2 Experimental Methods

2 Experimental Methods

2.1 Model Preparation

Strictly following sterile procedures, solid tumors are implanted. From the H22 tumor strain, a milky-white tumor fluid is extracted from the abdominal cavity of mice according to literature [14], diluted with physiological saline to a tumor cell count of 2×10⁶ cells/ml, and 0.2 ml of the tumor fluid is injected subcutaneously into the right anterior axillary region of the mice [14].

2.2 Animal Grouping and Dosage Administration

Seventy-two SPF-grade Kunming mice are randomly divided into 12 as the blank group based on weight and gender, while the remaining 60 mice are inoculated 24 hours later and then randomly assigned to the model control group; the Compound Cantharidin Capsules (BM) group; the Pei’s Soft Liver and Anti-Ascites Pill (PRGXP) low-dose group;

Research on Pei Zhengxue’s Series of Formulas and Medications

the PRGXP medium-dose group; and the PRGXP high-dose group. The specific dosages are as follows:

BM group: Clinical adult dosage is 3 capsules per dose, twice daily, 0.25 g per capsule. Based on a 60 kg body weight, the adult dosage is 0.025 g/kg·d, while the mouse dosage is equivalent to 10 times the adult dosage, i.e., 0.25 g/kg·d.

PRGXP group: Clinical adult dosage is 1 packet per dose, twice daily, 6 g per packet. Based on a 60 kg body weight, the adult dosage is 0.2 g/kg·d. The PRGXP low-dose group receives 1 g/kg (equivalent to 5 times the adult clinical dosage); the PRGXP medium-dose group receives 2 g/kg (equivalent to 10 times the adult clinical dosage); and the PRGXP high-dose group receives 4 g/kg (equivalent to 20 times the adult clinical dosage).

Mouse gavage volume: 0.2 ml per 10 g body weight.

Pei’s Soft Liver and Anti-Ascites Pill and Compound Cantharidin Capsules are each dissolved in distilled water before use to form suspensions. The experimental group mice are given equal volumes of the drugs via gavage, while the blank group and model control group mice receive equal volumes of distilled water, once daily, for 10 consecutive days.

2.3 Observation of General Condition of Mice

Observe the fur color, diet, activity level, and excretion of mice in each group during the experiment.

2.4 Determination of Tumor Inhibition Rate

Twenty-four hours after the last dose, weigh the mice, perform spinal cord dislocation euthanasia, dissect out the tumor tissue, weigh it, and calculate the tumor inhibition rate using the formula:

Tumor Inhibition Rate (%) = [(Average Tumor Weight of Model Group - Average Tumor Weight of Treatment Group) / Average Tumor Weight of Model Group] × 100%

2.5 Measurement of Thymus and Spleen Indices

Twenty-four hours after the last dose, weigh the mice, perform spinal cord dislocation euthanasia, remove the thymus and spleen, weigh them, and calculate the thymus and spleen indices using the formulas:

Thymus Index (mg/10 g) = Thymus Weight / (Final Body Weight - Tumor Weight) × 10

Spleen Index (mg/10 g) = Spleen Weight / (Final Body Weight - Tumor Weight) × 10

2.6 Determination of Serum TNF-α Concentration in Tumor-Bearing Mice Using ELISA Method