ASA VI inhibits TBHP-induced apoptosis and cartilage degradation in chondrocytes

Our experimental findings reveal the protective effects of ASA VI from Clematis chinensis on chondrocytes exposed to tert-Butyl hydroperoxide (TBHP). Initially, we assessed the viability of chondrocytes with the CCK-8 assay across various concentrations of Asperosaponin VI (0, 25, 50, 100, 200, 400 µM). The findings revealed that Asperosaponin VI preserved cell viability in a dose-dependent manner, particularly at concentrations above 50 µM, demonstrating significant protection against TBHP-induced cytotoxicity (Fig. 1A-B). Flow cytometry was used to detect the apoptosis of chondrocytes in each group, and it was found that TBHP treatment significantly induced the apoptosis of chondrocytes, and the apoptosis was inhibited in a concentration-dependent manner with the increase of ASA VI treatment concentration (P < 0.01 or P < 0.001, Fig. 1C). Further investigation using the TUNEL assay highlighted Asperosaponin VI's ability to inhibit apoptosis in chondrocytes. The results demonstrated that Asperosaponin VI rescued the apoptotis induced by TBHP in chondrocytes cells in concentrations dependent manner (50, 100, 200 µM) (Fig. 1D). The results showed that TBHP treatment significantly induced the expression of Bax and Caspase 3, and significantly inhibited the expression of Bcl-2. The expression of these three proteins was reversed in a concentration-dependent manner with the increase of ASA VI concentration (P < 0.01 or P < 0.001, Fig. 1E).This trend signifies the potent anti-apoptotic properties of Asperosaponin VI under oxidative stress conditions induced by TBHP. The protective influence of Asperosaponin VI extended to the ECM of chondrocytes, crucial for cartilage integrity and function. Western blot analysis confirmed that treatment with Asperosaponin VI significantly upregulated the expression of key cartilage matrix proteins, Collagen II and Aggrecan, while simultaneously downregulating the MMP-13, matrix metalloproteinase, which is often implicated in cartilage degradation (Fig. 1F). Notably, the effects were more pronounced at higher concentrations of Asperosaponin VI, showcasing its capability to suppress TBHP-induced ECM degradation effectively. In summary, Asperosaponin VI from Clematis chinensis exhibits significant protective effects against TBHP-induced cytotoxicity, apoptosis, and ECM degradation in chondrocytes, suggesting its potential as a therapeutic agent in mitigating cartilage damage associated with oxidative stress.

Fig. 1

ASA VI protects chondrocytes against TBHP-induced apoptosis and cartilage degradation. (A-B) Cell viability assessment using the CCK-8 assay in chondrocytes treated with varying concentrations of ASA VI (0, 25, 50, 100, 200, 400 µM). (C) T Flow cytometry shows the inhibition of apoptosis in chondrocytes treated with ASA VI at concentrations of 50, 100, and 200 µM. (D)TUNEL assay images displaying the inhibition of apoptosis in chondrocytes treated with ASA VI at concentrations of 50, 100, and 200 µM. (E) Western blot analysis showing the effects of ASA VI on the expression of apoptosis marker proteins in chondrocytes. (F) Western blot analysis showing the effects of ASA VI on the expression of key extracellular matrix proteins in chondrocytes. *Note: Experimental replicates (n = 3 for Western blot and n = 6 for CCK-8 and TUNEL assays) validate the statistical significance of the results (**P < 0.01, **P < 0.001, compared with Ctrl group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with TBHP group; data presented as mean ± SD)

ASA VI inhibits TBHP-induced ER stress and mitochondrial dysfunction

Building on the demonstrated protective effects of Asperosaponin VI from Clematis chinensis on apoptosis and cartilage degradation, our subsequent experiments delved into its impact on ER stress and mitochondrial dysfunction induced by TBHP. Western blot analysis revealed that TBHP treatment significantly upregulated ER stress markers including GRP78, CHOP, and ATF4. However, treatment with Asperosaponin VI significantly reduced the expression levels of these markers in a dose-dependent manner, illustrating its potential to alleviate ER stress (Fig. 2A). The phosphorylation status of PERK and eIF2α, critical components of the ER stress pathway, was also assessed. Asperosaponin VI treatment markedly reduced the phosphorylation of PERK and subsequently eIF2α (p-eIF2α/FoIF2α ratio), as shown in Fig. 2A, indicating reduced activation of this stress-induced pathway. These findings suggest that Asperosaponin VI's protective mechanisms may involve the modulation of key ER stress response elements.

