Phosphatase and tensin homolog accelerate the inflammatory injury and glycolysis of chondrocytes through TP53 protein-mediated p38MAPK pathway activation in a high-glucose environment

Culture and identification of primary chondrocytes

The primary chondrocytes used in this study were derived from previously preserved rat cartilage tissue. The cartilage tissue collection procedure was as follows. Wistar rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were euthanized through cervical dislocation. Bilateral hip joints were excised under sterile conditions, and the knee cartilage was quickly transferred to a sterilized container with an appropriate amount of preservation solution and stored in a −80°C refrigerator. The previously preserved rat cartilage samples were cut into small pieces that measured 3 mm³ and rinsed three times with phosphate-buffered saline (PBS) that contained 1% double antibody and precooled. Then, 0.25% trypsin (C0201, BiYunTian Biotechnology, Shanghai, China) was added, and the samples were digested in a 37°C incubator for 30 min. After the digestion solution was aspirated and discarded, 0.2% collagenase type II (LS004186, Worthington Biochemical Corporation, New Jersey, USA) was added for digestion at 37°C in a constant temperature shaker for 10 h. Then, 2 mL of the culture solution was added to terminate the digestion. Cartilage fragments were filtered using a 200-mesh filter screen, and the filtrate was centrifuged at 1000 rpm for 10 min. The supernatant was discarded and washed two times with Dulbecco’s modified Eagle’s medium (DMEM) (11965092, Gibco, Grand Island, New York, USA). Subsequently, cells were seeded in cell culture flasks and cultured in a carbon dioxide incubator. To ensure the absence of mycoplasma contamination, cells were tested using commercial mycoplasma detection kits (C0301S, BiYunTian Biotechnology, Shanghai, China) following the manufacturer’s instructions. Only mycoplasma-free cultures were used for subsequent experiments. In addition, primary chondrocytes were identified through morphological observation and cell surface marker detection. The morphology, growth characteristics, and cell density of chondrocytes were observed using an inverted phase contrast microscope. Chondrocytes were determined through the expression and localization of type II collagen and proteoglycan in cells.

SB203580 (10007974, Cayman Chemical, Ann Arbor, Michigan, USA), a selective inhibitor of the p38 mitogen-activated protein kinase (p38MAPK), was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 20 mM, which was stored at −20°C. For experimental treatments, the stock solution was further diluted to achieve a final concentration of 10 μM in a complete cell culture medium. Cells were pre-incubated with SB203580 for 30 min before being transfected.

Diabetic osteoarthritis (DOA) rat model

A total of 20 male Wistar rats (8 weeks old, 200–250 g; Beijing Vital River Laboratory Animal Technology Co., Ltd.) were housed in a clean and well-ventilated animal environment at 20 ± 2°C, with a relative humidity of 60–70% and a day/night cycle of 12 h/12 h. They had free access to water and food. The mice were randomly assigned to four groups, with five animals in each group, as follows: (1) control group: 6 weeks of normal diet, (2) DOA group: 30 mg/kg of streptozotocin (once a week for 2 weeks) was intraperitoneally injected after 4 weeks of a high-fat diet, (3) Scramble group: 20 μL of negative control was injected into the knee joint cavity of DOA rats every 7 days, and (4) small interfering (siRNA) against PTEN (si-PTEN) group: 20 μL of PTEN siRNA (3 μg/rat) were injected into the knee joint cavity of DOA rats once every 7 days. Considering animal welfare, the mice were euthanized 4 weeks after the injection of scramble or siRNA, and knee cartilage tissue samples were collected for subsequent research. Before euthanasia, the mice were deeply anesthetized through an isoflurane mask. The animals were euthanized through cervical dislocation when their respiratory rate slowed down, and they lost reflex movement.

