Fang-Hua Wu,Kui Chen,De-Kun Zhang,Chao Gong,Jiang-Yue Yu,Shen-Zhou Huang,Chen-Xin Huang,Li-Qun Wang＊

1Department of General Surgery,Fujian Medical University Affiliated Fuzhou First Hospital,Fuzhou 350009,China.

Abstract Objective: To examine the effects of Astragalus polysaccharide on immune function in B16 melanoma mice.Method: Forty male C57BL/6 mice were divided equally into a control group, model group, Astragalus polysaccharide low-dose group,and Astragalus polysaccharide high-dose group,with 10 mice per group.B16 cells were used to develop a mouse model of melanoma.After B16 engraftment, 40 mg/kg and 80 mg/kg Astragalus polysaccharide was administered by gavage every day to the Astragalus polysaccharide low-dose group and Astragalus polysaccharide high-dose group, respectively.Splenic index, thymic index, tumor growth curves, and tumor inhibition rates were measured.Flow cytometry was used to measure proportions of peripheral blood T lymphocyte subsets.Hematoxylin and eosin staining was used to examine histopathological changes in tumors.Immunofluorescence double staining was used to identify myeloid-derived suppressor cells in tumor tissues.Results:In the Astragalus polysaccharide high-dose group,splenic and thymic indices were significantly increased and tumor growth was inhibited in melanoma mice.Flow cytometry demonstrated increased CD4+and CD4+/CD8+T-cell ratios in the high-dose Astragalus polysaccharide group.HE staining demonstrated significantly decreased numbers of tumor cells among mice with melanoma in the high-dose Astragalus polysaccharide group.Immunofluorescence double staining demonstrated significantly decreased numbers of myeloid-derived suppressor cells in tumor tissues in the high-dose Astragalus polysaccharide group. Conclusion: Astragalus polysaccharide inhibits tumor growth, increases splenic and thymic indices, increases CD4+ and CD4+/CD8+ T-cell ratios, and decreases myeloid-derived suppressor cell numbers in melanoma mice.Our results indicate Astragalus polysaccharide may enhance immune function resulting in inhibition of tumorigenesis and tumor progression.

Keywords:Melanoma,Astragalus polysaccharide,Tumor,Immune function

Background

Melanomas are epithelial malignancies that originate from melanocytes in the neural crest, i.e., pigmented nevi that have undergone malignant transformation,and is characterized by high invasiveness and distal metastasis [1].The global incidence of melanoma continues to increase.The mean age at diagnosis is currently 57 years, with the incidence of melanoma increasing linearly between 25 and 50 years of age [2].Early-stage melanoma is typically insidious and difficult to detect.Hence, melanoma is predominantly diagnosed at middle to late disease stages when the optimal timing of surgical treatment has passed,thereby severely affecting mortality and morbidity [3].Despite progress in melanoma treatment,the prognosis of melanoma remains poor, with 5-year survival from stage IV metastatic melanoma reported to be 17% [4].Therefore, there is an urgent need to develop safe and effective treatments to improve patient outcomes following melanoma diagnosis.

Tumor immune evasion is one of the characteristics of tumorigenesis [5].Tumor cells often undergo a series of changes (such as loss of antigen expression)to inhibit T-cell activation, thereby evading immune surveillance and allowing the tumor to grow [6].Hence, promoting T-cell proliferation and activation can suppress tumor growth [7].Astragaluspolysaccharide (APS) is one of the important chemical components in Astragalus that can enhance immune function in the human body and is an ideal immune booster[8].At present,APS injection is widely used in clinical antineoplastic treatment as it boosts immunity,decreases the side effects of chemotherapy drugs, and improves the quality of life of patients [9, 10].In this study, we constructed a melanoma mouse model.We observed tumor growth curve and the pathological status of tumor tissues as well as the peripheral blood T-cell count and myeloid-derived suppressor cell(MDSC) count in tumor tissues to assess the immunoregulatory effects of APS on tumors in vivo.

Materials and methods

Reagents

APS was purchased from ShangHai YuanYe Biotechnology Co., Ltd (S27818).Erythrocyte lysis buffer was purchased from Beijing Solarbio Science&Technology Co., Ltd (R1010).Ly6G (ab25377) and CD11b (ab8878) antibodies were purchased from Abcam Inc.

