BCA Protein Concentration Assay Kit was provided from Shanghai Biyun Tian Biotechnology (Shanghai, China; Cat. No.: P0009). Western and IP Cell Lysis Buffer were obtained from Shanghai Biyun Tian Biotechnology (Shanghai, China; Cat. No.: P0013). HRP goat anti-mouse IgG (H + L) was acquired from abclonal (Wuhan, China; Cat. No.: AS003), and goat anti-rabbit IgG (H + L) HRP was provided from Affbiotech (Liyang, China; Cat. No.: S0001). P65 antibody was from HUABIO (Hangzhou, China; Cat. No.: HA721307). Molpure® Cell/Tissue Total RNA Kit was purchased from YEASEN (Shanghai, China; Cat. No.: 19221ES50). PrimeScript RT Reagent Kit were acquired from BaoRui Medical Biotechnology (Peking, China; Cat. No.: RR047A). TB Green TM Premix Ex TaqTM II (Tli RNaseH Plus) was from BaoRui Medical Biotechnology (Peking, China; Cat. No.: RR820A). DAPI staining reagent, Rat IL-1β ELISA Kit, and Rat TNF-α ELISA Kit were from Union-Bio(Shanghai, China).

Twenty-four male Sprague Dawley rats weighing between 230 and 250 g were purchased from Chengdu Dashuo Experimental Animal (Chengdu, China) with an animal experiment production licence number: SCXK (Sichuan) 2020-0030. The animals were housed in the animal facilities of Chengdu University Affiliated Hospital. These Sprague Dawley rats were fed standard laboratory chow and provided with water ad libitum, with the room temperature maintained between 25 °C and 28 °C. All housing and experimental procedures strictly adhered to the relevant regulations and guidelines for animal experiments and were conducted in accordance with ethical principles to ensure the welfare of the animals. The animal experiments received ethical approval from the Ethics Committee of Chengdu University Affiliated Hospital.

Twenty-four male SD rats were randomly divided into a normal group, a model group, and a treatment group. On the second day after modelling, The rats in the treatment group were gavaged daily with phellodendrine at a dosage of 48.2 mg/kg [12]. The normal control group and model control group received double-steamed water treatment, with both groups being administered 0.1 mL of it every day. During the adaptation period, the mice had free access to food and water. This treatment regimen was continued for 14 consecutive days.

The control group did not undergo modelling treatment, whereas the model group and treatment group Sprague Dawley rats underwent autologous transplantation of the nucleus pulposus from their tail to establish a NCLDH model. In accordance with the methods of He Yuancheng [13], this study adopted a technique for establishing a NCLDH model. First, the body weights of the rats were measured, and anaesthesia was induced via the intraperitoneal injection of 1% pentobarbital (40 mg/kg). After confirming the effect of anaesthesia, the intervertebral space between the spinous processes of lumbar vertebrae 4/5 was selected as the puncture center. The area of the back hair was shaved with a razor, with dimensions of 4 cm in length and 3 cm in width. The Sprague Dawley rat was subsequently immobilized on the operating table, and disinfection was performed at the root of the tail, approximately 1 cm from the base. The tail was then ligated and severed to expose the transparent gel-like nucleus pulposus tissue inside. Five pieces of nucleus pulposus tissue were collected and placed in a clean container. Next, 50 µL of physiological saline was added to the nucleus pulposus tissue, which was prepared as a suspension by grinding and stirring. Finally, 20 µL of autologous nucleus pulposus suspension and 30 µL of a 2% lidocaine solution were injected separately into the puncture center on the back. This method is used to establish a NCLDH model in rats for research purposes.

Neurological scoring is based on the method provided by Siegal, which assesses the motor coordination, limb sensation, reflex strength, balance ability and other aspects of the animals through behavioral observation of Sprague Dawley rats. Sprague Dawley rats were observed on the 2nd day after modelling and on the 14th day after intervention. The scoring principles are as follows: normal general condition, normal gait is recognized as level 0 and scored as 2 points; Sprague Dawley rats with basically normal gait are scored as level 1 and scored as 4 points; weakness of one or both hind limbs and mild difficulty in crawling are scored as level 2 and scored as 6 points; weakness of one or both hind limbs, weakness of hind limbs and obvious instability in crawling are scored as level 3 and scored as 8 points; instability while standing but able to move are scored as level 4 and scored as 10 points; and paralysis is scored as level 5 and scored as 12 points. Paralysis was classified as grade 5 with 12 points.

