The Effect of Pei's Shengxue Granules Combined with 5-Fu on Apoptosis of H₂ Tumor Cells and P27 Protein Expression in Mice Bearing Transplanted Tumors

The Effect of Pei's Shengxue Granules Combined with 5-Fu on Apoptosis of H₂ Tumor Cells and P27 Protein Expression in Mice Bearing Transplanted Tumors

Ding Jiexia

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Chinese Abstract

Objective: To explore the efficacy-enhancing and toxicity-reducing effects of Pei's Shengxue Granules (PG) on chemotherapy in H₂ tumor-bearing mice, as well as their impact on P27 protein expression in tumor tissues and the apoptosis rate of tumor cells.

Methods: An H₂ tumor-bearing mouse model was established. The in vivo antitumor activity of 5-Fu was analyzed by calculating the inhibition rate to determine PG's efficacy-enhancing effect. The toxicity-reducing effect of PG on 5-Fu-treated mice was assessed by measuring the thymus index and spleen index. Immunohistochemistry was used to determine the expression of P27 protein in H₂ tumor tissues, while flow cytometry was employed to measure the tumor cell cycle and apoptosis rate.

Results: ① The inhibition rate of 5-Fu was 42.47%. The combination of PG and 5-Fu exhibited an additive antitumor effect, with high and medium doses of PG combined with 5-Fu showing particularly significant results, achieving inhibition rates of 62.66% and 59.30%, respectively. ② PG alleviated the suppression of immune organs caused by 5-Fu in tumor-bearing mice, leading to significant improvements in both the thymus index and spleen index. ③ The expression of P27 protein in tumor tissues differed significantly among treatment groups compared with the model group (P<0.05), with PG upregulating P27 protein expression. ④ In the model group, the proportion of tumor cells in the G₀/G₁ phase was the lowest, while the S phase proportion was the highest. In all treatment groups, the proportion of cells in the G₀/G₁ phase increased markedly, with the high and medium dose PG + 5-Fu groups showing the most pronounced increase, which was significantly different from both the model group and the 5-Fu group (P<0.05). Conversely, the proportion of cells in the S phase decreased across all treatment groups, with the high-dose PG + 5-Fu group experiencing the most notable reduction, also significantly different from the model group and the 5-Fu group (P<0.05). ⑤ The apoptosis rate of tumor cells increased in all PG + 5-Fu dosage groups, with the high and medium dose PG + 5-Fu groups showing the most substantial rise, significantly different from the 5-Fu group (P<0.05).

Conclusion: PG demonstrates clear efficacy-enhancing and toxicity-reducing effects in tumor-bearing mice undergoing 5-Fu chemotherapy. It can upregulate P27 protein expression in tumor tissues and induce tumor cell apoptosis, thereby exerting an antitumor effect.

Keywords: Pei's Shengxue Granules (PG); efficacy enhancement; toxicity reduction; cell cycle; apoptosis; P27 protein

Research on Pei Zhengxue's Series of Formulas and Medicines

ABSTRACT

Objective: To research the efficacy enhancing and toxicity reducing of PG in H22 tumor-bearing mice on chemotherapy and the affect of tumor tissues of P27 protein expression and tumor cell apoptosis rate.

Methods: To establish the model of H22 tumor-bearing mice. To analyse antitumor activity of 5-Fu in vivo and judge efficacy enhancing of PG by calculating inhibition rate. To judge PG toxicity reducing on 5-Fu by measuring the thymus index and spleen index. To observe the expression of P27 protein by immunohistochemical method, tumor cell cycle and apoptosis rate by flow cytometry.

Results: ① The inhibition rate of 5-Fu was 42.47%. PG and 5-Fu combination had additive anti-tumor effect. High and medium doses PG and 5-Fu combination had significant anti-tumor effect and their inhibition rates were 62.66% and 59.30%. ② PG reduced the tumor-bearing mice by 5-Fu-induced suppression of immune organs, and the thymus index and spleen index improved. ③ P27 protein expression in the treatment groups compared with the model group were significantly different (p<0.05) and PG could induce the expression of P27 protein value. ④ Model group, the lowest proportion of cells in G0/G1 phase and the highest proportion of S phase. Medication in each group increased in the proportion of cells in G0/G1 phase. High and medium doses of PG + 5-Fu groups increased most significantly and compared with the model group and 5-Fu group were significantly different (p<0.05). Medication in each group the proportion of cells in S phase reduced. High-dose PG + 5-Fu group reduced the most obvious and compared with the model group and 5-Fu group were significantly different (p<0.05). ⑤ Various doses of PG combination of 5-Fu groups increased apoptosis of tumor cells. High and medium doses of PG + 5-Fu groups increased the most obvious, compared with 5-Fu group were significantly different (p<0.05).