Fig. 2

ASA VI alleviates TBHP-induced endoplasmic reticulum stress and mitochondrial dysfunction. (A) Western blot analysis showing the impact of ASA VI on TBHP-induced upregulation of ER stress markers (GRP78, CHOP, ATF4) and phosphorylation of PERK and eIF2α. (B) Western blot quantification of mitochondrial biogenesis proteins (PGC-1α, TFAM, and NRF-2). (C) Graph illustrating the restoration of ATP levels by Asperosaponin VI in TBHP-treated chondrocytes, measured via an ATP assay. (D) Fluorescence images from JC-1 staining assessing mitochondrial membrane potential. (E) Flow cytometry analysis corroborating the JC-1 staining results, with quantification of mitochondrial membrane potential preservation. *Note: Sample size for experimental replicates is n = 3 for Western blots and n = 6 for ATP assay, JC-1 staining, and flow cytometry. Statistical significance indicated as ***P < 0.001, compared with Ctrl group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with TBHP group;, data presented as mean ± SD

Further examining mitochondrial function, our results, demonstrated an increase in the expression of mitochondrial biogenesis markers such as PGC-1α, TFAM, and NRF-2 upon treatment with Asperosaponin VI (Fig. 2B). This suggests that Asperosaponin VI not only mitigates ER stress but also promotes mitochondrial biogenesis, potentially counteracting the mitochondrial dysfunction induced by TBHP. To corroborate these findings, we measured cellular ATP levels as an indicator of functional mitochondrial activity. We found that Asperosaponin VI treatment significantly restored ATP levels that were depleted by TBHP treatment, reinforcing its role in enhancing mitochondrial function and energy production (Fig. 2C). The mitochondrial membrane potential, a key indicator of mitochondrial health, was assessed using JC-1 staining, illustrated in Fig. 2D. Treatment with Asperosaponin VI led to a dose-dependent preservation of mitochondrial membrane potential compared to the TBHP-treated control, further corroborated by flow cytometry analysis (Fig. 2E), indicating improved mitochondrial function. Overall, our data strongly indicate that Asperosaponin VI from Clematis chinensis not only counters TBHP-induced apoptosis and cartilage degradation but also effectively inhibits ER stress and restores mitochondrial function in chondrocytes.

Chuanxuduan ASA VI alleviates mitochondrial dysfunction by inhibiting TBHP-mediated ER stress

Following the demonstration of ASA VI's efficacy in mitigating TBHP-induced apoptosis, cartilage degradation, ER stress, and mitochondrial dysfunction, we conducted further experiments to ascertain whether ASA VI alleviates mitochondrial dysfunction specifically by modulating ER stress. In these experiments, chondrocytes were pretreated with tunicamycin (TM), a known inducer of ER stress, to create a model of exacerbated mitochondrial dysfunction.

Western blot analysis revealed significant changes in protein expression associated with ER stress and mitochondrial biogenesis (Fig. 3A & B). Treatment with TM markedly elevated the levels of GRP78, CHOP, and ATF4, consistent with induced ER stress. However, subsequent treatment with ASA VI significantly reduced these elevations, thereby suggesting its role in attenuating the ER stress response. Moreover, the phosphorylation states of PERK and eIF2α, which are crucial for the initiation of ER stress responses, were similarly diminished under the influence of ASA VI. In addition to mitigating ER stress markers, Asperosaponin VI treatment notably increased the expression levels of mitochondrial biogenesis markers, such as PGC-1α, TFAM, and NRF-2, even in the presence of TM (Fig. 3B). This indicates that ASA VI not only reduces ER stress but also promotes mitochondrial biogenesis, likely contributing to the restoration of mitochondrial function. To specifically address mitochondrial function, we measured ATP production, a direct indicator of mitochondrial activity. Moreover, TM treatment significantly restored ATP levels elevated by ASA VI, supporting the hypothesis that ASA VI's protection of mitochondrial function is mediated through the attenuation of ER stress (Fig. 3C). Mitochondrial membrane potential was assessed using JC-1 staining (Fig. 3D). The results showed that ASA VI treatment preserved mitochondrial membrane potential, which was otherwise compromised by TM-induced ER stress, as corroborated by flow cytometry (Fig. 3E). These findings further affirm that the mitochondrial protective effects of ASA VI are closely linked to its ability to modulate ER stress.