Cell transfection

The logarithmic phase chondrocytes were seeded into six-well plates at 5 × 10⁵ cells/well, and the medium was discarded after 24 h of culture in DMEM. The glucose concentration of DMEM was 5.5 mmo/L in the control group and 25 mmo/L in the HG group.^[17-19] The si-PTEN, siRNA against tumor suppressor protein p53 (TP53) (si-TP53), and their negative controls (scramble) were provided by Santa Cruz Biotechnology (PTEN: sense: 5'-GCA CAA GAG GCC CUA GAU UTT-3' and antisense: 5'-AAU CUA GGG CCU CUU GUG CTT-3'; TP53, sense: 5'-CUA CUU CCU GAA AAC GTT-3'). The negative controls’ siRNA lacked significant sequence homology to any gene (sense 5'-UUC UCC GAACGU GUC ACG UTT-3', antisense 5'-ACG UGA CAC GUUCGG AGA ATT-3'). The plasmid DNA (pcDNA)-PTEN, pcDNA-TP53, and their negative controls (vector) were purchased from Thermo Fisher Scientific (Invitrogen, Carlsbad, California, USA). Cells were transfected in OptiMEM medium (Gibco, USA) using Lipofectamine 2000 transfection reagent (Invitrogen, USA) as recommended by the manufacturer.

Quantitative real-time polymerase chain reaction (RT-qPCR)

The total RNA in cells was isolated using TRIzol (Cat # 15596026, Invitrogen). Single-stranded complementary DNA was synthesized with the PrimeScript Reagent Kit (RR047A, Takara Bio Inc.). Then, qPCR (StepOnePlus, 4376305, Thermo Fisher Scientific) was conducted using an SYBR Premix Ex TaqTM Kit (RR420A, Takara Bio Inc.). The primers (Sangon Biotech) used in this study were as follows: PTEN forward, 5'-TGC AGT ATA GAG CGT GCA GA-3', reverse, 5'-TAG CCT CTG GAT TTG ACG GC-3'; TP53, 5'-AGA GAC CGC CGT ACA GAA GA-3', reverse, 5'-CTG TAG CAT GGG CAT CCT TT-3'; b-actin forward, 5'-ACA GGC ATC GTG ATG GAT TCT-3', reverse, 5'-CAG CAG TGG TGA AGT TAT-3. b-actin was used as the internal reference. The relative expression levels were normalized using the 2−ΔΔCt method.

Western blot

Radio immunoprecipitation assay lysis buffer (R0278, Sigma-Aldrich) was used to extract proteins from cells and tissues. Then, the proteins loaded on 10% polyacrylamide gel were separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE). The membrane was incubated with primary antibody (Abcam, UK) at 4°C overnight, and the membrane was blocked with 5% non-fat milk at room temperature for 2 h before incubation. The primary antibodies are as follows: β-actin (1:2500, ab8226), PTEN (1:10000, ab32199), MMP13 (1:3000, ab39012), collagen II (1:1000, ab34712), hexokinase2 (HK2) (1:1000, ab209847), lactate dehydrogenase A (LDHA) (1:5000, ab101562), TP53 (1:3000, ab32049) and p38 mitogen-activated protein kinase (p38MAPK) (1:1000, phospho T180, ab178867). Then, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:2000, ab6721) or goat anti-mouse IgG (1:3000, ab205719) for 1 h at room temperature. Electrochemiluminescence reagents (34580, Thermo Fisher Scientific) were dropped onto the membrane, and the membrane was exposed to a chemiluminescence imaging system (1708280, Bio-Rad Laboratories). Then, images were collected using Image Lab software (1700145EN, Bio-Rad Laboratories), and the bands were quantitatively analyzed with ImageJ software (version 1.x). Internal reference protein b-actin was selected for standardization to correct the difference in sample loading.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

The chondrocytes were seeded into 96-well cell culture plates with a cell density of 2 × 10⁴ cells/well, and the edges were filled with sterile PBS buffer. After 24 h of culture, 20 μL of MTT solution (96603, Takara Bio) was added to each well. After 4 h of culture, the supernatant of the medium was discarded, and 150 μL DMSO solution was added to each well. The blue-violet crystals in the cells were fully dissolved by shaking at room temperature in the dark for 10 min. The microplate reader (Evolution 201, Thermo Fisher Scientific) was set, and the absorbance value of each well was measured at a wavelength of 490 nm.

Enzyme-linked immunosorbent assay

The secretion levels of interleukin-6 (IL-6) (E10669m, BiYunTian Biotechnology, Shanghai, China) and tumor necrosis factor-alpha (TNF-a) (E10668m, BiYunTian Biotechnology, Shanghai, China) were determined using the corresponding kits. Standards were prepared in accordance with the manufacturer’s instructions. Then, they were diluted proportionally and labeled as different groups on 96-well plates. The 96-well plates were covered and kept in the dark for 30 min. Thereafter, the liquid was removed. After washing with a washing solution, the plates were left to stand for 30 s, and the liquid was removed again. This process was repeated five times. Subsequently, 150 μL of HRP-labeled specific detection antibodies were added to the loading well, and the plates were washed again five times after incubation for 30 min. Subsequently, chromogenic agents A and B (50 μL) were added, followed by the termination reagent after incubation for 15 min. After zeroing in on the blank hole of the microplate reader (Molecular Devices, M5290047, California, USA), a wavelength of 450 nm was selected to determine the absorbance value.

