1 Materials and Methods

1 Materials and Methods

1.1 Experimental Materials

1.1.1 Experimental Reagents

• 10% Formaldehyde (Shanghai Chemical Reagent Company)
• Hydrochloric Acid Alcohol (Shanghai Chemical Reagent Company)
• 0.5% H₂O₂ Solution (Shanghai Chemical Reagent Company)
• 1 ml EDTA (Ethylenediaminetetraacetic Acid) Repair Solution (Fujian Maxin Biotechnology Development Co., Ltd.)
• Rabbit Anti-Mouse CD4 and CD8 Monoclonal Antibodies (Changsha Yingrun Biological Products Co., Ltd.), Batch No.: GT200310

Phosphate Buffered Saline (PBS): Sodium Chloride 8.00 g, Disodium Phosphate Dihydrate 2.885 g, Potassium Chloride 0.20 g, Dipotassium Phosphate 0.20 g, prepared with triple-distilled water, filtered through a 0.22 μm membrane for sterilization, pH 7.2–7.4, stored at 4°C.

1.1.2 Experimental Instruments and Equipment

• Optical Microscope (Olympus BX60-F5)
• Linear Accelerator (Siemens Primus, Germany)
• Three-Category Blood Analyzer (Sysmex KX-21, Japan)
• Three-Category Blood Analyzer (Jitai CA-800, Japan)
• Ultra-Clean Workbench (Su Jing Group Suzhou Antai Air Technology Co., Ltd., VS-1300L model)
• Low-Temperature Refrigerator (Qingdao Haier Co., Ltd., BCD-213 model)
• Dehydrator (LEICA TP1020, Germany)
• Sectioning Machine (LEICA RM2135, Germany)
• Pathological Tissue Bleaching and Heating Treatment Device (Changzhou Zhongwei Electronic Instruments Factory, PHY-II model)
• Embedding Machine (Changzhou Zhongwei Electronic Instruments Factory, BMJ-I model)
• Staining Machine (Changzhou Zhongwei Electronic Instruments Factory, CM1900HE)
• Electronic Balance (Sartorius, Switzerland, BP211D model)

1.1.3 Experimental Drugs

Pei's Blood-Elevating Granules (composed of Sheng Di, Shan Yu Rou, Shan Yao, Dan Pi, Bei Sha Shen, Ren Shen Xu, Lu Dang Shen, Tai Zi Shen, Gui Zhi, Fu Xiao Mai, Jujube, Mai Dong, Wu Wei Zi, Zhi Gan Cao, Bai Shao, Mu Tou Hui, etc., manufactured by Gansu Provincial Academy of Medical Sciences as granular instant powder, each packet containing 36.25 g of crude herbs)

Zhenqi Fuzheng Granules (provided by Foci Pharmaceutical Co., Ltd., National Drug Approval Number Z62020416)

1.1.4 Experimental Animals

Ninety healthy BALB/C mice, half male and half female, weighing (20 ± 2) g, aged 8–10 weeks; ten DBA/2 mice, half male and half female, aged 8 weeks, provided by the Animal Experiment Center of Gansu Provincial Academy of Medical Sciences, Animal Qualification Certificate No.: Yi Dong Zi No. 14-009.

1.1.5 Preparation of Thymus and Lymph Node Cell Suspension

DBA/2 mice were euthanized by cervical dislocation, soaked in 75% alcohol for 5 minutes, routinely disinfected, then the thymus and lymph nodes from the neck, submandibular region, axilla, inguinal area, mesentery, etc., were aseptically removed. A small amount of physiological saline was added, the tissues were gently cut and crushed, then filtered through a 200-mesh nylon net, passed through a No. 4 needle to obtain a single-cell suspension. Trypan Blue was used to assess cell viability, with over 95% of cells remaining viable.

1.2 Experimental Methods

1.2.1 Animal Grouping and Model Establishment

1.2.1.1 Animal Grouping

Random grouping was adopted:

• Normal Control Group: 15 mice
• Aplastic Anemia Model Group: 60 mice (divided into Pei's Blood-Elevating Granules high-, medium-, and low-dose groups, as well as the model group, with 15 mice in each subgroup)
• Zhenqi Fuzheng Granules Control Group: 15 mice

1.2.1.2 Model Establishment

Following the aplastic anemia modeling methods of Yao Jun et al. and the improved immunologically mediated mouse aplastic anemia model proposed by Zhao Xin et al., except for the normal group, the remaining 75 BALB/C mice were subjected to whole-body irradiation with 6 MV gamma rays from a linear accelerator, at a height of 100 cm, for 1 minute, with a dose of 3.0 Gy. Within 4 hours after irradiation, 0.2 ml of thymus and lymph node cell suspension from DBA/2 mice was administered via tail vein injection, with a cell count of 1 × 10⁶ per mouse. One week after modeling, blood was drawn via tail vein, and the blood analysis results showed that, compared with the normal control group, the model group exhibited significant decreases in white blood cells, platelets, and hemoglobin (P < 0.01).

Bone Marrow Smear: One week after modeling, 5 mice were randomly selected from each group, decapitated, and the left femur was removed. Using a No. 4 needle, 1 ml of RPMI-1640 solution was injected to flush out the bone marrow, which was then spread onto a slide for HE staining. Under the optical microscope, the morphological features of the bone marrow tissue were observed. The results showed that the bone marrow smear of the model mice revealed hypoproliferation, with the granulocyte lineage averaging less than 15%, the erythroid lineage less than 7%, and megakaryocytes ranging from 0 to 7 per HP; in contrast, the normal group exhibited active bone marrow proliferation, with the granulocyte lineage averaging over 40%, the erythroid lineage over 14%, and megakaryocytes ranging from 45 to 72 per HP. There was a significant difference between the two groups (P < 0.05). These results confirmed that the immunologically mediated aplastic anemia mouse model replicated in this experiment was successful and could proceed according to the original protocol for subsequent experiments.

Table 1: Effects of Linear Accelerator Gamma Rays on Peripheral Blood HGB, RBC, WBC, and PLT in Aplastic Anemia Mice (x̄ ± S)

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

1.2.1.3 Administration Method

Pei's Blood-Elevating Granules and Zhenqi Fuzheng Granules were thoroughly dissolved in distilled water before use, while the model group received an equal volume of physiological saline via gavage. According to the human/mouse dosage equivalence conversion formula in the literature, the medium-dose group of traditional Chinese medicine corresponds to 20 times the adult clinical dosage. Therefore, Pei's Blood-Elevating Granules high-, medium-, and low-dose groups were administered 0.02 g/g·d, 0.01 g/g·d, and 0.005 g/g·d respectively, with 1 ml of medication each time (equivalent to 40 times, 20 times, and 10 times the adult dosage); Zhenqi Fuzheng Granules were given at 0.01 g/g·d (equivalent to 20 times the adult clinical dosage) with 1 ml each time. All groups were fed regular feed and received continuous gavage for 30 days.

Research on Pei Zhengxue's Series of Formulas and Medications

1.2.2 Specimen Collection and Processing

After administering the drugs for 20 days and 40 days, blood was collected twice via tail vein from each group, and the blood analysis results were statistically processed. All experimental animals were euthanized by cervical dislocation at the end of the experiment, their spleens were removed, and the specimens were fixed in 10% formaldehyde solution. After sectioning, HE staining was performed, and the pathological histological changes of the spleen were observed under the optical microscope.

2 Immune Function Evaluation