Primer synthesis, reverse transcription kit and real-time fluorescent quantitative polymerase chain reaction (PCR) kit (Takara Biotechnology Co., Ltd., Dalian, China), TRIzol kit (Ambion; Thermo Fisher Scientific, Dallas, TX, USA). Rabbit anti-human MMP-1, MMP-2, glyceraldehyde-3-phosphate dehy-drogenase (gAPDh) primary polyclonal antibodies and goat anti-rabbit horseradish peroxidase (hRP)-labeled secondary polyclonal antibody (cat nos. 10371-2-AP, 10373-2-AP, 10494-1-AP, SA00001-2; (Proteintech Group, Inc., Wuhan, China), bicinchoninic acid (BCA) protein assay kit and cell lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China).

A total of 50 DVT patients diagnosed in the Department of Vascular Surgery of People's Hospital of Jiyang from February 2016 to February 2017 were selected as the DVT group, including 27 males and 23 females aged 25–57 years, with an average age of 55.72±11.46 years. Inclusion criteria: i) patients who had DVT onset within 7 days, ii) patients who had similar disease conditions and iii) patients who had venous flow obstruction of the lower extremity which was confirmed by the anterograde venography of deep vein of lower extremity. Patients with the following conditions were excluded: pregnancy, infection, tumor, inherited anticoagulation factor deficiency, vascular abnormality or diabetes mellitus. Before treatment, all the patients had such clinical manifestations as high muscle tension and swelling in their affected extremities, and the DVT of unilateral lower extremity was verified via color Doppler ultrasound or venography examinations after admission to hospital. Another 50 healthy individuals receiving health examination in People's Hospital of Jiyang were enrolled as the normal control group which included 28 men and 22 women aged 26–58 years, with an average age of 52.35±10.38 years. They were all healthy adults without cardiac, hepatic, cerebral, renal and infectious diseases or diabetes mellitus. There were no statistically significant differences in the age, gender and weight between the two groups (P>0.05) (Table I).

The study was approved by the Ethics Committee of People's Hospital of Jiyang. Signed informed consents were obtained from the patients or guardians.

The patients with DVT of lower extremity were treated with continuous intravenous infusion of 200,000 units of urokinases for 5 days from the first day after the diagnosis was confirmed. Moreover, 4,000 units of low molecular weight heparin was injected subcutaneously twice daily, and 100 mg aspirin, an anti-platelet aggregation drug, was orally administered once daily.

A total of 5 ml fasting venous blood was collected in the morning from each patient in the DVT group before treatment and at 7 days after treatment as well as each healthy person in the normal control group, respectively. The blood was added into a vacuum blood collection tube without any anticoagulant and centrifuged at 4,000 × g 4°C for 15 min. After that, the supernatant was absorbed carefully and then stored in a refrigerator at −80°C for standby use.

Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of MMP-1, MMP-2, IL-6, IL-8 and TNF-α in the serum in accordance with the steps recommended in the kit instructions.

Before treatment and at 7 days after treatment, 10 ml fasting venous blood was drawn in the morning from each patient in the DVT group, and Ficoll-Paque PLUS was utilized to isolate the PBMCs. After the cells were cultured with Roswell Park Memorial Institute (RPMI)-1640 medium containing 10% serum in an incubator with 5% CO₂ at 37°C for 2 h, the suspended cells were washed out, and the adherent cells obtained were the PBMCs, which were adopted for subsequent experiments.

Detection of MMP-1 and MMP-2 protein expression levels in patient PBMCs via western blotting. Patient PBMCs were digested with trypsin and then collected, which were lysed using the lysis buffer and centrifuged at 3,000 × g for 10 min at 4°C to collect the supernatant protein. The protein concentration was determined by virtue of the BCA protein assay kit, and 30 µg protein loading in each specimen was taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a polyvinylidene fluoride (PVDF) membrane, sealing with blocking buffer for 2 h and addition of MMP-1, MMP-2 and GAPDH primary antibodies (dilution, 1:1,000) for incubation in the refrigerator at 4°C overnight. Next, the membrane was washed with Tris-buffered saline Tween-20 (TBST) 3 times, and then the secondary antibodies (dilution, 1:1,200) were added for incubation at room temperature for 2 h, followed by membrane washing with TBST 3 times. After that, the membrane was placed in enhanced chemiluminescence (ECL) liquid (MilliporeSigma, Burlington, MA, USA) for development in the dark, which was scanned and recorded by means of a gel imager for gray analysis. The densitometry was analyzed by Image J professional image analysis software (NIH, Bethesda, MD, USA).

The PBMCs of the patients collected after trypsin digestion were applied to extract the total RNA of the cells via the TRIzol kit. Then the samples with an absorbance ratio (A₂₆₀/A₂₈₀) of 1.8–2.0 were selected for reverse transcription, followed by PCR with complementary deoxyribonucleic acid (cDNA) as the template. The primer sequences are shown in Table II, and the reaction conditions are as follows: pre-denaturation at 94°C for 3 min and then at 95°C for 1 min, annealing at 50°C for 50 sec, extension at 72°C for 1 min, a total of 40 cycles of amplification and extension for 10 min. With GAPDH mRNA as the control, the experimental results were analyzed using the 2^−∆∆Cq method (10).

The circumferences at 15 cm above the knee and 10 cm below the knee of both extremities were measured while the blood samples of the DVT patients were collected. The difference between the circumference of affected extremity and unaffected extremity was calculated, and the circumference difference of the affected and unaffected extremities was compared before and after treatment.

Statistical Product and Service Solutions (SPSS) 17.0 software (IBM Corp., Armonk, NY, USA) was adopted for data processing. Measurement data were presented as mean ± standard deviation, and t-test was used for intergroup comparison. Linear (Pearson's) correlation analysis was utilized to analyze correlations, and P≤0.05 was considered to indicate a statistically significant difference.

The ELISA results indicated that the levels of MMP-1, MMP-2, IL-6, IL-8 and TNF-α in the serum of the DVT group were remarkably higher than those of the normal control group before treatment, and the differences were statistically significant (P<0.01) (Table III).

After 7 days of treatment, the levels of serum MMP-1, MMP-2, IL-6, IL-8 and TNF-α in the DVT group were markedly lower compared with those before treatment, showing statistically significant differences (P<0.01) (Table IV).

The results of western blotting revealed that compared with those before treatment, the expression levels of MMP-1 and MMP-2 proteins in the PBMCs were decreased notably after treatment, and the differences were statistically significant (P<0.01) (Fig. 1).

As shown in Fig. 2, the RT-qPCR results manifested that the expression levels of IL-6, IL-8 and TNF-α mRNAs in the PBMCs declined compared with those before treatment, with statistically significant difference (P<0.01).

The degree of limb swelling of the DVT patients was alleviated obviously after treatment, and the difference between the circumferences of unaffected extremity and affected extremity of the patients was decreased remarkably at 7 days after treatment in comparison with that before treatment, displaying statistically significant difference (P<0.01) (Table V).

The levels of IL-6, IL-8, TNF-α, MMP-1 and MMP-2 in the serum of DVT patients were recorded for Pearson's correlation analysis. It was indicated that the serum IL-6, IL-8 and TNF-α levels were positively correlated with MMP-1 and MMP-2 levels of patients in the DVT group, respectively (P<0.05 or P<0.01) (Table VI).