3.1 Significant Effects of Pei's Blood-Boosting Granules on Immune Organs of Tumor-Bearing Mice

3.1 Significant Effects of Pei's Blood-Boosting Granules on Immune Organs of Tumor-Bearing Mice

General observation shows that starting from the 5th day, mice in the tumor-bearing model group exhibit disheveled fur, and a tumor can be vaguely seen under the skin of the right anterior axillary region.

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Food and water intake decrease compared with before, body weight drops, spirits are low, activity is poor, and reactions are sluggish. Between the 8th and 10th days after modeling, the tumor grows rapidly, with an uneven surface and multiple nodules whose boundaries are unclear. By the 13th to 15th days, all mice in the group show cachexia, and some develop ascites. At the end of the experiment, dissection of the model group reveals vigorous tumor growth and high malignancy. In contrast, mice in the Pei's Blood-Boosting Granules groups at various doses show brighter fur, better spirits and diet, and slower tumor growth compared with the model group.

The effects of Pei's Blood-Boosting Granules on the immune organs of tumor-bearing mice are as follows: In the tumor-bearing model group, the thymus and spleen shrink in size and weight; some thymuses have unclear lobes and appear grayish-white, while the spleens turn pale red. However, in the Pei's Blood-Boosting Granules groups at various doses, the appearance of the thymus and spleen is not significantly different from that of the normal group. The thymus indices of the large-, medium-, and small-dose groups are all higher than those of the model group, with the large- and medium-dose groups having thymus indices of 18.9 mg/10 g and 18.0 mg/10 g respectively, representing increases of 36.0% and 29.5% compared with the model group (p<0.05). Compared with the normal group, the thymus index of the model group is significantly lower (p<0.01). The spleen indices of the Pei's Blood-Boosting Granules groups at various doses are also higher than those of the model group, with the large-dose group having a spleen index of 76.3 mg/10 g, which is 17.9% higher than that of the model group (p<0.05). The spleen index of the model group is significantly lower than that of the normal group (p<0.01). See Table 1 and Figures 1-1 and 1-2.

3.2 Effects of Pei's Blood-Boosting Granules on the Proliferation Activity of Splenic Lymphocytes in Tumor-Bearing Mice

The results show that the proliferation activity of splenic lymphocytes in the Pei's Blood-Boosting Granules groups at various doses is higher than that of the model group, with the large-dose group having an A₄₉₀ value of 0.102, which is significantly higher than that of the model group (p<0.05). Compared with the normal group, the proliferation activity of splenic lymphocytes in the model group is significantly lower (p<0.05). See Table 2 and Figure 2.

Table 1: Effects of Pei's Blood-Boosting Granules on the Thymus and Spleen Indices of Tumor-Bearing Mice (n=10, x̄±S)

Note: *p<0.05 compared with the model group; ▲p<0.05 compared with the normal group

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Figure 1-1: Effects of Pei's Blood-Boosting Granules on the Thymus Index of Tumor-Bearing Mice (n=10, x̄±S)

(*p<0.05 compared with the model control group; ▲p<0.05 compared with the normal group)

Figure 1-2: Effects of Pei's Blood-Boosting Granules on the Spleen Index of Tumor-Bearing Mice (n=10, x̄±S)

(*p<0.05 compared with the model control group; ▲p<0.05 compared with the normal group)

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Table 2: Effects of Pei's Blood-Boosting Granules on the Proliferation Activity of Splenic Lymphocytes in Tumor-Bearing Mice (n=8, x̄±S)

Note: *p<0.05 compared with the model group; ▲p<0.05 compared with the normal group

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Figure 2: Effects of Pei's Blood-Boosting Granules on the Proliferation Activity of Splenic Lymphocytes in Tumor-Bearing Mice (n=8, x̄±S)

(*p<0.05 compared with the model control group; ▲p<0.05 compared with the normal group)

3.3 Effects of Pei's Blood-Boosting Granules on the IL-2 Concentration in the Supernatant of Splenic Cells in Tumor-Bearing Mice

The results show that the IL-2 concentration in the supernatant of splenic cells in the Pei's Blood-Boosting Granules groups at various doses is higher than that of the model group, with the large-dose group having an IL-2 concentration of 61.6 pg/mL, which is significantly higher than that of the model group (p<0.05). The IL-2 concentration in the supernatant of splenic cells in the model group is significantly lower than that of the normal group (p<0.01). See Table 3 and Figure 3.

