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American Journal of Translational Research logoLink to American Journal of Translational Research
. 2021 Dec 15;13(12):13555–13566.

Silencing of TLR7 protects against lipopolysaccharide-induced chondrocyte apoptosis and injury by blocking the p21-mediated JAK2/STAT3 pathway

Dan Liu 1, Wei Liu 2, Limin Jiang 1, Shengjie Dong 1, Weihua Ma 1, Shijun Wang 1, Chao Wan 1
PMCID: PMC8748165  PMID: 35035696

Abstract

Involvement of toll-like receptor 7 (TLR7) in the immune response has been reported in diverse inflammatory diseases. However, the role of TLR7 in the pathogenesis of osteoarthritis (OA) is poorly understood. In this study, we sought to investigate the contribution of TLR7 in regulating chondrocyte apoptosis, inflammation, and degradation of the extracellula matrix (ECM), and its underlying mechanisms. We found that TLR7 expression was increased in cartilage tissues of OA patients and in lipopolysaccharide (LPS)-induced chondrocytes. Silencing of TLR7 alleviated LPS-induced chondrocyte apoptosis, inflammation, and ECM degradation. Mechanistically, TLR7 silencing inhibited the JAK2/STAT3 signaling pathway by inducing p21 expression. Moreover, p21 knockdown and colivein (an activator of JAK2/STAT3 signaling) partially rescued the suppressive role of TLR7 silencing on chondrocyte apoptosis, the inflammatory response, and ECM underproduction. Taken together, our data revealed that knockdown of TLR7 attenuated chondrocyte apoptosis and injury by blocking the p21-mediated JAK2/STAT3 pathway, suggesting that TLR7 may be a therapeutic target in OA.

Keywords: Toll receptor 7, apoptosis, injury, chondrocyte, osteoarthritis

Introduction

Osteoarthritis (OA) is a common chronic disease that affects the joint spaces and muscle strength, and is a leading cause of disability [1]. OA is characterized by degeneration of articular cartilage, and its pathogenic mechanisms include a disorder of anabolic and catabolic gene expression and chondrocyte apoptosis, which results in irreversible joint damage, pain, and synovial inflammation [2]. Chondrocytes, as the only cell type present in cartilage, play a vital role in OA initiation and progression. Synthesis of structural components of the extracellular matrix (ECM) and matrix-degrading proteases occurs in chondrocytes. Damage of the ECM, increased expression of matrix-degrading proteases, and chondrocyte apoptosis are the main causes of degeneration of articular cartilage [3,4]. Furthermore, accumulation of pro-inflammatory cytokines, including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and IL-6, has been found in the synovial tissues and cartilage of OA patients. These cytokines affect cartilage matrix degradation by regulating the production of matrix metalloproteinases (MMPs) in OA [5]. Therefore, it is important to investigate the mechanism of chondrocyte dysfunction for OA therapy.

Toll-like receptor 7 (TLR7), an important member of the TLR family, plays a vital role in immune-mediated inflammatory disorders by triggering the production of downstream signaling cascades [6,7]. Studies show that TLR7 signaling triggers the secretion of chemokines and pro-inflammatory cytokines by immune cell activation, thereby promoting the development of inflammatory disorders, such as systemic lupus erythematosus [8], glomerulonephritis [9], and rheumatoid arthritis [10]. Moreover, TLR7 ligation can affect the expression of cytokines, such as p21 and p-STAT3, and induces pancreatic tumorigenesis progression by regulating stromal inflammation [11]. In acute kidney injury, TLR7 also promotes apoptosis and inflammatory responses [12]. On the other hand, TLRs have been reported to regulate anabolic and catabolic pathways and chondrocyte apoptosis of OA by activating innate immune responses [13], namely TLR4 [14], and TLR2 [15]. Notably, TLR7 expression is upregulated in the lipopolysaccharide (LPS)-induced inflammatory response of post-traumatic OA [16]. Higher expression of TLR7 has been found in synovial membrane samples and blood samples of OA [17]. Furthermore, TLR7 signaling activation by miR-21 results in OA knee pain [18]. However, the molecular mechanism by which TLR7 regulates apoptosis and chondrocyte injury in OA is poorly understood.

