Attached Figures

Attached Figures

Page 88: Immunohistochemical photo of mutant P53 (immunohistochemical staining, ×400)

• A Model group
• B Low-dose PG group
• C Medium-dose PG group
• D High-dose PG group
• E ZG group

Page 89: Pathological tissue photo of tumors (HE staining, ×400)

• A Model group
• B Low-dose PG group
• C Medium-dose PG group
• D High-dose PG group
• E ZG group

Page 90: Caspase-3 flow cytometry diagram

• A Model group
• B Low-dose PG group
• C Medium-dose PG group
• D High-dose PG group
• E ZG group

Research on Pei Zhengxue’s Series of Formulas

Discussion

A tumor is a new growth formed when local tissue cells lose normal growth regulation at the genetic level under the influence of various carcinogenic factors, leading to abnormal cell proliferation. Tumors are genetic diseases, with their biological basis being genetic abnormalities. Carcinogenic factors cause somatic cell gene mutations, resulting in abnormal gene expression and disrupting cellular bioactivity, thereby forming tumor cells that differ from normal cells in morphology, metabolism, and function. Two types of genes directly participate in tumor development: oncogenes and tumor suppressor genes. Oncogene products positively regulate cell proliferation; when they mutate or are overexpressed, they can lead to excessive cell growth. Conversely, tumor suppressor gene products inhibit cell proliferation. [17][30]

Studies have shown that the occurrence and development of liver cancer are related to multiple apoptosis-related genes. P53 is a tumor suppressor gene that encodes a nuclear phosphoprotein with a relative molecular mass of 53,000, existing in two forms: wild-type (Wt) and mutant-type (Mt). In normal cells, the half-life of wild-type p53 protein is short, its metabolism is unstable, and its content is low, making it difficult to detect using conventional immunohistochemistry. Therefore, the P53 protein detected by immunohistochemistry is usually the mutant-type P53 protein. [18] As a tumor suppressor gene, the protein encoded by P53 can inhibit DNA synthesis and prevent cells from transitioning from G phase to S phase. In tumor tissue, however, this gene mutates into a mutant form, causing the encoded protein to lose its tumor-suppressing function. Studies have shown that mutations or inactivation of the P53 gene are important events in the development of many tumors. The loss of P53 function is mainly due to gene mutations; when the P53 gene mutates and loses its ability to inhibit cell proliferation and division, it works together with oncogenes such as ras and c-myc to transform normal cells into cancerous ones. Mutations in the P53 gene exhibit obvious heterogeneity, manifesting as mutations at different levels, including gene structure, transcription, and translation. Structural mutations typically include base substitutions, frameshift mutations, rearrangements, or gene deletions. Among these, base substitution (point mutation) is the main mutation type in the P53 gene, with mutation hotspots often concentrated in several codon positions within exons 5–8, such as 173, 178, 248, 249, and 278. Although base substitutions involve only one or two bases, because they occur in the functional regions of the gene, they can alter the amino acid composition and thus change the protein's function. Many studies on human malignant tumors have found that over 98% of P53 gene mutations occur within exons 5–8. Therefore, the mutation rate in this region can basically reflect the overall mutation rate of the P53 gene. Accumulation of mutant P53 protein inside cells promotes cell transformation and proliferation, ultimately leading to tumor formation. [18] Controlling the occurrence of mutant P53 through medication is the ultimate goal of this experiment. This experiment shows that in all Pei’s Shengxue Granules dosage groups, the expression of mutant P53 protein in mice is lower than in the model group, with the large- and medium-dose groups showing the best effect (p<0.05). This indicates that Pei’s Shengxue Granules have a relatively obvious inhibitory effect on H tumors in mice, thereby enhancing their anti-tumor efficacy.

Caspase is a family of apoptosis proteases. In recent years, people have discovered that caspase-3 plays a central role in apoptosis signal transduction, serving as a downstream factor in the apoptosis protease cascade. If its activity is inhibited, it can lead to impaired apoptosis, disrupting the dynamic balance between apoptosis and proliferation and potentially further promoting tumor development. [19] The role of caspase-3 in apoptosis: it inactivates intracellular substances that block apoptosis, directly enzymatically degrades cell structures to promote apoptosis, and enzymatically degrades certain proteins involved in cytoskeleton regulation, such as lectins, FAK, and kinase II activated by P21 (PAK2), causing cell apoptosis when these proteins are degraded and inactivated. The starting point of caspase-3’s action is the separation of its regulatory and effector regions. When participating in apoptosis, caspase-3 has a retrospective nature, able to execute each step systematically, sever connections with surrounding cells, reorganize the cytoskeleton, and block DNA replication and repair. [20] Experiments show that the precursor of caspase-3 is widely expressed in various cells, effectively and rapidly inducing apoptosis, and it persists longest in nerve cells. Complex proteolytic systems are often composed of kinases, cofactors, and feedback factors that work in sequence to regulate the activity of effector kinases. So how does apoptosis get initiated? Extensive genetic analysis and biochemical evidence suggest that caspase-3 activation follows a cascading pattern: death signals activate the initiating caspase-3, which then activates the effector caspase, leading to cell breakdown. [21][22][23] However, death receptors and mitochondria first process and integrate death signals before activating different initiating caspase-3. Studies have shown that caspase-3 is expressed in many normal human tissues and tumors. Caspase-3 participates in regulating hepatocyte proliferation and the transformation of cells during apoptosis; low expression of caspase-3 contributes to the development of HCC, and low caspase-3 expression plays an important role in the occurrence and progression of HCC. The results of this study show that Pei’s Shengxue Granules can promote the expression of caspase-3 protein, thereby inducing apoptosis through upregulation of caspase-3 expression and achieving an anti-tumor effect, promoting apoptosis of tumor cells and thus realizing the anti-tumor function of Pei’s Shengxue Granules.

