4.2 Establishment of the Animal Model

4.2 Establishment of the Animal Model

This experiment adopted the classic drug-induced tumor modeling method—the H₂₂ liver cancer transplantation model in mice. According to different experimental needs, there are three types of transplantation models: orthotopic transplantation model (transplanting liver cancer cells or tissue directly into the animal’s liver), subcutaneous-orthotopic transplantation tumor model (first implanting intact liver cancer tissue under the animal’s skin to form a transplantable tumor, then transplanting the tumor into the animal’s liver), and ectopic transplantation model (transplanting liver cancer cells or tissue into other parts of the animal’s body, such as the subcutaneous tissue). This experiment used the third method, which is the most commonly used. The H₂₂ tumor strain (solid liver cancer) was introduced by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, in 1963, and later transformed into an ascitic type. Tests revealed that it still retains epithelial tissue characteristics, classifying it as poorly differentiated liver cancer.

Research on Pei Zhengxue’s Series of Formulas and Medicines

Moreover, previous senior colleagues who established the model have all indicated that this method achieves a tumor formation rate of 100%, with stable biological characteristics, rapid growth, and ease of operation. Therefore, the drug’s metabolism and distribution within the body can be better reflected.

4.3 Selection of Positive Control Drugs

When selecting positive control drugs, certain principles must be followed. First, comparability: Compound Cantharidin Capsules also have effects such as breaking blood stasis, clearing heat and detoxifying, and softening hard masses and dispersing nodules. Clinically, they are used to treat primary liver cancer and other gastrointestinal tumors, with similar functions, indications, and routes of administration as Pei’s Soft Liver and Anti-Bloating Pill. Second, legality: Compound Cantharidin Capsules are legally produced medicines manufactured in accordance with the Pharmacopoeia standards. Third, this drug is currently recognized as an effective anti-cancer medication. Experiments have shown that cantharidin in cantharidin can inhibit DNA and RNA synthesis in tumor cells, induce apoptosis, and block cancer cell division at the M phase, disrupting cell structure [9]. Therefore, this experiment selected Compound Cantharidin Capsules because they share considerable similarities with Pei’s Soft Liver and Anti-Bloating Pill in terms of function and pharmacological research, making them suitable as positive control drugs.

4.4 Antitumor Effects of Pei’s Soft Liver and Anti-Bloating Pill

The experimental results indicate that the average tumor weight in the three dosage groups of Pei’s Soft Liver and Anti-Bloating Pill was lower than that in the model group, at (0.914±0.073) g, (0.777±0.084) g, and (0.966±0.079) g, respectively. All these differences were statistically significant compared with the model control group (p<0.05). The corresponding tumor inhibition rates were 32.2%, 42.4%, and 28.2%, proving that Pei’s Soft Liver and Anti-Bloating Pill can significantly inhibit tumor growth, with the medium dose showing the best effect.

4.5 Effects of Pei’s Soft Liver and Anti-Bloating Pill on Immune Organs

The body’s immune function is closely related to the development and progression of tumors. A strong immune system enhances the body’s immune response against tumors, thereby inhibiting their growth. The spleen and thymus are the most important immune organs. The spleen is the largest peripheral immune organ, housing various immune cells and serving as the site for generating immune responses to antigens, as well as the collection point for multiple immune effectors. The thymus is a central and crucial immune organ, playing a major role in cellular immunity and serving as the key site for the production, differentiation, and maturation of T cells. It can mediate cellular immunity against tumors. Therefore, changes in the thymus and spleen can to some extent reflect the strength of the body’s immune status, and improving the quality of the thymus and spleen is considered one of the objective indicators for enhancing immune function.

In the model group, the weight of the thymus and spleen in mice decreased, with smaller volumes. A few thymuses appeared grayish-white, while the spleens were pale red. In contrast, the thymus index in all dosage groups of Pei’s Soft Liver and Anti-Bloating Pill was higher than that in the model group, with the medium- and large-dose groups showing statistically significant increases compared with the model control group (p<0.05). Similarly, the spleen index in all dosage groups was higher than that in the model group, with the medium- and large-dose groups showing statistically significant increases compared with the model control group (p<0.05). There was no significant difference between the dosage groups and the cantharidin group (p>0.05). The experimental results demonstrate that Pei’s Soft Liver and Anti-Bloating Pill can improve the immune organs—thymus and spleen—in H₂₂ tumor-bearing mice, indicating that the drug can enhance the non-specific immune function of tumor-bearing mice.

4.6 Effects of Pei’s Soft Liver and Anti-Bloating Pill on Selected Indicators