Fig. 3

ASA VI mitigates TBHP-mediated ER stress and promotes mitochondrial biogenesis. (A) Western blot analysis showing the effects of TM and ASA VI on ER stress markers in chondrocytes. Proteins analyzed include GRP78, CHOP, ATF4, phosphorylated PERK (p-PERK), and phosphorylated eIF2α (p-eIF2α). (B) Western blot evidence detailing the influence of Asperosaponin VI on mitochondrial biogenesis markers, including PGC-1α, TFAM, and NRF-2, in the presence of TM. (C) ATP production assay results confirming that ASA VI restores ATP levels, which are compromised by TM-induced ER stress, indicating improved mitochondrial activity. (D) JC-1 staining images displaying the mitochondrial membrane potential. (E) Flow cytometry analysis validating the protective effects of ASA VI on mitochondrial membrane potential under stress conditions. *Note: Sample size for Western blot and flow cytometry experiments is n = 3, and for ATP assay and JC-1 staining, n = 6. Statistical significance indicated by **P < 0.01, **P < 0.001, data presented as mean ± SD

ASA VI activates AMPK and upregulates SIRT3 protein expression

In further elucidating the molecular mechanisms underlying the protective effects of ASA VI from Clematis chinensis, we investigated its influence on AMPK activation and the regulation of SIRT3 protein expression. Our experiments aimed to determine if ASA VI directly influences these critical cellular pathways.

Initially, we analyzed the effect of ASA VI on the phosphorylation of AMPK in chondrocytes exposed to TBHP. The results showed that TBHP treatment alone reduced the phosphorylation of AMPK. However, the addition of ASA VI notably increased AMPK phosphorylation, even in the presence of TM, which typically exacerbates stress responses and would be expected to suppress AMPK activity. This indicates that ASA VI not only restores but actually enhances AMPK activity under stress conditions. Simultaneously, we observed that ASA VI significantly upregulated the protein levels of SIRT3, which plays a key role in mitochondrial biogenesis and function. To further validate the specific roles of AMPK and SIRT3 in the mechanism of action of ASA VI, we employed shRNA to knock down AMPK and SIRT3 in chondrocytes. The results indicate that the effects of ASA VI on both AMPK phosphorylation and SIRT3 protein levels were significantly diminished when either AMPK or SIRT3 was knocked down (Fig. 4B). Collectively, these data provide compelling evidence that ASA VI activates AMPK and upregulates SIRT3, forming a functional axis that likely contributes to its protective effects against TBHP-induced oxidative stress and mitochondrial dysfunction.

Fig. 4

AMPK-SIRT3 axis activation by ASA VI in chondrocytes under oxidative stress. (A) Western blot analysis illustrating the impact of ASA VI on the phosphorylation of AMPK (p-AMPK) and expression of SIRT3 in chondrocytes treated with TBHP. (B) Effects of AMPK and SIRT3 knockdown on ASA VI-induced responses. *Note: Experiment replicates, n = 3. Data are presented as mean ± SD with statistical significance indicated **P < 0.01, **P < 0.001

ASA VI reduces ER stress via the AMPK-SIRT3 pathway, thereby preserving mitochondrial function

We further elucidated the mechanism by which ASA VI from Clematis chinensis alleviates ER stress-induced mitochondrial dysfunction, specifically exploring the role of the AMPK-SIRT3 pathway.

Western blot analysis demonstrated that treatment with ASA VI results in a significant reduction in the expression of ER stress markers such as GRP78, CHOP, and ATF4 in TBHP-treated chondrocytes. This reduction was further enhanced by the activation of the AMPK pathway, suggesting that AMPK activation is a critical mediator of the protective effects of ASA VI against ER stress (Fig. 5A). In addition to reducing ER stress markers, ASA VI also restored mitochondrial function (Fig. 5B), where the expression of mitochondrial biogenesis markers PGC-1α, TFAM, and NRF-2 was significantly increased, even in the presence of AMPK knockdown. This finding indicates that while the AMPK pathway contributes to the mitigation of ER stress, ASA VI may also activate alternative pathways that support mitochondrial biogenesis and function.

