Clinical significance of serum GPX2 levels in patients with osteoarthritis was suppressed and reduced inflammation of osteoarthritis through STAT3 function

Study design for clinical research

This study was approved by the Ethics Committee of First Teaching Hospital of Tianjin University of TCM and all participants gave their informed consent. Patients with knee OA (N = 24) and healthy normal (N = 24) were recruited from December 2017 to February 2018. Diagnosis of osteoarthritis was performed according to criteria of the American College of Rheumatology. Demographic characteristics of included patients were showed at Table 1.

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Table 1.

Demographic characteristics of included patients.

Items	Osteoarthritis (n = 54)	Normal (n = 60)
Age (mean ± SE, years)	63.9 ± 5.5	62.7 ± 5.7
Sex (female/male, n)	31/23	30/30
BMI (mean ± SE, kg/m2)	25.9 ± 2.1	26.2 ± 2.6
K/L grade (2/3/4)	17/21/16

qPCR validation and Microarray experiments

RNA was reverse transcribed into cDNA using a PrimeScript OneStep RT-qPCR kit. cDNA was then transcribed using SuperScript III First strand cDNA system (Invitrogen). The detailed RT-qPCR procedure is described as follows: 95°C for 10 min; followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 15 s, 99°C for 10 s, 59°C for 35 s, and 95°C for 5 s; and then cooled to 4°C. The relative expression levels were calculated with the 2^−ΔΔCq method.

Total RNA was extracted from serum samples, and the amount of RNA was quantified by use of NanoDrop 1000. Total RNA of each sample was used for reverse transcription using an Invitrogen SuperScript double stranded cDNA synthesis kit. Double stranded cDNA was executed with a NimbleGen one-color DNA labeling kit and then executed for array hybridization using NimbleGen hybridization system and washing with the NimbleGen wash buffer kit. Axon GenePix 4000B microarray scanner (Molecular Devices) was used for scanning.

Animal experiment for vivo model

All experiments were approved by Animal Care and Use Committee of First Teaching Hospital of Tianjin University. All C57BL/6 mice were fed in a 25  ±  1°C temperature and 50  ±  5% humidity with a 12  h light/dark cyclic schedule and free access to standard diet. The medial anterior meniscotibial ligament of the right knee was cut using a surgical scissor in the patellar tendon in the middle and tendon of the tibial plateau. Number of model mice = 10, number of sham mice = 10, and number of GPX2 = 10. Model mice were injected with 1 μg/3 days/mice for 2 weeks.

Histological analysis

The bone tissues were fixed with 4% paraformaldehyde for 24 h. Tissue samples were embedded in paraffin, and the 5 μm thick sections were stained with Safranin O and Fast Green. Tissue samples were obtained using fluorescence microscopy (Nikon Eclipse TE2000-U, Japan).

Osteoarthritis was assessed using Mankin OA Scoring System: 0–11, changes in articular cartilage structure; 0–8, safranin O staining; 0–2, hypertrophic chondrocytes.

Enzyme-linked immunosorbent assay

The serum IL-1β level was quantified using an IL-1β kits and absorbance were read at a 450 nm wavelength using a microplate reader (Model 3550; Thermo Fisher Scientific, Inc.). Cell levels of TGF-β, TNF-α, IL-1β, IL-6, and IL-18 was quantified using TGF-β, TNF-α, IL-1β, IL-6, and IL-18 kits and absorbance were read at a 450 nm wavelength using a microplate reader (Model 3550; Thermo Fisher Scientific, Inc.).

Cell culture for vitro model and cell transfection

THP-1 cell is purchased from ATCC (Porton Down, Salisbury, UK) and cultured in RPMI 1640 (HyClone; GE Healthcare Life Sciences, UT, USA) containing 10% heat-inactivated fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.). Cells are stimulated by 100 nM PMA for 48 h into macrophage-like cells. GPX2, si-gpx2, and negative mimics were purchased from Genepharma Co., Ltd. (Shanghai, China) and transfected into macrophage-like cells using Lipofectamine 2000 (Invitrogen, USA). After 48 h, macrophage-like cells was induced with LPS (500 ng/mL) for 4 h.

