1 Materials and Methods

1 Materials and Methods

1.1 Experimental Materials

1.1.1 Experimental Drugs

Pei's Blood-Boosting Granules: Composed of ginseng root, northern ginseng, prince ginseng, and Lu Dangshen, provided by the Gansu Provincial Academy of Medical Sciences, each package containing 36.25g of crude herbs.

Zhenqi Fuzheng Granules: Provided by Dingxi Pharmaceutical Factory, batch number 003021.

1.1.2 Experimental Animals

BALB/C mice, half male and half female, weighing 22±2g, clean grade, provided by the Experimental Animal Center of the Gansu Provincial Academy of Medical Sciences. Animal qualification certificate number: Yi Dong Zi No. 14-009.

1.1.3 Tumor Strain

Mouse-transplanted hepatocellular carcinoma (H₂₂) tumor strain, introduced by the Beijing Institute of Pharmaceutical Research and preserved by the Pharmacology and Toxicology Center of the Gansu Provincial Academy of Medical Sciences.

1.1.4 Experimental Reagents

1.1.5 Experimental Instruments

1.2 Experimental Methods

1.2.1 Tumor Inoculation

H₂₂ tumor inoculation was conducted according to the "National Anti-Cancer Drug Screening Procedures." Under sterile conditions, ascitic fluid from H₂₂ hepatocarcinoma-bearing mice was diluted with 0.9% normal saline to a concentration of 2 × 10⁷ tumor cells per milliliter, and 0.2 ml was injected subcutaneously into the right axilla of each mouse.

1.2.2 Dose Design and Grouping

The clinical adult dose of Pei's Shengxue Granules is 30 g/day, equivalent to 0.5 g/kg. Based on the drug equivalence calculation method for human/mouse dosage in the literature, different dose groups were designed:

• Low-dose Pei's Shengxue Granules group: 2.5 g/kg (equivalent to 5 times the adult dose)
• Medium-dose Pei's Shengxue Granules group: 5 g/kg (equivalent to 10 times the adult dose)
• High-dose Pei's Shengxue Granules group: 10 g/kg (equivalent to 20 times the adult dose)

In addition, the following groups were established:

• Model group
• Zhenqi control group: 1.67 g/kg (equivalent to 10 times the adult dose)
• Normal control group

Each group consisted of 10 animals.

1.2.3 Administration Method

Medication was initiated the day after tumor inoculation. The experimental groups received 0.2 ml of the drug per 10 g of body weight by gavage, while the normal and model groups received an equal volume of distilled water. Medication was administered once daily for 15 consecutive days.

1.2.4 Tumor Inhibition Rate Determination [4]

The day after medication cessation, mice were euthanized by cervical dislocation, the tumors were completely excised, and their weights were measured using an electronic balance.

Calculation of tumor inhibition rate:

Tumor inhibition rate (%) = (1 - T/C) × 100%

(T/C: average tumor weight of the treatment group / average tumor weight of the control group)

1.2.5 Macrophage Phagocytic Function Determination

According to reference [5], peritoneal macrophages were prepared as follows: the abdominal skin was gently stripped, 10 ml of PBS solution was injected into the peritoneal cavity of each mouse, the peritoneal macrophages were collected, transferred to a 10 ml centrifuge tube, and centrifuged at 4°C, 1000 rpm for 5 minutes. The supernatant was discarded, the cell concentration was adjusted to 2 × 10⁶/ml using 1640 culture medium, and 200 μl of the cell suspension was added to each well of a 96-well cell culture plate (two replicates per sample). The plates were incubated at 37°C, 5% CO₂ for 2 hours. After discarding the culture medium, 100 μl of 0.072% neutral red was added, and the plates were incubated at 37°C, 5% CO₂ for 30 minutes. The neutral red was then discarded, and the plates were washed three times with warm PBS. Next, 150 μl of 0.1 mol/L acidic isopropanol was added to each well, thoroughly mixed, and left overnight. The next day, the A value was measured using a microplate reader.

1.2.6 NO Induction and Detection

The previously prepared peritoneal macrophages were added to a 96-well plate, 200 μl per well, and incubated at 37°C, 5% CO₂ for 2 hours. The culture medium was then discarded, and the non-adherent cells were washed away with PBS. Fifty micrograms per milliliter of LPS solution was added, 200 μl per well, and the plates were incubated at 37°C, 5% CO₂ for 24 hours. The supernatant was the NO sample to be tested. The test samples were then added to a 96-well plate, 100 μl per well, with four replicates per sample—two blank wells and two wells containing Griess reagent. The plates were left at room temperature for 10 minutes, and the A values were measured using a microplate reader.

