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. 2024 Jul 9;17(7):e13881. doi: 10.1111/cts.13881

Protective effect of clusterin against interleukin‐1β‐induced apoptosis and inflammation in human knee osteoarthritis chondrocytes

Tachatra Ungsudechachai 1, Jiraphun Jittikoon 1, Sittisak Honsawek 2, Wanvisa Udomsinprasert 1,✉
PMCID: PMC11233271  PMID: 38982592

Abstract

Chondrocyte apoptosis is recognized as one of the pathological features involved in cartilage degeneration driving the onset and progression of knee osteoarthritis (OA). This study aimed to determine the molecular mechanism underlying the effect of clusterin (CLU), anti‐apoptotic molecule, in human knee OA chondrocytes. Primary knee OA chondrocytes were isolated from the cartilage of knee OA patients and divided into five groups: (1) the cells treated with interleukin (IL)‐1β, (2) CLU alone, (3) a combination of IL‐1β and CLU, (4) LY294002 (PI3K inhibitor) along with IL‐1β and CLU, and (5) the untreated cells. Production of apoptotic, inflammatory, anabolic, and catabolic mediators in knee OA chondrocytes was determined after treatment for 24 h. Our in vitro study uncovered that CLU significantly suppressed the production of inflammatory mediators [nitric oxide (NO), IL6, and tumor necrosis factor (TNF)‐α] and apoptotic molecule (caspase‐3, CASP3). CLU significantly upregulated messenger ribonucleic acid (mRNA) expressions of anabolic factors [SRY‐box transcription factor‐9 (SOX9) and aggrecan (ACAN)], but significantly downregulated mRNA expressions of IL6, nuclear factor kappa‐B (NF‐κB), CASP3, and matrix metalloproteinase‐13 (MMP13). Anti‐apoptotic and anti‐inflammatory effects of CLU were mediated through activating PI3K/Akt signaling pathway. The findings suggest that CLU might have beneficial effects on knee OA chondrocytes by exerting anti‐apoptotic and anti‐inflammatory functions via PI3K/Akt pathway, making CLU a promising target for potential therapeutic interventions in knee OA.

Study highlights.

  • WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Apoptosis in chondrocytes plays a crucial role in knee OA pathogenesis, leading to joint inflammation and eventually to cartilage degeneration. Clusterin exerts its anti‐apoptotic and anti‐inflammatory actions in various cells.

  • WHAT QUESTION DID THIS STUDY ADDRESS?

This study investigated the molecular mechanism underlying the effect of CLU, anti‐apoptotic molecule, in human knee OA chondrocytes.

  • WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Anti‐apoptotic and anti‐inflammatory effects of CLU were mediated through PI3K/Akt signaling pathway by suppressing production of various inflammatory and catabolic mediators at both protein and mRNA levels including caspase‐3, NO, IL6, TNF‐α, NF‐κB, as well as MMP13, while also upregulating mRNA expressions of anabolic factors including SOX9 and ACAN.

  • HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

The beneficial effect of CLU mediated through PI3K/Akt could potentially offer a promising avenue for discovering a novel therapeutic target aimed at slowing down cartilage degradation in knee OA.

INTRODUCTION

Knee osteoarthritis (OA), a chronic degenerative joint disease, is one of the most common forms of arthritis in the elderly where the patients will develop severe pain as well as declined physical activity and eventually require joint replacement. The increasing prevalence of knee OA means that there is a rising need for improving diagnosis and treatment of the disease. 1 Considering there remains a lack of specific indicators and treatments that are effective in stopping or reversing the degenerative process, there is still a significant group of knee OA patients in whom these treatments do not provide adequate pain relief. 2 From this, it is important to note that elucidating molecular mechanisms underlying the pathogenesis of knee OA may pave the way for the discovery of new therapeutic targets for knee OA. Despite extensive research efforts, the specific order of pathologic alterations occurring in knee OA is still uncertain. Of various pathological mechanisms known to induce knee OA etiology, chondrocyte apoptosis is recognized as a critical pathological feature of knee OA. 3 , 4 It is noteworthy that treating key aspect of apoptosis in chondrocytes holds great promise for painlessness and structure modification in knee OA patients.

Of various molecules playing regulatory roles in apoptosis, clusterin (CLU) acting as a protective molecule against cellular apoptosis and inflammatory response would have great potential as a novel therapeutic molecule. As to its general role, CLU, also known as apolipoprotein J, is an ATP‐independent chaperon protein that inhibits protein aggregation and precipitation. 5 On the basis of its property, CLU has been functionally implicated in several physiological processes including cell growth, lipid transportation, cell differentiation, and cellular senescence. Through activation of several signaling molecules, including complement mediators, immunoglobulins, transforming growth factor‐beta (TGF‐β), phosphorylated nuclear factor kappa‐B inhibitor‐alpha (IκBα), and activated B‐cell lymphoma (BCL)‐2‐like protein (BAX), and phosphoinositide 3‐kinase (PI3K)/protein kinase B (Akt) pathway, CLU plays a regulatory role in cellular proliferation, inflammation, and immune response. 5 , 6 , 7 , 8 , 9 Given its overexpression in various types of tissues including the articular cartilage and the synovium, 10 , 11 there are numerous studies demonstrating increased circulating CLU levels in several pathological conditions. 12 , 13 , 14 With regard to knee OA pathology, a previous study has drawn attention to search biomarkers for identifying OA progression and has demonstrated that CLU levels in serum and joint fluid of OA patients were significantly associated with joint space narrowing. 15 Supporting this, previous studies have depicted that reduced circulating CLU levels were significantly associated with knee OA severity. 16 , 17 In human knee OA synoviocytes, an experimental study has demonstrated that expression and secretion of CLU was regulated by tumor necrosis factor‐alpha (TNF‐α), 17 a pro‐inflammatory cytokine. In addition to this, CLU has been shown to alleviate interleukin (IL)‐1β‐induced inflammation via suppressing PI3K/Akt pathway in human knee OA synoviocytes, thereby confirming the possible role of CLU in inflammatory response. 18

