3.1 Effect of Pei's Shengxue Granules on Reducing Toxicity of 5-Fu Chemotherapy in Tumor-Bearing Mice

3.1 Effect of Pei's Shengxue Granules on Reducing Toxicity of 5-Fu Chemotherapy in Tumor-Bearing Mice

3.1.1 General Condition

General observation showed that starting from the fifth day, mice in the model group exhibited disheveled fur, with faintly visible tumors under the skin of the right anterior axillary region. The mice displayed poor mental state, reduced appetite, sluggish movement, huddling, lethargy, drowsiness, dull and brittle fur, and darkened fur color. Between the eighth and tenth days after modeling, the tumors grew rapidly, with uneven surfaces and multiple nodules, unclear boundaries. At the end of the experiment, dissection of the model group mice revealed that the tumors had grown more vigorously than those in the treatment groups. The 5-Fu group showed slower tumor growth compared to the model group, with poorer mental state and appetite. In contrast, mice in the Pei's Shengxue Granules groups combined with 5-Fu exhibited brighter fur, better mental

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state, better appetite, and slower tumor growth.

3.1.2 Effect of Pei's Shengxue Granules on Immune Organs—Thymus and Spleen—in Tumor-Bearing Mice Undergoing 5-Fu Chemotherapy

The thymus and spleen of mice in the model group shrank in size and weight. The 5-Fu group showed even more pronounced shrinkage in thymus and spleen size and weight. The thymus index of the 5-Fu group was lower than that of the model group, at 15.2±3.9 (mg/10g), showing a significant difference compared to the model group (p<0.05). However, the Pei's Shengxue Granules groups combined with 5-Fu significantly increased the thymus index of the mice: the high- and medium-dose groups had thymus indices of 23.3±2.6 (mg/10g) and 21.1±2.8 (mg/10g), respectively, both significantly higher than the 5-Fu group (p<0.05). In contrast, the low-dose group combined with 5-Fu did not show a significant difference compared to the 5-Fu group (p>0.05). Similarly, the spleen index of the 5-Fu group was lower than that of the model group, at 33.7±6.4 (mg/10g), also showing a significant difference compared to the model group (p<0.05). The Pei's Shengxue Granules groups combined with 5-Fu all had higher spleen indices than the 5-Fu group: the high- and medium-dose groups had spleen indices of 48.8±9.8 (mg/10g) and 43.2±5.4 (mg/10g), respectively, both significantly higher than the 5-Fu group (p<0.05), while the low-dose group combined with 5-Fu did not show a significant difference compared to the 5-Fu group (p>0.05). (See Table 1, Figure 1, and Figure 2)

Table 1: Effects of Pei's Shengxue Granules Combined with 5-Fu on Immune Organs of Tumor-Bearing Mice (n=12, X̄±s)

Note: △p<0.05

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Figure 1: Effects of Pei's Shengxue Granules Combined with 5-Fu on Thymus Index of Tumor-Bearing Mice (n=12, X̄±s)

(△p<0.05 compared with the model group; *p<0.05 compared with the 5-Fu group)

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Figure 2: Effects of Pei's Shengxue Granules Combined with 5-Fu on Spleen Index of Tumor-Bearing Mice (n=12, X̄±s)

(△p<0.05 compared with the model group; *p<0.05 compared with the 5-Fu group)

3.2 Effect of Pei's Shengxue Granules on Enhancing the Anti-Tumor Efficacy of 5-Fu Chemotherapy in Tumor-Bearing Mice

The effect of Pei's Shengxue Granules combined with 5-Fu on the tumor inhibition rate in tumor-bearing mice was as follows: Both the 5-Fu group and the various doses of Pei's Shengxue Granules combined with 5-Fu significantly inhibited tumor growth in the mice, with the average tumor weight being significantly lower than that of the model group (p<0.05). Among them, the high- and medium-dose groups of Pei's Shengxue Granules combined with 5-Fu had average tumor weights of 1.044±0.354(g) and 1.138±0.214(g), respectively, both significantly lower than the 5-Fu group (p<0.05), with inhibition rates of 62.66% and 59.30%, respectively. (See Table 2 and Figure 3)

Table 2: Effects of Pei's Shengxue Granules Combined with 5-Fu on Tumor Inhibition Rate in Tumor-Bearing Mice (n=12, %, X̄±s)

Note: △p<0.05 compared with the model group; *p<0.05 compared with the 5-Fu group.

