Reagents

Rehmanniae Radix Praeparata (22070801, Henan, China), Angelicae Sinensis Radix (21061101, Gansu, China), Paeoniae Radix Alba (21122601, Anhui, China) and Chuanxiong Rhizoma (095210801, Sichuan, China) were purchased from Beijing Tongrentang Co.,ltd. Mouse catalase (CAT) (MM-44125M1), malondialdehyde (MDA) (MM-0897M1), beta-Nicotinamide adenine dinucleotide trihydrate (NAD +) (MM-1010M1), interleukin (IL)-6 (MM-0163M1), Janus activated kinase 1 (JAK1) (MM-46967M1), JAK2 (MM-46942M1), signal transducer and activator of transcription (STAT3) (MM-45741M1), cyclin-dependent kinase inhibitor 2A (CDKN2A/p16) (MM-44525M1) enzyme linked immunosorbent assay (ELISA) kits were purchased from Jiangsu Meimian Industrial Co., Ltd. Mouse taurine ELISA kit (JM-12220M2) was provided by Jiangsu Jingmei Biotech Co., Ltd. Bicinchoninic acid assay (BCA) protein quantitative kit (ZJ102) was from Shanghai Yaenzyme Biotechnology Co., Ltd. β-galactosidase microplate assay kit (ADS1062W) was purchased from Jiangsu Addison Biotechnology Co., Ltd. Hifair® III 1st Strand cDNA Synthesis Super Mix for qPCR (11141ES60), Hieff UNICON® Universal Blue qPCR SYBR Master Mix (11141ES) were from Yeasen Biotechnology (Shanghai) Co., Ltd. Animal tissue/cell genome DNA extraction kit (8028011) as well as Absolute Mouse Telomere Length Quantification qPCR Assay Kit (M8918, ScienCell™) purchased from akewe Biotech Co., Ltd. p16INK4a Polyclonal Antibody (PA5-20379) and beta Galactosidase Polyclonal Antibody (PA5-102503) were from Invitrogen (US). Antibodies used in flow cytometry were listed in Table 1.

Table 1 Antibodies used in flow cytometryAnimals and IR

According to the Taiping Huimin Hejiju Prescription, the composition of SWD includes 15 g of Rehmanniae Radix Praeparata, 10 g of Angelicae Sinensis Radix, 10 g of Paeoniae Radix Alba, and 6 g of Chuanxiong Rhizoma, totaling 41 g per day. The equivalent dose ratio between mice and humans is 9.1 [14]. Using the standard human body weight of 70 kg for conversion, the equivalent dose of SWD for mice can be calculated as follows:

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SPF C57BL/6 J male mice, 21–24 g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The mice were raised in the Animal Center of the Academy of Military Medical Sciences (AMMS) and randomly divided into 5 groups: normal control group (NC), irradiation group (Model), low-dose SWD administration group (SWD-L, 5 g/kg/d i.g.), medium-dose SWD administration group (SWD-M, 10 g/kg/d i.g.), high-dose SWD administration group (SWD-H, 20 g/kg/d i.g.), which equivalent to 1, 2, 4 times the clinical equivalent dose (drawing from our two decades of research experience on SWD, which is most appropriate), with 7 mice in each group. After 7 days of adaptive feeding, the mice, except for NC, were exposed to 6.0 Gy 60Co γ-ray at a dose rate of 102.63 cGy/min to induce hematopoietic-immune injury. The mice received a continuous administration of drugs for 30 days 8 weeks after irradiation. The experiment was performed in accordance with the guidelines of the European Community and approved by the Institution of animal Care and Use Committee of AMMS: IACUC-DWZX-2023–547.

Decocting and preparation of SWD

410 g of SWD (Rehmanniae Radix Praeparata 150 g, Angelicae Sinensis Radix 100 g, Paeoniae Radix Alba 100 g, Chuanxiong Rhizoma 60 g) was added to Soxhlet extraction device, soaked in 3 L of ultra-pure water overnight, steamed 3 times the next day, 1.5 h each time, combined with the decoction, concentrated, poured into the freeze dryer. The freeze-dried powder of SWD was prepared by cryogenic freeze-drying for 35 h. At last, the total of 410 g of raw herbs yielded 221.7511 g of freeze-dried powder, with a yield rate of 54%. The corresponding concentration of drug was prepared by freeze-dried powder and ultra-pure water.

Mice behavioral experiment

Open field test (OFT): The experimental animal is quickly placed in the central area of the experimental box and immediately left, and the animal behavior analysis software is opened 2 min later to automatically record the activities of the animal in the box for 5 min.