JAK2 signaling pathways, especially the classical JAK2/STAT3 pathway are involved in orthopaedic diseases, such as OA of the knee [19], osteosarcoma [20], and rheumatoid arthritis [21]. The related genes of the JAK2/STAT3 signaling pathway (JAK2 and STAT3) are usually activated in chondrocytes of various orthopaedic diseases [22,23]. Studies suggested that the JAK2/STAT3 pathway can regulate apoptosis and the production of MMPs and pro-inflammatory cytokines in chondrocytes to mediate OA progression [24,25]. JAK2/STAT3 signaling activated by IL-1 induces chondrocyte apoptosis and MMP expression in OA [26]. In addition, the JAK2/STAT3 pathway induced by angiotensinogen regulates IL-6-mediated inflammatory responses in OA [27]. Therefore, targeted inhibition of the JAK2/STAT3 pathway is expected to maintain normal physiologic function of chondrocytes and to improve OA.

In our research, we first studied the function of TLR7 in LPS-induced chondrocyte apoptosis, inflammation and ECM degradation, and then investigated the underlying mechanisms. Our results showed that TLR7 was involved in LPS-induced chondrocyte apoptosis, inflammation, and ECM degradation through regulating the p21-mediated JAK2/STAT3 signaling pathway. Our findings may provide a new solution for OA treatment.

Materials and methods

Ethics statement

All procedures were strictly performed accordance with the Animal Care and Use Committee of Yantaishan Hospital (ethic code: wydw20140129). The collection of human articular cartilage tissue was approved by the Ethical Committee of Yantaishan Hospital and followed the guidelines of the Declaration of Helsinki.

Cartilage tissues of patients

OA human articular cartilages were collected from 24 patients who underwent knee arthroplasty surgery (15 women and 9 men aged 64-72 years old). The normal human articular cartilages from 24 donors with no significant clinical and imaging features of OA were obtained from femoral condyles and tibial plateaus at autopsy (10 women and 14 men aged 56-68 years old). Informed consent was obtained from all participants and the study was approved by the Ethic Committee of Yantaishan Hospital.

Cell culture

Human chondrocytes (C28/I2) were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China) and incubated in DMEM/F-12 culture medium (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS: Gibco, Rockville, MD) using a humidified incubator (37°C, 5% CO2). When cells reached 70-80% confluence they were treated with 1, 5 and 10 μg/ml of LPS (Sigma, St. Louis, MO, USA) for 6, 12 and 24 h.

Transfection

TLR7 siRNA plasmid, p21 siRNA plasmid and control siRNA plasmid were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc, California, USA).

TLR7 siRNA sequence: 5’-AACTTCTTGGGCTTAGAACA-3’; p21 siRNA sequence: 5’-GGGTCGAAAACGGCGGCAGACC-3’; siRNA negative control sequence: 5’-UCUUCCGAACGUGUCACGUTT-3’. C28/I2 cells (1×106 cells/well) were cultured in a six-well plate for 24 h at 37°C, and then 2 μg of plasmids transcribing siRNA were transiently transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Besides, cells were transfected with the TLR7 overexpression plasmid (Addgene plasmid #13022) according to the method of Chamberlain et al. [28].

RNA extraction and RT-qPCR

Total RNA was extracted from cartilage tissues and chondrocytes using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, USA). The cDNA was synthesized by reverse transcription reaction and the quantitative reverse transcription PCR (RT-qPCR) was performed using PrimeScriptTM RT-PCR Kit (TaKaRa; Dalian) in QuantStudioTM 12K Flex system (Life Tech, USA). The cycling conditions were as follows: 95°C for 10 min for 1 cycle and 95°C for 15 s and 60°C for 1 min repeated for 40 cycles, followed by a dissociation stage. Relative gene expression was quantified by the cycle threshold (Ct) 2-ΔΔCt method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. Each sample was analyzed in triplicate. Primer sequences are listed in Table S1.