Fig. 5

ASA VI Reduces ER stress and restores mitochondrial function via the AMPK-SIRT3 pathway. (A) Western blot analysis of the effects of ASA VI on ER stress-related proteins GRP78, CHOP, ATF4, and the phosphorylation levels of PERK and eIF2α in chondrocytes treated with TBHP and tunicamycin. (B) Western blot showing changes in mitochondrial biogenesis markers PGC-1α, TFAM, and NRF-2 in response to ASA VI in the presence of AMPK knockdown. (C): Measurement of ATP levels demonstrating the effect of ASA VI on cellular energy production in TBHP-treated chondrocytes. (D) JC-1 staining images depicting the influence of ASA VI on mitochondrial membrane potential in TBHP and tunicamycin-treated chondrocytes. (E): Flow cytometry analysis quantifying mitochondrial membrane potential changes in response to ASA VI treatment in the context of ER stress. *Note: n = 3 for western blot and 6 for ATP assay, JC-1 staining, and flow cytometry. Data are represented as mean ± SD. Statistical significance is indicated by ***P < 0.001

The role of AMPK and SIRT3 in mediating the effects of ASA VI was further assessed through shRNA-mediated knockdown experiments. The findings revealed that knockdown of AMPK significantly attenuates the beneficial effects of ASA VI on ER stress markers and mitochondrial function markers. However, when ER stress was chemically inhibited using 4-PBA, some mitochondrial functions, such as ATP production and mitochondrial membrane potential (measured by JC-1 staining), were restored with AMPK activity. This suggests a complex regulatory network where ASA VI may exert protective effects through multiple pathways, although the AMPK-SIRT3 axis plays a central role. Mitochondrial health was further assessed through ATP measurement and MitoSOX Red staining to evaluate oxidative damage (Fig. 5C). ASA VI significantly improved ATP levels and reduced mitochondrial oxidative stress in chondrocytes, underscoring its potential to enhance cellular energy metabolism and reduce oxidative damage under stress conditions. Flow cytometry analysis (Fig. 5E) confirmed the protective effect of ASA VI on mitochondrial membrane potential, highlighting its role in maintaining mitochondrial integrity during ER stress. These comprehensive analyses indicate that ASA VI not only mitigates TBHP-induced ER stress but also enhances mitochondrial function through a synergistic action involving the AMPK-SIRT3 pathway and potentially other signaling mechanisms.

Overall, these results substantiate the multifaceted protective effects of ASA VI against oxidative stress and provide a robust foundation for its potential therapeutic use in diseases characterized by ER stress and mitochondrial dysfunction.

ASA VI weakens ER stress via the AMPK-SIRT3 pathway to inhibit chondrocyte apoptosis

Continuing our investigation into the therapeutic potential of Asperosaponin VI from Clematis chinensis, we focused on its ability to mitigate ER stress and consequent apoptotic and degenerative processes in chondrocytes via the AMPK-SIRT3 signaling pathway. This segment of the study aimed to elucidate the protective effects of Asperosaponin VI against TBHP-induced cellular damage. ASA VI treatment significantly enhanced cell viability in TBHP-treated chondrocytes, as quantified by the CCK-8 assay (Fig. 6A). This improvement in cell survival was closely linked to the upregulation of AMPK, suggesting that activation of this kinase is a pivotal mechanism by which ASA VI alleviates ER stress. The decrease in cell apoptosis was not only due to direct effects on viability but also connected to reductions in apoptotic cell counts (Fig. 6B). Here, ASA VI substantially decreased the number of TUNEL-positive cells, implicating a strong anti-apoptotic effect mediated through the modulation of the AMPK pathway under oxidative stress conditions. Further exploring the impact on cartilage integrity, demonstrates that ASA VI effectively inhibited the degradation of key extracellular matrix proteins such as Collagen II and Aggrecan, while also reducing the expression of MMP-13 (Fig. 6C). This preservation of ECM components underscores the comprehensive protective effects of ASA VI against TBHP-induced stress, extending beyond cell survival to include maintaining structural integrity of the cartilage tissue. Overall, the results presented clearly demonstrate that ASA VI leverages the AMPK-SIRT3 pathway to confer robust protection against oxidative stress-induced apoptosis and degradation in chondrocytes.

