Rabbit monoclonal antibodies targeting LRP1 (#2703-1; 1:1,000) were obtained from Epitomics, Inc. (Burlingame, CA, USA), while monoclonal rabbit antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #2118; 1:1,000), phosphorylated NF-κB subunit p65/RelA (#3033; 1:1,000) and mouse monoclonal antibodies against IκB (#4814; 1:1,000) and were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). In addition, rabbit polyclonal antibodies targeting Akt (#BS2987; 1:700), phospho (p)-Akt (#BS4009; 1:600), B-cell lymphoma 2 (Bcl-2; #BS1511; 1:600), Bcl-2-associated X protein (Bax; #BS1030; 1:600) and caspase-3 (#BS1518; 1:700) were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA), and rabbit polyclonal antibodies against MMP-13 (#sc-30073; 1:600) and nitric oxide synthase (iNOS; #sc-67003; 1:600) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Dulbecco's modified Eagle's medium (DMEM)/F-12 nutrient mixture and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-goat IgG secondary antibodies were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Recombinant rat TNF-α was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). An NF-κB inhibitor (Bay 11–7082) was purchased from the Beyotime Institute of Biotechnology (Haimen, China). This study was approved by the Ethics Committee of Wuhan University (Wuhan, China).

Primary chondrocytes were isolated from the knee joint of rats (age, 7 days) using a sequential proteinase and collagenase digestion technique (13), and the cells released by enzymatic digestion were cultured to a density of 3.0×10⁶ cells/ml in DMEM/Ham's F-12 media (1:1) supplemented with 10% FBS (complete media). The cells were cultured at 37°C in humidified air with 5% CO₂. The cells were maintained at 20–80% confluency, and cells between passages 2 and 3 were used for the following experiments. Subsequently, the cells were plated onto a six-well plate at a density of 2.0×10⁵ cells/ml. The chondrocytes were starved in DMEM overnight, after which pretreatment with the Bay 11–7082 NF-κB inhibitor (10 µM) was conducted 30 min prior to the addition of TNF-α. With regard to the signaling experiments, chondrocytes were treated with TNF-α (30 ng/ml) for 30 min. For the quantitative polymerase chain reaction (qPCR) experiments, the cells were treated with TNF-α (30 ng/ml) for 12 h.

Knockdown of LRP1 expression in the chondrocytes was achieved using a lentivirus vector expressing a rat LRP1-targeting shRNA (shLRP1; Genechem, Shanghai, China), according to the manufacturer's instructions. The target nucleotide sequence of the oligoduplexes was as follows: 5′-GCA UUG GCG UGC AGC UUA AUU-3′. A non-targeting control (NTC) oligoduplex was used as a negative control. At a multiplicity of infection of 20:1 for 12 h after transfection, the medium was exchanged for fresh medium, and the cells were cultured for a further 48–72 h. mRNA was harvested after 48 h and protein was harvested after 72 h. The effects of LRP1 knockdown on the expression of various indices were determined using qPCR and western blot analysis.

Protein was extracted from stably transfected cells using a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology), after which 30 µg protein per lane was loaded onto a 10% SDS-PAGE gel. Proteins were transferred to a polyvinylidene difluoride membrane, and blocked with 5% non-fat dry milk in Tris-buffered saline and Tween 20 (TBST). The membranes were incubated with the primary antibodies overnight at 4°C, and subsequently washed three times with TBST. Next, the membranes were incubated with the HRP-conjugated secondary antibodies (1:5,000) in TBST for 2 h at room temperature. After washing with TBST buffer, the western blots were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA), and exposed to Kodak X-ray film (Kodak, Rochester, NY, USA).

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. First-strand cDNA synthesis was conducted using an RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's instructions. Target gene mRNA expression levels were determined using a SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) qPCR system, with the following protocol: Denaturation for 10 sec at 95°C, followed by 40 cycles of 15 sec at 95°C and 60 sec at 60°C. Forward and reverse primer sequences are presented in Table I. GAPDH from the same sample was used as the internal control, and the relative contents of the copy numbers of the target gene mRNA were calculated. The qPCR experiments were conducted independently and in triplicate.