Western blot analysis

The chondrocytes were lysed in RIPA lysis buffer (Sigma, St. Louis, MO, USA) for 15 min. Total protein concentration in chondrocytes was detected using a BCA protein assay kit (Solarbio, Beijing, China). The total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with 5% skimmed milk at room temperature for 1 h. The membranes were incubated with the primary antibody overnight at 4°C and then incubated with goat anti-rabbit horseradish peroxidase antibody (Sigma, St. Louis, MO, USA). The protein complexes were detected by enhanced chemiluminescence (Luminata Forte, Millipore, USA). Chemiluminescent signal was quantified by BandScan 5.0 software. The primary antibodies human anti-TLR7 (1:100, #sc-57463), MMP-3 (1:1000, #sc-21732), MMP-13 (1:1000, #sc-515284), aggrecan (1:1000, #sc-33695) and GAPDH (1:1000, #sc-47724) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p21 (1:1000, #2947), iNOS (1:1000, #39898), STAT3 (1:2000, #4904), phospho-STAT3 (1:2000, #9131), JAK2 (1:1000, #3230), phospho-JAK2 (1:1000, #3771), Bcl-2 (1:1000, #3498), Bax (1:1000, #2772) and cleaved caspase-3 (1:1000, #9661) were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH was used as an internal control.

MTT assay

Human chondrocyte proliferation was measured using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Dojindo Laboratories, Kumamoto, Japan). Chondrocytes (5×103/ml) were inoculated in 96-well plates, cultivated to logarithmic growth phase and then added to the DMEM/F-12 medium supplemented with 5 mg/ml of MTT reagent. MTT formazan crystals were mixed with dimethyl sulfoxide (Sangon, Shanghai, China) and oscillated for 10 min at a low speed. Cell viability was measured using a microplate reader (Thermo Fisher Scientific, Massachusetts, USA) by spectrophotometry at 570 nm.

Apoptosis assay

Human chondrocyte apoptosis was detected using the Cell Apoptosis Detection Kit (Qiagen, Valencia, CA, USA). Briefly, after 24 h cultured with LPS, chondrocytes transfected with plasmids were incubated with 10 μl of annexin V-FITC and propidium iodide (Becton, Heidelberg, Germany) at room temperature for 15 min and the annexin V-FITC staining protocol was followed to measure apoptosis.

ELISA assay

The corresponding ELISA kits were used to measure the contents of IL-6, TNF-α and IL-1β (BioSite, Paris, France). The experimental steps were performed in accordance with the manufacturer’s instructions.

Measurement of nitrite oxide production

The content of nitrite oxide (NO) in human chondrocytes was detected using the Nitric Oxide assay kit (Thermo Fisher Scientific, Massachusetts, USA). The experimental steps were carried out according to the manufacturer’s instructions. The absorbance of NO at 550 nm was determined.

Statistical analysis

Statistical analyses of all data were achieved by using the GraphPad Prism 6 (GraphPad Software, Inc.). Statistical significance was performed using one-way or two-way ANOVA, with Tukey’s post hoc test used for comparisons between the two groups. The data were represented as means ± SEM and each experiment was performed in triplicate in this study. P<0.05 was considered significant.

Results

TLR7 was upregulated in cartilage tissues of OA patients and LPS-induced chondrocytes

Expression of TLR7 was detected in cartilage tissues of OA patients and healthy participants. The results showed that mRNA expression of TLR7 was dramatically higher in cartilage tissues of OA patients than that in healthy participants (Figure 1A). Chondrocytes were treated with different concentrations of LPS (0, 1, 5, and 10 μg/ml) for 6, 12, and 24 h. The results indicated that LPS significantly suppressed the cell viability of chondrocytes in a dose- and time-dependent manner (Figure 1B). Moreover, LPS treatment significantly increased mRNA and protein expression of TLR7 in chondrocytes (Figure 1C, 1D).