Flow cytometry

Chondrocytes were digested with trypsin and collected into Eppendorf (EP) tubes, and the collected cells were rinsed 2 times with PBS. Then, 500 μL of binding buffer was added to resuspend the cells, and 1 μL of annexin V-fluorescein isothiocyanate (FITC) (556547, BD Biosciences) was added to allow the cells to react in the dark for 15 min at room temperature. After the reaction, 5 μL of phycoerythrin was added, and the cells were incubated for 15 min. Apoptosis was detected through flow cytometry (BD Biosciences).

Glucose uptake determination

When the growth density of primary chondrocytes in the six-well plate reached 80%, they were cultured in sugar-free DMEM for 30 min. Then, 50 μM of 2-NBDG (KFM511, Dojindo Molecular Technologies) was added, and the chondrocytes were placed in an incubator for 30 min. The culture medium was aspirated, and precooled PBS was added to adhere to the wall and wash the cells. Subsequently, the cells were digested with trypsin on ice, and the cell suspension was centrifuged at 500 × g for 5 min in a 4°C centrifuge. After discarding the supernatant, the cell pellet was resuspended with PBS and centrifuged at 500 × g for 5 min at 4°C. Subsequently, the cells were resuspended with PBS and transferred to a new 1.5 mL EP tube. BD Accuri^TM C6 plus flow cytometry FITC channel was used to detect cellular glucose uptake.

Lactic acid content determination

Lactate detection kits (MAK065-1KT, Sigma-Aldrich) were used to detect lactate content in cells and tissues. Chondrocytes with about 85% confluence were seeded in six-well plates, and the same amount of cell culture medium was added. Then, 20 μL of distilled water was added to the blank tube, 20 μL of standard was added to the standard tube, 20 μL of the cell culture solution of each group was added to the corresponding experimental tube, and 1 mL of enzyme working solution and 200 μL of chromogenic agent were added to each tube. The contents of each tube were shaken for 1 min to mix them evenly. After incubation in a 37 °C incubator for 10 min, 2 mL of stop solution was added to each tube. The liquid of each group was transferred to a 96-well plate, and the absorbance of each sample was detected at 530 nm using a microplate reader (Molecular Devices, M5290047, California, USA).

Reactive oxygen species (ROS) content determination

The cells were collected and counted. Then, 1 × 10⁶ cells were aspirated and added to 10 μmol/L of dichlorodihydrofluorescein diacetate (DCFH-DA) working solution (701201, Cayman Chemical) and incubated in a cell incubator for 30 min in the dark. Subsequently, the cells were washed three times with PBS to fully remove the DCFH-DA that did not enter the cells. The cells were resuspended with PBS and transferred to 96-well plates for observation using a fluorescence microscope (488 nm, 525 nm). 4',6-Diamidino-2-phenylindole (D9542, Sigma-Aldrich) is a blue fluorescent dye that can strongly bind to DNA, and it is used for the staining of nuclei. Green fluorescence indicates the level of intracellular ROS. ImageJ software (version 1.53c, National Institutes of Health, USA) was used to analyze fluorescence intensity.

Co-immunoprecipitation (Co-IP) assay

First, 50 μL of protein A/G-beads, 100 μL of PBS, 50 μL of 0.1% bovine serum albumin, and an appropriate amount of corresponding antibody or IgG were prepared into a mixed solution and incubated for 24 h. Then, the mixed solution was centrifuged at 12000 rpm at 4°C for 5 min. The supernatant was aspirated and washed two times with PBS. Then, cell protein lysate was added and incubated on ice for 8 h. After incubation, the samples were centrifuged at 12000 rpm at 4°C for 5 min. The supernatant was aspirated and washed three times with blank protein lysate. Then, the blank lysate was aspirated, and a loading buffer was added. The sample was boiled for 5 min, and SDS–PAGE was performed.

