This study was performed at the Medical Science Experimentation Center of the Zhongshan School of Medicine of Sun Yat-Sen University (Guangzhou, China). Male specific pathogen-free (SPF) Lewis rats (age, 3–4 weeks; weight, 70±15 g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Male SPF BALB/c nude rats (age, 4–6 weeks; weight, 15±2 g) were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China). All animals were housed according to the national animal treatment guidelines (http://www.gov.cn/gongbao/content/2011/content_1860757.htm) and all experimental procedures were approved by the Committee on the Use of Live Animals for Teaching and Research of Sun Yat-Sen University and the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University [Approval no. Ethical application (2013) No. 149]. The Walker 256 cell line was acquired from the Cell Bank of the Laboratory Center of Sun Yat-Sen University. Glauber's salt (Guangzhou Pharmaceuticals Corporation, Guangzhou, China) containing 99.0% Na₂SO₄ was dissolved in ultraviolet (UV)-disinfected saline to a concentration of 2 g/ml.

A Glauber's salt solution was prepared, as described above. Concentrated JPJD cream was prepared, which was composed of 30 g Codonopsis (root), 15 g Poria, 15 g Atractylodes (root), 6 g liquorice (Glycyrrhiza glabra), 12 g Bupleurum (root) 15 g Curcuma (root) and 30 g Scutellaria barbata (stem and leaf), which were obtained from China Resources Sanjiu Medical & Pharmaceutical Co., Ltd., Beijing, China). The sources, which were identified according to the first part of the 1998 Chinese Pharmacopoeia and combined according to the established ratio (12), were concentrated using water extraction and volatile oil collection. These procedures were performed at the Science and Technology Industrial Park of Guangzhou University of Chinese Medicine (Guangzhou, China). Finally, the JPJD formulation was diluted into a concentrated aqueous cream that contained 2 g crude components per ml. TP5 solution (10 mg; license no. H20058462; cat no. 20130806; Beijing ShuangLu Pharmaceutical Co., Ltd., Beijing, China) was utilized for injection.

TRIzol® reagent (15596-026; Thermo Fisher Scientific, Shanghai, China); polymerase chain reaction (PCR) amplification kit (DR011; Takara Biotechnology Co., Ltd., Dalian, China); Hi-Di Formamide (Lot no. 1305031; serial no. 4404307), GeneScan™ 600 LIZ® (Lot no. 1206023; serial no. 4408399) and an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific); and GeneMarker® Genotyping software, version 2.2 (SoftGenetics LLC, State College, PA, USA) were used.

The primer sequences were as reported in a previous study by Douillard et al (15). They were synthesized using an ABI 3900 desktop high-throughput DNA synthesizer (Thermo Fisher Scientific).

Male Lewis rats (age, 3–4 weeks), which were housed under a temperature of 24–26°C at a 12-h light/dark cycle, were randomized into five groups (n=15 per group) as follows: i) Control group (group A), animals received 1 ml/100 g normal saline per day intragastrically; ii) FRL-OHC group (group B), animals received treatment to establish the FRL-OHC model; iii) low dose JPJD group (group C), animals received treatment to establish the FRL-OHC model and the intragastric administration of 37.5 g/kg JPJD per day; iv) high dose JPJD group (group D), animals received treatment to establish the FRL-OHC model and the intragastric administration of 75 g/kg JPJD per day; and v) TP5 group (group E), animals received treatment to establish the FRL-OHC model, an intramuscular injection of 5 mg TP5 every 48 h and 1 ml/100 g normal saline per day intragastrically. All rats were fed simultaneously. The FRL model establishment procedure was terminated for animals in groups B-E after 29 days. Following 7 days of free feeding, the OHC model was then established in the relevant groups. Similarly, there was 17 days of free feeding and observation. Liver, thymus and hepatocellular carcinoma samples were collected under anesthesia immediately after the rats with OHC reached the ethical limits for animal experimentation (lethargy, erect back hair, relative body mass of 80%, fever or ascites). Anesthesia was induced via an intraperitoneal injection of 10% chloral hydrate (3.5 ml/kg; Sigma-Aldrich Shanghai Trading Co., Ltd., Shanghai, China). Samples were stored at −80°C prior to analysis. Apparent FRL scale scores and body mass were recorded daily in each group. Hepatocellular carcinoma volume was calculated as follows: Maximal diameter (mm) × minimal diameter (mm)²/2. Visceral indices were calculated as follows: Weight of the cancer-bearing liver or thymus (g) or hepatocellular carcinoma volume × 100/final body weight (g). Following the completion of the study, three rats from each group were selected and their thymus, liver and hepatocellular carcinoma tissues were harvested from the anesthetized rats to analyze the spectral-type diversity of TCRVβCDR3 repertoire. For group A, the three rats were selected at random, while for the other groups, three rats with a maximal hepatocellular carcinoma diameter ≥10 mm were selected from each group.

