes under IL−1β treatment (Fig. 2E and G). Not only that, chondrocytes treated with IL−1β displayed an increased positive staining rate of senescence-associated-galactosidase (SA-β-gal) (Fig. 2L and M) and rising γ-H2AX foci number (an indicator of DNA damage response) (Fig. 2N and O). Taken together, these findings implied that SIRT6 may serve as a crucial moderator in the senescence phenotype of chondrocytes during the development of OA.
FST suppresses IL−1β-induced chondrocyte ECM catabolism and DMM-mediated cartilage degradation
Subsequently, we investigated the effect of FST on the inflammatory cytokines by western blot analysis. As shown in Fig. 3A and D, IL−1β upregulated the expressions of inflammatory mediators iNOS and COX−2, whereas the rising trends were reversed by the administration of FST in a dose-dependent manner. It was indicated that FST remarkably rescued the downregulation of COL2A and aggrecan caused by IL−1β at the protein level compared to the IL−1β group (Fig. 3A and B), which is implicated in ECM anabolism. In addition, IL−1β dramatically increased the expressions of ECM catabolic proteins MMP13 and ADAMTS5, while treatment with FST reduced the expressions of these proteins (Fig. 3A and C). The results from immunofluorescence analysis for COL2A and MMP13 further confirmed that FST inhibited ECM degradation and promoted ECM synthesis in vitro (Fig. 3E-H). Degradation and fissures of articular cartilage were observed by H&E staining analysis. SO/FG staining was performed to assess the proteoglycan loss of cartilage tissues. It was exhibited that DMM surgery induced post-traumatic OA (PTOA) gave rise to significant cartilage erosion and loss of proteoglycan. Intra-articular injection of FST ameliorated these changes to some extent (Fig. 3I). The OARSI score confirmed that the OA severity was alleviated by FST treatment (Fig. 3J). Histological immunofluorescence staining revealed that COL2A level was remarkably decreased and MMP13 expression was observably upregulated in articular cartilage caused by DMM, which was prominently reversed by injected FST intra-articularly (Fig. 3K-N). Collectively, it is suggested that FST exerted its anti-osteoarthritis effects by inhibiting inflammatory response and decreasing cartilage matrix degradation.
FST inhibits senescence of chondrocytes and cartilage tissues induced by IL−1β and DMM surgery
Next, the effects of FST on chondrocyte senescence in vitro and in vivo were investigated. The western blot assay showed that FST treatment upregulated the lowered expression of SIRT6 after IL−1β stimulation (Fig. 4A and B). The result from immunofluorescence staining was in keeping with this change (Fig. 4E and F). Upon IL−1β induction, the expressions of the two critical biomarkers of cellular senescence P21 and P16 were visibly enhanced, however, such abnormal increases of the expressions could be inhibited by FST treatment in a dose-dependent manner (Fig. 4A, C, and D). Likewise, IHC analysis indicated that FST attenuated the decline of SIRT6 and the increase of P16 induced by DMM surgery in vivo (Fig. 4G-J). Meanwhile, the increased number of SA-β-gal positive chondrocytes caused by IL−1β intervention was partially reversed by the application of FST (Fig. 4K and L). Furthermore, a marker γ-H2AX indicating DNA double-strand breaks was detected. The western blot results exhibited a markedly high expression of γ-H2AX followed by IL−1β treatment relative to the control group, which was downregulated upon FST administrated (Fig. 4M and N). The immunofluorescence staining revealed that FST decreased the accumulation of γ-H2AX foci in chondrocyte nuclei stimulated by IL−1β, which was in good agreement with the western blot result (Fig. 4O and P). Additionally, as expected, the articular cartilage in rats treated with FST showed a lower expression of γ-H2AX compared with that of DMM group, which was consistent with the results of the in vitro experiments (Fig. 4Q and R). These findings revealed that FST suppressed senescence phenotype and DNA damage in chondrocytes in vitro and in vivo.