Although significant involvement of circulating CLU in knee OA severity and its role in inflammation in knee OA synoviocytes have already been explored, 15 , 16 , 17 , 18 no studies are currently available on its protective effect against inflammation and apoptosis in knee OA chondrocytes. Accordingly, the purpose of this study was to elucidate the molecular mechanism underlying the protective effect of CLU against inflammation and apoptosis in chondrocytes isolated from knee OA patients.

METHODS

Cell isolation and culture

The articular cartilage specimens were obtained from 10 knee OA patients (age range: 58–70 years, 60% females) who underwent total knee replacement between 2019–2020 at the Department of Orthopedics, Faculty of Medicine, Chulalongkorn University. All knee OA patients did not have any additional underlying conditions, such as diabetes mellitus, advanced liver or renal diseases, histories of long‐term steroid treatment, other forms of arthritis, previous knee injury and/or infection, malignancy, or other chronic inflammatory diseases. The articular cartilage pieces were carefully dissected from the femoral condyles, minced in a sterile hood, and then digested in a 1% pronase (Sigma, USA) solution for 1 hour (h) at 37 degree Celsius (°C) and 5% carbon dioxide (CO2). The pronase solution was removed after the designated incubation time, and the digested pieces of cartilage were rinsed with culture media. Subsequently, they were subjected to digestion with a 3.5% collagenase solution (Sigma, USA) overnight. The digestion of cartilage was filtered through a 40 micrometers (μm) cell strainer and then centrifuged at 112 g for 10 minutes (mins). This resulted in a chondrocyte pellet that was suspended in Dulbecco's modified Eagle medium (DMEM) F/12 (Hyclone® Laboratories, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone® Laboratories, USA), streptomycin, penicillin G, and ascorbic acid (Sigma, USA). The suspension was transferred to a 75 square centrimeter (cm2) tissue culture flask (Nunc, Roskilde, Denmark) containing 10 milliliter (mL) of supplemented DMEM F/12. The cells were maintained at 37°C in humidified atmosphere with 5% CO2. The culture media was changed every 3 days until the cells reached confluence. The cells from the initial passage (P1) were utilized for subsequent experiments. To determine the signaling mechanism behind the effect of CLU on chondrocyte apoptosis, chondrocytes at the first or second passage (P1‐P2) were stimulated in the following manner: The experiment involved different treatments on chondrocytes: untreated chondrocytes, chondrocytes treated with IL‐1β at a concentration of 10 ng/mL, chondrocytes treated with recombinant CLU at various concentrations [1, 5, 10, or 20 nanogram (ng)/mL], chondrocytes treated with a combination of IL‐1β and CLU, and chondrocytes pretreated with a PI3K inhibitor (LY294002) at a concentration of 10 μM, followed by treatment with IL‐1β and CLU.

3‐(4,‐dimethylthiazol‐2‐y)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay

To investigate the effect of CLU on cell viability and its potential to mitigate IL‐1β‐induced cytotoxicity in knee OA chondrocytes, an MTT assay was performed. In brief, knee OA chondrocytes were seeded at a concentration of 5.0 × 104 cells/mL into 96‐well plates (Thermo Fisher Scientifics, Waltham, MA, USA), cultured in serum‐free DMEM F/12 (Hyclone® Laboratories, USA), and then incubated at 37°C, 5% CO2, and 95% humidity for 24 h. Following the treatment based on the experimental grouping, the culture medium was removed, and the adherent chondrocytes were washed twice with 100 microliter (μL) of phosphate‐buffered saline (PBS) in each well. Following that, 90 μL of culture medium and 10 μL of 5 milligram (mg)/mL MTT solution (Sigma, USA) were added into each well. The 96‐well plate was then incubated at 37°C for 4 h or until the color developed. After the incubation period, the MTT solution was removed, and 100 μL of dimethylsulfoxide (DMSO) (Sigma, USA) was subsequently added. The well plate was placed on the horizonal shaker for 10 mins until the purple crystals were completely dissolved. The absorbance of each well was measured using a microplate reader (Infinite M200 pro, Tecan, Switzerland).

Caspase‐3 activity assay

To determine the effect of CLU on apoptosis in chondrocytes, the activity of caspase‐3 in cell lysate derived from knee OA chondrocytes at a concentration of 2.0 × 104 cells/mL in 24‐well plates after treatment for 24 h was measured using Caspase‐3 Fluorometric Assay Kit (Raybiotech, Georgia), as per the manufacturer's instructions. The absorbance of fluorescence was measured using a microplate reader (Infinite M200 pro, Tecan, Switzerland), with an excitation wavelength of 400 nm and an emission wavelength correction at 505 nm.