Control and shLRP1 chondrocytes were pretreated with Bay 11–7082 (10 µM) for 2 h and incubated with or without TNF-α (30 ng/ml) for a further 24 h. The effect of TNF-α on the expression levels of MMP-13 and COL2A1 secreted by the chondrocytes in the culture medium was assessed using a rat MMP-13 and COL2A1 ELISA kit (Wuhan Elabscience Biotechnology Co., Ltd., Wuhan, China), according to the manufacturer's instructions.

Control and shLRP1 chondrocytes were incubated with or without TNF-α (30 ng/ml) for 24 h. Apoptotic cells were detected using a TUNEL assay (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's instructions. Briefly, the cells were fixed in 4% paraformaldehyde solution at 4°C for 1 h and washed three times with fresh phosphate-buffered saline (PBS). The slides were incubated with 50 µl terminal deoxynucleotidyl transferase and TUNEL Reaction mixture for 1 h at 37°C in the dark. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (Beyotime Institute of Biotechnology) solution was added to stain cell nucleus for 5 min. After washing with PBS for 5 times with 3 min interval, slides were covered with anti-fade mounting medium. Three fields were obtained from each group using a fluorescence microscope (Nikon Corporation, Tokyo, Japan).

Statistical evaluations were conducted using SPSS software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. The statistical significance between the differences in the mean values was assessed using one-way analysis of variance, where P<0.05 was considered to indicate a statistically significant difference.

In order to knockdown LRP1 expression, RNA interference technology was used to transfect the chondrocytes with a lentivirus vector expressing an LRP1-targeting shRNA. The transfection efficiency was ~80%, as determined by fluorescence microscopy. Thus, the expression levels of LRP1 protein and mRNA were substantially knocked down to 70% of the control (control, 1.020±0.01826; NTC, 0.9875±0.02175; shLRP1, 0.3125±0.0175; Fig. 1A and B). Furthermore, the cell-surface mRNA expression levels of TNFR1 were measured using qPCR, and the TNFR1 mRNA expression levels were determined to be higher in the shLRP1 cells when compared with the control cells (control, 1.010±0.01958; NTC, 1.003±0.01931; shLRP1, 1.560±0.057; Fig. 1C). These results indicated that the mRNA knockdown was specific for LRP1.

NF-κB is a key mediator of TNF-α signal transduction in chondrocytes (4). Therefore, the effect of LRP1 on TNF-α-induced NF-κB activation and IκB phosphorylation was evaluated in chondrocytes using western blot analysis. IκB binds NF-κB, retaining it in the cytoplasm. Phosphorylation of IκB promotes IκB the degradation and release of activated NF-κB. In order to evaluate the role of the IKK-NF-κB signaling pathway in mediating inflammation, the basal condition of the IKK-NF-κB pathway was determined by treating the chondrocytes with an NF-κB-specific inhibitor (Bay 11–7082) for 30 min, with no exogenous stimulants. No statistically significant differences were observed between the control and shLRP1 groups with regard to the basal levels of the phosphorylated NF-κB subunit p65/RelA and IκB protein (Fig. 2A). Next, the activity of the IKK-NF-κB pathway was analyzed in chondrocytes treated with Bay 11–7082 for 30 min prior to TNF-α stimulation. Protein expression of phosphorylated p65/RelA was observed in the control and LRP1-knockdown cells exposed to TNF-α, although increased levels of the phosphorylated NF-κB subunit p65/RelA were observed and IκB was undetectable in the shLRP1 cells. Furthermore, Bay 11–7082 was demonstrated to inhibit the upregulation of phosphorylated p65/RelA in the shLRP1 chondrocytes treated with TNF-α (Fig. 2B).