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Figure 3: Effects of Pei's Shengxue Granules Combined with 5-Fu on Tumor Inhibition Rate in Tumor-Bearing Mice (n=12, %, X̄±s)

3.3 Effect of Pei's Shengxue Granules Combined with 5-Fu on P27 Protein Expression in Tumor Tissues of Tumor-Bearing Mice

The expression of P27-positive cells in tumor tissues across all treatment groups was significantly higher than that of the model group, with a significant difference compared to the model group (p<0.05). The 5-Fu group had 10.52±1.24 P27-positive cells in tumor tissues, while the Pei's Shengxue Granules groups combined with 5-Fu showed varying degrees of higher P27-positive cell expression than the 5-Fu group, with the high- and medium-dose groups being the most prominent: their P27-positive cell counts were 18.65±1.19 and 15.12±1.33, respectively, both significantly higher than the 5-Fu group (p<0.05). (See Table 3 and Appendix 1)

Table 3: Effects of Pei's Shengxue Granules Combined with 5-Fu on P27 Expression in H22 Mouse Tumor Tissues (n=6, X̄±s)

Note: △p<0.05, compared with the model group; *p<0.05, compared with the 5-Fu group.

3.4 Effects of Pei's Shengxue Granules Combined with 5-Fu on the Cell Cycle and Apoptosis Rate of H22 Tumor Cells in Mice

The groups receiving different doses of Pei's Shengxue Granules combined with 5-Fu showed varying degrees of influence on the proportion of tumor cell cycle distribution. In the 5-Fu group, the G0/G1 phase was 42.25±15.16(%), which differed significantly from the model group (p<0.05). However, all groups receiving Pei's Shengxue Granules combined with 5-Fu exhibited a higher G0/G1 phase compared to the 5-Fu group, with the highest effect observed in the high- and medium-dose PG+5-Fu groups, where the G0/G1 phase increased to 57.83±10.70(%) and 54.07±8.98(%), respectively, both showing significant differences compared with the 5-Fu group (p<0.05). The S phase in both the 5-Fu group and all groups receiving Pei's Shengxue Granules combined with 5-Fu was lower than that in the model group.

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The S phase decreased, with the most significant reduction seen in the high-dose PG+5-Fu group, dropping to 30.53±6.24(%), which was significantly different from the 5-Fu group (p<0.05). In the model group, no subdiploid peak of apoptotic cells was observed; however, after treatment with various doses of PG+5-Fu and 5-Fu, a subdiploid peak of apoptotic cells appeared, with the highest effect seen in the high-dose group. 5-Fu can induce apoptosis in tumor cells, with an apoptosis rate of 25.03±3.23(%), which differed significantly from the model group (p<0.05); all groups receiving Pei's Shengxue Granules combined with 5-Fu were able to increase the apoptosis rate of tumor cells, with the highest effects observed in the high- and medium-dose PG+5-Fu groups, where the apoptosis rates reached 53.48±3.89(%) and 48.52±5.79(%), both showing significant differences compared with the 5-Fu group (p<0.05). This indicates that PG can synergistically enhance 5-Fu-induced apoptosis in tumor cells, thereby increasing the anti-tumor efficacy of 5-Fu. (See Table 4, Appendix 2)

Table 4: Effects of Pei's Shengxue Granules Combined with 5-Fu on the Tumor Cell Cycle and Apoptosis Rate in Tumor-Bearing Mice (n=6, x̄±s)

Note: △p<0.05, compared with the model group; *p<0.05, compared with the 5-Fu group.