FST regulates anabolism and catabolism in osteoarthritic chondrocytes by targeting SIRT6 both in vitro and in vivo
To further ascertain whether the underlying mechanism of FST on OA chondrocyte metabolism was mediated by SIRT6, chondrocytes were treated with IL−1β in the presence or absence of MDL and OSS. Compared to IL−1β-treated chondrocytes, in the protein level, there was alleviation of high expression of inflammatory cascade cytokines iNOS and COX−2 induced by IL−1β in the high dose of FST (20 µM) group and MDL group, while the effect of FST was blocked by OSS in the FST + OSS group (Fig. 5A and D). Simultaneously, FST displayed the same anti-osteoarthritis effects as MDL manifested by ameliorating the decline of ECM synthesis markers COL2A and aggrecan and the elevation of ECM degradation enzymes MMP13 and ADAMTS5 stimulated by IL−1β. Conversely, the OSS exerted the opposite function, which antagonized the effect of FST (Fig. 5A-C). As expected, intra-articular injection of FST and MDL relieved the OA-like features of cartilage degeneration in vivo, as evidenced by loss of proteoglycan, rough cartilage surface, and cartilage erosion or fissures with a high OARSI score, and the effect of FST was blocked by OSS in the DMM + FST + OSS group (Fig. 5E and F). Additionally, histological immunofluorescence showed that FST and MDL restored ECM homeostasis disrupted by DMM operation, revealed by upregulating the reduced expression of anabolic marker COL2A and lowering enhanced expression of catabolic marker MMP13 (Fig. 5G-J). These findings demonstrated that the regulation of FST on the cartilage matrix metabolism and SASP factors secretion was closely associated with SIRT6 activation. Subsequently, molecular docking was conducted to further determine whether FST could activate SIRT6 and the affinity of FST against SIRT6. As shown in Fig. 5K-N, the docking results uncovered that FST could bind with inner sites of the structural domain of FST through forming conventional hydrogen bonds, Pi-Pi T-shaped interactions, Pi-Pi stacked interactions, and Pi-alkyl interactions. The energy of affinity of FST binding to SIRT6 was−8.9 kcal/mol, implying a favorable binding affinity. Conclusively, it was further confirmed that FST exerted its anti-OA influences by maintaining ECM homeostasis via modulating SIRT6.
FST activates SIRT6 to ameliorate chondrocyte senescence during the OA development
Next, we continued to ascertain the mechanism of how FST prevents cartilage senescence. Firstly, in vitro, the western blot result demonstrated that SIRT6 was pharmacologically activated by FST just as the effect of the widely accepted SIRT6 agonist MDL, which was antagonized by OSS intervention (Fig. 6A and B). Again, the in vivo experiment for the SIRT6 immunohistochemical staining of articular cartilage verified the above phenomenon (Fig. 6E and F). The increasing expressions of senescence-related markers P16 and P21 induced by IL−1β were decreased by the administration of FST and MDL, which was counteracted by SIRT6 inhibition using OSS (Fig. 6A, C, and D). For P16 expression in the articular cartilage, FST had the same effect as MDL, indicated by equally attenuating the higher level of P16 in rat cartilage tissues after DMM surgery, which was partially offset by OSS (Fig. 6E and G). SA-β-gal staining uncovered that the administration of FST and MDL led to a notable reduction of SA-β-gal positive rate relative to IL−1β intervention, while this effect was blocked by OSS (Fig. 6H and I). In light of the postulated direct downregulation of FST upon senescent phenotype, then we investigated the role of FST-mediated SIRT6 regulation in modulating the IL−1β and DMM-induced DNA damage response. A variety of external stimuli can cause DNA damage, lead to cell cycle arrest and ultimately induce senescence. H2AX is a histone H2A variant, and detection of H2AX foci can reflect DNA double-strand breaks (DSBs), genomic instability, and telomere dysfunction. The generation of phosphorylated histone H2AX (γ-H2AX) is well recognized as a DSBs marker. As expected, in the protein level, the effect of FST on contributing to the significant attenuation of the increased level of γ-H2AX induced by IL−1β treatment was antagonized by OSS and MDL had the same effect as FST (Fig. 6J and K). Consistently, the cellular immunofluorescence staining for γ-H2AX also confirmed that both FST and MDL could alleviate DNA damage response after chondrocytes were subjected to IL−1β to mimic in vitro OA condition, which was repressed by OSS intervention (Fig. 6L and M). In addition, likewise, the immunohistochemical staining of cartilage for γ-H2AX was in complete accordance with the in vitro experiments (Fig. 6N and O). In conclusion, these findings revealed that the administration of FST produced an ameliorative effect on senescent phenotype and DNA damage response in chondrocytes treated with IL−1β and rats with post-traumatic OA by activating SIRT6.