Immunohistochemical assay

The cartilage tissue sections were washed two times with distilled water. Subsequently, 3% hydrogen peroxide was dropped onto the samples, and then the samples were kept in a dark environment for 10 min and washed once with distilled water. Next, the sections were blocked in 10% goat serum for 1 h and then rinsed two times with PBS. The rinsed clean sections were incubated overnight with anti-PTEN antibody (ab31392, Abcam, 1:50) and anti-TP53 antibody (ab32049, Abcam, 1:50) at 4°C. The sections were rinsed three times with PBS and incubated with HRP-labeled secondary antibody (ab6721, Abcam, 1/1000). Thereafter, the sections were rinsed and incubated with 3,30-diaminobenzidine (36201ES03, Sigma-Aldrich) substrate for 2 min. Under the high-power field of a light microscope (DM4000B, Leica Microsystems, Wetzlar, Germany), the brown-yellow particles in cells are considered positive cells. The quantification of positive cells was performed using CellProfiler software (version 4.0.7). Pipelines were designed to detect and classify cells based on staining intensity. Data are presented as the percentage of positive cells relative to the total cell count.

Hematoxylin and eosin (HE) staining

The cartilage tissue sections were dewaxed with absolute ethanol, fixed with 95% ethanol again for 5 min, and then rinsed with distilled water and dried. Subsequently, hematoxylin was added. After staining for 3 min, the sections were rinsed with distilled water and dried. Then, the sections were placed in 1% hydrochloric acid for 3 s and washed with tap water for 10 min to prepare the nucleus blue. After blotting the water with filter paper, eosin (HT110232-5G, Sigma-Aldrich, St. Louis, Missouri, USA) was added for 1 min and then fixed with 95% ethanol for 3 s. Finally, the slides were dehydrated with absolute ethanol, dried, and sealed with a sealing solution. The morphology of the cartilage tissue was observed and compared under a microscope (Olympus IX71; Tokyo, Japan).

Online bioinformatics database analysis

The protein-protein interaction network is a bioinformatic tool for representing and analyzing physical and functional interactions between proteins within cells or between different cells. To further screen out proteins that may interact with PTEN, we searched and analyzed the online bioinformatic database (https://genemania.org/s; https://thebiogrid.org/) in this study.

Statistical analysis

All statistical analyses were performed using the Statistical Package for the Social Sciences software (SPSS) (version 21.0; SPSS, Chicago, Illinois, USA). Quantitative data derived from three independent experiments were expressed as mean ± standard deviation. The Shapiro–Wilk test was utilized to verify the data’s normal distribution, while Levene’s test was employed to assess the homogeneity of variance. Comparisons among multiple groups were conducted using analysis of variance (ANOVA) with a least significant difference post hoc analysis. P < 0.05 indicated statistical significance.

PTEN silencing inhibits HG-induced injury in chondrocytes

Compared with the control group, the protein expression of PTEN was increased in the HG-treated primary chondrocytes, while transfection of si-PTEN inhibited the increase of PTEN messenger RNA (mRNA) (P < 0.05), [Figure 1a] and protein (P < 0.05), [Figure 1b and 1c] expression caused by HG. The results of the cell behavior study showed that under an HG environment, the viability of chondrocytes was weakened (P < 0.05), [Figure 1d], apoptosis was increased (P < 0.05), [Figure 1e and f], and the secretion of inflammatory factors IL-6 and TNF-a was increased (P < 0.05), [Figure 1g]. In addition, the expression of MMP13 protein was increased, and the expression of collagen II protein was decreased in chondrocytes stimulated by HG (P < 0.05), [Figure 1h and i], but the knockdown of PTEN expression significantly reversed cell damage caused by HG. These results suggest that PTEN aggravates the inflammatory injury of chondrocytes in an HG environment.

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Figure 1:

Effect of PTEN on the injury of chondrocytes in an HG environment. The HG-stimulated primary chondrocytes were transfected with scramble or small interfering PTEN for 48 h. (a) mRNA expression of PTEN. (b and c) PTEN protein expression. (c and d) Cell viability. (e and f) Cell apoptosis. (g) IL-6 and TNF-a secretion levels. (h and i) Protein expression of MMP13 and collagen II. n = 3. ^✶P < 0.05. HG: High glucose, PTEN: Phosphatase and tensin homolog, IL-6: Interleukin-6, TNF-a: Tumor necrosis factor-alpha, MMP13: Matrix metalloproteinase 13, mRNA: Messenger RNA, FITC: Fluorescein isothiocyanate, PI: Propidium iodide.