1.2.7 IL-1 Induction and Detection [6]

The previously prepared peritoneal macrophages were added to a 48-well plate, 1 ml per well, and incubated at 37°C, 5% CO₂ for 2 hours. The culture medium was then discarded, and the non-adherent cells were washed away with PBS. Five micrograms per milliliter of LPS solution was added, 1 ml per well, and the plates were incubated at 37°C, 5% CO₂ for 24 hours. The supernatant was collected as the IL-1 sample to be tested.

Operation according to kit instructions:

Reagent preparation: ① Take the kit out of the refrigerator 20 minutes in advance to allow it to reach room temperature. ② Dilute the concentrated washing solution with double-distilled water (1:20). ③ Standard solution: Add 1.0 ml of standard diluent to the lyophilized standard, wait until completely dissolved, then let it sit for 15 minutes to mix well (2000 pg/ml). Further dilute according to the required concentration for the standard curve. (Standard curve concentrations: 7.8, 15.625, 31.25, 62.5, 125, 250 pg/ml.) ④ Biotinylated antibody working solution: Dilute the concentrated biotinylated antibody with biotinylated antibody diluent (1:100). ⑤ Enzyme conjugate working solution: Dilute the concentrated enzyme conjugate with enzyme conjugate diluent (1:100).

Operating steps: ① Take out the required plates from the sealed bags that have already been brought to room temperature.

Research on Pei Zhengxue Series Formulas

1.2.7 Induction and Detection of IFN-γ in Splenic Cells

The spleen was aseptically removed, rinsed once in PBS solution, gently crushed with a slide, and 3 ml of PBS solution was used to rinse the slide and petri dish. The mixture was filtered through a 200-mesh sieve into a small green bottle, then slowly poured along the wall of a 10 ml glass centrifuge tube pre-filled with 3 ml of lymphocyte separation solution. The tube was centrifuged at 4°C, 3000 rpm for 20 minutes, and the lymphocyte separation layer was aspirated into a 5 ml glass centrifuge tube. PBS solution was added up to the 5 ml mark, and the tube was centrifuged again at 4°C, 1000 rpm for 10 minutes. The supernatant was discarded, and the cell concentration was adjusted to 1 × 10⁶/ml using 1640 solution. The cells were then added to a 48-well plate, 1 ml per well, with 5 μg/ml of LPS added as the final concentration. The plates were incubated at 37°C, 5% CO₂ for 24 hours, and the supernatant was collected as the IFN-γ sample to be tested.

Operate according to the kit instructions:

Reagent preparation: ① Take the kit out of the refrigerator 20 minutes in advance to allow it to reach room temperature. ② Dilute the concentrated washing solution with double-distilled water (1:20). ③ Standard solution: Add 1.0 ml of standard diluent to the lyophilized standard, wait until completely dissolved, then let it sit for 15 minutes to mix well (2000 pg/ml). Further dilute according to the required concentration for the standard curve. (Standard curve concentrations: 7.8, 15.625, 31.25, 62.5, 125, 250 pg/ml.) ④ Biotinylated antibody working solution: Dilute the concentrated biotinylated antibody with biotinylated antibody diluent (1:100). ⑤ Enzyme conjugate working solution: Dilute the concentrated enzyme conjugate with enzyme conjugate diluent (1:100).

Operating steps: ① Take out the required strips from the sealed bags that have already been brought to room temperature. ② Except for the blank wells, add the samples or different concentrations of standard solutions (100 μl per well) to the corresponding wells, seal the reaction wells with sealing tape, and incubate at 37°C for 90 minutes. ③ Manual washing of the plates: Shake off all the liquid in the wells, add 350 μl of washing solution to each well, let it sit for 30 seconds, then shake off the liquid again and pat dry on absorbent paper. Wash the plates five times. ④ Except for the blank wells, add the biotinylated antibody working solution (100 μl per well). Seal the reaction wells with sealing tape, and incubate at 37°C for 60 minutes. ⑤ Wash the plates five times. ⑥ Except for the blank wells, add the enzyme conjugate working solution (100 μl per well). Seal the reaction wells with sealing tape, and incubate at 37°C for 30 minutes. ⑦ Wash the plates five times. ⑧ Add the chromogenic reagent (100 μl per well), avoid light, and incubate at 37°C for 15–20 minutes. ⑨ Add the stop solution (100 μl per well), mix well, and immediately measure the OD450 value (within 5 minutes).