The spleen is one of the important peripheral immune organs, while the thymus is an important central immune organ, both playing crucial roles in the body’s immune regulation; therefore, observing changes in the thymus and spleen can help evaluate the body’s immune status. [24] This experiment shows that Pei’s Shengxue Granules can enhance the non-specific immune function of tumor-bearing mice. The "Inner Canon" first introduced the disease name of "accumulation and mass," and explored its formation and treatment principles. Liver cancer falls under the category of "accumulation" in traditional Chinese medicine; "accumulation" refers to a tangible, fixed, immovable mass that causes localized, stabbing pain, often affecting the internal organs and belonging to the blood level, with a relatively long disease course.[2] The disease of liver cancer has been documented as early as in the "Inner Canon"; throughout history it has also been referred to as "fat qi," "stagnant qi," and "accumulated qi." For example, the "Classic of Difficult Questions" records: "The accumulation of the spleen is called 'stagnant qi.' It occurs in the epigastric region, causing the abdomen to swell like a plate, and if left untreated for a long time, it leads to weakness in the limbs, jaundice, and loss of appetite." The "Essential Principles of Disease Origin and Treatment" states: "The abdomen becomes hard and firm upon palpation." The "Treatise on Causes and Symptoms of Various Diseases—Accumulation and Mass" notes: "Upon diagnosis, liver accumulation is found, with a taut and fine pulse and pain in both hypochondria."[26] These records outline the diagnostic key points of liver cancer. Traditional Chinese medicine refers to tumors as "liu jia," "accumulation and mass," "goiter tumor," "breast cancer," "loss of vitality," "organ toxicity," "obstructive dysphagia," and other terms, believing that tumor formation arises from both internal pathogenic factors (deficiency of vital energy and emotional imbalance) and external pathogenic factors (pathogenic factors and dietary/nutritional factors). The main pathological mechanisms include qi stagnation and blood stasis, phlegm-dampness accumulation, heat toxin infiltration, and deficiency of vital energy. Modern medicine primarily treats tumors through surgery, radiotherapy, and chemotherapy, whereas traditional Chinese medicine, based on syndrome differentiation and treatment, mainly employs therapeutic principles such as reinforcing vital energy and nourishing the root, clearing heat and detoxifying, activating blood circulation and removing stasis, softening hard masses and dispersing nodules, transforming phlegm and eliminating dampness, and using poison to attack poison.

Traditional Chinese medicine holds that, in terms of human pathogenesis, "internal deficiency of vital energy" is the fundamental cause of disease. The "Plain Questions" states: "When vital energy is present within, pathogenic factors cannot invade; where pathogenic factors gather, it must be due to deficiency of vital energy."[27] The "Authentic Surgery" says: "The formation of accumulation is due to deficiency of vital energy; only when vital energy is deficient can accumulation form," directly applying the "vital energy deficiency leading to disease" concept from the "Plain Questions" to tumor pathogenesis. Only when vital energy is insufficient can pathogenic factors invade the body, causing imbalances in qi, blood, yin, and yang, ultimately leading to tumor formation.[2] The "Spiritual Pivot—On True and False Pathogens" states: "True qi is what one receives from heaven and is combined with food qi to fill the body." True qi encompasses both innate and acquired aspects; throughout history, physicians have attributed the innate aspect to the two kidneys, calling it "yuan qi" (original qi), and the acquired aspect to the spleen and stomach, calling it "zhong qi" (food qi). The spleen governs zhong qi, while the kidneys govern yuan qi; therefore, in the "reinforcing vital energy and consolidating the root" principle, "strengthening the spleen" and "tonifying the kidneys" are the most important components. Experimental studies have demonstrated that the "spleen" and "kidneys" in traditional Chinese medicine play significant roles in the immune system, endocrine system, metabolic system, autonomic nervous system, and gastrointestinal-pancreatic endocrine system, among others; thus, "strengthening the spleen and tonifying the kidneys" can improve the function of these systems. The "reinforcing vital energy and consolidating the root" approach aims to enhance and mobilize the body's disease-resistance capabilities, which aligns well with modern immunology.

