Chinese Abstract

Chinese Abstract

Objective: Through experimental research on Peishengxue Granule, this study aims to demonstrate the drug's impact on peripheral blood, CD4⁺ and CD8⁺ levels in the spleen, and the CD4⁺/CD8⁺ ratio in AA mice, while observing pathological changes in the spleen before and after medication. The goal is to understand the role of Peishengxue Granule in the immune system of AA mice, explore its possible mechanisms, and provide scientific and microscopic evidence for its widespread clinical application.

Methods: Ninety BALB/C mice, weighing (20±2) g, were randomly divided into six groups: high-, middle-, and low-dose Peishengxue Granule groups (hereafter referred to as Groups I, II, and III), Zhenqi Fuzheng Granule control group (Group IV), model group (Group V), and normal control group (Group VI), with 15 mice in each group. Except for the normal control group, the remaining 75 mice were subjected to whole-body irradiation with 3.0 Gy of gamma rays from a linear accelerator, followed by intravenous infusion of thymus and lymph node cell suspensions derived from DBA/2 mice within four hours of irradiation. One week after irradiation, successful modeling was confirmed through blood and bone marrow examinations. Each group received medication for 20 and 40 days, after which blood samples were collected for analysis. At the end of the experiment, mouse spleens were harvested, and immunohistochemical methods were used to measure CD4⁺ and CD8⁺ levels as well as the CD4⁺/CD8⁺ ratio, while also observing pathological histological changes in the spleen of each group.

Results: Peishengxue Granule significantly increased white blood cell, platelet, and hemoglobin levels in the peripheral blood of AA model mice, though the increase in red blood cells was not significant. Spleen CD4⁺ levels showed an upward trend, while CD8⁺ levels showed a downward trend, resulting in an increased CD4⁺/CD8⁺ ratio. Pathological examination of the spleen indicated that Peishengxue Granule could alleviate pathological changes in the spleen of model mice, significantly reducing the apoptosis rate of splenic lymphocytes and promoting the recovery of germinal centers.

Conclusion: The mechanism of action of Peishengxue Granule may involve multiple aspects: blood analysis shows that the granule can significantly increase white blood cell, platelet, and hemoglobin levels in the peripheral blood of AA mice; immune function evaluation indicates that Peishengxue Granule can regulate the body's disrupted immune state, increasing CD4⁺ and decreasing CD8⁺ levels and activity, thereby adjusting the CD4⁺/CD8⁺ ratio and reducing the release of negative regulatory factors such as IL-2, IFN-γ, and TNF-α that inhibit bone marrow hematopoiesis; spleen pathology sections suggest that Peishengxue Granule has a significant ameliorative effect on the pathological condition of the spleen in AA mice.

Keywords: Aplastic anemia; blood analysis; CD4⁺; CD8⁺; spleen pathology

Research on Pei Zhengxue’s series of prescriptions Methods: 90 BALB/C mice, weighing (20±2)g, were divided into six groups at random: high dose group of PG (group I is the abbreviation); medium dose group of PG (group II is the abbreviation); low dose group of PG (group III is the abbreviation); Zhenqifuzheng Granule group (ZG) (group IV is the abbreviation), model group (group V is the abbreviation), and natural group (group VI is the abbreviation). Each group has 15 mice. 75 mice were irradiated by 3.0GY γ ray from Beeline accelerator for whole body except those of group VI. Then they were injected with cell suspension from thymus and lymphocyte of DBA/2 mice within four hours. The AA model was proved successful by analysing the hemogram and medulla a week later. Treat them with different medicine for 20 days and 40 days, hemogram was measured separately. When the experiment finished, the content of CD4, CD8 and the value of CD4 over CD8 in the spleen was measured. The pathologic histology sections of the spleen were observed.

Results: PG improved the value of WBC, PLT and HGB in the peripheral blood significantly. The content of CD4 increased and the content of CD8 decreased, so the value of CD4 over CD8 increased. Pathologic histology sections of the spleen showed they were improved evidently. The apoptosis rate of the lymphocyte reduced, and the accruement center revived.