Total Akt/phosphorylated Akt enzyme‐linked immunosorbent assay (ELISA)

To investigate the potential protective effect of CLU against apoptosis on knee OA chondrocytes via the activation of the PI3K/Akt pathway, the levels of total Akt and phosphorylated Akt in cell lysates derived from knee OA chondrocytes at a concentration of 2.0 × 104 cells/mL in 24‐well plates after treatment for 24 h were assessed using a commercial ELISA kit (Abcam, Cambridge, UK), following the manufacturer's instructions.

Total nitric oxide (NO) assay

To assess the effect of CLU on reactive oxygen species (ROS) production, the levels of total NO in the culture medium from knee OA chondrocytes at a concentration of 2.0 × 104 cells/mL in 24‐well plates after stimulation for 24 h were measured using the Nitrate/Nitrite Fluorometric Assay Kit, following the manufacturer's instructions (Abnova, San Francisco, CA, USA). The fluorescence of each sample was measured using an automated microplate reader (Infinite M200 pro, Tecan, Switzerland), with excitation at 375 nm and emission at 417 nm.

Enzyme‐linked immunosorbent assay (ELISA)

After treatment for 24 h, the culture medium was collected from knee OA chondrocytes seeded at a concentration of 2.0 × 104 cells/mL into 24‐well plates. Levels of inflammatory mediators including IL6 and TNF‐α were quantitatively measured using a commercially available sandwich ELISA kits (Biolegend, San Diego, CA), according to the manufacturer's instructions.

Quantitative real‐time polymerase chain reaction (qPCR)

After treatment for 24 h, total ribonucleic acid (RNA) was extracted from cell lysate from knee OA chondrocytes at a concentration of 2.0 × 104 cells/mL in 24‐well plates using RNeasy Mini kit (Qiagen, Hilden, Germany), and complementary deoxyribonucleic acid (cDNA) was reverse transcribed using a TagMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions. To examine relative mRNA expressions of anabolic, catabolic, inflammatory, and apoptotic genes including SRY (Sex‐Determining Region Y)‐Box 9 (SOX9), aggrecan (ACAN), IL6, nuclear factor kappa‐B (NF‐κB), matrix metalloproteinase‐13 (MMP13), caspase‐3 (CASP3) in knee OA chondrocytes, qPCR was performed using qPCR Green Master Mix HRox (Biotechrabbit GmbH, Hennigsdorf, Germany) on StepOnePlus Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers used for SOX9, ACAN, IL6, NF‐κB, MMP13, and CASP3 and glyceraldehyde 3‐phosphate dehydrogenase (GADPH) amplification are demonstrated in Table S1. Relative mRNA expressions of those genes normalized to the GADPH gene as an internal control were determined using 2−∆∆Ct method.

Statistical analysis

The statistical analyses were performed using statistical package for the social sciences (SPSS) Statistics version 26.0 (SPSS, Chicago, IL, USA) and GraphPad Prism 9.0 (GraphPad Software, CA, USA). For normally distributed variables, Student's t‐test and ANOVA with a Tukey post hoc test were used for comparing two independent groups and more than two groups, respectively. On the other hand, for abnormally distributed variables, the Mann–Whitney U test and Kruskal–Wallis H test were employed for comparing two independent groups and more than two groups, respectively. A Chi‐square test was conducted to assess the presence of statistically significant differences among groups for categorical variables. The data are presented using mean ± standard deviation (SD). Statistical significance was determined by a p‐value of less than 0.05 for all analyses.

Ethics statement

The experimental protocol was approved by the Ethical Committee on Human Research of the Faculty of Dentistry/Faculty of Pharmacy, Mahidol University and conducted in conformity with the guidelines of the Declaration of Helsinki (MU‐DT/PY‐IRB 2019/074.2511). In the present study, all participants were adults and were provided with information about the purpose of the study. Written informed consents were obtained from all participants before their participation.

RESULTS

Effect of CLU on cell viability on knee OA chondrocytes

Cell viability was investigated following treatment with different concentrations of CLU in knee OA chondrocytes. As depicted in Figure 1a, there were no significant differences observed among knee OA chondrocytes treated with various concentrations of CLU (1, 5, 10, 20 μg/mL), in comparison to the untreated cells. More specifically, the viability percentages of knee OA chondrocytes after CLU treatment at concentrations of 1, 5, 10 and 20 μg/mL were 105.40% ± 0.74, 112.70% ± 3.91, 129.30% ± 19.94, and 123.30% ± 0.07, respectively. According to the results, knee OA chondrocytes showed the highest viability when treated with 10 μg/mL CLU. This treatment was then used for the subsequent experiments.

FIGURE 1.

FIGURE 1

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Effect of CLU on cell viability on knee OA chondrocytes. (a) Viability of knee OA chondrocytes following treatment with various concentrations of CLU. (b) Viability of knee OA chondrocytes after treatment with 10 ng/mL IL‐1β, both with and without the addition of 10 μg/mL CLU (**p < 0.01).

The effect of CLU on IL‐1β‐induced cytotoxicity in knee OA chondrocytes was assessed by measuring cell viability through the MTT assay. The analysis revealed that the viability of knee OA chondrocytes treated with CLU was significantly higher than that of the cells treated with IL‐1β (p = 0.0078) (Figure 1b).

