Abstract

Abstract

Objective: By observing the effects of Peishi Ruangan Xiaopi Pills (PRGXP) on the expression of Ang-2 in the tumor tissue of H22-bearing mice and the level of IL-12 in their serum, this study takes tumor angiogenesis as the research focus to explore the inhibitory effect of Peishi Ruangan Xiaopi Pills on tumor angiogenesis and their immunomodulatory effects on mice, thereby providing a theoretical basis for their anti-tumor activity. Methods: According to the principle of randomization, 72 tumor-bearing mice were evenly divided into six groups, with 12 mice in each group: Group A—blank control group; Group B—model control group; Group C—low-dose Peishiruanganxiaopiwan group; Group D—medium-dose Peishiruanganxiaopiwan group; Group E—high-dose Peishiruanganxiaopiwan group; and Group F—Fufang Banmao Capsule (BM) group. Tumor tissue was inoculated subcutaneously in the right anterior axilla of the mice to establish the H22 (hepatocellular carcinoma) solid tumor-bearing mouse model for Groups B–F. The mice were administered the respective treatments by gavage for 12 consecutive days. After a 24-hour drug-free interval, the mice were weighed, and blood was collected from the eyeballs using clean test tubes. Following centrifugation, the serum was collected and stored at −20°C. After euthanasia by cervical dislocation, the tumor tissues, thymus, and spleen were excised and weighed to calculate the tumor inhibition rate and the indices of the immune organs (thymus and spleen). Histological sections of the hepatocellular carcinoma tumors were prepared and subjected to HE staining to observe pathological changes. Immunohistochemistry was used to determine the expression of Ang-2 in the tumor tissues of the tumor-bearing mice, while ELISA was employed to measure the concentration of IL-12 in the serum of the tumor-bearing mice.

Results:

1. All dose groups of Peishiruanganxiaopiwan exhibited significant inhibitory effects on the tumor tissues of the H22 tumor-bearing mice: the tumor weight in the high-dose group was (0.914±0.073) g, with a tumor inhibition rate of 32.2%; the medium-dose group had a tumor weight of (0.777±0.084) g, with an inhibition rate of 42.4%; and the low-dose group showed a tumor weight of (0.966±0.079) g, with an inhibition rate of 28.2%. The tumor weights in all dose groups were significantly lower than those in the model control group, with statistically significant differences compared to the control group (p<0.05).

2. HE staining revealed that necrosis of tumor cells was more pronounced in all dose groups of Peishiruanganxiaopiwan than in the model control group.

3. The thymus index (TI) and spleen index (SI) in all dose groups of Peishiruanganxiaopiwan were higher than those in the model group. Specifically, the TI and SI of the medium-dose group were (52.8±11.6) mg/10g and (64.1±7.8) mg/10g, respectively, showing statistically significant differences compared with the model control group (p<0.05).

4. The serum IL-12 levels in all dose groups of Peishiruanganxiaopiwan were higher than those in the model control group. The IL-12 concentrations in the medium- and high-dose groups were (21.15±3.33) pg/ml and (20.95±3.58) pg/ml, respectively, both of which were statistically significant when compared with the model group (p<0.05). The IL-12 concentration in the Fufang Banmao Capsule (BM) group was (21.04±3.70) pg/ml, also showing a statistically significant difference compared with the model control group (p<0.05).

5. The expression of Ang-2-positive cells in the tumor tissues of all dose groups of Peishiruanganxiaopiwan was lower than that in the model group, with significant differences observed between the high- and medium-dose groups and the model group (p<0.05).

Research on the Pei Zhengxue Series of Formulas

Conclusion

Peishiruanganxiaopiwan exhibits a clear inhibitory effect on tumors in H22 tumor-bearing mice, enhances the body’s non-specific immune function, and increases the indices of the thymus and spleen in these mice. Furthermore, this formulation suppresses the expression of Ang-2 in the tumor tissues of H22 tumor-bearing mice while simultaneously elevating the serum IL-12 levels, suggesting that one of its anti-tumor mechanisms may involve inhibiting tumor angiogenesis and thereby suppressing tumor growth.

Keywords: Peishiruanganxiaopiwan; H22 (hepatocellular carcinoma); Angiopoietin-2 (Ang-2); Interleukin-12 (IL-12); Angiogenesis

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ABSTRACT

Objective: By observing the effect of Peishiruanganxiaopi pills (PRGXP) on the expression of Ang-2 in tumor and IL-12 in serum on H₂₂ bearing mice, in order to research the tumor angiogenesis, we have discussed Peishiruanganxiaopi Pill on tumor angiogenesis inhibition, as well as regulating on the immune system of tumor-bearing mice, and provide a scientific basis for the anti-tumor effects of the drug.

Methods: According to the principle of randomization, 72 of tumor-bearing mice model was divided into six groups, each group 12. Group A: blank group, and group B: model group, group C: little dose group of PRGXP, group D: middle dose group of PRGXP, group E: great dose group of PRGXP, group F: Fufang Banmao capsule (BM) group. By hypodermic inoculation in the right front axilla of mice, we established the tumor-bearing mice model for H₂₂. Every group was dosed for twelve days. After 24h of the last dose, the eyeballs of the mice were extirpated for blood collection with clean test tube, collected after centrifugation serum in -20°C refrigerator. Mice were killed after peeling tumor tissue, thymus, spleen weighing, calculating the tumor inhibiting rates, the thymus index (TI) and the spleen index (SI), make the slide of the H₂₂ tumor from the mice and to observe by HE technique; then we assessed effects of PRGXP on Ang-2 in tumor by immunohistochemistry method. Detected the concentration of IL-12 in serum by ELISA.

Results:

1. Each dose group of PRGXP on H₂₂ tumor-bearing mice showed significant inhibition of tumor weight. The weight of tumor in E, D and C group separately are (0.914±0.073) g, (0.777±0.084) g, (0.966±0.079) g, which lower than the model group (p<0.05), corresponding to the antitumor rate are 32.2%, 42.4% and 28.2%;
2. HE staining showed: There was more necrosis in each dose group of PRGXP than in the model group;
3. The SI and TI of the each dose group of PRGXP are higher than the model group. The SI and TI of the middle group are (52.8±11.6) mg/10g, (64.1±7.8) mg/10g, compared with the model group, there was a significantly difference (p<0.05);
4. The density of the IL-12 in each dose group of PRGXP are higher than the model group, the density of the IL-12 of the middle and great group separately are (21.15±3.33) pg·ml⁻¹, (20.95±3.58) pg·ml⁻¹, which has statistic significance in compare with the model group (p<0.05).
5. The expression of Ang-2 in tumor on PRGXP each dose groups have less than the model group.

Research on the Pei Zhengxue Series of Formulas

The PRGXP high- and medium-dose groups compared with the model group were significantly different (p<0.05).

Conclusion: Each dose group of Peishiruanganxiaopiwan (PRGXP) on H22 tumor-bearing mice showed significant inhibition of tumor weight. PRGXP can improve the body's non-specific immune function, increase the weight of spleen and thymus in mice. PRGXP inhibit the expression of Ang-2 in H22 tumor-bearing mice, while elevated serum IL-12 content. PRGXP anti-tumor effect maybe one of the mechanisms by inhibiting the expression of artery.

KEY WORDS: Peishiruanganxiaopiwan; H22 (transplanted liver cancer); Ang-2; IL-12; Angiogenesis.

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Preface