Figure 1.

Figure 1

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TLR7 expression is upregulated in cartilage tissues of OA-patients and LPS-stimulated chondrocytes. Chondrocytes were treated with different concentrations of LPS (0, 1, 5, and 10 μg/ml) for 6, 12, and 24 h and cell supernatants were collected after culturing for 48 h. A. TLR7 expression in cartilage tissues of OA patients was detected by RT-qPCR. *P<0.05 compared with healthy controls. B. The viability of chondrocytes after LPS treatment. C. mRNA expression of TLR7 in LPS-induced chondrocytes was detected by RT-qPCR. D. Expression of TLR7 in LPS-induced chondrocytes was detected by western blot assay. C28/I2 chondrocytes were treated with different concentrations of LPS for 12 h; *P<0.05 and **P<0.01 compared with 0 μg/ml LPS treatment; #P<0.05 compared with 5 μg/ml LPS treatment. Columns present the mean ± SEM (n≥3).

Silencing of TLR7 inhibited LPS-induced chondrocyte apoptosis and ECM degradation

To investigate the effect of TLR7 on LPS-induced chondrocytes, cells were transfected with TLR7 siRNA (si-TLR7) or control siRNA (si-NC) for 48 h and then treated with 5 μg/ml LPS for 12 h. The results showed that protein expression of TLR7 was increased significantly after LPS treatment, whereas silencing of TLR7 suppressed the increase of TLR7 expression (Figure 2A, 2B). As expected, the downregulation of TLR7 significantly increased cell viability (Figure 2C). Flow cytometry showed that transfection of si-TLR7 inhibited apoptosis of chondrocytes compared with the si-NC group (Figure 2D). TLR7 knockdown significantly reduced the protein expressions of cleaved caspase-3 (cle-caspase3) and BAX, and enhanced Bcl-2 expression in LPS-induced chondrocytes (Figure 2E). In addition, we further investigated whether TLR7 regulates the ECM degradation in LPS-induced chondrocytes. The results showed that the expressions of MMP-3 and MMP-13 were upregulated by LPS, which were significantly inhibited by TLR7 silencing. Moreover, the inhibiting effect of LPS on aggrecan expression was compensated by TLR7 knockdown (Figure 2F).

Figure 2.

Figure 2

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TLR7 silencing inhibited LPS-induced cell apoptosis and ECM degradation. Chondrocytes were transfected with control siRNA (si-NC, 1 μg/ml) and TLR7 siRNA (si-TLR7, 1 μg/ml) for 48 h and then cells were treated with 5 μg/ml LPS for 12 h. A, B. TLR7 expression in chondrocytes analyzed by western blot assay. C. The viability of chondrocytes evaluated by MTT assay. D. Apoptosis rate detected by flow cytometry analysis. E. Expression of cle-caspase3, BAX and Bcl-2 assayed by western blot analysis. F. Expression of MMP-3, MMP-13 and aggrecan assayed by western blot analysis. GAPDH was used as an internal control. Columns present the mean ± SEM (n≥3); *P<0.05 versus control; #P<0.05 versus LPS group.

Cells were transfected with TLR7 overexpression plasmids (Figure S1A). Overexpression of TLR7 further reduced the LPS-inhibited cell viability (Figure S1B). The upregulation of TLR7 significantly elevated the apoptosis rate in LPS-treated chondrocytes, together with an increase of apoptosis-related protein expression (Figure S1C-E). Furthermore, TLR7 overexpression up-regulated the expressions of MMPs proteins and decreased aggrecan expression in LPS-induced cells (Figure S1F).