Rats were housed individually at 23±1°C with a 12–12 h light-dark cycle and a feeding regimen of tap water ad libitum and alternate-day food restriction (11). Rats received food between 9:00 a.m. one day to 9:00 a.m. the following day. For the following 24 h, the rats received water only. The rat diet accorded with the National Standard of China, and consisted of water (10%), crude protein (18%), crude fat (4%), crude fiber (5%), crude ash (8%), calcium (1.2%) and phosphorus (1%). For each feeding period, 200 g food was administered and the remaining food was measured on the next day to calculate the food intake per 100 g body mass. Glauber's salt solution (0.25 g/ml) (12) was administered daily (1 ml/100 g) via oral gavage for 29 days prior to feeding. The effect of the FRL modeling was evaluated according to the apparent FRL scale (Table I) based on factors including the degree of weight loss, tail cleanliness and hair color and aggregation.

In the apparent FRL model scale, the grading criterion for relative body mass was developed according to the limitation of 20% human weight loss (16). During the establishment of the FRL model, rats in groups A and B were matched according to weight (weight difference, ±5 g) for calculation of the relative body mass (FRL rat weight/normal rat weight as a percentage). During the period of FRL-OHC model establishment, the rats were 8–9 weeks old and their weight gain reduced, thus another equation was required: Relative body mass = final weight/weight prior to establishment of the model. A total score of ≤6 on the apparent FRL model scale was considered to be asymptomatic, 7–12 was mildly symptomatic, 13–18 was typically symptomatic and 19–24 was severely symptomatic.

The OHC model was established according to previously described procedures (13,14). In brief, Walker-256 cells (1×10⁷) were transplanted subcutaneously by an injection made in the neck of nude BALB/c rats. Tumors were harvested after reaching a diameter of >1 cm. First, the animals were anesthetized via an intraperitoneal injection of 10% chloral hydrate (3.5 ml/kg; Sigma-Aldrich Shanghai Trading Co., Ltd., Shanghai, China), then the thoracic and abdominal cavities were opened and the tissues or organs were carefully removed. Following the removal of necrotic tissue, the tumor tissues were cut into 1-mm³ pieces. The tissue fragments were then implanted into the FRL model Lewis rats to establish the OHC model. Under inhalation anesthesia, a vertical incision under the xiphoid was cut after sterilization. Inhalation anesthesia was induced as follows: 4 ml ether (Tianjin Damao Chemical Reagent Factory, Tianjin, China) were transferred to a 15-ml centrifuge tube (Becton Dickinson Medical Devices, Shanghai, Co Ltd, Shanghai, China) and a cotton ball (Winner Medical Co. Ltd., Shenzhen, China) was added and left to soak. Following soaking, the cotton ball was moved close to the nose of the animal inducing anesthesia. The anesthetic procedure lasted for a maximum of 3 min. Subsequently, the liver of the rat was exposed and cancer tissues were implanted using a 1-mm coarse needle (Cat. no. 305198; Becton Dickinson Medical Devices Shanghai Co., Ltd., Shanghai, China). Finally, the abdominal cavity was closed layer by layer after hemostasis was achieved.

Total RNA extraction, PCR analysis and TCRVβCDR3 repertoire detection were performed according to methods described by Douillard et al (15) and Venturi et al (17). An ABI 3500xL Genetic Analyzer was used for fragment analysis of the TCRVβCDR3 repertoire according to the manufacturer's instructions (18).

TCRVβCDR3 fragment analysis data obtained using the ABI 3500xL and GeneScan™ 600 LIZ® was imported into GeneMarker software, version 2.2 (19). The spectral diagram and related data of the 20 gene fragments of the TCRVβCDR3 subfamily were obtained.

The diversities of the TCRVβCDR3 repertoire in the thymus, liver and cancerous tissues in each group were compared with the normal repertoire diversity and fragment sizes in the thymus tissue in the control group (17,20). Fluorescence peaks and their data that did not correspond to the sites of the various TCRVβCDR3 subfamilies of the normal thymus tissue were deleted to retain comparability between the groups.

Clonal types of the TCRVβCDR3 subfamily were confirmed visually by three independent researchers. The normal spectral-type of the TCRVβCDR3 subfamilies is a bell-shaped quasi-Gaussian distribution; however, other non-Gaussian distributions may appear, including a skewed-peak type, a no clonal type (no peak detected) and a monoclonal type (one peak detected).

Three samples were analyzed for each group. The results indicated that for each group, the TCRVβCDR3 subfamily spectral-types, expressed and unexpressed, of the thymus, liver and cancerous tissues of the three samples were identical. During data analysis, the number of the TCRVβCDR3 subfamilies, expressed and unexpressed, was used as the raw data. Medians of the numbers of the fluorescence peaks and clonal types (quasi-Gaussian distribution, skewed-peak and monoclonal type) were used, while means of Simpson's diversity index (Ds), area under the fluorescence peak and relative fluorescence intensity of the each peak were used for the analysis. The relative fluorescence intensity (RI) was determined using the following formula: RI (%) = (100 × area under the fluorescence peak of the target fragment)/(total area under the fluorescence peak of the complete subfamily). In the calculations, the area under the fluorescence peak was expressed as 1×10⁻³ of the original value, and the Ds value was expressed as 100-fold of the original value. As these data involved only three samples, they were not analyzed using statistical tests.

Data were analyzed using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA). Continuous measurement data were expressed as the mean ± standard deviation. Analysis of variance was used for the normally distributed measurement data. Rank sum tests and Kaplan-Meier survival analysis were used for the non-normally distributed measurement data. P<0.05 was considered to indicate a statistically significant difference.