Cell culture

The B16 mouse melanoma cell line was purchased from Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences.During thawing, B16 cells that had been stored in liquid nitrogen were thawed in a water bath at 37 ° C.B16 cells were cultured in Dulbecco’s modified Eagle medium high-glucose medium containing 10% fetal bovine serum (89% Dulbecco’s modified Eagle medium high-glucose culture medium + 10% fetal bovine serum + 1% penicillin/streptomycin).Cells were centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded.Cell pellets were resuspended in complete culture medium and seeded in T25 culture flasks.Culture flasks were incubated in a cell culture incubator at 37°C and 5%CO2, and media were changed every 24 hours.Cells were passaged when cell density reached approximately 80%confluence by using trypsin digestion.Cells in the logarithmic growth phase were used for cell engraftment.

Development of a melanoma mouse model

Male C57BL/6 mice weighing (20 ± 2) g were were provided by Fujian Institute of Traditional Chine Medicine (license number: SCXK (Min) 2019-0010,China).Animals of specific-pathogen-free grade were used and housed at room temperature (22°C ± 2°C)with sterile feed and sterile water.B16 cells in the logarithmic growth phase were collected, and physiological saline was used to adjust cell density to 1.0 × 107/mL.Skin at the right forelimb axilla was disinfected with 75% alcohol and inoculated with 0.2 mL of the B16 cell suspension.Tumor formation was observed approximately 7 days after inoculation.All animal experiments were approved by the Animal Ethics Committee at Fujian Medical University.

Experiment grouping and dosing regimen

Forty C57BL/6 mice were divided equally into control group, model group, APS low-dose group, and APS high-dose group.After routine feeding for 1 week,melanoma cells were engrafted into mice in all groups except those in the control group.After successful engraftment, 40 mg/kg or 80 mg/kg APS was administered by oral gavage every day to the APS low-dose group and APS high-dose group,respectively.Each group was dosed continuously for 14 days(Table 1).APS doses were obtained by converting the daily dose in humans to an equivalent dose for mice based on weight, with the dose used in the low-dose group equivalent to that used in humans.

Monitoring of antineoplastic markers

During the dosing period,a Vernier caliper was used to measure the long and short axes of mouse tumors to calculate tumor volume and plot tumor growth curves for tumor-bearing mice.After dosing, axillary subcutaneous tumor tissues were excised and weighed and tumor inhibition rates were calculated.Further, the splenic and thymic indices were calculated.

Tumor volume calculation formula: Tumor volume(mm3) = Long axis (mm) × Short axis (mm)2/2.Formula for calculation of tumor inhibition rate(MTW,mean tumor weight):Tumor inhibition rate=(MTW in model group - MTW in experimental group)/MTW in model group×100%.

Measurement of peripheral blood T-cell count

After tumor engraftment and APS administration,peripheral blood was collected from mice and erythrocyte lysis buffer was used to remove erythrocytes.Flow cytometry was used to measure peripheral blood CD4+T-cell and CD8+T-cell percentages.All flow cytometry results were analyzed using FlowJo.

Hematoxylin and eosin staining

After tumor engraftment and APS admini stration,axillary subcutaneous tumor tissues were excised and fixed in 10% formalin followed by dehydration,paraffin embedding, and continuous sectioning.Routine hematoxylin and eosin staining was then performed.An optical microscope (ECLIPSE E100,Nikon Corporation) was used to image tumor immunohistochemical analyses.

Immunofluorescence staining

After tumor engraftment and APS administration,axillary subcutaneous tumor tissues were excised and fixed in 10% formalin followed by dehydration,paraffin embedding, and continuous sectioning.Dewaxing, hydration using an ethanol gradient,antigen retrieval, and blocking for 30 minutes were then performed.Primary incubation was performed with Ly6G (1:2000) and CD11b (1:4000) antibodies.Sections were incubated at 4℃ overnight.Secondary antibodies were added, and sections were incubated at room temperature.A fluorescence microscope(Olympus Corporation) was used to calculate the percentage of green fluorescence-positive and red fluorescence-positive cells in each field.

Results

APS inhibited B16 melanoma proliferation

We used the B16 melanoma cell line to develop a melanoma mouse model and administered different doses of APS by intraperitoneal injection.Splenic and thymic indices were significantly lower in the model group compared to the control group(P＜0.05 andP＜0.01, respectively).APS administration led to significantly increased splenic and thymic indices in the melanoma model (P＜ 0.05 andP＜ 0.01,respectively, Table 1).APS administration led to decreased tumor volume in the melanoma mouse model(P＜0.05,Figure 1),with tumor inhibition rates of 32.36%and 47.89%in low-dose and high-dose APS groups,respectively(Table 1).