Silencing of TLR7 inhibited LPS-induced inflammatory response in chondrocytes

Inflammation plays an essential role in the pathogenesis of OA [31]. Thus, we further verified the effect of TLR7 on inflammatory factors in chondrocytes. As shown in Figure 3A and 3B, the results confirmed that the NO content and the protein level of iNOS were elevated in LPS-induced cells; however, it was markedly reduced by TLR7 knockdown. Moreover, the downregulation of TLR7 significantly suppressed the mRNA expressions of IL-1β, TNF-α, and IL-6 in LPS-stimulated chondrocytes (Figure 3C). the increased levels of IL-1β, TNF-α and IL-6 in LPS-treated chondrocytes were dramatically inhibited by TLR7 silencing (Figure 3D-F). In contrast, overexpression of TLR7 further increased the production of these inflammatory mediators and cytokines induced by LPS in cells (Figure S2).

Figure 3.

Figure 3

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Production of inflammatory mediators and cytokines in LPS-stimulated chondrocytes were reduced by TLR7 knockdown. Chondrocytes were transfected with si-NC and si-TLR7 for 48 h and then cells were treated with 5 μg/ml LPS for 12 h. A. The content of NO was detected in cells. B. The protein level of iNOS in cells was analyzed by western blot assay. C. mRNA expressions of IL-1β, TNF-α, and IL-6 in chondrocytes were assessed by RT-qPCR. D-F. The contents of IL-1β, TNF-α, and IL-6 in chondrocytes were detected through ELISA assay. Columns present the mean ± SEM (n≥3); *P<0.05 versus control; #P<0.05 versus LPS group.

TLR7 modulated the JAK2/STAT3 pathway by suppressing p21 expression

Accumulating evidence demonstrated that p21 and the JAK2/STAT3 pathway were closely associated with OA progression [19,32]. Our results showed that mRNA expression of p21 in cartilage tissues of OA patients was lower than in healthy participants (Figure 4A). LPS treatment decreased p21 expression, whereas TLR7 silence reversed the inhibition effect of LPS on p21 expression (Figure 4B). Pearson correlation analysis indicated that a negative correlation existed between TLR7 expression and p21 expression (Figure 4C). Furthermore, expressions of p-JAK2 and p-STAT3 were upregulated by LPS treatment, but they was suppressed significantly by TLR7 knockdown (Figure 4D, 4F, 4G). These results suggested that TLR7 knockdown elevated the phosphorylation levels of JAK2 and STAT3 and reduced p21 expression in LPS-treated chondrocytes.

Figure 4.

Figure 4

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TLR7 modulated the JAK2-STAT3 pathway by regulating p21 expression. Chondrocytes were transfected with si-NC, si-TLR7, and p21 siRNA (si-p21). Samples were collected after transfection for 48 h. A. mRNA expression of p21 in cartilage tissues of OA patients detected by RT-qPCR. B. p21 expression in LPS-induced chondrocytes detected by western blot assay. C. Relation between TLR7 and p21 evaluated by Pearson analysis. D-G. Protein expressions of p21, p-JAK2/JAK2 and p-STAT3/STAT3 assayed by western blot. GAPDH was used as an internal control. *, stands for P<0.05. Columns present the mean ± SEM (n≥3).

To investigate the relationship between TLR7, p21 and JAK2/STAT3 signaling, p21 siRNA and TLR7 siRNA were co-transfected into LPS-treated cells. The results showed that p21 expression was upregulated in LPS-induced cells transfected with si-TLR7, which was significantly inhibited by p21 knockdown (Figure 4D, 4E). Importantly, the suppressive effect of TLR7 knockdown on the phosphorylation levels of JAK2 and STAT3 was reversed by p21 silencing (Figure 4F, 4G).

