A total of 72 male Sprague Dawley rats (weight, 215±15 g; age, 8 weeks) were purchased from the Animal Center of the West China Medical College of Sichuan University (Chengdu, China). All animals were housed in the animal laboratory under controlled, conventional conditions (temperature, 24±1°C; humidity, 60±10%; 12-h light-dark cycle), and were given free access to food and water during the experimental session. Following a week of adaptation, rats were randomly divided into three groups (n=24/group): The sham operation, model and exercise intervention groups. The experimental protocol for the care and use of laboratory animals was approved by the Experimental Animal Ethics Committee of West China Hospital of Sichuan University (Chengdu, China). The associated permit number is 2018/66A.

The lumbar disc herniation model was established in the rats as previously reported (15,16). All rats were anesthetized by an intraperitoneal injection of 10% chloral hydrate (350 mg chloral hydrate/kg rat body weight; Animal Center of the West China Medical College of Sichuan University). The hair on the rats' abdomen and lower back was clipped. The left side of the body was incised and routine disinfection was performed to avoid wound infection. The incision was 3–3.5 cm long. The nucleus pulposus (NP) was harvested between the 2nd and 3rd coccygeal intervertebral disc of rat tails. In the model and exercise intervention groups, the L5 and L6 nerve roots were cut after exposing the ventral posterior wall. Harvested NP was then placed on the top of the left L5 and L6 nerve roots. Next, the wound was washed with saline and bandaged with sterile gauze. In the sham group, the intervertebral disc was snipped without NP transplantation. Rats were separately fed in a single cage and fasted for solids and liquids following surgery. Animals were administered with penicillin for 3 successive days. The rats had normal appetite without obvious infection signs, peritonitis or death following administration of 10% chloral hydrate.

Rats in the exercise intervention group were treated on day 5 post-surgery. The rats were placed in a pool sized 100×70×60 cm with a water depth of 50 cm. The water temperature was set to 30±2°C. Initially, the animals were subjected to exercise twice for 10 min. Next, animals trained for 20, 30 and 45 min on days 1, 2 and 3, respectively. The training occupied 60 min a day, 6 days a week for 4 weeks.

The hind paw withdrawal threshold was detected as previously described (17,18). Rats were placed in the test box of a heat pain checker (PL-200) with thermal light exposure. The thermal light was focused on the back half of one side of the rat foot through the bottom glass plate, and the spot diameter was 5 mm. The time from exposure to withdrawal was recorded. The interval between tests lasted for a minimum of 5 min, and the mean value was measured three times.

Rats were sacrificed via cervical dislocation following anesthesia by intraperitoneal injection of 10% chloral hydrate (350 mg chloral hydrate/kg rat body weight) at 7, 14 and 28 days following exercise intervention. After being soaked in 70% ethanol for 5 min, the skin of rats was cut along the posterior median line, paravertebral muscle was separated and the lumbar vertebral segment was removed. Then, intervertebral disc fiber ring was cut open with a sharp knife under microscopy, and the NP tissue was removed, fixed with 4% paraformaldehyde at 4°C for 48–72 h, and the remaining NP tissues were preserved at −80°C.

The relative expression of PLA2, TNF-α, IL-6 and β-actin were evaluated by RT-qPCR. Total RNA was extracted from NP tissue using TRIzol reagent (Thermo Fisher Scientific, Inc.) and cDNA was synthesized using PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.) at 37°C for 15 min and 85°C for 5 sec, in accordance with the manufacturer's protocol. The expression levels of PLA2, TNF-α, IL-6 and β-actin were then detected with SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd). The thermocycling conditions were as follows: 3 min at 95°C; 40 cycles of 95°C for 5 sec and 60°C for 30 sec; followed by 72°C for 30 sec. The data was analyzed using Bio-Rad CFX Manager software (version 3.0; Bio-Rad Laboratories, Inc.). The 2^−∆∆Cq method was used for comparative quantitation (19). β-actin was used as endogenous controls. Primer sequences are presented in Table I.

NP tissues were fixed with 4% paraformaldehyde for 48–72 h at room temperature. NP was then embedded in paraffin and 5-µm-thick paraffin sections were prepared. Apoptotic cells were detected using an in situ cell death detection kit (Roche Applied Science) at 37°C for 1 h in the dark, in accordance with the manufacturers protocol. Samples were then rinsed in 0.1 M PBS three times for 5 min, incubated in Converter peroxidase (POD) for 30 min at 37°C, rinsed in 0.1 M PBS three times for 5 min and color-developed with 3,3′-diaminobenzidine for 5 min at room temperature. Samples were subsequently counterstained with hematoxylin at room temperature for 1–3 sec. Following complete washing with distilled water, sections were mounted with Rhamsan gum (Beijing Solarbio Science & Technology Co., Ltd.) and observed under a fluorescence microscope (Olympus Corporation; magnification, ×400). Apoptotic cells exhibited brown staining within the nucleus.