Effect of CLU on Akt phosphorylation in knee OA chondrocytes

Total Akt and phosphorylated Akt levels in knee OA chondrocytes among different treatment groups are depicted in Figure 2. As expected, there were no significant differences in total Akt levels among the different treatment groups (Figure 2a). In the CLU‐treated cells, phosphorylated Akt levels were observed to be significantly higher than those of the untreated group (p < 0.001) (Figure 2b). Following treatment with LY294002 (PI3K inhibitor) in combination with IL‐1β and CLU, phosphorylated Akt levels in knee OA chondrocytes were significantly lower than those in the CLU‐treated group (p = 0.0124) (Figure 2b).

FIGURE 2.

FIGURE 2

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Effect of CLU on Akt phosphorylation in knee OA chondrocytes after treatment with various substances. (a) Total Akt in knee OA chondrocytes. (b) Phosphorylated Akt in knee OA chondrocytes (*p < 0.05, ***p < 0.001).

Protective effect of CLU against IL‐1β‐induced caspase‐3 activity

As illustrated in Figure 3a, knee OA chondrocytes treated with IL‐1β exhibited the highest levels of relative caspase‐3 activity, which were significantly greater than those of the untreated cells and knee OA chondrocytes treated with CLU, a combination of IL‐1β and CLU, and LY294002 together with IL‐1β and CLU (p < 0.0001, p < 0.0001, p < 0.001, p = 0.0462, respectively).

FIGURE 3.

FIGURE 3

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Effect of CLU on caspase‐3 activity and production of inflammatory mediators in knee OA chondrocytes after treatment with various substances. (a) Caspase‐3 activity in knee OA chondrocytes. (b) Total NO levels in knee OA chondrocytes. (c) IL6 levels in knee OA chondrocytes. (d) TNF‐α levels in knee OA chondrocytes. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Protective effect of CLU against IL‐1β‐induced production of inflammatory mediators

To determine the production of inflammatory mediators in knee OA chondrocytes induced by IL‐1β, levels of total NO (an indicator of ROS production), IL6, and TNF‐α in the culture medium of knee OA chondrocytes after a 24‐h treatment were measured using Nitrate/Nitrite Fluorometric Assay Kit and ELISA, respectively. As illustrated in Figure 3b, the cells treated with IL‐1β had the highest levels of total NO, which were significantly greater than the levels observed in the CLU‐treated group (p = 0.0013). Besides this, it was observed that the IL‐1β‐treated group showed the highest levels of IL6, which were significantly higher than those of the untreated cells, CLU‐treated group, and IL‐1β‐CLU combination‐treated group (Figure 3c). On the other hand, the CLU treatment resulted in a significant reduction in IL6 levels compared with the combination of IL‐1β and CLU or LY294002 with IL‐1β and CLU (Figure 3c). In addition to IL6, TNF‐α levels in the cells treated with CLU were found to be significantly lower than in those of IL‐1β‐treated group (p = 0.0447) (Figure 3d).

Protective effect of CLU against IL‐1β‐induced upregulation of mRNA expressions of anabolic, catabolic, inflammatory, and apoptotic genes

Relative mRNA expressions of anabolic genes

Relative mRNA expressions of anabolic genes, such as SOX9 and ACAN, in knee OA chondrocytes were investigated after a 24‐h treatment. In Figure 4a, the cells treated with CLU showed a significant increase in relative SOX9 mRNA expression, compared with the untreated cells and the cells stimulated with IL‐1β, IL‐1β‐CLU combination, and LY294002 combined with IL‐1β and CLU (p < 0.0001, p < 0.0001, p < 0.0001, p = 0.0014, respectively).

FIGURE 4.

FIGURE 4

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Effect of CLU on mRNA expressions of anabolic, catabolic, inflammatory, and apoptotic genes in knee OA chondrocytes after treatment with various substances. (a) Relative mRNA expressions of anabolic genes including SOX9 and (b) ACAN. (c) Relative mRNA expression of the catabolic gene including MMP13. (d) Relative mRNA expressions of inflammatory genes including IL6 and (e) NF‐κB. (f) Relative mRNA expression of apoptotic gene including CASP3. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Relative ACAN mRNA expression is depicted in Figure 4b. After treatment with CLU, there was a significant increase in relative ACAN mRNA expression in knee OA chondrocytes compared with the untreated cells and the IL‐1β‐treated group (p = 0.0097, p < 0.0001, respectively). The cells treated with both IL‐1β and CLU showed a significant decrease in ACAN mRNA expression compared with the cells treated with CLU alone (p = 0.0024). In a similar manner, knee OA chondrocytes pretreated with LY294002 showed a significant decrease in ACAN mRNA expression compared with the cells treated with CLU alone (p < 0.001).

Relative mRNA expression of catabolic gene

Considering the significant role of catabolic molecules in cartilage degradation, MMP13 mRNA expression was further examined in knee OA chondrocytes following a 24‐h treatment. As revealed in Figure 4c, the analysis showed a significant increase in relative MMP13 mRNA expression in the IL‐1β‐treated cells compared with both the untreated group and the CLU‐treated cells (p = 0.024, p < 0.001, respectively). Besides this, knee OA chondrocytes treated with LY294002, IL‐1β, and CLU showed a significant rise in relative MMP13 mRNA expression when compared with the cells treated with CLU alone (p = 0.047) (Figure 4c).

