2 Experimental Methods

2.1 Model Preparation

White tumor fluid was extracted from the abdominal cavity of H22 tumor-strain-propagated mice, and according to reference [11], it was diluted with physiological saline to a tumor cell count of 2 × 10⁷ cells/ml [12]. Except for the normal group of 12 mice, 0.2 ml of tumor fluid was inoculated subcutaneously into the right anterior axillary region of each remaining mouse.

2.2 Animal Grouping and Dosage

Twenty-four hours after inoculation, the animals were randomly divided into high-dose Pei’s Shengxue Granules (PG) + 5-Fu group (10 g/kg, equivalent to the adult dosage of

Research on Pei Zhengxue’s series of prescriptions

20 times)+5-Fu group, medium-dose (5 g/kg, 10 times)+5-Fu group, low-dose (2.5 g/kg, 5 times)+5-Fu group, 5-Fu (0.02 g/kg) control group, and model control group, with 12 mice in each group. Pei’s Shengxue Granules were thoroughly dissolved in distilled water before use and administered via gavage at equal volumes (0.2 ml/10 g) once daily [13]; the 5-Fu dosage was uniformly 0.02 g/kg, administered via intraperitoneal injection once daily; the normal group and model group received equal volumes of distilled water once daily, continuously for 10 days [2].

2.3 Determination of Tumor Inhibition Rate

The next day after stopping medication, the mice were weighed, euthanized, and their tumor tissues were dissected and weighed. The tumor inhibition rate was calculated using the following formula:

Tumor Inhibition Rate (%) = (Average Tumor Weight of Control Group - Average Tumor Weight of Treated Group) / Average Tumor Weight of Control Group × 100%.

2.4 Blood Routine Examination

The tails of the mice were disinfected with 75% alcohol cotton balls, and 20 μl of peripheral blood was drawn from the tail vein. The blood was mixed with 0.5 ml of blood cell diluent, and then analyzed automatically by a blood cell analyzer.

2.5 Measurement of Mouse Bone Marrow DNA Content

After blood collection, mice in each group were euthanized. One intact right femur from each mouse was excised, the external muscle tissue was removed, and all bone marrow was flushed into a centrifuge tube with 10 mL of 0.05 mol/L CaCl₂ solution. After refrigeration at 4°C for 30 minutes, the mixture was centrifuged at 2,500 rpm for 15 minutes, the supernatant was discarded, and the pellet was resuspended in 5 mL of 0.2 mol/L HClO₄, thoroughly mixed, heated in a water bath for 15 minutes, cooled, centrifuged again at 1,000 rpm for 5 minutes, and the OD value was measured at 268 nm using a UV spectrophotometer.

2.6 Radioimmunoassay for Determining Serum TNF-α Concentration

The eyeballs of mice were enucleated to collect blood, which was then collected in clean test tubes. The blood was allowed to clot at room temperature for 2 hours, followed by centrifugation at 1,500 rpm for 10 minutes to obtain serum, which was aliquoted and stored at −20°C. According to the instructions of the ¹²⁵I-TNF-α radioimmunoassay kit, the calibration standards provided in the kit were sequentially added to the test tubes, while the tracer, antibody, and other reagents were added to their respective tubes as specified in the protocol. The mixtures were thoroughly mixed and incubated in a 4°C refrigerator for 24 hours. Then, PR separator was added, the mixtures were thoroughly mixed again, and left at room temperature for 20 minutes. Before centrifugation, two tubes were selected to measure the total radioactivity T. The samples were then centrifuged at 3,500 rpm for 25 minutes at 4°C, the supernatant was discarded, and the radioactivity counts of each tube were measured using a γ counter. A standard curve was plotted on logit-log coordinate paper, and the concentration of each sample was determined by reading the corresponding value from the standard curve based on the B/B₀% ratio.

2.7 ELISA Method for Determining Serum IFN-γ Concentration

The eyeballs of mice were enucleated to collect blood, which was then collected in clean test tubes. The blood was allowed to clot at room temperature for 2 hours, followed by centrifugation at 1,500 rpm for 10 minutes to obtain serum, which was aliquoted and stored at −20°C. For the determination of IFN-γ concentration, the procedure was carried out according to the kit instructions.