Fig. 6

ASA VI inhibits chondrocyte apoptosis and ECM degradation via AMPK-SIRT3 pathway activation. (A): Cell viability measured by CCK-8 assay to assess the protective effects of ASA VI against TBHP-induced cytotoxicity (n = 6 replicates). (B): TUNEL assay conducted to evaluate the anti-apoptotic properties of ASA VI in response to TBHP exposure (n = 6 replicates). (C): Western blot analysis used to determine the influence of ASA VI on the expression levels of key extracellular matrix proteins including Collagen II, Aggrecan, and MMP-13 in TBHP-treated chondrocytes (n = 3 replicates). *Note: Data are presented as mean ± SD. Statistical significance is indicated by **P < 0.01, ***P < 0.001

ASA VI inhibits ER stress via activating the AMPK pathway, mitigating the progression of OA

Our continued exploration of ASA VI's therapeutic potential against OA has revealed its significant role in modulating ER stress and the AMPK-SIRT3 signaling pathway in an in vivo model. Findings illustrated the effect of ASA VI on the expression levels of AMPK and SIRT3. In our DMM rat model, treatment with ASA VI markedly increased the expression of both AMPK and SIRT3, highlighting its role in activating these critical pathways associated with cellular stress responses and mitochondrial function (Fig. 7A). Interestingly, the inhibition of AMPK, achieved through the administration of Compound C, resulted in a significant downregulation of SIRT3, suggesting a regulatory relationship where AMPK activation is essential for SIRT3 expression and function. By examining the ATP and ROS levels in the knee joint, it was found that ASA VI treatment could significantly improve the DMM induced ATP reduction and ROS accumulation, which were offset by Compound C (P < 0.05, Fig. 7B).

Fig. 7

ASA VI ameliorates osteoarthritis progression via the AMPK-SIRT3 pathway in a DMM rat model. (A) Quantitative PCR assessing AMPK and SIRT3 expression changes due to ASA VI treatment (n = 6). (B) Assay kits assessing ATP levels and ROS accumulation treated with ASA VI (n = 6). (C-D) Safranin O and H&E staining to examine cartilage integrity in osteoarthritic rats treated with ASA VI (n = 6, Bar = 50 μm). (E) Evaluation of OARSI scores to measure osteoarthritis progression with ASA VI intervention (n = 6). (F) PCR to monitor ER stress marker levels in response to ASA VI (n = 6). (G) Western Blots to monitor ER stress marker levels in response to ASA VI (n = 6). *Note: Data are presented as mean ± SD. Statistical significance is indicated by ***P < 0.001, compared with Sham group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with DMM group; &P < 0.05, &&P < 0.01, &&&P < 0.001, compared with DMM + ASA VI group

Histological assessments depicted the impact of ASA VI on joint tissue. Safranin O and H&E staining revealed notable improvements in joint architecture with ASA VI treatment compared to the DMM control groups (Figs. 7C, D). These images clearly show reduced cartilage degradation and better preservation of cartilage structure, indicating the efficacy of ASA VI in protecting joint integrity in OA. In terms of clinical parameters, the changes in OARSI scores was observed to quantify the severity of cartilage destruction. Treatment with ASA VI led to significantly lower OARSI scores compared to the DMM-only group, demonstrating its potential to mitigate the structural progression of OA (Fig. 7E).

Further molecular analysis (Fig. 7F) confirmed that ASA VI reduces the levels of key ER stress markers, including GRP78 and CHOP. Figure 7G shows the expression levels of related proteins, which are consistent with the mRNA levels, suggesting that ASV VI treatment inhibits ER stress through AMPK and STRT3. These results, obtained through PCR, provide a biochemical basis for the observed improvements in tissue pathology and OARSI scores, linking the reduction of ER stress to the therapeutic effects of ASA VI. Overall, these findings substantiate that ASA VI acts through the AMPK-SIRT3 pathway to effectively reduce ER stress, thus attenuating the progression of OA. The comprehensive data from histological, molecular, and clinical assessments underscore the promise of ASA VI as a potential therapeutic agent in treating OA by targeting key pathways involved in disease pathogenesis and progression.