Table 3: Effects of Pei's Blood-Boosting Granules on the IL-2 Concentration in the Supernatant of Splenic Cells in Tumor-Bearing Mice (n=6, x̄±S)

Note: *p<0.05 compared with the model group; ▲p<0.05 compared with the normal group

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Figure 3: Effects of Pei's Blood-Boosting Granules on the IL-2 Concentration in the Supernatant of Splenic Cells in Tumor-Bearing Mice (n=6, x̄±S)

(*p<0.05 compared with the model control group; ▲p<0.05 compared with the normal group)

3.4 Effects of Pei's Shengxue Granules on the mRNA Transcription Levels of IL-2 and IFN-γ in Splenic Lymphocytes of Tumor-Bearing Mice

The electrophoresis images show that the mRNA expression of the internal control β-actin remains constant, with consistent band intensities across all samples; in each dosage group of Pei's Shengxue Granules, the bands for IL-2 and IFN-γ mRNA are brighter than those in the model group, with particularly pronounced increases in band intensity for IL-2 and IFN-γ mRNA in the high- and medium-dose groups. See Figures 4-2 and 4-3.

The results of the ratio between the optical density parameters of the mRNA bands in each sample and those of the internal reference gene (β-actin) mRNA bands indicate that the expression levels of IL-2 and IFN-γ mRNA in splenic cells of mice in all dosage groups of Pei's Shengxue Granules are higher than in the model group, with significant increases observed in the high- and medium-dose groups (p<0.05). Compared with the normal group, the expression levels of IL-2 and IFN-γ mRNA in splenic lymphocytes of mice in the model group are significantly reduced (p<0.01). See Table 4 and Figure 4-1.

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Table 4: Effects of Pei's Shengxue Granules on the Expression of IL-2 and IFN-γ mRNA in Tumor-Bearing Mice (n=6, x̄±S)

Note: Compared with the normal group, *p<0.05, **p<0.01

Figure 4-1: Effects of Pei's Shengxue Granules on the Expression of IL-2 and IFN-γ mRNA in Splenic Lymphocytes of Tumor-Bearing Mice (n=6, x̄±S) (*p<0.05 compared with the model control group; ▲p<0.05 compared with the normal group)

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Figure 4-2: IL-2 Electrophoresis Image

1. Normal Group, 2. Model Group, 3. Low-Dose Group, 4. Medium-Dose Group, 5. High-Dose Group, 6. Zhenqi Group

Figure 4-3: IFN-γ Electrophoresis Image

1. Normal Group, 2. Model Group, 3. Low-Dose Group, 4. Medium-Dose Group, 5. High-Dose Group, 6. Zhenqi Group

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Discussion

Pei's Shengxue Granules are primarily composed of Liuwei Dihuang Tang combined with Shengmai San, supplemented with Taizishen, Beishashen, Dangshen, and other herbs—all of which are tonifying and reinforcing formulas. The formula emphasizes Taizishen, Dangshen, and Beishashen to strengthen the spleen, while Liuwei Dihuang Tang is used to tonify the kidneys, fully embodying the traditional Chinese medicine principle of "tonifying the body and consolidating the root."