After Sprague Dawley rats were euthanized via decapitation on the 14th day of intervention, the L5 nerve and surrounding tissues were dissected. Total RNA was extracted via the Molpure® Cell/Tissue Total RNA Kit (YEASEN, Shanghai, China; Cat. No.: 19221ES50). The purity of the RNA was assessed, with A260/A280 ratios ranging between 1.8 and 2.0. The total RNA was then mixed in a 20 µL reaction mixture and reverse transcribed into cDNA. Real-time quantitative PCR amplification was conducted via the QuantStudioTM 3 Real-Time PCR System (Thermo Fisher Scientific, US ). The PCR amplification conditions were as follows: initial denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing at 55 °C for 30 s, and extension at 72 °C for sufficient elongation, with a total of 45 cycles. The QuantStudioTM Design & Analysis SE Software from Thermo Fisher Scientific (US) is used to analyze the CT (Threshold cycle) values of each sample during the PCR process, and the relative mRNA expression level of P65 is calculated using the 2^-ΔΔCT method. Table 1 lists the rat-specific sequences of primers used for detecting p65 and mRNA via PCR analysis.

After centrifugation of rat left atrial arterial blood, serum samples were obtained, and the levels of IL-1β and TNFα were detected via an ELISA s following the protocols of the manufacturer.

After Sprague Dawley rats were euthanized via decapitation on the 14th day of intervention, the L5 nerve and surrounding tissues were dissected. The samples were then transferred to 2 mL grinding tubes, The sample was then removed and put into a 2 mL grinding tube, followed by the addition of 2 pieces of 3 mm grinding beads and RIPA lysate (Shanghai Biyun Tian, Shanghai, China; Cat. No.: P0013) with a mass ratio of sample to lysate at 1:10. The tubes were placed in a high-speed low-temperature tissue grinder (temperature: -20°C) and subjected to grinding four times for 60 s each time. After grinding, the tubes were removed and allowed to sit at 4°C for 30 minutes for lysis. After 30 minutes, the tubes were centrifuged (temperature: 4°C, speed: 12000 rpm) for 10 minutes. Following centrifugation, the supernatant was collected, and the protein concentration was determined via a Bicinchoninic AcidAssay (BCA) protein quantification kit from Shanghai Biyun Tian Biotechnology Co., Ltd (Shanghai, China; Cat. No.: P0009). The samples were then subjected to high-temperature denaturation at 95°C for 15 minutes and stored at -80°C after denaturation. The samples were loaded onto a 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel (PAGE) (saiguobio, Guangzhou, China; Cat. No.: 3250GR500) and electrophoresed to transfer the proteins onto a polyvinylidene fluoride(PVDF)(Sigmaaldrich, Wuxi, China; Cat. No.: ISEQ00010) membrane. The PVDF membrane was blocked with 5% skim milk for 2 hours. After blocking, the PVDF membrane was incubated with primary antibodies (dilution ratio: p65 1:1000; Tubulinβ 1:2000) overnight at 4°C with gentle shaking. The PVDF membrane was then washed three times with Tris-buffered saline containing Tween-20 (TBST) for 5 minutes each. Next, the PVDF membrane was incubated with secondary antibodies (dilution ratio: 1:5000) for 2 hours at room temperature with gentle shaking. After incubation, the PVDF membrane was washed three times with TBST for 10 minutes each time. The PVDF membrane was then fully immersed in a mixture containing ‘Torchlight’ Hypersensitive ECL Western HRP Substrate (zen-bio, Chengdu, China; Cat. No.: 17046) in a culture dish under dark conditions. The bands were visualized via Tanon Fluorescence Imaging System software V2.0 (Shanghai, China), and the results were scanned and analysed via Gel-Pro Analyser4 software (Media Cybernetics, US), with the integrated optical density (IOD) representing the target protein.