Experiments were conducted to determine whether the knockdown of LRP1 promoted the enhanced transcription of iNOS, as a result of NF-κB activation. The results revealed that the mRNA expression levels of iNOS were elevated in the shLRP1 chondrocytes treated with TNF-α, as compared with the control cells. Furthermore, the mRNA and protein expression levels of iNOS were significantly reduced in the Bay 11–7082-pretreated shLRP1 chondrocytes exposed to TNF-α (shLRP1 + TNF-α, 2.29±0.12; control + TNF-α, 1.737±0.03; shLRP1 + TNF-α + Bay 11–7082, 1.67±0.084; Fig. 2C–E), providing evidence for the activation of NF-κB in the LRP1 knockdown cells.

The IKK-NF-κB pathway is one of a number of key TNF-α signal transducers in chondrocytes, and the signaling pathway reportedly regulates the transcription of MMP-13 (4). Therefore, the effects of LRP1 on TNF-α-induced MMP-13 expression were investigated. In the absence of exogenous stimulants, no statistically significant alterations were observed in the base protein expression levels of MMP-13 between the control and shLRP1 groups. However, MMP-13 expression was substantially increased in the shLRP1 chondrocytes stimulated with TNF-α (Fig. 3A). ELISA analysis was used to determine the MMP-13 expression levels in the chondrocyte culture medium. As shown in Fig. 3B, the extracellular levels of MMP-13 were increased in the TNF-α-induced shLRP1 chondrocyte medium. Furthermore, the results indicated that the levels of MMP-13 were significantly reduced in the Bay 11–7082-pretreated shLRP1 cells that were exposed to TNF-α (control, 1.125±0.54; shLRP1 + TNF-α, 2.675±0.088; control + TNF-α, 1.533±0.059; shLRP1 + TNF-α + Bay 11–7082, 1.221±0.05). In addition, extracellular levels of COL2A1 were significantly downregulated in the TNF-α-induced shLRP1 chondrocytes (control, 145.8±6.85; shLRP1 + TNF-α, 57.44±4.12; control + TNF-α, 94.26±3.754; shLRP1 + TNF-α + Bay 11–7082, 89.01±3.1; Fig. 3C), which was reversed with Bay 11–7082 pretreatment. These results indicated that LRP1 inhibited TNF-α-induced MMP-13 expression and indirectly upregulated the extracellular levels of COL2A1 via the inhibition of NF-κB.

Since LRP1 was demonstrated to markedly affect TNF-α-induced inflammation, the effects of LRP1 on apoptosis were investigated by analyzing the levels of Akt, Bcl-2, Bax and caspase-3 using western blot analysis and a TUNEL assay. Activation of Akt and Bcl-2 has been previously associated with the inhibition of the apoptotic cleavage of caspase-3 (14). To clarify this mechanism, chondrocytes were cultured in serum free medium for 12 h and treated with TNF-α or DMEM for an additional 2 h. The results indicated that TNF-α-induced shLRP1 chondrocytes exhibited significantly upregulated expression levels of caspase-3 (control, 0.1197±0.002; shLRP1 + TNF-α, 0.294±0.006; control + TNF-α, 0.117±0.003; shLRP1, 0.224±0.003) and reduced ratios of p-Akt/Akt (control, 1.5±0.049; shLRP1 + TNF-α, 0.71±0.051; control + TNF-α, 1.326±0.061; shLRP1, 0.963±0.05; Fig. 4A) and Bcl-2/Bax (control, 3.145±0.05; shLRP1 + TNF-α, 1.309±0.048; control + TNF-α, 2.745±0.056; shLRP1, 1.574±0.041; Fig. 4B). These results indicated that LRP1 knockdown enhanced Bax and caspase-3 expression, while reducing Bcl-2 expression, which may serve an antiapoptotic function in chondrocytes.

To further confirm the antiapoptotic effect of LRP1 knockdown in vitro, control and shLRP1 chondrocytes were starved in DMEM overnight, after which the cells were treated with TNF-α (30 ng/ml) or DMEM for 24 h. The percentage of TUNEL-positive cells was subsequently determined microscopically. As shown in Fig. 5A and B, the proportion of apoptotic cells increased significantly in the shLRP1 chondrocytes (control, 3±0.96%; shLRP1 + TNF-α, 35.11±4.53%; control + TNF-α, 6.55±0.96%; shLRP1, 21.25±2.01%).