1.2.8 Induction and Detection of IFN-γ in Splenic Cells

The spleen was aseptically removed, rinsed once in PBS solution, gently crushed with a slide, and 3 ml of PBS solution was used to rinse the slide and petri dish. The mixture was filtered through a 200-mesh sieve into a small green bottle, then slowly poured along the wall of a 10 ml glass centrifuge tube pre-filled with 3 ml of lymphocyte separation solution. The tube was centrifuged at 4°C, 3000 rpm for 20 minutes, and the lymphocyte separation layer was aspirated into a 5 ml glass centrifuge tube. PBS solution was added up to the 5 ml mark, and the tube was centrifuged again at 4°C, 1000 rpm for 10 minutes. The supernatant was discarded, and the cell concentration was adjusted to 1 × 10⁶/ml using 1640 solution. The cells were then added to a 48-well plate, 1 ml per well, with 5 μg/ml of LPS added as the final concentration. The plates were incubated at 37°C, 5% CO₂ for 24 hours, and the supernatant was collected as the IFN-γ sample to be tested.

Operate according to the kit instructions:

Reagent preparation: ① Take the kit out of the refrigerator 20 minutes in advance to allow it to reach room temperature. ② Dilute the concentrated washing solution with double-distilled water (1:20). ③ Standard solution: Add 1.0 ml of standard diluent to the lyophilized standard, wait until completely dissolved, then let it sit for 15 minutes to mix well (2000 pg/ml). Further dilute according to the required concentration for the standard curve. (Standard curve concentrations: 7.8, 15.625, 31.25, 62.5, 125, 250 pg/ml.) ④ Biotinylated antibody working solution: Dilute the concentrated biotinylated antibody with biotinylated antibody diluent (1:100). ⑤ Enzyme conjugate working solution: Dilute the concentrated enzyme conjugate with enzyme conjugate diluent (1:100).

Operating steps: ① Take out the required strips from the sealed bags that have already been brought to room temperature. ② Except for the blank wells, add the samples or different concentrations of standard solutions (100 μl per well) to the corresponding wells, seal the reaction wells with sealing tape, and incubate at 37°C for 90 minutes. ③ Manual washing of the plates: Shake off all the liquid in the wells, add 350 μl of washing solution to each well, let it sit for 30 seconds, then shake off the liquid again and pat dry on absorbent paper. Wash the plates five times. ④ Except for the blank wells, add the biotinylated antibody working solution (100 μl per well). Seal the reaction wells with sealing tape, and incubate at 37°C for 60 minutes. ⑤ Wash the plates five times. ⑥ Except for the blank wells, add the enzyme conjugate working solution (100 μl per well). Seal the reaction wells with sealing tape, and incubate at 37°C for 30 minutes. ⑦ Wash the plates five times. ⑧ Add the chromogenic reagent (100 μl per well), avoid light, and incubate at 37°C for 15–20 minutes. ⑨ Add the stop solution (100 μl per well), mix well, and immediately measure the OD450 value (within 5 minutes).

1.2.9 2,4-Dinitrochlorobenzene-induced Mouse DTH (Ear Swelling Method)

The experimental grouping and administration method were the same as before. Five days before euthanasia, the abdominal skin of each mouse was depilated with barium sulfide over an area of about 3 cm × 3 cm, and 50 μl of 1% DNCB solution was evenly applied to sensitize the skin. One day before euthanasia, 10 μl of DNCB solution was evenly applied to both sides of the right ear of each mouse for challenge. Twenty-four hours after the challenge, the mice were euthanized by cervical dislocation, and the left and right ear shells were cut off. The diameter of the ear pieces, 8 mm, was punched out using a stainless steel punch, and the weight (in mg) was recorded. The difference between the weights of the left and right ears represented the degree of DTH.

1.3 Statistical Analysis

All experimental data are expressed as mean ± standard deviation (x̄±s). Statistical analysis was performed using SPSS 10.0 software, and one-way ANOVA was used for comparing multiple group means.

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2. Results