P21-mediated JAK2-STAT3 signaling is involved in TLR7-knockdown alleviation of chondrocyte apoptosis and injury

To investigate the role of p21-mediated JAK2-STAT3 signaling in TLR7-knockdown induced alleviation of chondrocyte apoptosis, ECM degradation and inflammation, the cells were transfected with si-p21 and treated with colivelin (an activator of JAK2/STAT3 signaling). The results indicated that cell viability increased by downregulation of TLR7 was significantly reduced by p21 silencing or colivelin (Figure 5A), and that p21 silencing or colivelin significantly increased the apoptosis rate compared with TLR7 silence in LPS-treated cells (Figure 5B). Expressions of BAX and caspase-3 in cells transfected with si-TLR7 were increased by p21 knockdown and colivelin, respectively. Bcl-2 expression was upregulated by TLR7 knockdown in LPS-stimulated cells, but this was reversed by si-p21 or colivelin (Figure 5C, 5D). Expressions of MMP-3 and MMP-13 were reduced notably by TLR7 silencing, whereas p21 silencing or colivelin reversed the suppressive roles of TLR7 knockdown in MMP expression. Furthermore, the expression of aggrecan was decreased by downregulation of p21 and colivelin, (Figure 5E, 5F) respectively. In addition, mRNA expression and the levels of IL-1β, TNF-α, and IL-6 were decreased by silencing of TLR7 in LPS-treated cells, which were recovered by p21 knockdown or colivelin (Figure 5G-J).

Figure 5.

Figure 5

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TLR7 silencing protected against chondrocyte apoptosis and injury by suppressing the p21-mediated JAK2/STAT3 pathway. Chondrocytes were transfected with si-NC, si-TLR7 and si-p21 and pre-treated with 0.25 µmol l-1 colivelin for 2 h. Samples were collected after transfection for 48 h. A. The viability of chondrocytes evaluated by MTT assay. B. Apoptosis rate of chondrocytes detected by apoptosis assay. C, D. Expressions of cle-caspase3, BAX and Bcl-2 assayed by western blot. E, F. Expressions of MMP-3, MMP-13 and aggrecan assayed by western blot analysis. G. mRNA expressions of IL-1β, TNF-α, and IL-6 in chondrocytes assessed by RT-qPCR. H-J. The levels of IL-1β, TNF-α, and IL-6 in chondrocytes were detected by ELISA. GAPDH was used as an internal control. Columns present the mean ± SEM (n≥3); *P<0.05 versus control; #P<0.05 versus LPS; $P<0.05 versus LPS+siTLR7.

Discussion

Degeneration of articular cartilage is the main cause of OA pathogenesis. The only cells present in cartilage are chondrocytes, which play a vital role in the development of cartilage damage and joint dysfunction [33,34]. In normal conditions, anabolic and catabolic functions of chondrocytes exist in a state of equilibrium. The loss of phenotypic stability of chondrocytes contributes to chondrocyte hypertrophy, ultimately resulting in the appearance of OA [35]. Therefore, investigating the function of chondrocytes in OA pathogenesis has received much attention. LPS-stimulated chondrocytes have been widely used as an in vitro model for OA [36,37]. In our study, we also constructed an LPS-stimulated chondrocyte model as an in vitro method for investigating OA pathogenesis. The results showed that LPS treatment caused chondrocyte apoptosis, an inflammatory response, and ECM underproduction.

TLR activation results in the production of chemokines and increased pro-inflammatory cytokines in synovium or cartilage, thereby promoting OA progression [38,39]. Moreover, TLRs can cause articular cartilage dysfunction by involving chondrocyte ECM degradation or apoptosis [40,41]. In this study, we investigated the role of TLR7 in chondrocyte apoptosis, inflammation, and ECM degradation. We found that TLR7 expression was upregulated significantly in cartilage tissues of OA patients and in LPS-induced chondrocyte model. Similarly, it was reported that expression of TLR7 was upregulated in synovial membrane samples of OA patients and LPS-treated joint tissue of mice [16,17]. Furthermore, previous studies confirmed that TLR7 induced the production of TNF-αand type 1 interferons, thereby promoting an inflammatory response and apoptosis of regulatory T cells [42]. TLR7 knockout attenuated cell apoptosis in enterovirus 71-infected mice [43]. Our results demonstrated that LPS-induced inflammation was alleviated by TLR7 knockdown by decreasing the production of pro-inflammatory factors IL-1β, TNF-α and IL-6. TLR7 silencing rescued the LPS-induced chondrocyte apoptosis by regulating apoptosis-related genes. Besides, it has been reported that TLR7 induced the expression of MMPs, such as MMP-9 [44]. In our study, TLR7 aggravated LPS-induced chondrocyte catabolic events by inducing the expression of ECM-related genes. Therefore, we speculated that TLR7 is involved in the OA progression by regulating chondrocyte apoptosis, inflammation, and ECM degradation.