Reagent Preparation: ① Take the reagent kit out of the refrigerator 30 minutes in advance to allow it to equilibrate to room temperature. ② Dilution of standard solutions: Add 1 mL of sample diluent to each vial of lyophilized standard, dissolve thoroughly, and then perform serial two-fold dilutions. ③ Preparation of biotin-labeled antibody working solution: Calculate the total volume required based on 0.1 mL per well, and prepare the solution by adding 10 μL of biotin-labeled antibody to 990 μL of antibody diluent. ④ Preparation of avidin–peroxidase complex (ABC) working solution: Calculate the total volume required based on 0.1 mL per well, and prepare the solution by adding 10 μL of avidin–peroxidase complex (ABC) to 990 μL of ABC diluent.

Operating Steps: ① Take out the required number of microplates from the sealed bags that have been equilibrated to room temperature. ② Sequentially add 0.1 mL of each serially diluted standard solution to one row of seven wells, leaving one well filled only with sample diluent as a control. Then, add 100 μL of the processed sample to each well. ③ Seal the reaction wells with sealing tape and incubate in a 37°C incubator for 90 minutes. ④ Manual plate washing: Carefully discard the liquid in each well, add 350 μL of washing solution to each well, let it sit for 30 seconds, then discard the liquid again, pat dry on thick absorbent paper, and repeat the washing process twice. ⑤ Sequentially add the prepared biotin antibody working solution at 0.1 mL per well. Seal the reaction wells with sealing tape and incubate at 37°C for 60 minutes. ⑥ Wash three times with 0.01 M TBS, soaking for about 1 minute each time. ⑦ Sequentially add the prepared ABC working solution at 0.1 mL per well. Seal the reaction wells with sealing tape and incubate at 37°C for 30 minutes. ⑧ Wash five times with 0.01 M TBS, soaking for about 1–2 minutes each time. ⑨ Sequentially add 0.09 mL of TMB developing solution that has been equilibrated at 37°C for 30 minutes to each well, and incubate in the dark at 37°C. During the incubation, observe frequently; when you can clearly see a distinct gradient of blue color in the first 3–4 wells of the standard solution, but the last 3–4 wells show little difference, add 0.1 mL of TMB stop solution to each well. (The developing reaction should not exceed 30 minutes.) Measure the OD value at 450 nm using an enzyme-linked immunosorbent assay reader, and determine the corresponding concentration based on the absorbance values of the samples on the coordinate graph.

2.8 Statistical Methods

All experimental data are expressed as mean ± standard deviation (x̄±s). SPSS 16.0 statistical software was used for data analysis, and one-way ANOVA was applied for comparing multiple groups.

3 Experimental Results

3.1 General Condition

During the first three days after tumor cell inoculation, the mice showed no obvious differences in appearance compared with the normal group; their food intake was normal, they were active, and their fur was shiny. Starting from the fifth day after inoculation, the model group became sluggish, with a flat mass palpable under the skin of the right forelimb axilla, its surface uneven and showing multiple nodules. In the 5-Fu group and the Pei’s Shengxue Granules combined with 5-Fu groups, the masses felt smaller, but the animals appeared more energetic than those in the previous two groups. After one week, the model group’s food intake decreased, the masses grew larger, their movement slowed down, their fur lost its luster, piloerection was obvious, their reactions were sluggish, and they preferred to gather in groups. The 5-Fu group’s condition was significantly worse than both the model group and the normal group: their fur was dull and sparse, they moved less, preferred to gather in groups, were lazy to forage, and these conditions worsened day by day. In the Pei’s Shengxue Granules combined with 5-Fu groups, the general condition was still relatively good: the fur remained shiny, there was no hair loss, they were still active, and their food intake was normal.

3.2 Effects of Pei’s Shengxue Granules Combined with 5-Fu on Tumor Weight and Tumor Suppression Rate in Tumor-Bearing Mice

As shown in Table 1, the tumor weights in all treatment groups were lower than those in the model group, with statistically significant differences (p<0.05). The tumor weights in the low-, medium-, and high-dose Pei’s Shengxue Granules combined with 5-Fu groups were all lower than those in the 5-Fu group, with the medium-dose group showing a statistically significant difference compared with the 5-Fu group (p<0.05). The tumor suppression rates for the low-, medium-, and high-dose Pei’s Shengxue Granules combined with 5-Fu groups and the 5-Fu group were 51.17%, 61.07%, 56.03%, and 47.93%, respectively. See Table 1 and Figure 1 for details.

Table 1: Effects of Pei’s Shengxue Granules on Mouse Tumor Weight and Tumor Suppression Rate (n=10, x̄±s)

Note: *p<0.05 compared with the model group, △p<0.05 compared with the 5-Fu group