The collected NP cells were resuspended in 100 µl Annexin V binding buffer (1×10⁵ cells) with 5 µl Annexin V-fluorescein isothiocyanate and 5 µl PI (cat. no. 556547; BD Biosciences). Subsequently, cell apoptosis was detected using a FACSCalibur™ flow cytometer (BD Biosciences) within 1 h. Data were analyzed using FlowJo 10.07 software (Tree star, Inc.).

NP tissue (100 mg) was rinsed with 1×PBS, homogenized in 1 ml of 1×PBS and stored overnight at −20°C. After two freeze-thaw cycles at −20°C overnight were performed to break cell membranes, homogenates were centrifuged at 4°C for 5 min at 5,000 × g. The supernatant was removed and assayed immediately. sPLA2 (cat. no. CSB-E13206r; Cusabio Technology LLC) activity, TNF-α content (cat. no. PT516; Beyotime Institute of Biotechnology) and IL-6 content (cat. no. PI328; Beyotime Institute of Biotechnology) was detected via the colorimetry method using a microplate reader (Thermo Fisher Scientific, Inc.) at 450 nm, according to kit instructions.

Protein samples were prepared from NP tissues using radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology, Ltd.). Protein concentration was measured using a bicinchoninic acid Protein assay kit (Wuhan Boster Biological Technology, Ltd.). Protein samples (20 µg) were then separated using 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). The membrane was subsequently blocked with 5% skimmed milk powder for 1 h at room temperature and incubated with primary anti-nuclear factor (NF)-κBp65 (cat. no. 8242) and β-actin (cat. no. 4970) antibodies (Cell Signaling Technology, Inc.) at a dilution of 1:1,000 overnight at 4°C. Samples were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Protein bands were visualized using an ECL chemiluminescence kit (EMD Millipore). Protein levels were calculated relative to β-actin and Image-ProPlus software (version 6.0; Media Cybernetics, Inc.) was used for densitometry analysis.

SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. Quantitative data are presented as the mean ± standard deviation. One-way analysis of variance and Dunnett's test were used to determine significance among groups, and P<0.05 was considered to indicate a statistically significant difference.

Prior to surgery, the hind paw withdrawal threshold was ~10 sec in all three groups. Compared with the model group, the hind paw withdrawal threshold was significantly increased in the exercise intervention group on days 14 and 28 following surgery. As compared with the sham operation group, the hind paw withdrawal threshold of the model group was significantly reduced (Fig. 1). These data indicated that swimming could alleviate nerve root pain in LDH rats as the training time increased.

The sPLA2 expression in the exercise intervention group was decreased on day 14, as compared with that in the model group. sPLA2 expression in the model group was significantly increased on days 7 and 14, as compared with that in the sham group (Fig. 2A). As compared with the model group, the IL-6 and TNF-α content was reduced on days 14 and 28 following surgery. As compared with the sham group, the IL-6 and TNF-α content clearly increased on days 7, 14 and 28 in the model group (Fig. 2B and C).

The mRNA expression level of PLA2 in the exercise intervention group significantly decreased on days 14 and 28 following surgery, when compared with the sham group, and the same expression in the model group was higher than that in the sham group from day 7 following surgery (Fig. 3A). The mRNA expression level of IL-6 in the exercise intervention group was significantly decreased on days 14 and 28, when compared with that in the model group, and that of the model group was increased from day 7 following surgery, when compared with that in the sham group (Fig. 3B). The mRNA expression level of TNF-α in the exercise intervention group was significantly lower than that in the model group on day 14, while that in the model group was clearly increased from day 7 following surgery, compared with the sham group (Fig. 3C).

Apoptotic cells were detected by TUNEL and Annexin V/PI. The TUNEL staining results demonstrated that TUNEL-positive cells were observed in the model and exercise intervention groups, but those in the exercise intervention group were markedly decreased, as compared to those in the model group (Fig. 4). The flow cytometry results demonstrated that the apoptotic rate in the exercise intervention group was significantly decreased from day 7 following surgery, as compared with that in the model group. The rate in the model group was significantly increased from day 7 following surgery, as compared with that in the sham group (Fig. 5).

Western blotting results demonstrated that the NF-κBp65 protein expression level in the model group was significantly increased, when compared with that in the sham group on days 7, 14 and 28 following surgery, and was significantly higher on day 14, as compared with day 7. In the exercise intervention group, the NF-κBp65 protein expression was significantly decreased, as compared with that in model group, and was significantly lower on day 28, as compared to day 7 (Fig. 6).