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PTEN silencing inhibits HG-induced glycolysis in chondrocytes

Compared with the control group, the glucose uptake of primary chondrocytes in an HG environment was significantly increased (P < 0.05), [Figure 2a], the content of lactate production in cells was increased (P < 0.05), [Figure 2b], and the knockdown of PTEN inhibited the excessive glycolysis and lactate accumulation of chondrocytes. In addition, HG-induced a large amount of ROS production in chondrocytes, but ROS generation was decreased under the action of PTEN siRNA (P < 0.05), [Figure 2c and d]. The expression of the glycolytic molecules HK2 (P < 0.05), [Figure 2e and f] and LDHA protein (P < 0.05), [Figure 2g and h] was increased in chondrocytes treated with HG, and PTEN silencing reversed the promoting effect of an HG environment on the increased expression of HK2 and LDHA protein.

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Figure 2:

Effect of PTEN on the glycolysis of chondrocytes in an HG environment. The HG-stimulated primary chondrocytes were transfected with scramble or small interfering PTEN for 48 h. (a) Glucose uptake by cells was detected using a 2-NBDG fluorescent probe. (b) Lactate detection kits were used to detect lactic acid content. (c and d) ROS generation (DCFH-DA fluorescent probe). (e and h) Glycolytic molecules HK2 and LDHA protein expression. n = 3. ^✶P < 0.05. HG: High glucose, PTEN: Phosphatase and tensin homolog, ROS: Reactive oxygen species, HK2: Hexokinase2, LDHA: Lactate dehydrogenase A, DAPI: 4’,6-diamidino-2-phenylindole, DCFH-DA: 2’,7’-dichlorofluorescin diacetate. Scale bar = 25 μm (400×).

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PTEN upregulates TP53 expression

By searching the online bioinformatics database, we found that TP53 may be a binding protein of PTEN [Figure 3a] and further determined the binding relationship between PTEN and TP53 through Co-IP experiments [Figure 3b]. The qPCR results showed that the overexpression of PTEN promoted TP53 mRNA expression, while the transfection of small interfering PTEN inhibited TP53 mRNA expression (P < 0.05), [Figure 3c]. These results suggest that PTEN can bind to TP53 and positively regulate the expression of TP53.

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Figure 3:

TP53 is a binding protein of PTEN. (a) Potential binding proteins of PTEN. (b) Binding relationship of PTEN and TP53 was validated through Co-IP. (c) Primary chondrocytes were transfected with pcDNA-PTEN or small interfering -PTEN for 48 h. Then, qPCR was performed to detect TP53 mRNA expression. n = 3. ^✶P < 0.05. PTEN: Phosphatase and tensin homolog, TP53: Tumor suppressor protein p53, mRNA: Messenger RNA, Co-IP: Co-immunoprecipitation, qPCR: Quantitative real-time polymerase chain reaction, pcDNA: Plasmid DNA, IP: Immunoprecipitation, IB: Immunoblotting.

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TP53 silencing inhibits HG-induced injury and glycolysis in chondrocytes

The qPCR and Western blot results showed that the mRNA (P < 0.05), [Figure 4a] and protein (P < 0.05), [Figure 4b and c] expression of TP53 was increased in HG-treated primary chondrocytes, while the transfection of si-TP53 reversed the expression of TP53. In addition, we observed that in the HG group, cell viability was decreased (P < 0.05), [Figure 4d], apoptosis was increased (P < 0.05), [Figure 4e and f], inflammatory factor secretion was increased (P < 0.05), [Figure 4g], MMP13 protein expression was increased (P < 0.05), [Figure 4h and i], and collagen II protein expression was decreased (P < 0.05), [Figure 4h and i]. Meanwhile, TP53 silencing significantly improved HG-induced inflammatory injury. In HG-treated chondrocytes, glucose uptake was increased (P < 0.05), [Figure 4j], lactate production was increased (P<0.05), [Figure 4k], ROS generation was increased (P < 0.05), [Figure 4l and m], glycolytic molecule HK2 and LDHA protein expression was increased (P < 0.05), [Figure 4n and o], and the transfection of si-TP53 reversed the effect of HG treatment.