Table 1 Effects of Astragalus polysaccharide on splenic index, thymic index, and tumor inhibition rate in melanoma mice

Figure 1 Tumor growth curve of melanoma mice.

APS increased CD4+ T-cell ratio and CD4+/CD8+T-cell ratio

Flow cytometry results demonstrated significantly decreased CD4+T-cell and CD4+/CD8+T-cell ratios in the model group compared to the control group(P＜0.05 andP＜0.01,respectively).CD4+T-cell and CD4+/CD8+T-cell ratios were significantly increased in the APS groups compared to the control group(P＜0.05,Figure 2,Table 2).

APS decreased tumor cell count

Hematoxylin and eosin staining demonstrated that tumor cells in the model group had good morphology,deep nuclear staining, a high degree of malignancy,and tight cell connections.Compared with the model group, tumor cell borders in the APS groups were unclear, cell connections were disrupted, connective tissue fibrous hyperplasia was present,cell nuclei were pyknotic, cytoplasmic acidophilia was increased, and interstitial liquefaction was observed to form blurry granular substances, all of which are features of tumor cell growth inhibition(Figure 3).

APS decreased MDSC count

Immunofluorescence results showed decreased Ly6G+(green fluorescence) and CD11b+(red fluorescence)expression in the APS groups compared to the model group (Figure 4), indicating APS decreases MDSC numbers in tumor tissues.

Table 2 Effects of Astragalus polysaccharide on peripheral blood lymphocyte subsets in melanoma mice

Figure 3 Effects of Astragalus polysaccharide on tumor tissues hematoxylin and eosin staining from melanoma mice.M

Figure 2 Effects of Astragalus polysaccharide on peripheral blood lymphocyte subsets in melanoma mice.

Figure 4 Effects of Astragalus polysaccharide on Ly6G+CD11b+ tumor tissues from melanoma mice (400×and 600×) and quantitation of APS.

Discussion

In the present study, we used B16 cells to construct a melanoma mouse model and administered APS as an intervention.Analysis of tumor growth curves and tumor inhibition rates demonstrated APS had significant inhibitory effects on melanoma growth.Tumor inhibition rates in the low-dose and high-dose APS groups were 32.36% and 47.89%, respectively.The spleen and thymus are two major immune organs,and the splenic index and thymic index can directly reflect the immune status of the body [11].The results of the present study demonstrate significantly decreased splenic and thymic indices in the model group compared with the control group, indicating relative immunosuppression in melanoma mice.Splenic and thymic indices in the low-dose and high-dose APS groups were significantly increased compared to the model group, indicating APS improved immune function in melanoma mice.

T-cell mediated immune responses recognize and destroy abnormal cells, such as pathogen-infected cells and tumor cells [12].Therefore, changes in T-cell subset counts can reflect tumorigenesis and tumor progression [13].The results of the present study demonstrate significantly decreased CD4+T-cell and CD4+/CD8+T-cell ratios in melanoma model mice;however, CD4+T-cell and CD4+/CD8+T-cell ratios were significantly increased in the low-dose and high-dose APS groups, indicating APS can correct T lymphocyte subset counts in melanoma mice, thereby ameliorating immunosuppression and increasing immunity.

MDSCs are immature bone marrow-derived cells that can inhibit T-cell proliferation and activation [14].In mice, MDSCs co-express the myeloid differentiation antigens Ly6G and CD11b [15].Immunofluorescence analyses in the present study demonstrate increased Ly6G+and CD11b+expression in melanoma model mice, indicating increased MDSC counts that may induce melanoma occurrence and progression.Low-dose and high-dose APS was found to inhibit Ly6G+and CD11b+expression in melanoma mice, indicating APS decreases MDSC counts to varying degrees thereby enhancing immunity and inhibiting tumor growth.

In summary, APS inhibits tumor growth and increases splenic and thymic indices in this melanoma mouse model.The effects of APS may be mediated by increased CD4+T-cell and CD4+/CD8+T-cell ratios and decreased MDSC counts, thereby enhancing immunity resulting in inhibition of tumorigenesis and tumor progression.