Research on Pei Zhengxue’s series of prescriptions

There is considerable commonality between this approach and modern immunology, molecular biology, and related fields. Patients with liver cancer at early, middle, and late stages all exhibit signs of vital energy deficiency, often presenting with symptoms such as fatigue, dizziness, lower back pain, palpitations, and shortness of breath. Such medications can replenish deficiencies in the body's qi, blood, yin, and yang, alleviate symptoms, reduce the toxic side effects of radiotherapy and chemotherapy, protect the spleen and stomach as well as bone marrow, and effectively inhibit tumor growth and development by strengthening vital energy and nourishing the root, thereby extending patients' lives. Modern research indicates that medicines used to reinforce vital energy and nourish the root have the following effects in treating tumors: ① they can promote immune function, enhancing lymphocyte proliferation and reticuloendothelial system activity; ② they can protect and improve bone marrow hematopoietic function, increasing blood cell components; ③ they can enhance endocrine fluid regulation, promoting pituitary-adrenal cortical function; ④ they can adjust the ratio of cyclic adenosine monophosphate to cyclic guanosine monophosphate within the cancerous organism, 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 efficacy; ⑧ certain tonic formulas have direct anti-cancer effects, controlling cancer cell invasion and metastasis. Commonly used herbs include: codonopsis, princeps codonopsis, astragalus, polygonatum, ligustrum fruit, morinda, curculigo, white peony, rehmannia, eucommia, psoralea, turtle shell, dodder seed, licorice, and so on. In terms of drug treatment for tumors, comprehensive therapy combining chemotherapy, immunotherapy, biological therapy, and traditional Chinese medicine has shown synergistic effects and reduced toxicity, significantly improving tumor remission rates, quality of life, and survival rates. However, current tumor treatments still face challenges such as multi-drug resistance, severe toxic side effects of chemotherapy, and damage to immune and bone marrow hematopoietic functions, making tumor treatment quite difficult. Therefore, finding safe and effective drugs remains an important research topic in oncology today. Modern research shows that traditional Chinese medicine can exert anti-tumor effects by boosting the body's immunity, inducing tumor cell differentiation and apoptosis, influencing tumor cell cycles, inhibiting tumor angiogenesis, combating tumor metastasis, and reversing multi-drug resistance to chemotherapeutic agents, particularly demonstrating overall therapeutic advantages in terms of enhancing efficacy and reducing toxicity.

Professor Pei Zhengxue, 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; reinforcing vital energy and nourishing the root is the basic principle for treating malignant tumors, with "strengthening the spleen and tonifying the kidneys" as its essence. Based on this, he formulated the "Lanzhou Formula," which, after more than forty years of continuous practice, refinement, and reorganization, eventually became "Pei’s Blood-Boosting Granules." Its composition mainly includes Liuwei Dihuang Decoction, Shengmai Powder, North Sand Ginseng, Codonopsis, Princeps Codonopsis, Cinnamon Twig, Jujube, Schisandra, Honey-roasted Licorice, White Peony, and other ingredients. The "Four Ginsengs" represent the culmination of strengthening the spleen and benefiting qi, serving as a powerful force for nourishing postnatal qi; combined with Shengmai Powder to benefit qi and nourish the lungs, the Four Ginsengs’ effect of strengthening the spleen and benefiting qi is further enhanced. "Six Flavor Rehmannia Decoction" is a specialized formula for tonifying kidney qi, with a heavy use of Cornus fruit (30g) to further emphasize its ability to nourish innate qi. "Cinnamon Twig Decoction": Cinnamon Twig, White Peony, Ginger, Licorice, and Jujube constitute a complete Cinnamon Twig Decoction, which "harmonizes exterior and interior, stabilizes the organs," and was once hailed as the "crown of all formulas." Professor Pei believes that the Cinnamon Twig Decoction in the "Lanzhou Formula" not only greatly strengthens both innate and acquired qi but also enhances the harmonization of exterior and interior, stabilizing the organs, thereby actually strengthening the regulation of the autonomic nervous system, metabolic system, endocrine system, and immune system. Floating wheat, licorice, and jujube make up the Sweet Wheat and Jujube Decoction, which ancient people used to treat women's depression with remarkable efficacy. Professor Pei believes that the Sweet Wheat and Jujube in the "Lanzhou Formula" not only regulate the autonomic nervous system but also have calming and sedative effects, regulating thought processes, thus supplementing the previously mentioned major unfulfilled functions. In summary, the "Lanzhou Formula" strengthens the body's vital energy through the overarching principle of reinforcing vital energy and nourishing the root, thereby achieving the effect of reinforcing the righteous and dispelling the evil. Clinically, however, it is necessary to tailor treatment according to each patient's specific condition, adjusting dosage and administration to achieve the desired therapeutic effect.

In conclusion, this experiment, by establishing a H22-bearing mouse model and utilizing molecular biology techniques, further observes the mechanism by which Pei’s Blood-Boosting Granules induce tumor cell apoptosis, as well as the protein expression levels of apoptosis-related genes p53 and Caspase-3. Further in-depth research and exploration will provide scientific evidence for the application of this medication in treating malignant tumors, as well as for traditional Chinese medicine and integrated Chinese-Western medicine approaches to tumor treatment, and offer scientific, microscopic foundations for the widespread clinical application of Pei’s Blood-Boosting Granules.

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Research on Pei Zhengxue’s Series of Prescriptions

Conclusion