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Figure 4:

Effects of TP53 on the injury and glycolysis of chondrocytes in an HG environment. The HG-stimulated primary chondrocytes were transfected with scramble or small interfering-TP53 for 48 h. (a) mRNA expression of TP53. (b and c) Protein expression of TP53. (d) Cell viability. (e and f) Cell apoptosis. (g) Content of IL-6 and TNF-a. (h and i) Protein expression of MMP13 and collagen II. (j) Glucose uptake (2-NBDG fluorescent probe). (k) Lactate detection kits were used to detect lactic acid content. (l and m) ROS generation. (n and o) Protein expression of HK2 and LDHA (glycolytic molecules). n = 3. ^✶P < 0.05. HG: High glucose, TP53: Tumor suppressor protein p53, IL-6: Interleukin-6, TNF-a: Tumor necrosis factor-alpha, MMP13: Matrix metalloproteinase 13, ROS: Reactive oxygen species, HK2: Hexokinase2, LDHA: Lactate dehydrogenase A, mRNA: Messenger RNA, IL: Interleukin, DAPI: 4’,6-diamidino-2-phenylindole, FITC: Fluorescein isothiocyanate, PI: Propidium iodide. Scale bar = 25 μm (400×).

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PTEN promotes HG-induced injury and glycolysis in chondrocytes through TP53

The HG-treated primary chondrocytes were transfected with either si-PTEN alone or together with pcDNA-TP53 to further verify whether PTEN plays a regulatory role through TP53. The Western blot results showed that compared with that in the HG group, the expression of TP53 protein in primary chondrocytes treated with HG transfected with siPTEN alone was decreased, while pcDNA-TP53 reversed the inhibitory effect of si-PTEN on TP53 protein expression (P < 0.05), [Figure 5a and 5b]. Compared with the HG group, cell viability was enhanced (P < 0.05), [Figure 5c] in the primary chondrocytes treated with HG transfected with si-PTEN alone, apoptosis was reduced (P < 0.05), [Figure 5d and e], IL-6 secretion was decreased (P < 0.05), [Figure 5f], and MMP13 protein expression was reduced (P < 0.05), [Figure 5g and h]. Meanwhile, TP53 overexpression significantly reversed the effect of si-PTEN, resulting in further aggravation of cell inflammatory injury. In addition, compared with the HG group, the primary chondrocytes treated with HG transfected with si-PTEN alone exhibited reduced glucose uptake (P < 0.05), [Figure 5i], lactate production (P < 0.05), [Figure 5j], ROS generation (P < 0.05), [Figures 5k and l], and glycolytic molecule HK2 protein expression (P < 0.05), [Figure 5m and n]. Meanwhile, TP53 overexpression significantly reversed the effect of si-PTEN and caused excessive glycolysis in chondrocytes.

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Figure 5:

PTEN affects the injury and glycolysis of chondrocytes by upregulating TP53 in an HG environment. (a and b) Protein expression of TP53. (c) Cell viability. (d and e) Cell apoptosis. (f) IL-6 secretion levels. (g and h) MMP13 protein expression. (i) Glucose uptake. (j) Lactate detection kits were used to detect lactic acid content. (k and l) ROS generation. (m and n) Glycolytic molecules HK2 protein expression. n = 3. ^✶P < 0.05. HG: High glucose, PTEN: Phosphatase and tensin homolog, TP53: Tumor suppressor protein p53, IL-6: Interleukin-6, MMP13: Matrix metalloproteinase 13, ROS: Reactive oxygen species, HK2: Hexokinase2, DAPI: 4’,6-diamidino-2-phenylindole, PI: Propidium iodide. Scale bar = 25 μm (400×).

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PTEN promotes the HG-induced injury and glycolysis of chondrocytes by activating the p38MAPK pathway through the upregulation of TP53

Compared with the control group, the phosphorylation level of p38MAPK protein in the HG-treated primary chondrocytes was significantly increased, and the transfection of si-PTEN reduced the phosphorylation level of p38MAPK protein, but the overexpression of TP53 reversed the inhibitory effect of PTEN silencing. Under the action of SB203580, which is a p38 pathway inhibitor, the phosphorylation level of p38MAPK protein was decreased again (P < 0.05), [Figure 6a and b]. In addition, after the inhibition of the p38MAPK pathway by SB203580, TNF-a secretion (P < 0.05), [Figure 6c], apoptosis (P < 0.05), [Figure 6d and e], glucose uptake (P < 0.05), [Figure 6f], lactate production (P < 0.05), [Figure 6g], ROS generation (P < 0.05), [Figure 6h and i], and LDHA protein expression were decreased (P < 0.05), [Figure 6j and k], whereas collagen II protein expression was increased (P < 0.05), [Figure 6l and m] in HG-treated cartilage. The above results suggest that PTEN promotes HG-induced injury and glycolysis of chondrocytes by activating the p38MAPK pathway through the up-regulation of TP53.

