2 Experimental Methods

2 Experimental Methods

2.1 Model Preparation

Strictly follow aseptic techniques. According to the literature, extract milky white tumor fluid from the abdominal cavity of H22 tumor-strain-transferred mice, dilute it with physiological saline to a tumor cell count of 2×10⁶ cells/mL, disinfect the skin at the right anterior axillary region of each mouse with iodine tincture and 75% ethanol, and then subcutaneously inoculate 0.2 mL of tumor fluid at the disinfected site.

2.2 Animal Grouping, Dosage, and Administration Method

Randomly select 12 SPF-grade Kunming mice based on body weight and gender as the blank group; weigh the remaining 60 mice 24 hours after subcutaneous tumor inoculation, then randomly divide them into: model control group; Pei’s Soft Liver and Anti-Bloating Pill (PRGXP) administration group (divided into small, medium, and large dose groups); and Compound Cantharidin Capsule group (BM group). Administer drugs as follows:

PRGXP group: Adults take 1 bag per dose, twice daily, 6 g per bag. Based on a body weight of 60 kg, the adult dosage is 0.2 g/kg·d. The dosages for mice are:

• PRGXP small-dose group: 1 g/kg body weight, equivalent to 5 times the adult clinical dosage
• PRGXP medium-dose group: 2 g/kg body weight, equivalent to 10 times the adult clinical dosage
• PRGXP large-dose group: 4 g/kg body weight, equivalent to 20 times the adult clinical dosage

BM group: Adults take 3 capsules per dose, twice daily, 0.25 g per capsule. Based on a body weight of 60 kg, the adult dosage is 0.025 g/kg·d. The dosage for mice is equivalent to 10 times the adult dosage, i.e., 0.25 g/kg·d.

Mice gastric gavage volume: 0.2 mL per 10 g.

Pei’s Soft Liver and Anti-Bloating Pill and Compound Cantharidin Capsules are dissolved in distilled water to prepare suspensions for later use. Mice in the experimental groups receive equal volumes of the drug by gastric gavage, while mice in the model control group receive an equal volume 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 starting from the time of tumor cell suspension inoculation.

2.4 Determination of Tumor Inhibition Rate

Twenty-four hours after the last drug administration, euthanize the mice by spinal dislocation, weigh them, quickly excise the tumors, weigh the tumors using an electronic balance, calculate the average tumor weight for each group, compare the tumor weights of the treatment groups with those of the control group, and calculate the tumor inhibition rate.

Tumor inhibition rate (%) = [(Average tumor weight of the model group - Average tumor weight of the treatment group) / Average tumor weight of the model group] × 100%

Research on Pei Zhengxue’s series of prescriptions

2.5 Determination of Thymus and Spleen Indices

As above, 24 hours after the last drug administration, euthanize the mice by spinal dislocation and weigh them, then open the abdomen to remove the thymus and spleen and weigh them separately.

Calculate the thymus and spleen indices according to the formula:

Thymus index (mg/10 g) = Thymus weight / (Mouse final body weight - Tumor weight) × 10

Spleen index (mg/10 g) = Spleen weight / (Mouse final body weight - Tumor weight) × 10

2.6 Detection of P27 and Bcl-2 Protein Expression in Tumor Tissue

Euthanize the mice by cervical dislocation, excise the tumors, then strip off the connective tissue attached to the tumor mass to obtain the tumor tissue, fix it with 10% formaldehyde, dehydrate it, and embed it in paraffin.