The inhibitory effect of FST on apoptosis in chondrocytes and articular cartilage tissues was mediated by SIRT6
Apoptosis is a cell fate that is distinct from cellular senescence. It is widely known that mitochondria are not only essential for apoptosis but the major regulators of the SASP. Apoptosis largely resulted from widespread mitochondrial outer membrane permeabilization (MOMP), which occurring in a subset of mitochondria is found to be a fundamental characteristic of cellular senescence and mitochondrial apoptotic stress may be a significant driver of the SASP from a recently published literature [51]. Given the evidence providing the linkage between senescence and apoptosis, it could be concluded that similar mitochondria-dependent mechanisms are involved in the regulation of both cellular apoptosis and senescence. Consequently, we next proceeded to explore the role of FST on apoptosis and mitochondrial oxidative stress and their association with SIRT6. The western blot analysis indicated that FST exhibited anti-apoptotic effects by upregulating the suppressive expression of Bcl−2 and decreasing the higher expression of Bax in apoptotic rat chondrocytes induced by IL−1β in a dose-dependent manner. Meanwhile, the effect of the high dose of FST (20 µM) was equivalent to MDL, which was counteracted by OSS intervention in contrast (Fig. 7A-E). Consistently, in vivo, the cartilage IHC staining for Bcl−2 again confirmed that intra-articular injection of FST and MDL reversed the decline of Bcl−2 level compared with that of the DMM group and that was blocked by OSS (Fig. 7J and K). Mechanistically, reversible redox homeostasis mechanisms acting as specific signaling systems could trigger crucial physiological responses. Disruption of the redox homeostasis leads to the overproduction of ROS, which commits cells to die ultimately. The ROS levels detected by DCFH-DA fluorescent probe suggested that FST exerted an obviously inhibitory effect on the abnormal mass production of ROS aroused by IL−1β treatment in a dose-dependent manner (Fig. 7F and G). Given that mitochondria are the major site of ROS generation, we then went on investigating the effect of FST on mitochondrial function, mainly focused on the alterations in mitochondrial membrane potential (MMP). The increase of green JC−1 monomers and reduction of red JC−1 aggregates signify mitochondrial dysfunction and the drop of MMP. Notably, The JC−1 assay displayed that the restored effect of FST on the abnormal decrease of MMP arising from IL−1β intervention was antagonized by OSS, oppositely, the beneficial effect of FST was extremely similar to MDL (Fig. 7H and I). Together, these data support the conclusion that FST exerts its protective effects against apoptosis in chondrocytes and articular cartilage tissues through maintaining mitochondrial homeostasis by targeting SIRT6.
FST exerts its beneficial effects and activates the Nrf2/HO−1 signaling pathway through SIRT6 in vitro and in vivo
Nuclear factor erythroid−2-related factor 2 (Nrf2) is identified as a master antioxidant transcription regulators and a critical redox sensor. Under the imbalance of redox homeostasis, Nrf2 is prone to translocate to the nucleus from the cytoplasm and bind to the antioxidant response element (ARE), thereby modulating its downstream target genes to protect cells from oxidative stress-induced DNA damage and apoptosis. Haem oxygenase 1 (HO−1), a pivotal antioxidant enzyme and Nrf2-dependent cytoprotective effector, involved in catalyzing the breakdown of free haemoglobin and maintaining its cellular content at a stable level, contributing to the release of antioxidant factors that perform its anti-oxidative, anti-inflammatory, and anti-apoptotic activities. Accumulating evidence suggests that the activation of the Nrf2/HO−1 signaling pathway meditates chondroprotective effects during OA progression [52, 53]. In this study, there existed no significant difference in the protein expression of Nrf2 and HO−1 in the IL−1β group compared with that of the control group. Nevertheless, the administration of FST upregulated the expression of Nrf2 in the nucleus and the level of HO−1 in the whole cell relative to the IL−1β-induced rat chondrocytes in a dose-dependent manner (Fig. 8A-D). To further thoroughly investigate the interaction between SIRT6 and Nrf2 under FST stimuli, IL−1β treated chondrocytes were subjected to OSS and MDL in the presence or absence of FST. Unsurprisingly, the results of western blot revealed that the stimulative effects of FST on the expressions of Nrf2 of the nucleus and the whole-cell HO−1 were antagonized by OSS and MDL had the same effect as FST, which demonstrated that FST facilitated the SIRT6-dependent nuclear translocation of Nrf2 (Fig. 8E-H). Meanwhile, it was further unveiled that intra-articular injection of FST enhanced the percentage of Nrf2 nuclear positive cells in the DMM + FST group compared with that of the DMM group in articular cartilage, while SIRT6 inhibition by OSS partially reversed the promotion of FST on Nrf2 and this beneficial effect of FST was equivalent to DML (Fig. 8I and J). These data suggested that SIRT6 was required for the activation of the Nrf2/HO−1 signaling pathway in response to oxidative stress. Overall, these findings revealed that FST protected chondrocytes and articular cartilage against oxidative stress, apoptosis, and senescence, and consequently attenuated OA development through the induction of SIRT6/Nrf2/HO−1 signaling (Fig. 8K).