Discussion

Professor Pei Zhengxue, drawing on his more than forty years of clinical experience, believes that "deficiency of vital energy" is the fundamental cause of the occurrence and development of malignant tumors; strengthening vital energy and consolidating the root is the basic principle for treating malignant tumors, with "tonifying the spleen and nourishing the kidney" as its essence. Based on this principle, he formulated the "Lanzhou Formula," which has been continuously refined, enriched, and reorganized over more than forty years to finally become "Pei's Shengxue Granules." Its main components include Liuwei Dihuang Decoction, Shengmai Powder, Beishashen, Dangshen, Taizishen, Guizhi, Jujube, Wuweizi, Zhigancao, Baishao, and others. The "Four Ginsengs" represent the culmination of tonifying the spleen and replenishing qi, serving as the powerful force for reinforcing the postnatal foundation; when combined with Shengmai Powder to tonify qi and nourish the lungs, their spleen-tonifying and qi-replenishing effects are further enhanced. Liuwei Dihuang Decoction is a specialized formula for tonifying kidney qi, with a heavy use of Shan Yurou (30g) to further strengthen its innate tonifying power. Guizhi Tang—Guizhi, Baishao, ginger, gancao, and jujube—forms a complete Guizhi Tang composition, a formula that "harmonizes ying and wei externally and stabilizes the internal organs," often referred to by predecessors as the crown of all formulas. Old Master Pei believes that Guizhi Tang in the "Lanzhou Formula" not only greatly strengthens both innate and postnatal qi but also enhances its ability to harmonize ying and wei and stabilize the internal organs, effectively strengthening the regulation of the autonomic nervous system, metabolic system, endocrine system, and immune system. Fumai, gancao, and jujube form Ganmai Dazao Tang, a formula traditionally used to treat women's depressive disorders with remarkable efficacy. Old Master Pei believes that Ganmai Dazao in the "Lanzhou Formula," in addition to regulating the autonomic nervous system, also has calming and sedative effects, as well as the ability to regulate thinking, thus supplementing the previously mentioned functions that were not fully addressed. In summary, the "Lanzhou Formula" strengthens the body's vital energy through the great principle of reinforcing vital energy and consolidating the root, thereby achieving the effect of reinforcing the righteous and dispelling the evil, with excellent clinical efficacy in anti-tumor treatment and reducing toxicity while enhancing effectiveness.

Traditional Chinese Medicine holds that in terms of human pathogenesis, "internal deficiency of vital energy" is the root cause of disease. The "Plain Questions" states, "When vital energy is preserved internally, evil cannot invade; where evil gathers, it must be due to deficiency of vital energy." The "Orthodox Surgery" says, "Accumulation arises from deficiency of vital energy; only when vital energy is deficient can accumulation form," directly applying the "deficiency of vital energy causes disease" view from the "Plain Questions" to tumor pathogenesis. Only when vital energy is insufficient can evil invade the body, leading to imbalances in qi, blood, yin, and yang, ultimately forming tumors. The "Spiritual Pivot·Chapter on Puncturing True Evil" says, "True qi is what one receives from heaven and is supplemented by food qi to fill the body," indicating that true qi encompasses both innate and postnatal aspects. Throughout history, medical practitioners have attributed the innate aspect to the two kidneys, calling it "yuanqi" (original qi), and the postnatal aspect to the spleen and stomach, calling it "zhongqi" (food qi). The spleen governs zhongqi, while the kidneys govern yuanqi; therefore, in the principle of "reinforcing vital energy and consolidating the root," "tonifying the spleen" and "nourishing the kidney" are the two most important pillars. Experimental studies have proven that the "spleen" and "kidneys" in TCM have significance in areas such as the immune system, endocrine system, metabolic system, autonomic nervous system, and gastrointestinal-pancreatic endocrine system, and that "tonifying the spleen and nourishing the kidney" can improve the function of these systems. Modern research shows that tonifying and reinforcing medicines used to treat tumors have the following effects: ① they can promote the body's immune function, increasing lymphocyte proliferation and reticular endothelial system activity; ② they can protect and improve bone marrow hematopoietic function, increasing blood cell components; ③ they can enhance the regulatory function of endocrine fluids, promoting the function of the hypothalamus–pituitary–adrenal cortex axis; ④ they can adjust the ratio of cyclic adenosine monophosphate to cyclic guanosine monophosphate within cancer patients, which helps inhibit cancer cell growth; ⑤ they have a dual-regulating effect; ⑥ they can improve the body's metabolism; ⑦ they can reduce the toxic side effects of radiotherapy and chemotherapy while enhancing their effectiveness; ⑧ some tonifying formulas have direct anti-cancer effects, controlling cancer cell infiltration and metastasis.

This experiment further explores the anti-tumor mechanism of Pei's Shengxue Granules by measuring the expression of P27 protein in tumor tissues of each group and the status of tumor cell apoptosis. Through observing the tumor suppression rate and immune organ indices in tumor-bearing mice, we investigate its synergistic and detoxifying effects on chemotherapy drugs.