Relative mRNA expressions of inflammatory genes

Given that the production of inflammatory mediator is one of the pathological mechanisms driving knee OA progression, the effect of CLU on mRNA expressions of inflammatory genes including IL6 and NF‐κB in knee OA chondrocytes after 24 h of stimulation was further determined. Figure 4d shows that treatment with IL‐1β resulted in a significant increase in relative IL6 mRNA expression compared with the untreated cells (p = 0.0470). On the other hand, CLU stimulation led to a remarkable decrease in relative IL6 mRNA expression when compared with IL‐1β stimulation (p = 0.0155) (Figure 4d). In comparison to the cells treated with CLU alone, knee OA chondrocytes pretreated with LY294002 followed by IL‐1β and CLU showed a significant increase in relative IL6 mRNA expression (p = 0.0172) (Figure 4d). Alternatively, the cells treated with IL‐1β and CLU combination exhibited a significant decrease in relative IL6 mRNA expression compared with the cells treated with LY294002 combined with IL‐1β and CLU (p = 0.0262) (Figure 4d).

Apart from relative IL6 mRNA expression, this study also examined relative mRNA expression of NF‐κB in knee OA chondrocytes after 24 h of treatment. A significant increase in relative NF‐κB mRNA expression was found in knee OA chondrocytes treated with IL‐1β compared with the untreated cells (p = 0.0077) (Figure 4e). It is worth noting that the cells treated with CLU showed a notable decrease in relative NF‐κB mRNA expression in comparison to the cells treated with IL‐1β (p = 0.0075) (Figure 4e). Besides this, relative mRNA expression of NF‐κB was found to be significantly lower in knee OA chondrocytes treated with a combination of IL‐1β and CLU than that of the cells treated with only IL‐1β (p = 0.0077) (Figure 4e).

Relative mRNA expression of gene‐related programmed cell death

As caspase‐3 serves as a crucial mediator of programmed cell death playing a pivotal role in chondrocyte apoptosis, CASP3 mRNA expression in knee OA chondrocytes following a 24‐h stimulation was additionally examined. As depicted in Figure 4f, the cells treated with IL‐1β had the highest relative CASP3 mRNA expression, which was significantly greater than the untreated cells, the CLU‐treated cells, and the cells treated with LY294002 along with IL‐1β and CLU (p < 0.0001, p < 0.0001, p < 0.0001, respectively). Consistent with this, knee OA chondrocytes treated with LY294002 in combination with IL‐1β and CLU exhibited a significant increase in relative CASP3 mRNA expression compared with the cells treated with CLU alone (p < 0.0001) (Figure 4f).

DISCUSSION

Although the molecular mechanism underlying chondrocyte apoptosis in knee OA is not well‐defined, several studies have used inhibitors to prevent cell apoptosis. 19 , 20 , 21 Consequently, research on a new molecule with promising properties as a therapeutic target, such as CLU, might halt disease development and improve knee OA outcomes. The protective effects of CLU were mediated by several signaling pathways. Through PI3K/Akt pathway activation, CLU reportedly protected cells against apoptosis and increased survival. 22 , 23 , 24 , 25 In line with these prior studies, this current study is the first attempt to demonstrate the protective effect of CLU against IL‐1β‐induced cytotoxicity in knee OA chondrocytes. Notably, CLU significantly enhanced the viability of knee OA chondrocytes after treatment with IL‐1β. Although the protective effect of CLU against chondrocyte apoptosis and inflammation through PI3K/Akt signaling pathway has not been previously elucidated, its anti‐apoptotic effects in various cell types have been well‐documented. 22 , 25 , 26 The aforementioned findings suggest the protective role of CLU in knee OA chondrocytes via PI3K/Akt signaling pathway. It is widely acknowledged that mitochondria regulate cell apoptosis by activating caspase family members, including caspase‐3. 27 Typically, caspase‐3 is an executioner for the programmed cell death, which is a downstream effector of inhibiting PI3K/Akt signaling pathway. Liu et al. 8 have uncovered that CLU significantly diminished caspase‐3 activities in H2O2‐induced cardiomyocytes, indicating that CLU can prevent apoptosis via caspase‐3 activation. Similar to this previous study, our study demonstrated that CLU treatment deteriorated caspase‐3 activity in knee OA chondrocytes when compared with the IL‐1β‐treated group. Moreover, PI3K inhibition prominently reduced caspase‐3 activity in the cells co‐treated with IL‐1β and CLU. Our findings suggest that CLU might protect knee OA chondrocytes induced by IL‐1β against apoptosis via reducing caspase‐3 activation. The activation of the PI3K/Akt pathway by CLU in knee OA chondrocytes led to protection against apoptosis. This, in turn, triggered phenotypic changes in the joint, ultimately contributing to the pathogenesis of knee OA. The changes in the articular cartilage are caused by an elevated build‐up of ROS, leading to the generation of NO and inflammatory cytokines such as IL6. 28 , 29 This study showed that total NO levels were significantly elevated in knee OA chondrocytes treated with IL‐1β. Our findings support clinical and experimental data showing that inflammatory stress caused mitochondrial dysfunction, leading to NO overproduction and oxidative stress, chondrocyte apoptosis, and cartilage degeneration in knee OA. 30 Based on this premise, CLU is proposed to protect chondrocytes from inflammatory stress‐induced apoptosis under oxidative stress conditions via activating the PI3K/Akt pathway. In addition to NO production, our findings revealed a significant elevation in IL6 levels in knee OA chondrocytes treated with IL‐1β, as compared with the untreated cells. However, CLU treatment significantly reduced IL6 levels in knee OA chondrocytes compared with IL‐1β treatment. In addition, the IL‐1β‐treated group had significantly higher TNF‐α levels than the CLU‐treated group. Given that IL‐1β is a potential inducer of cartilage deterioration, 31 , 32 , 33 our data indicate that CLU might act as an anti‐inflammatory activator of chondrocyte death.