In the study of human pathogenesis, traditional Chinese medicine holds that "internal deficiency of vital energy" is the fundamental cause of disease. As stated in the "Suwen Yipian · Cifa Lun": "When vital energy is preserved within, pathogenic factors cannot invade," and in the "Suwen · Ping Re Bing Lun": "Wherever pathogenic factors gather, vital energy must be deficient." In terms of treatment, the principle of "tonifying the body and consolidating the root" has been proposed. The body's "vital energy," also known as "true qi," is described in the "Lingzhu · Ci Jie Zhen Xie Pian" as "the true qi received from heaven, which combines with the qi from food to nourish the body." True qi encompasses both innate and acquired aspects: ancient physicians attributed the innate aspect to the two kidneys, calling it "yuan qi," while the acquired aspect was assigned to the spleen and stomach, referred to as "zhong qi" (food qi). The spleen governs zhong qi, and the kidneys govern yuan qi; therefore, in the principle of "tonifying the body and consolidating the root," strengthening the spleen and tonifying the kidneys represent the two most important components. Experimental studies have demonstrated that the "spleen" and "kidneys" in traditional Chinese medicine play crucial roles in the immune system, endocrine system, metabolic system, autonomic nervous system, and gastrointestinal–pancreatic endocrine system, and that "strengthening the spleen and tonifying the kidneys" can improve the functions of these systems. The concept of "tonifying the body and consolidating the root," which aims to enhance and mobilize the body's resistance to disease, shares significant similarities with modern immunology. In general, the "vital energy" mentioned in traditional Chinese medicine represents the body's intrinsic ability to resist disease, equivalent to the physiological functions of the human immune system. The immune system comprises non-specific immunity and specific immunity. Zhong qi and wei qi in traditional Chinese medicine correspond to non-specific immunity in modern immunology, while yuan qi corresponds to specific immunity; specific and non-specific immunity mutually reinforce each other to achieve the body's overall immune response, and zhong qi and yuan qi likewise complement each other to form the body's vital energy. Thus, "tonifying the body and consolidating the root" can enhance the body's immune function. With the advancement of immunology and molecular biology, research has confirmed that the occurrence and progression of malignant tumors are closely linked to a decline in the body's immune function. In cancer treatment, local tumor eradication through surgery, radiotherapy, chemotherapy, and other methods often fails to benefit patients, as their immune function is severely compromised, leading to distant metastasis and a significant reduction in quality of life and survival time. Consequently, the role of immune function in tumor initiation, progression, metastasis, reversal, and regression has increasingly attracted attention.

Professor Pei Zhengxue, a renowned expert in integrated traditional Chinese and Western medicine and mentor, drawing on his more than 40 years of clinical experience, believes that deficiency of vital energy is the root cause of malignant tumor development; tonifying the body and consolidating the root is the fundamental principle for treating malignant tumors, with "strengthening the spleen and tonifying the kidneys" as its essence. Based on this principle, he formulated the "Lanzhou Formula," which underwent over 30 years of continuous practice, refinement, and reorganization before finally becoming "Pei's Shengxue Granules." Its main ingredients include Liuwei Dihuang Tang, Shengmai San, Beishashen, Dangshen, Taizishen, Guizhi, Dazao, Wuweizi, Zhigancao, Baishao, and others. Zhou Xiaomian et al. have shown that Liuwei Dihuang soft capsules can improve immune dysfunction caused by cyclophosphamide in adult and juvenile mice, as evidenced by enhanced T-cell proliferation, increased T-cell counts—especially CD8-positive cells—markedly elevated natural killer activity in splenic cells, significantly reduced delayed-type hypersensitivity reactions, and strengthened defensive functions. Zhao Changqing and Wang He et al. have experimentally demonstrated that Liuwei Dihuang soft capsules possess certain anti-tumor and anti-aging effects, suggesting that enhancing the body's immune function may be one of the mechanisms underlying these benefits. Studies have also shown that American ginseng polysaccharides affect calcium-dependent potassium channels in mouse T lymphocytes; using patch-clamp techniques to record the current through these channels, researchers found that American ginseng polysaccharides can increase the probability of opening, prolong the duration of opening, and shorten the closing time of ConA-activated calcium-dependent potassium channels, whereas they alone cannot activate the channels. Research has further revealed that Ophiopogon polysaccharides can increase the weight of the thymus and spleen in mice, promote phagocytosis by macrophages, and exert effects on...