The role of p21 in OA has been reported widely. p21 is not only involved in chondrocyte regeneration and differentiation [45], but also regulates apoptosis, inflammation, and catabolic response in chondrocytes [32,46,47]. In this study, we found that p21 expression was decreased in cartilage tissues of OA patients and in LPS-induced chondrocytes. Importantly, p21 expression was inhibited by TLR7. A study shows that p21 expression is regulated by TLR7 ligation in pancreatic ductal adenocarcinoma [11]. Moreover, our results further indicated that the downregulation of p21 expression reversed the effect of TLR7 knockdown on LPS-induced apoptosis, the inflammatory response, as well as ECM degradation. Thus, we believed that silencing of TLR7 alleviated chondrocyte apoptosis and injury by regulating p21 expression.

The JAK2/STAT3 pathway plays an important role in the processes of OA [24] and is associated with p21 or TLR7 in various diseases [11,32]. In OA patients and rat model, expression of JAK family members JAK1 and JAK3 was significantly upregulated [48]. Inhibition of JAK2/STAT3 effectively protects against OA in mice [49]. Our results indicated that LPS activated the JAK2/STAT3 pathway in chondrocytes. On the other hand, JAK2/STAT3 signaling is activated by TLR7 or p21 to mediate catabolic and inflammatory responses and apoptosis. Evidence suggested that TLR7 ligation regulated the expressions of p21, STAT3, and p-STAT3, and accelerated inflammatory response and apoptosis of pancreatic cancer [11]. p21 silencing caused the increased STAT3 phosphorylation and thereby upregulated the expression MMPs and accelerated the chondrocyte catabolic response [32]. In this study, silencing of TLR7 inhibited LPS-induced JAK2/STAT3 signaling by regulating p21 expression. Importantly, colivelin, an activator of JAK2/STAT3 signaling, reversed the effect of TLR7 knockdown on apoptosis, the inflammatory response and ECM underproduction. Based on the above analysis, we uncovered novel findings that silencing of TLR7 protected against LPS-induced chondrocyte apoptosis, inflammation and ECM degradation by the p21-mediated JAK2/STAT3 pathway (Figure 6). Although our results confirmed the effect of TLR7 on LPS-induced chondrocytes, limitations still existed in this study. How TLR7 reduced the p21 expression requires further study. Besides, the regulation mechanism of TLR7 in in vivo models needs to be investigated to support the results from in vitro experiments. These limitations will be solved in our further research.

Figure 6.

Figure 6

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The molecular mechanism of TLR7 regulating apoptosis, ECM degradation, and inflammation in the LPS-induced chondrocytes.

Conclusion

We identified that TLR7 expression was increased in cartilage tissues of OA patients and in LPS-induced chondrocytes. Silencing of TLR7 attenuated LPS-induced chondrocyte apoptosis, the inflammatory response, and ECM degradation. Additionally, p21 was involved in TLR7 regulation of the JAK2/STAT3 signaling pathway. Taken together, these results suggested that silencing of TLR7 protected against LPS-induced chondrocyte apoptosis, inflammation, and ECM degradation by blocking the p21-mediated JAK2/STAT3 pathway. This finding provides new insights into mechanisms of OA.

Acknowledgements

Wei Liu, Limin Jiang and Shengjie Dong: Conceptualization, Software, Dan Liu and Chao Wan: Data curation, Methodology, Writing-Original draft preparation. Shijun Wang: Investigation. Weihua Ma: Software.

Disclosure of conflict of interest

None.

Supporting Information

ajtr0013-13555-f7.pdf (501.5KB, pdf)

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