Aside from the release of NO and inflammatory mediators, the CLU effect mediated through the PI3K/Akt pathway might affect gene expressions of molecules involving knee OA pathogenesis. Sex‐determining region Y‐Box 9 (SOX9) is a key transcription factor regulating gene expressions of cartilage matrix molecules such as aggrecan and type II collagen. 34 , 35 Our results revealed a significant upregulation of SOX9 mRNA expression in knee OA chondrocytes treated with CLU compared with the untreated cells, whereas SOX9 mRNA expression in the cells pretreated with PI3K inhibitor were significantly reduced compared with the CLU‐treated group. Considering knee OA pathogenesis, SOX9 expression has been shown to be related to expressions of cartilage matrix genes including ACAN and MMP13, possibly due to its action as a transcription factor for activation of transcriptional expressions of cartilage matrix genes. 35 In addition to SOX9 mRNA expression, the present study uncovered that ACAN mRNA expression in knee OA chondrocytes treated with CLU was significantly upregulated when compared with the untreated chondrocytes and the cells pretreated with LY294002. Our finding is similar to previous studies denoting that aggrecan expression was activated via PI3K/Akt signaling pathway. 36 , 37 Apart from the upregulation of anabolic genes, the cells treated with CLU showed a significant decrease in MMP13 mRNA expression. These results can be explained by the fact that inflammation can stimulate the production of matrix‐degrading enzymes responsible for cartilage degradation. In the cells pretreated with LY294002, there was a significant increase in relative MMP13 mRNA expression compared with the CLU‐treated group. In accordance with our findings, previous studies have revealed that the PI3K/Akt signaling pathway plays an important role in promoting the production of cartilage matrix molecules such as aggrecan and inhibiting matrix‐degrading enzymes including MMP13. 19 , 38

Apart from measuring total NO and IL6 protein levels, mRNA expressions of inflammatory mediators were further determined in the current study. In accordance with IL6 protein levels, relative IL6 mRNA expression in IL‐1β‐treated chondrocytes was observed to be significantly higher than that in the untreated cells. On the contrary, the chondrocytes treated with CLU had significantly downregulated IL6 mRNA expression compared with IL‐1β‐treated chondrocytes. This finding suggests that CLU could potentially suppress the production of inflammatory cytokine at mRNA and protein levels in knee OA chondrocytes. More specifically, IL6 production was completely decreased after treatment with PI3K inhibitor, thereby suggesting that CLU might have a protective effect against inflammation in knee OA chondrocytes through the PI3K/Akt pathway. Our findings are supported by a previous study, which noted an increase in CLU concentrations in circulation in hamsters during inflammation. 39 Furthermore, accumulating evidence has denoted that CLU reportedly suppressed expressions of inflammatory cytokines including IL6 and NF‐κB. 39 , 40 Supporting this, our additional result uncovered that relative NF‐κB mRNA expression in chondrocytes after treatment with IL‐1β was significantly greater than that in the untreated chondrocytes and CLU‐treated chondrocytes. This finding suggests that CLU could repress NF‐κB mRNA expression in knee OA chondrocytes under inflammatory conditions.

In addition to inflammatory genes, relative CASP3 mRNA expression in IL‐1β‐treated group was significantly greater than that in the cells treated with LY294002 in combination with IL‐1β and CLU, as well as CLU alone. Knee OA chondrocytes treated with LY294002 along with IL‐1β and CLU showed an increase in relative CASP3 mRNA expression compared with the CLU‐treated group. Supporting the observed result of caspase‐3 activity, this can be explained by the fact that CLU might inhibit CASP3, an apoptotic molecule, through the PI3K/Akt signaling pathway. Based on the results derived from our in vitro study, CLU might possess anti‐inflammatory action in knee OA chondrocytes via modulating the production of various inflammatory, anabolic, catabolic, and apoptotic mediators. This regulation was mediated through the activation of PI3K/Akt signaling pathway, thereby highlighting the anti‐inflammatory and anti‐apoptotic effects of CLU in knee OA chondrocytes.

Despite notable findings presented herein, it is important to acknowledge its limitations. The protective role of CLU through PI3K/Akt signaling in knee OA chondrocytes was investigated by isolating the cells from the articular cartilage derived from knee OA patients who underwent total knee replacement. It is possible that the age of patients who underwent knee operations for OA could have an impact on the growth of chondrocytes. Although the isolated yield and growth of chondrocytes were concerned, previous study has normally reported the potential results. 19 In the same way, this study can normally isolate chondrocytes from knee OA cartilage and observe the normal growth of chondrocytes, which were used in our experiments providing intriguing results.

In summary, our study provided supporting evidence on the protective role of CLU against inflammation and apoptosis in knee OA chondrocytes through the PI3K/Akt signaling pathway. The beneficial effect of CLU mediated through PI3K/Akt could potentially offer a promising avenue for discovering a novel therapeutic target aimed at slowing down cartilage degradation in knee OA. Further investigation is required to uncover the additional signaling mechanisms that contribute to its protective role.

