3.7 Effects of Pei’s Shengxue Granules on the Apoptosis Rate of Tumor Cells in Tumor-Bearing Mice

3.7 Effects of Pei’s Shengxue Granules on the Apoptosis Rate of Tumor Cells in Tumor-Bearing Mice

As shown in Table 5, compared with the model group, the large-, medium-, and small-dose Pei’s Shengxue Granules groups as well as the Zhenqi Fuzheng Granules group all increased the apoptosis rate of tumor cells (P<0.05). The medium-dose PG group showed the most pronounced effect, with an apoptosis rate of 33.46%, indicating that PG has the ability to promote or induce apoptosis in tumor cells. See Table 5 and Figure 5.

Table 5: Effects of Pei’s Shengxue Granules on the Apoptosis Rate of Tumor Cells in Tumor-Bearing Mice (%; X±S)

Note: Compared with the model group, **P<0.01 *P<0.05

Discussion

Traditional Chinese Medicine regards “internal deficiency of vital energy” as the fundamental cause of disease. According to the “Suwen · Yipian Cifa Lun,” “When vital energy is preserved within, pathogenic factors cannot invade”; and according to the “Suwen · Pingre Bing Lun,” “Wherever pathogenic factors gather, vital energy must be deficient.” The “Yizong Bida” also states, “The formation of accumulations occurs when vital energy is insufficient and pathogenic factors take hold.” Zhang Jingyue further noted, “Anyone with spleen-kidney deficiency or general weakness is prone to accumulation-related diseases.” All these principles indicate that the development and progression of tumors is a process of vital energy deficiency and pathogenic factor excess, with vital energy depletion being the root cause. Therefore, the treatment of tumors in TCM focuses on comprehensively regulating the balance of qi, blood, yin, and yang in the body, strengthening vital energy to expel pathogenic factors. Consequently, the therapeutic principle of “strengthening vital energy and consolidating the foundation to drive out pathogenic factors” has been proposed. Pei’s Shengxue Granules mainly consist of Liuwei Dihuang Tang combined with Shengmai San, plus Taizishen, Beishashen, Dangshen, and other ingredients—all of which are considered tonifying and reinforcing herbs. The formula emphasizes Taizishen, Dangshen, and Beishashen to strengthen the spleen, while Liuwei Dihuang Tang is used to tonify the kidneys, fully embodying the TCM principle of “strengthening vital energy and consolidating the foundation.”

This view has also been confirmed by modern immunology: the occurrence, development, and prognosis of tumors are closely related to the body’s immune status. If the body’s immune function is weakened, it may lead to the onset and aggravation of tumors; moreover, the higher the degree of tumor differentiation and malignancy, the more likely it is that tumor antigens will suppress the body’s immune function or allow tumor cells to evade immune surveillance, resulting in a poorer prognosis. Thus, the importance of the therapeutic principle of “strengthening vital energy and consolidating the foundation” in tumor treatment is evident. Following this guiding principle, many domestic oncologists have conducted extensive research on the antitumor effects of traditional Chinese herbal medicines based on their clinical experience. Experiments have shown that Chinese herbal medicines can effectively improve deficiencies in vital energy and enhance the body’s immune function, while also exerting antitumor effects such as killing tumor cells and blocking the cell cycle. Due to the complex composition of Chinese herbal medicines, compound formulations often possess multiple mechanisms of action, which is the advantage of Chinese medicine in targeting multiple aspects and pathways in antitumor therapy.

Professor Pei Zhengxue, a renowned expert in integrated Chinese and Western medicine and mentor, drawing on his more than forty years of clinical experience, believes that vital energy deficiency is the fundamental cause of malignant tumor occurrence and development, and that strengthening vital energy and consolidating the foundation is the basic principle for treating malignant tumors. Based on this insight, he formulated the “Lanzhou Formula,” which, after more than thirty years of continuous practice, refinement, and reorganization, eventually evolved into “Pei’s Shengxue Granules.”