Grasp force measurement: Place the grasp force tester in a horizontal position and position the mouse on it. Grasp the tail of the mouse and gently apply even force to pull back, causing the mouse to release its claw. Record the maximum grasp force achieved. Each mouse was measured 3 times, and the maximum value was used to evaluate the muscle strength of the mouse.

Treadmill stress test: First at the speed of 10 m/min (5 min) and 20 m/min (5 min) adaptive training 10 min, and then 30 m/min until the mice exhaustion. Runway angle was set 15°, current 0.5 mA. It was considered as exhaustion while the mice were shocked 5 times consecutively within 2 min.

Novel-object preference test (NOP): On the first day, two identical objects were placed in the apparatus, and mice were allowed to freely explore for 10 min. On the second day, one of the identical objects was replaced with a different object in the apparatus, and the mice were also allowed to explore for 5 min. The duration of exploration for each object was measured. The recognition index (RI) is calculated as follows: RI = T2/(T1 + T2) × 100%, where T1 is the time for mice to explore familiar objects and T2 is the time to explore new objects.

Blood cell counts

At the end of the administration, blood samples were collected and a hematology analyzer (Sysmex XN-1000 V) was used to determine blood cell counts. The content of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and the proportion of neutrophils (NEUT%), lymphocytes (LYMPH%), eosinophils (EO%) and monocytes (MONO%) were counted.

Calculation of organ index

The mice in each group were weighed, and the spleen and thymus tissues were removed and weighed after euthanasia.

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Flow cytometry

Single cell samples from the BM and spleen were labeled with corresponding fluorescent antibodies to detect mouse HSPCs as well as immune cells. The primary HSPCs in mice, including lymphoid and myeloid progenitors, were identified based on the expression of LIN, Sca-1, c-kit, CD34, CD16/32, and CD127. T cells were characterized by the presence of CD3, CD4, and CD8, while B cells were identified using CD19 and B220. Natural killer (NK) cells were distinguished by the expression of NK1.1 [15]. This approach was utilized to assess the impact of IR on the composition of hematopoietic and immune cell populations over time.

ELISA

BM and spleen proteins were quantified in which the contents of IL6, JAK1, JAK2, STAT3 and p16 proteins were detected by ELISA kits. All operations were carried out according to the manufacturer’s instructions, through the steps of sample addition, enzyme addition, warming, washing, color development, termination and finally the absorbance is measured in sequence at 450 nm.

Detection of the activity of β-galactosidase

The BM samples were adjusted to the same concentration level according to the results of protein quantification. The temperature and wavelength of the microplate reader were set at 37 ℃ and 405 nm. Each measuring tube (A1) was provided with a contrast one (A0), ΔA = A1-A0.

Quantitative real-time PCR (qPCR)

Total RNA from mice BM was extracted and reverse-transcribed into cDNA. The mRNA levels of different genes were quantitatively analyzed by 2−△△Ct using β-actin as the internal reference. Primers p21, GLB1, Socs3 and β-actin were synthesized in Beijing Tianyi Huiyuan Biotechnology Co., Ltd. The sequences are shown in Table 2.

Table 2 Primer sequencesDetermination of telomere length

Genomic DNA of mice BM cells was extracted and two qPCR reaction systems were prepared, one was telomere primer stock and the other was single copy reference gene primer stock, and the results were calculated by 2−△△Ct method.

Histological analysis

The brain, thymus and spleen tissues of mice (n = 3) were fixed with 4% paraformaldehyde, dehydrated and embedded with paraffin to prepare paraffin sections with a thickness of about 5 µm.

Hematoxylin–eosin staining (H&E): Sections were placed in hematoxylin staining solution, soaked for 10–15 min, washed with distilled water and then added to eosin dyeing solution for 15 s and washed with distilled water. After which, the sections were dehydrated with 75, 85, 95 and 100% ethanol gradient for 2 min, respectively and were sealed with xylene transparent and neutral glue.

Immunofluorescence staining (IF): The fluorescence intensity of p16 protein and β-galactosidase in spleen and thymus of mice was detected through the steps of permeability, sealing, antibody incubation, etc. Image J 2.0 was used to calculate the mean density of fluorescence.

Statistical analyses

All the data in this experiment were repeated for more than 3 times, and the data were represented by mean ± standard deviation (\(\overline\)± s). GraphPad Prism 8 was used to plot the data. One-way ANOVA was used to analyze the data between multiple groups, and t test was used to analyze the data between two groups. p < 0.05 was considered significant.