AUTHOR CONTRIBUTIONS

T.T., J.J., and W.U. wrote the manuscript. W.U. designed the research. T.T., J.J., and W.U. performed the research. T.T., J.J., and W.U. analyzed the data. J.J., S.H., and W.U. contributed new reagents/analytical tools.

FUNDING INFORMATION

This research project has been funded by Mahidol University [Fundamental Fund: fiscal year 2024 by National Science Research and Innovation Fund (NSRF)].

CONFLICT OF INTEREST STATEMENT

The authors declared no competing interests for this work.

Supporting information

Table S1.

CTS-17-e13881-s001.docx (15KB, docx)

Ungsudechachai T, Jittikoon J, Honsawek S, Udomsinprasert W. Protective effect of clusterin against interleukin‐1β‐induced apoptosis and inflammation in human knee osteoarthritis chondrocytes. Clin Transl Sci. 2024;17:e13881. doi: 10.1111/cts.13881

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

REFERENCES

  • 1. GBD 2021 Osteoarthritis Collaborators . Global, regional, and national burden of osteoarthritis, 1990–2020 and projections to 2050: a systematic analysis for the global burden of disease study 2021. Lancet Rheumatol. 2023;5(9):e508‐e522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Imani F, Patel VB. Therapeutic challenges for knee osteoarthritis. Anesth Pain Med. 2019;9(3):e95377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Salucci S, Falcieri E, Battistelli M. Chondrocyte death involvement in osteoarthritis. Cell Tissue Res. 2022;389(2):159‐170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Chen B, Wang L, Xie D, Wang Y. Exploration and breakthrough in the mode of chondrocyte death ‐ a potential new mechanism for osteoarthritis. Biomed Pharmacother. 2024;170:115990. [DOI] [PubMed] [Google Scholar]
  • 5. Wilson MR, Easterbrook‐Smith SB. Clusterin is a secreted mammalian chaperone. Trends Biochem Sci. 2000;25(3):95‐98. [DOI] [PubMed] [Google Scholar]
  • 6. Falgarone G, Chiocchia G. Chapter 8 Clusterin: a multifacet protein at the crossroad of inflammation and autoimmunity. Adv Cancer Res. 2009;104:139‐170. [DOI] [PubMed] [Google Scholar]
  • 7. Choi B, Kang SS, Kang SW, et al. Secretory clusterin inhibits osteoclastogenesis by attenuating M‐CSF‐dependent osteoclast precursor cell proliferation. Biochem Biophys Res Commun. 2014;450(1):105‐109. [DOI] [PubMed] [Google Scholar]
  • 8. Liu X, Meng L, Li J, et al. Secretory clusterin is upregulated in rats with pulmonary arterial hypertension induced by systemic‐to‐pulmonary shunts and exerts important roles in pulmonary artery smooth muscle cells. Acta Physiol (Oxf). 2015;213(2):505‐518. [DOI] [PubMed] [Google Scholar]
  • 9. Xiu P, Dong X, Dong X, et al. Secretory clusterin contributes to oxaliplatin resistance by activating Akt pathway in hepatocellular carcinoma. Cancer Sci. 2013;104(3):375‐382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Connor JR, Kumar S, Sathe G, et al. Clusterin expression in adult human normal and osteoarthritic articular cartilage. Osteoarthr Cartil. 2001;9(8):727‐737. [DOI] [PubMed] [Google Scholar]
  • 11. Min BH, Jeong SY, Kang SW, et al. Transient expression of clusterin (sulfated glycoprotein‐2) during development of rat pancreas. J Endocrinol. 1998;158(1):43‐52. [DOI] [PubMed] [Google Scholar]
  • 12. Kropáčková T, Mann H, Růžičková O, et al. Clusterin serum levels are elevated in patients with early rheumatoid arthritis and predict disease activity and treatment response. Sci Rep. 2021;11(1):11525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13. Jongbloed W, van Dijk KD, Mulder SD, et al. Clusterin levels in plasma predict cognitive decline and progression to Alzheimer's disease. J Alzheimers Dis. 2015;46(4):1103‐1110. [DOI] [PubMed] [Google Scholar]
  • 14. Miners JS, Clarke P, Love S. Clusterin levels are increased in Alzheimer's disease and influence the regional distribution of Aβ. Brain Pathol. 2017;27(3):305‐313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Ritter SY, Collins J, Krastins B, et al. Mass spectrometry assays of plasma biomarkers to predict radiographic progression of knee osteoarthritis. Arthritis Res Ther. 2014;16(5):456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Kropackova T, Sleglova O, Ruzickova O, Vencovsky J, Pavelka K, Senolt L. Lower serum clusterin levels in patients with erosive hand osteoarthritis are associated with more pain. BMC Musculoskelet Disord. 2018;19(1):264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Ungsudechachai T, Honsawek S, Jittikoon J, Udomsinprasert W. Clusterin is associated with systemic and synovial inflammation in knee osteoarthritis. Cartilage. 2021;13(1_suppl):1557S‐1565S. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Ungsudechachai T, Honsawek S, Jittikoon J, Udomsinprasert W. Clusterin exacerbates interleukin‐1β‐induced inflammation via suppressing PI3K/Akt pathway in human fibroblast‐like synoviocytes of knee osteoarthritis. Sci Rep. 2022;12(1):9963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. Lopez‐Armada MJ, Carames B, Cillero‐Pastor B, et al. Phosphatase‐1 and ‐2A inhibition modulates apoptosis in human osteoarthritis chondrocytes independently of nitric oxide production. Ann Rheum Dis. 2005;64(7):1079‐1082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Nuttall ME, Nadeau DP, Fisher PW, et al. Inhibition of caspase‐3‐like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro. J Orthop Res. 2000;18(3):356‐363. [DOI] [PubMed] [Google Scholar]