After Sprague Dawley rats were euthanized by decapitation on the 14th day of intervention, the L5 nerve and surrounding tissues were dissected and fixed in 4% formaldehyde. The samples were then embedded in paraffin and sectioned, and the sections were immersed in citrate buffer (pH 6.0) for antigen retrieval, followed by microwave treatment for 20 min. After cooling, the sections were washed three times with phosphate-buffered saline (PBS) (zsbio, Peking, China; Cat. No.: ZLI-9062) for 5 min each. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide at room temperature for 25 min, followed by washing the slides in PBS (pH 7.4) on a shaker 3 times, each time for 5 min. Serum blocking was performed using bovine serum albumin at room temperature for at least 30 min. Primary antibody was applied by gently removing the blocking solution and adding prediluted primary antibody to the sections, which were then placed flat in a humidified chamber and incubated overnight at 4 °C. After incubation, the sections were washed three times with PBS for 5 min each, followed by the addition of secondary antibody for 30 min at 37 °C. The sections were subsequently washed again with PBS three times for 5 min each. The 4’,6-diamidino-2-phenylindole (DAPI) (Servicebio, Wuhan, China; Cat. No.: G1012) staining was performed by incubating the sections with DAPI at room temperature for 10 min, followed by washing with PBS three times for 5 min each. Finally, the sections were mounted with anti-fade mounting medium. OlyVIA scanning software (Olympus, Japan) was used to capture images of the sections. Each section was observed at low magnification to visualize the entire tissue, followed by capturing images at 200x magnification from three different fields. DAPI staining was used to label the cell nuclei in blue, while positive expression of p65 was visualized in green. The Halo Data Analysis System (lndica Labs, US) was used to calculate the number of positive cells in each image.

The samples were embedded in paraffin and made into sections for storage. First, the paraffin sections were dewaxed, a process that included the sequential use of xylene I, xylene II and xylene III (zhiyuanhx, Tianjin, China; Cat. No.: 202150101), each solvent for 15 min, followed by treatment of the sections with anhydrous ethanol I and anhydrous ethanol II (haixinghuagong, Chengdu, China; Cat. No.: GB678-90), each continuously for 5 min, 85% alcohol and 75% alcohol for 5 min each, and finally, washing of the treated sections with distilled water. The next stage was the antigen repair stage, where the sections were placed in citrate buffer (zsbio, Peking, China; Cat. No.: ZLI-9065) with a pH of 6.0 and repaired via a repaireer at 80 °C for 20 min. After repair, the sections were washed three times with PBS solution for 5 min each. Finally, to block endogenous peroxidase activity, the sections were soaked in 3% hydrogen peroxide solution for 10 min at room temperature, followed by three washes with PBS solution for 5 min each. The tissue samples were washed for 5 min; next, to seal possible nonspecific binding sites, goat serum was added to the samples for sealing, and this step lasted for 20 min at room temperature. After that, the first antibody was added, and the samples were incubated overnight at 4 °C. The next day, the samples were washed three times with PBS for 5 min each time. To continue, the second antibody was added, and the samples were incubated at 37 °C for 30 min. The samples were again washed three times with PBS for 5 min each. Next, the DAB (zsbio, Peking, China; Cat. No.: ZLI-9018) color development process was carried out by adding drops of freshly prepared DAB color development solution to the tissue samples and controlling the time of color development at room temperature until the appearance of brownish‒yellow color positivity, after which the sections were washed with distilled water to stop color development. Finally, to highlight the nuclei of the samples, the sections were immersed in hematoxylin for 3 min, followed by rinsing with water and waiting for the sections to return blue in water before rinsing thoroughly with running water. Dehydration was then performed by placing the sections in anhydrous ethanol at concentrations of 75%, 85%, and 95% and immersing them at each concentration for 10 min. The sections were subsequently transferred to xylene and immersed for 10 min to complete the dehydration step, after which they were finally sealed with neutral gum. Images of the sections were captured via CaseViewer(3DHISTECH, Hungary), a digital section viewing software, and the average protein concentration was determined via ImageJ software (National Institutes of Health, US).