In recent years, animal experiments have demonstrated that Pei’s Shengxue Granules can significantly increase hemoglobin, white blood cell, and platelet levels in aplastic anemia model mice (established by whole-body irradiation with a 3.0 Gy linear

Research on Pei Zhengxue Series Formulas

accelerator), and can markedly restore bone marrow hematopoietic function in these mice [8]. Histopathological examination of mouse spleen sections shows that Pei’s Shengxue Granules can alleviate pathological changes in the spleen of aplastic anemia model mice, reduce lymphocyte apoptosis, promote the recovery of germinal centers, expand the white pulp area, thin the capsule, and essentially normalize dilated sinusoids. Immunohistochemical analysis of spleen CD4 and CD8 levels indicates that Pei’s Shengxue Granules can increase CD4 levels and decrease CD8+ levels in the spleen of aplastic anemia model mice, thereby increasing the CD4/CD8 ratio and enhancing the immune function of these mice [9]. MTT colorimetric assays show increased lymphocyte proliferation; double-antibody sandwich ELISA reveals an upward trend in IL-2 concentration in splenic lymphocyte culture supernatants; and reverse transcription-polymerase chain reaction (RT-PCR) demonstrates increased expression levels of IL-2 mRNA and IFN-γ mRNA in splenic lymphocytes [10].

This experiment found that, compared with the control group, the large-, medium-, and small-dose Pei’s Shengxue Granules groups all had higher thymus and spleen indices than the model group. This suggests that Pei’s Shengxue Granules have a significant weight-increasing effect on the thymus and spleen—immune organs—in tumor-bearing mice, indicating that Pei’s Shengxue Granules can enhance the non-specific immune function of these mice. The experiment also observed that all three dose groups of PG significantly inhibited tumor growth in tumor-bearing mice, with PG’s tumor inhibition rate markedly higher than that of the model group, especially in the medium-dose group, demonstrating that Pei’s Shengxue Granules have excellent therapeutic effects against tumors. Morphologically, PG was also found to induce apoptosis in tumor cells of tumor-bearing mice; transmission electron microscopy revealed that some tumor cells had condensed chromatin arranged in a crescent shape, with chromatin condensation and solidification, nuclear deformation, intact mitochondrial and other organelle structures, and some cells even showed apoptotic bodies, while macrophages were observed phagocytosing apoptotic cells in localized tumor tissues.

Wang He et al. [29] demonstrated that Liuwei Dihuang soft capsules have certain antitumor and anti-aging effects, and suggested that enhancing the body’s immune function might be one of the mechanisms behind these effects. Research has shown that American ginseng polysaccharides affect calcium-dependent potassium channels in mouse T lymphocytes; patch-clamp recordings revealed that American ginseng polysaccharides can increase the open probability, prolong the opening time, and shorten the closing time of calcium-dependent potassium channels activated by ConA, but cannot activate the channels on their own [30]. Studies have also found that Ophiopogon polysaccharides can increase the weight of the thymus and spleen in mice, promote macrophage phagocytosis, and strongly counteract the decline in white blood cell counts caused by cyclophosphamide, thus having a clear promoting effect on all aspects of the body’s immune system and enhancing humoral immunity and adaptability. Zhao Guohua et al. [31] reported that 150 mg/kg of yam polysaccharides has the best inhibitory effect on Lewis lung cancer and B16 melanoma.