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Cyclophosphamide-induced leukopenia is markedly counteracted by these compounds; they also significantly antagonize leukopenia caused by Co irradiation and can increase erythrocyte aggregation rates, exerting obvious promotional effects on various aspects of the body's immune function, thereby enhancing humoral immunity and improving the body's adaptability. Zhao Guohua et al. [38] reported that yam polysaccharides at a dose of 150 mg/kg exhibit optimal inhibitory effects against Lewis lung cancer and B16 melanoma.

The thymus is an important central immune organ and the site where T cells differentiate and mature, playing a crucial role in immune regulation; the spleen is another key peripheral immune organ, serving as the habitat for various immune cells and the place where immune responses are generated, as well as an important site for synthesizing immune-active substances such as interferons, complements, and cytokines. Therefore, observing changes in the thymus and spleen allows us to assess the body's immune status. In this experiment, the thymus and spleen of mice in the tumor-bearing model group showed shrinkage in volume and weight, with some thymuses exhibiting unclear lobulation and a grayish-white color, while the spleens became paler red. In contrast, the appearance of the thymus and spleen in mice treated with Pei's Shengxue Granules at various doses was not significantly different from that of the normal group. The thymus index and spleen index in the high-, medium-, and low-dose groups were all higher than in the model group, with the high-dose group showing thymus indices of 18.9 mg/10 g and spleen indices of 76.3 mg/10 g, representing increases of 36.0% and 17.9%, respectively, compared with the model group (p<0.05); the medium-dose group had a thymus index of 18.0 mg/10 g, up 29.5% from the model group; compared with the normal group, the model group's thymus and spleen indices were significantly lower (p<0.01), while the high-dose group's thymus and spleen indices were close to those of the normal group (p>0.05). These findings suggest that Pei's Shengxue Granules have a clear weight-increasing effect on the thymus and spleen—the immune organs of tumor-bearing mice—indicating that the granules can enhance the non-specific immune function of these animals.

Lymphocytes constitute the main cell population of the body's immune system and can be divided into T cells, B cells, NK cells, and other subgroups. The spleen is a large lymphoid organ, primarily housing T lymphocytes and B lymphocytes. The proliferative capacity of splenic lymphocytes stimulated by ConA is an important indicator of the body's lymphocyte function. This experiment shows that the proliferative activity of splenic lymphocytes in the Pei's Shengxue Granules group was higher than in the model group, with the highest effect observed in the high-dose group (p<0.05). This indicates that Pei's Shengxue Granules can promote the proliferative capacity of ConA-stimulated splenic lymphocytes, enhance the cellular immune function of tumor-bearing mice, and thereby improve their anti-tumor efficacy.

Within the body's immune system, immune cells, immune molecules, and interactions between immune cells and immune molecules influence and regulate each other, forming a delicate and complex regulatory network that maintains immune balance and self-stability. The IL-2–IFN-γ–NKC immune regulatory network is one of the most important networks and is closely related to the MΦ–IL-1–Th1 immune regulatory network. Cytokines can directly or indirectly affect multiple processes, including antigen presentation, lymphocyte recognition of antigens, lymphocyte differentiation and maturation, and antibody production, playing a crucial role in immune regulation. IL-2 and IFN-γ are important regulatory factors in the cytokine network, playing a significant role in inhibiting tumor cell growth and regulating the immune system. Changes in the expression of cytokines such as IL-2 and IFN-γ are often used as an important indicator for evaluating immune function. The results of this study show that the concentration of IL-2 in the supernatant of splenic cell cultures in all dosage groups of Pei's Shengxue Granules was higher than in the model group, with the high-dose group reaching 61.6 pg/ml, significantly higher than the model group (p<0.05); furthermore, the expression levels of IL-2 and IFN-γ mRNA in splenic cells of mice in all dosage groups were also higher than in the model group, with particularly pronounced increases in the high- and medium-dose groups (p<0.05). These findings suggest that Pei's Shengxue Granules can promote the secretion of IL-2 as well as the expression of IL-2 mRNA and IFN-γ mRNA, thereby regulating the body's immune function through its effects on cytokines—a key mechanism by which Pei's Shengxue Granules exerts its anti-tumor effects.

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Conclusion