Conclusion: PG has obvious efficacy enhancing and toxicity reducing on tumor-bearing mice who are treated with 5-Fu. PG can induce the expression of P27 protein and apoptosis in tumor cells to show the antitumor function.

Keywords: PG; efficacy enhancing; toxicity reducing; cell cycle; apoptosis; P27 protein

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Preface

Pei's Shengxue Granules is a specialized formula for treating leukemia, formulated by Professor Pei Zhengxue, a nationally renowned expert in integrated traditional Chinese and Western medicine, based on over 40 years of clinical experience and the theory of "supporting vital energy and consolidating the root." The formula mainly consists of Liuwei Dihuang Tang combined with Shengmai San, plus ginseng root, Prince Ginseng, North Sand Ginseng, Codonopsis pilosula, etc., with emphasis on using ginseng root, Prince Ginseng, Codonopsis pilosula, and North Sand Ginseng to strengthen the spleen, while Liuwei Dihuang Tang is used to tonify the kidneys.

Research on Pei Zhengxue's Series of Formulas and Medicines

This formula was named the "Lanzhou Formula" at the 1974 National Hematology Conference in Suzhou after completely and thoroughly curing a patient with acute monocytic leukemia (M₂), Ma Changsheng, thirty years ago. Since then, Professor Pei has used this formula, with modifications, to treat various blood diseases and, primarily in combination with radiotherapy and chemotherapy, to treat tumors, achieving remarkable therapeutic effects. In 2003, Pei's Shengxue Granules successfully cured another leukemia patient (L₂), Liu Ligang, which was subsequently reported in the Nanchang Daily and the Gansu Daily. In addition to treating leukemia, Professor Pei has achieved significant therapeutic effects with this formula in treating malignant tumors, especially when used in conjunction with radiotherapy and chemotherapy to treat various malignancies. When used together with radiotherapy and chemotherapy drugs, it can enhance efficacy and reduce toxicity, improve the body's immunity and anti-tumor treatment outcomes, prolong patients' survival, and improve their quality of life. In 1997, this formula was designated as an internal preparation of the Gansu Provincial Academy of Medical Sciences and named "Pei's Shengxue Granules." Some experimental studies on this drug have passed achievement appraisal and even won the First Prize for Huangfu Mi Scientific and Technological Achievements in Gansu Province. Given its remarkable clinical efficacy, Pei's Shengxue Granules has become a well-known specialized remedy for blood diseases and tumors both within and outside the province.

During my apprenticeship, according to incomplete statistics, this formula has cured three cases of esophageal cancer, one case of gastric cancer, three cases of leukemia, three cases of aplastic anemia, and three cases of primary liver cancer. Among them, the therapeutic effects on esophageal cancer, gastric cancer, liver cancer, leukemia, and aplastic anemia are particularly significant. Xue used the "Lanzhou Formula" to treat primary liver cancer, resulting in a noticeable reduction in the patient's tumor mass, complete recovery of liver function, improved quality of life, and prolonged survival. Li and others used the Lanzhou Formula in combination with chemotherapy to treat ten cancer cases, and the observation showed that the short-term efficacy of the Lanzhou Formula group was superior to that of the chemotherapy-only group (p<0.05); in terms of adverse reactions, the Lanzhou Formula group experienced significantly fewer side effects than the chemotherapy-only group (p<0.01). Pei's Shengxue Granules were used in combination with radionuclides to treat fifteen liver cancer cases, with the treatment group receiving radionuclides plus Pei's Shengxue Granules (PG) and the control group receiving only radionuclides. It was found that the treatment group experienced significantly milder side effects than the control group, and there was also a significant difference in therapeutic efficacy between the two groups.