Numerous studies have also shown that the uncontrolled proliferation of malignant tumors is associated with reduced apoptosis and increased cell division. Many genes have now been identified as being involved in tumor cell apoptosis, among which nuclear transcription factor NF-κB is a key regulator of gene transcription, participating in the regulation of numerous genes related to cell proliferation and apoptosis, and playing an important role in the occurrence, development, and prognosis of tumors [33–35]. Nuclear Factor-κB (NF-κB) has been proven to be widely present in various types of cells. Activation of NF-κB can regulate both anti-apoptotic and pro-apoptotic protein expression, as well as the expression of cell cycle regulatory proteins at checkpoints, including P53, Bcl-2, IAPs, CyclinD1, and GADD45, among others. To date, as many as 150 genes have been found to be regulated by NF-κB [35–38]. Studies have found that NF-κB is highly expressed in many types of tumors, including gastric cancer, breast cancer, colorectal cancer, liver cancer, ovarian cancer, multiple myeloma, prostate cancer, and others; inhibiting NF-κB expression can promote apoptosis and block the cell cycle [39]. In recent years, pharmaceutical research institutions both domestically and internationally have conducted extensive research on NF-κB, discovering a series of compounds that act on the NF-κB pathway, primarily inhibitors of NF-κB activity. These inhibitors can block NF-κB activation at different stages, inducing apoptosis, inhibiting cell proliferation, and exerting antitumor effects. NF-κB plays an extremely important role in regulating pathological physiological processes such as apoptosis and the cell cycle [40–46]. However, numerous experimental studies have shown that the role of NF-κB varies depending on the type of tumor cell and the specific drug-induced apoptosis pathway; furthermore, even for the same type of drug, the role of NF-κB in different cell types’ apoptosis pathways, the relationship between P53 and NF-κB, and the impact of different levels of NF-κB inhibition on apoptosis remain unclear, requiring further research [47–48]. Further studies have shown that one of the main characteristics of malignant tumor cells is uncontrolled autonomous proliferation. Abnormal regulation of tumor cell proliferation, however, results from disruptions in cell cycle checkpoints, leading to unlimited proliferation of tumor cells and a relative or absolute reduction in cell death [49,50]. DNA serves as the fundamental basis for cell proliferation, differentiation, and genetic material; if the synthesis of DNA and RNA is inhibited, cell proliferation and differentiation will inevitably be affected. During the cell cycle, the G1 phase is the pre-DNA synthesis phase, during which RNA, proteins, and necessary precursor substances are synthesized. The S phase is the DNA synthesis phase, where DNA self-replication occurs along with the synthesis of key substances such as histones, resulting in a doubling of DNA content. This is followed by the G2 phase, the post-DNA synthesis phase, which prepares the cell for the M phase. The strong proliferative capacity of tumor cells is mainly reflected in the abnormally active DNA synthesis during the S phase, with vigorous DNA replication. Each phase of the cell cycle must prepare for the next to ensure the production of two daughter cells with identical genetic material. Therefore, cells must undergo checks before entering the next phase, and each checkpoint is crucial for maintaining the cell cycle and genomic stability. If the "checking" function of these checkpoints fails, cancer may develop. Among the four checkpoints in the cell cycle—G1/S, S/G2, G2/M, and M/G1—the G1/S and G2/M checkpoints are the most important [1]. Antitumor drugs that block or delay a specific point or part of the tumor cell cycle can affect the progression of the cell cycle, thereby slowing down or even halting tumor cell proliferation and causing changes in the distribution of cell cycle phases.

The results of this experiment indicate that PG inhibits NF-κB expression and increases the proportion of tumor cells in the G0/G1 phase while decreasing the proportion in the S phase, with the medium-dose group showing the most pronounced effect. Therefore, it is believed that the antitumor mechanism of PG lies in inhibiting NF-κB expression, affecting gene transcription function, and blocking the transition of tumor cells from the G1 phase to the S phase, resulting in a large accumulation of G1-phase tumor cells. Meanwhile, DNA synthesis and replication in S-phase tumor cells are inhibited and blocked, leading to a decrease in the S-phase ratio and reduced cell proliferation, thus suppressing tumor growth and proliferation. Consequently, Pei's Shengxue Granules exhibit good antitumor effects, particularly the medium-dose group, which demonstrates excellent antitumor efficacy, providing ample scientific theoretical support for its widespread clinical application. However, how PG regulates gene transcription remains to be further investigated.

Conclusion