  • 21. Zhang Y, Cai W, Han G, et al. Panax notoginseng saponins prevent senescence and inhibit apoptosis by regulating the PI3KAKTmTOR pathway in osteoarthritic chondrocytes. Int J Mol Med. 2020;45(4):1225‐1236. [DOI] [PubMed] [Google Scholar]
  • 22. Trougakos IP, Lourda M, Antonelou MH, et al. Intracellular clusterin inhibits mitochondrial apoptosis by suppressing p53‐activating stress signals and stabilizing the cytosolic Ku70‐Bax protein complex. Clin Cancer Res. 2009;15(1):48‐59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Hoter A, Rizk S, Naim HY. The multiple roles and therapeutic potential of molecular chaperones in prostate cancer. Cancers (Basel). 2019;11(8):1194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Ranney MK, Ahmed ISA, Potts KR, Craven RJ. Multiple pathways regulating the anti‐apoptotic protein clusterin in breast cancer. Biochim Biophys Acta. 2007;1772(9):1103‐1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Ammar H, Closset JL. Clusterin activates survival through the phosphatidyl‐inositol 3‐kinase/Akt pathway. J Biol Chem. 2008;283(19):12851‐12861. [DOI] [PubMed] [Google Scholar]
  • 26. Jun HO, Kim DH, Lee SW, et al. Clusterin protects H9c2 cardiomyocytes from oxidative stress‐induced apoptosis via Akt/GSK‐3beta signaling pathway. Exp Mol Med. 2011;43(1):53‐61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Musumeci G, Loreto C, Carnazza ML, Martinez G. Characterization of apoptosis in articular cartilage derived from the knee joints of patients with osteoarthritis. Knee Surg Sports Traumatol Arthrosc. 2011;19(2):307‐313. [DOI] [PubMed] [Google Scholar]
  • 28. Glyn‐Jones S, Palmer AJR, Agricola R, et al. Osteoarthritis. Lancet. 2015;386(9991):376‐387. [DOI] [PubMed] [Google Scholar]
  • 29. Rasheed Z, Akhtar N, Haqqi TM. Advanced glycation end products induce the expression of interleukin‐6 and interleukin‐8 by receptor for advanced glycation end product‐mediated activation of mitogen‐activated protein kinases and nuclear factor‐κB in human osteoarthritis chondrocytes. Rheumatology. 2010;50(5):838‐851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Komori T. Cell death in chondrocytes, osteoblasts, and osteocytes. Int J Mol Sci. 2016;17(12):2045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Wang C, Tan Z, Niu B, et al. Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia. elife. 2018;7:e37673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Sun J, Wei X, Wang Z, et al. Inflammatory milieu cultivated Sema3A signaling promotes chondrocyte apoptosis in knee osteoarthritis. J Cell Biochem. 2018;119(3):2891‐2899. [DOI] [PubMed] [Google Scholar]
  • 33. Litherland GJ, Dixon C, Lakey RL, et al. Synergistic collagenase expression and cartilage collagenolysis are phosphatidylinositol 3‐kinase/Akt signaling‐dependent. J Biol Chem. 2008;283(21):14221‐14229. [DOI] [PubMed] [Google Scholar]
  • 34. Zhang Q, Ji Q, Wang X, et al. SOX9 is a regulator of ADAMTSs‐induced cartilage degeneration at the early stage of human osteoarthritis. Osteoarthr Cartil. 2015;23(12):2259‐2268. [DOI] [PubMed] [Google Scholar]
  • 35. Fukui N, Ikeda Y, Ohnuki T, et al. Regional differences in chondrocyte metabolism in osteoarthritis: a detailed analysis by laser capture microdissection. Arthritis Rheum. 2008;58(1):154‐163. [DOI] [PubMed] [Google Scholar]
  • 36. Beier F, Loeser RF. Biology and pathology of rho GTPase, PI‐3 kinase‐Akt, and MAP kinase signaling pathways in chondrocytes. J Cell Biochem. 2010;110(3):573‐580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37. Yin W, Park J‐I, Loeser RF. Oxidative stress inhibits insulin‐like growth factor‐I induction of chondrocyte proteoglycan synthesis through differential regulation of phosphatidylinositol 3‐kinase‐Akt and MEK‐ERK MAPK signaling pathways. J Biol Chem. 2009;284(46):31972‐31981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE. Interleukin‐1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c‐jun N‐terminal kinase, and nuclear factor κB: differential regulation of collagenase 1 and collagenase 3. Arthritis Rheum. 2000;43(4):801‐811. [DOI] [PubMed] [Google Scholar]
  • 39. Hardardóttir I, Kunitake ST, Moser AH, et al. Endotoxin and cytokines increase hepatic messenger RNA levels and serum concentrations of apolipoprotein J (clusterin) in Syrian hamsters. J Clin Invest. 1994;94(3):1304‐1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. Won JC, Park CY, Oh SW, Lee ES, Youn BS, Kim MS. Plasma clusterin (ApoJ) levels are associated with adiposity and systemic inflammation. PLoS One. 2014;9(7):e103351. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1.

CTS-17-e13881-s001.docx (15KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

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