Animal experiments have demonstrated that Pei's Shengxue Granules can significantly inhibit mutant p53 and increase Caspase-3 protein expression, suggesting that the drug can induce tumor cell apoptosis by downregulating mutant p53 and upregulating Caspase-3 protein expression, indicating that Pei's Shengxue Granules possess significant anti-tumor effects. Inhibiting tumor cell growth and proliferation is one of the important molecular mechanisms behind Pei's Shengxue Granules' anti-tumor action. Pei's Shengxue Granules inhibit NF-κB expression, affecting gene transcription function and blocking the progression of tumor cells from the G₁ phase to the S phase, causing a large accumulation of G₁ phase tumor cells. At the same time, DNA synthesis and replication in the S phase are inhibited and blocked, leading to a decrease in the S phase ratio and a decline in cell proliferation ability, thereby suppressing tumor growth and proliferation. Pei's Shengxue Granules can increase the apoptosis rate of tumor cells, inducing tumor cell apoptosis, which further demonstrates their significant anti-tumor effects. Pei's Shengxue Granules have a clear weight-increasing effect on the immune organs of tumor-bearing mice, namely the thymus and spleen, thereby enhancing the specific immune function of these mice. They can promote the proliferative capacity of splenic lymphocytes stimulated by ConA, strengthening the cellular immune function of tumor-bearing mice and enhancing their anti-tumor effects. They can also promote the secretion of IL-2 and the expression of IL-2 and IFN-γ mRNA, suggesting that Pei's Shengxue Granules can regulate the body's immune function by influencing cytokines. Pei's Shengxue Granules can regulate the body's disordered immune state, enhancing delayed allergic reactions in tumor-bearing mice and increasing their cellular immune function. They can also enhance the phagocytic function of peritoneal macrophages, with trends of increased levels of IL-1, IFN-γ, NO, and other cytokines. Pei's Shengxue Granules have a clear inhibitory effect on tumors in H₂₂ tumor-bearing mice. Enhancing immune function is an important mechanism behind Pei's Shengxue Granules' anti-tumor action. Pei's Shengxue Granules can significantly raise the peripheral blood hemoglobin, white blood cell, and platelet counts in aplastic anemia model mice (established through whole-body irradiation with a 3.0 Gy linear accelerator), effectively restoring the bone marrow hematopoietic function of these mice.

Pathological tissue sections of mouse spleens show that Pei's Shengxue Granules can alleviate pathological changes in the spleens of aplastic anemia model mice, reduce the apoptosis rate of lymphocytes, promote the recovery of germinal centers, expand the white pulp area, thin the capsule, and restore dilated sinusoids to normal. Immunohistochemical analysis of CD4 and CD8 levels in the spleen shows that Pei's Shengxue Granules can increase the CD4⁺ content in the spleens of aplastic anemia model mice while decreasing the CD8⁺ content, raising the CD4⁺/CD8⁺ ratio and thus improving the immune function of these mice. In recent years, the immunomodulatory and anti-cancer effects of traditional Chinese medicine on the body have received increasing attention. The presence of tumors not only suppresses cellular immune function but also inhibits humoral immune function, while the use of chemotherapy drugs can further exacerbate immune suppression. Therefore, enhancing the immune function of cancer patients, especially preventing and treating severe immune suppression caused by chemotherapy, has become a major focus in anti-tumor therapy. To further investigate the synergistic and toxicity-reducing effects of Pei's Shengxue Granules on the anti-tumor efficacy of chemotherapy drugs and their underlying mechanisms, we built upon our previous research and employed a mouse H₂₂ tumor-bearing model to study the synergistic and toxicity-reducing effects of Pei's Shengxue Granules following 5-Fu chemotherapy in H₂₂ tumor-bearing mice, as well as the associated mechanisms. By observing the growth of H₂₂ tumors in mice treated with different doses of PG combined with 5-Fu, we determined its anti-tumor efficacy. We also investigated the anti-tumor mechanism of PG from the perspective of inducing tumor cell apoptosis, examining P27 protein expression in H₂₂ tumor cells at both the protein and gene levels, and exploring the molecular mechanisms by which Pei's Shengxue Granules induce tumor cell apoptosis. This research aims to provide scientific evidence for the clinical application of this drug in the treatment of malignant tumors, as well as for the integration of traditional Chinese medicine and Western medicine in oncology, thereby offering a scientific and microscopic basis for the widespread clinical use of Pei's Shengxue Granules.

Experimental Study