Conclusion: The mechanism in treatment of PG may has several aspects. It has the function of improving the value of WBC, PLT and HGB in the peripheral blood; it may reduce IL-2, TNF-α, IFN-γ which were negative factors for hematopoiesis by increasing the quantity and activity of CD4 and decreasing the quantity and activity of CD8; it can ameliorate the pathologic histology changes of the spleen.

Keywords: AA; hemogram; CD4; CD8; the spleen pathology

Foreword

Since Paul first reported aplastic anemia (AA, abbreviated as aplasia) in 1898, people have gradually deepened their understanding of this disease through more than 100 years of exploration. Aplasia is caused by chemical, physical, biological, and some unknown factors that lead to the reduction or failure of bone marrow hematopoietic function. This disease is characterized by damage to hematopoietic stem cells and a decrease in all blood cells in peripheral blood (red blood cells, granulocytes, platelets).

Research on Pei Zhengxue's series of prescriptions

According to the speed of onset and the severity of the condition, it can be divided into acute and chronic types. The acute type is characterized by initially not obvious anemia, often with bleeding, infection, and high fever as the initial symptoms, but the condition progresses rapidly, with progressive worsening of anemia, severe and extensive bleeding—bleeding not only occurs on the skin, mucous membranes, and fundus of the eye, but also often involves internal organ bleeding. Infections are commonly seen in the oral cavity, pharynx, skin, and around the anus, as well as high fever caused by pneumonia, sepsis, etc. The course of the disease is dangerous, and patients often die within a short period due to infection or internal organ bleeding. The chronic type has a slow onset, and symptoms such as anemia, infection, and bleeding are relatively mild [1].

Modern medicine has not yet fully elucidated the causes of this disease, which can be caused or induced by toxins, drugs, radioactive substances, severe infections, bone marrow tumors, or leukemia, leading to impairment or destruction of bone marrow hematopoietic function. Clinically, it often manifests as severe anemia, bleeding, and infection, with simultaneous reduction of all three cell lines, and usually without hepatosplenomegaly or lymphadenopathy. Most scholars believe that the pathogenesis of this disease is due to multiple factors, including defects in bone marrow hematopoietic stem cells, abnormalities in immune mechanisms, and disturbances in the hematopoietic microenvironment. The main pathological change is fatty infiltration of red bone marrow, where red bone marrow with hematopoietic function is replaced by fat; the greater the extent of replacement, the more severe the anemia. Based on the speed of onset, severity of the condition, degree of bone marrow destruction, and prognosis, it is divided into acute and chronic types. According to surveys conducted in some regions of China, there are 1.87–2.1 cases per 100,000 people, similar to the incidence reported in Japan. All age groups can be affected, but it is more common among young and middle-aged adults, with males being more frequently affected than females. The ratio of acute to chronic cases is 1:4.6.