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Figure 6:

PTEN affects injury and glycolysis of chondrocytes by up-regulating TP53 to activate the p38MAPK pathway in an HG environment. SB203580, a p38 pathway inhibitor. (a and b) Phosphorylation level of p38 protein. (c) TNF-a secretion level. (d and e) Cell apoptosis. (f) Glucose uptake. (g) Lactic acid content. (h and i) ROS generation. (j and k) Glycolytic molecule LDHA protein expression. (l and m) Collagen II protein expression. n = 3. ^✶P<0.05. HG: High glucose, PTEN: Phosphatase and tensin homolog, TP53: Tumor suppressor protein p53, TNF-a: Tumor necrosis factor-alpha, ROS: Reactive oxygen species, LDHA: Lactate dehydrogenase A, p38MAPK: p38 mitogen-activated protein kinase, FITC: Fluorescein isothiocyanate, PI: Propidium iodide. Scale bar = 25 μm (400×).

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PTEN silencing attenuates cartilage damage in DOA rats

The immunohistochemical results showed that compared with that in the control group, the number of PTEN and TP53 positive cells in the knee cartilage tissue of DOA rats was significantly increased, while PTEN silencing reduced the expression of PTEN and TP53 (P < 0.05), [Figure 7a and b]. In addition, the phosphorylation level of p38MAPK was increased (P < 0.05), [Figure 7c and d], the protein expression of collagen II was decreased (P < 0.05), [Figure 7e and f], and the protein expression of HK-2 was increased (P < 0.05), [Figure 7e and g] in the knee cartilage tissue of DOA rats. Interfering with PTEN expression significantly reversed the aforementioned changes. Furthermore, the secretion of IL-6 (P < 0.05), [Figure 7h], glucose uptake (P < 0.05), [Figure 7i, and lactate production (P < 0.05), [Figure 7j] were reduced in the knee cartilage tissue of DOA rats with reduced PTEN expression. The HE staining results of the knee cartilage tissue showed that the distribution of cartilage matrix was disordered in the DOA group, multiple chondrocytes were crowded together, and some matrices appeared to be fibrosis. Meanwhile, PTEN silencing significantly alleviated damage to chondrocytes (P < 0.05), [Figure 7k].

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Figure 7:

Effects of PTEN on the inflammatory injury and glycolysis of cartilage tissue in rats with DOA. (a and b) Representative images of immunohistochemistry for PTEN and TP53. Scale bar = 50 μm (×200) and 25 μm (×400). (c and d) Phosphorylation level of p38MAPK protein. (e and f) Protein expression of collagen II. (e and g) Glycolytic molecule HK-2 protein expression. (h) IL-6 secretion level. (i) Glucose uptake. (j) Lactic acid content. (k) Representative images of the hematoxylin and eosin staining of the knee cartilage tissue. ^✶P < 0.05. DOA: Diabetic osteoarthritis, PTEN: Phosphatase and tensin homolog, TP53: Tumor suppressor protein p53, IL-6: Interleukin-6, HK2: Hexokinase2, p38MAPK: p38 mitogen-activated protein kinase. Scale bar = 50 μm and 25 μm.

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Supplementary results 1. Identification of primary chondrocytes

Under an inverted microscope, the primary chondrocytes were oval in shape, clear in outline, uniform in size, and arranged singly. After incubation for 48 h, the chondrocytes were relatively larger, more closely arranged, remained round or oval, and exhibited stronger adhesion to the cell wall. After 96 h of incubation, the cells grew rapidly, and their morphology gradually turned into a unified irregular “paving stone”-like structure, while cell morphology was more 3D [Supplementary Figure 1a]. In addition, the expression and localization of collagen II and aggrecan in the cells further determined the chondrocytes. As shown in the figure, the red fluorescence-labeled collagen II [Supplementary Figure 1b] and green fluorescence labeled aggrecan [Supplementary Figure 1c] were abundantly expressed in the cells.