Western blotting

Total proteins of cell samples were by using RIPA lysis buffer and protease inhibitor cocktail (Beijing Solarbio Science Technology Co., Ltd., Beijing, China). The protein concentration was determined by using BCA Kit (Beyotime). 50 μg protein samples were loaded to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto polyvinyl difluoride (PVDF, Thermo Scientific™, USA) membranes. Membranes was incubated with GPX2, STAT3, p-STAT3, and GAPDH at 4°C overnight after blocking with 5% non-fat milk in tris-buffered saline with 0.1% tween 20 (TBST). The membrane was washed with TBST and then incubated with anti-rabbit secondary antibody (1:5000) for 2 h at room temperature. Immunoreactive bands were visualized using the ECL kit (Thermo Scientific™, USA) and integrated density of the bands was quantified by Quantity One software (Bio-Rad).

Statistical analysis

Data were analyzed using SPSS version 17.0 (SPSS, IBM, Chicago, IL, USA, N = 3). The level of significance (p) was set at 0.05. The two-tailed unpaired Student’s t-tests were used for comparisons of the two groups. One-way analysis of variance followed by Tukey’s post hoc test was used for multiple groups.

Clinical significance of serum GPX2 levels in patients with osteoarthritis

First, we determined the serum levels of GPX2 in OA and healthy population. Compared with healthy normal group, the mRNA expression of GPX2 was decreased while the serum level of IL-1β was increased in patients with late stage of OA (Figure 1(a) and (b)). GPX2 levels were negatively correlated with IL-1β levels in patients with osteoarthritis (Figure 1(c)). ROC analysis showed that the serum GPX2 levels could be used to screen osteoarthritis patients from healthy normal, with an ROC curve area of 0.9167 (Figure 1(d)). In mice with osteoarthritis, both mRNA and protein levels of GPX2 in bone tissue were suppressed (Figure 1(e)–(g)).OPEN IN VIEWER

GPX2 regulated inflammation levels in in vitro model of osteoarthritis

over-expression of GPX2 decreased the levels of TGF-β, TNF-α, IL-1β, IL-6, and INF-γ, and induced IL-10 cell levels in vitro model of osteoarthritis (Figure 2(a)–(g)). si-gpx2 plasmid reduced GPX2 expression, promoted the cellular levels of TGF-β, TNF-α, IL-1β, IL-6, and INF-γ, and suppressed the cellular level of IL-10 in in vitro model of osteoarthritis (Figure 2(h)–(n)).OPEN IN VIEWER

GPX2 presented osteoarthritis in mouse model

The study assessed the function of GPX2 in mouse model of osteoarthritis. GPX2 reduced OARSI score, presented bone cell damage, decreased the levels of TGF-β, TNF-α, IL-1β, IL-6 and INF-γ, and increased IL-10 level in mouse model of osteoarthritis (Figure 3(a)–(h)). Moreover, we examined the mechanism of GPX2 in model of osteoarthritis using microarray analysis (Figure 3(i)). GPX2 suppressed the protein expression of p-STAT3 in bone tissue of mice with osteoarthritis (Figure 3(j)–(f)).OPEN IN VIEWER

GPX2 combined and regulated STAT3 protein expression

We evaluated the mechanism of GPX2 on inflammation of osteoarthritis. Over-expression of GPX2 induced the protein expression of GPX2, and suppressed that of p-STAT3 in vitro (Figure 4(a)–(b)). Over-expression of GPX2 suppressed p-STAT3 expression in vitro (Figure 4(c)). IP assay showed that GPX2 combined STAT3 protein expression (Figure 4(d)). Then, over-expression of GPX2 promoted Ubiquitination-STAT3 protein expression in vitro (Figure 4(e)). Down-regulation of GPX2 suppressed the protein expression of GPX2, and induced that of p-STAT3 in vitro (Figure 4(f)–(g)).OPEN IN VIEWER

The inhibition of STAT3 reduced the anti-inflammation effects of si-GPX2 in osteoarthritis.

We further evaluated the role of STAT3 in the anti-inflammation effects of GPX2 in osteoarthritis. The administration of STAT3 inhibitor (5 μM, Alantolactone) reduced the anti-inflammation effects of si-GPX2 on the induction of p-STAT3 protein expression, and the inhibition of TGF-β, IL-1β, and IL-6 levels in in vitro model of osteoarthritis (Figure 5).OPEN IN VIEWER