Aplastic anemia falls under the categories of "blood deficiency," "blood syndrome," "deficiency fatigue," "deficiency damage," and "blood depletion" in traditional Chinese medicine. Acute aplasia belongs to "acute fatigue," "heat fatigue," and "blood syndrome," while chronic aplasia belongs to "deficiency fatigue," "blood deficiency," and "blood syndrome." Traditional Chinese medicine believes that there is a close relationship between blood and qi, and between blood and essence. Qi is yang, blood is yin; the generation of blood depends on qi—qi can generate blood, blood can transform into qi, and essence and blood can mutually transform. Essence is the root of life, stored in the kidneys; kidney essence generates marrow, and marrow essence can produce blood. As stated in "Various Causes and Symptoms of Diseases—Symptoms of Deficiency Fatigue": "The kidneys store essence, and essence is what blood is made of." In "Plain Questions—On the Great Principles of Yin and Yang": "The kidneys govern bone marrow production and marrow formation," and "Blood is transformed from essence," illustrating the relationship among kidneys, bone marrow, and blood, especially the direct connection between bone marrow and hematopoiesis. The strength or weakness of kidney function affects marrow's ability to generate essence and blood, so traditional Chinese medicine considers bone marrow involved in hematopoiesis to be closely related to the kidneys. In "Spiritual Pivot—On the Decree of Vital Energy": "The middle jiao receives qi, extracts juice, transforms it, and turns it red—this is called blood." In "Plain Questions—On the Subtle Essence of Pulse": "The pulse is the residence of blood." There are two pathways for blood generation: transformation of food and water essence into blood, and transformation of kidney essence into blood. In "Spiritual Pivot—On the Meeting of Ying and Wei": "The middle jiao secretes waste,蒸s fluids, transforms its essence, sends it up to the lung pulse, then transforms it into blood to nourish the body—nothing is more precious than this." In "Lushan Hall Classifications—On Blood": "Blood is the juice of the middle jiao, flows out and becomes essence, nourishes the heart, transforms into red blood, and becomes blood." Blood circulates endlessly, irrigating the entire body. In "Plain Questions—On the Differentiation of Meridians": "Food qi enters the stomach, turbid qi returns to the heart, essence permeates the pulse, pulse qi flows through the meridians, meridian qi returns to the lungs, the lungs serve hundreds of pulses, sending essence to the skin and hair." In "Zhang's Medical Compendium—On Various Blood Doors": "If qi does not dissipate, it returns to the kidneys to become essence; if essence does not leak, it returns to the liver to transform into clear blood." The liver stores blood to generate qi and blood, and can store the essence and nutrients from food as raw materials for hematopoiesis, so the liver is also related to hematopoiesis. In summary, the generation of blood is mainly related to the kidneys, spleen, and liver.

This experiment studies the impact of Pei's Hematopoietic Granules on the immune system of aplastic anemia mice and preliminarily explores its mechanism of action. Pei's Hematopoietic Granules are a granular preparation developed by Professor Pei Zhengxue, a nationally renowned expert in integrated traditional Chinese and Western medicine, based on many years of clinical experience. They have the effects of tonifying the kidneys and generating marrow, strengthening the spleen and benefiting qi, nourishing blood and harmonizing the stomach, and have achieved satisfactory therapeutic effects in clinical practice. To further confirm these effects and explore the mechanism, this experimental study was initiated.

Research Progress

The research progress on the immune regulation mechanism of traditional Chinese medicine in treating aplastic anemia [4] is as follows: Yao et al. [5] successfully developed an animal model of immune-mediated aplasia by irradiating BALB/C mice with sub-lethal doses of radiation and injecting thymus lymph node cells taken from DBA/2 mice. This animal model closely matches the immune pathogenesis of aplasia, so many TCM researchers use it for their studies. Animal experiments have verified that TCM treatment of aplasia works by adjusting T-cell immune function, stimulating hematopoietic colony formation, and influencing the proliferation and differentiation of hematopoietic cells. To further study the mechanism of TCM treatment of aplasia, Zhou et al. [6], after successfully improving and replicating the immune-mediated aplasia mouse model, observed the effect of blood-tonifying compounds on this animal model. The results showed that blood-tonifying compounds could reduce the mortality rate of aplastic anemia mice, increase peripheral blood counts and nucleated cells in the bone marrow, raise the percentage of Th (CD4) cells, the Th/Ts (CD4/CD8) ratio, IL-3, and IL-2R levels, while lowering Ts (CD8) cells, IL-2, IFN-γ levels, and IFN-γ gene expression in splenic cells. Shi et al. [7] used this aplasia model and administered decoctions of qi-tonifying, blood-activating, spleen-and-kidney-warming, and righteous-strengthening, detoxifying traditional Chinese medicines via gavage. The results showed that these formulas could, to varying degrees, increase Th cells, decrease Ts cells, raise the Th/Ts ratio, alleviate the suppression and damage caused by immune abnormalities to bone marrow hematopoietic cells, and promote the repair and reconstruction of bone marrow hematopoiesis in aplastic anemia.

1 Etiology and Pathogenesis