Objective: This study was to investigate the effect of the Chinese herbal compound Kanggan granule (KG) on immune function in a mouse model of immunosuppression and its possible mechanism of action. Method: ICR mice were randomly divided into a normal control group (untreated non-immunosuppressed, Control), untreated immunosuppressed group (Model), positive control group (immunosuppressed and treated with 1.6 g/kg astragalus granule [AG]), high-dose KG group (immunosuppressed and treated with 24 g/kg, KG-24), and low-dose KG group (immunosuppressed and treated with 6 g/kg, KG-6). Each group received intragastric administration once daily for 7 days. Immunosuppression was induced by an intraperitoneal injection of dexamethasone (25 mg/kg) once daily beginning on day 1 for 3 days. To illuminate the mechanism of immunomodulatory, we studied the effects of KG on nonspecific immunity, humoral immunity and cellular immunity in mice respectively. Results: KG improved organ weights and improved the phagocytic ability of mononuclear macrophages in immunosuppressed mice (p < 0.01 or p < 0.05). The proliferation of spleen lymphocytes and number of peripheral blood leukocytes were enhanced after KG treatment in immunosuppressed mice (p < 0.05). KG increased the CD4+/CD8+ ratio in immunosuppressed mice (p < 0.01) and increased serum IL-2 and IgG levels (p < 0.01). Conclusions: KG can improve immune function in immunosuppressed mice. Nonspecific immunity, humoral immunity, and cellular immunity were all enhanced.
The immune system is indispensable for our body. It protects against the invasion of foreign pathogens through nonspecific immunity, humoral immunity, and cellular immunity [
The clinical practice of traditional Chinese medicine (TCM) has a long history that has resulted in a considerable number of useful preparations, and TCM is still widely used today [
Specific-pathogen-free male ICR mice (weighing 20 - 22 g) were supplied by Chengdu Dossy Experimental Animals Co. Ltd. (Chengdu, Sichuan, China). All of the animals were raised in plastic cage at 25˚C ± 2˚C and had free access to food and water. The animals were raised under a 12 h/12 h light/dark cycle. The animal studies were conducted in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. All of the procedures were approved by the Animal Research Committee of West China School of Pharmacy.
Kanggan granule (Sichuan Gooddoctor-Panxi Pharmaceutical Co., Ltd., Xichanng, Sichuan, China; batch No. 160703), Astragalus granule (AG; Sichuan Baili Pharmaceutical Co., Ltd., Chengdu, Sichuan, China; batch No. 161248), dexamethasone sodium phosphate injection (Tianjin Kingyork Group Co., Ltd., Tianjin, China; batch No. 1610092), RPMI 1640 Medium (Thermo Fisher Scientific, Waltham, MA, USA), Concanavalin A (ConA; Sigma-Aldrich, St. Louis, MO, USA), lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA), MTT (Amresco Co., Solon, Ohio, USA), the mouse interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Yaji Biotechnology Co., Ltd., Shanghai, China), the mouse immunoglobulin G (IgG) ELISA kit (Nanjing SenBeijia Biotechnology Co., Ltd., Nanjing, Jiangsu, China), anti-CD3 (fluorescein isothiocyanate [FITC]; Thermo Fisher Scientific Co., Waltham, MA, USA), anti-CD4 (P-phycoerythrin [PE]; Thermo Fisher Scientific Co., Waltham, MA, USA), and anti-CD8 (PE-cy5; Thermo Fisher Scientific Co., Waltham, MA, USA) were used.
Based on the previous reports [
Macrophage phagocytosis was determined using a previously described method with modifications [
The detection was guided by previous reports [
Serum was obtained by centrifuging peripheral blood at 1500× g for 15 min according to the instructions of the IL-2 and IgG ELISA kits. The OD was measured at a wavelength of 450 nm (Varioskan Flash, Thermo Fisher Scientific Co., Waltham, MA, USA), and the concentrations of IL-2 and IgG were measured.
Detection of T-lymphocyte subsets was performed as described previously with modifications [
Following the previous reports [
The mice were transcardially perfused with phosphate-buffered saline (PBS). The spleen and thymus were removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Paraffin sections (5 μm) were prepared for hematoxylin-eosin (HE) staining.
Using SPSS17.0 statistical software for experimental data processing and data were expressed as mean ± SEM. The difference of data between groups was examined by one-way analysis of variance, and p < 0.05 as the difference was statistically significant.
At the beginning of the experiment, the appetite, fluid consumption, activity, and appearance of the mice were normal. A few days after KG administration, the appetite, fluid consumption, and appearance of the mice did not apparently change, but activity decreased compared with the normal control group. Immunosuppressed mice typically drink less, exhibit a decrease in appetite, and have low activity.
The body weights of the mice in each group were generally the same before administration. Seven days after KG administration, body weight, the spleen index, the thymus index, the K-value, and the α-value of the immunosuppressed mice significantly decreased compared with the normal control group (p < 0.01). The spleen index, K-value, and α-value in the AG group, KG-24 group, and KG-6 group were higher compared with the untreated immunosuppressed group (p < 0.01 or p < 0.05). The thymus index in the AG group, KG-24 group, and KG-6 group were not significantly different from the untreated immunosuppressed group (p > 0.05;
Hematoxylin-eosin staining revealed changes in the morphology of the spleen and thymus. Compared with the normal control group, the boundary of white pulp and red pulp was unclear in the untreated immunosuppressed group. The area of white pulp was lower in the untreated immunosuppressed group. More apoptotic cells appeared, and macrophages were rare in white pulp in the untreated immunosuppressed group. After treatment with AG and KG, the boundary between white and red pulp was clear. Fewer apoptotic cells were observed, and the number of macrophages increased in white pulp (
Groups | Dosage (g/kg) | Bodyweight (g) | Organ index (mg/g) | K | α | |||
---|---|---|---|---|---|---|---|---|
Day 1 | Day 8 | Spleen | Thymus | |||||
Control | ― | 20.78 ± 0.80 | 25.13 ± 1.25 | 3.826 ± 0.514 | 2.357 ± 0.325 | 0.056 ± 0.014 | 7.357 ± 0.875 | |
Model | ― | 20.43 ± 0.53 | 23.18 ± 0.82## | 2.527 ± 0.610## | 0.785 ± 0.158## | 0.037 ± 0.010## | 5.831 ± 0.829## | |
AG | 1.6 | 20.57 ± 0.51 | 24.68 ± 1.21** | 3.507 ± 0.842** | 0.800 ± 0.220 | 0.050 ± 0.014* | 6.832 ± 0.790* | |
KG-24 | 24 | 20.60 ± 0.83 | 24.28 ± 0.94* | 3.300 ± 0.973* | 0.793 ± 0.287 | 0.048 ± 0.010* | 6.637 ± 0.522* | |
KG-6 | 6 | 20.77 ± 1.34 | 24.48 ± 1.00* | 3.357 ± 0.723* | 0.806 ± 0.085 | 0.050 ± 0.008** | 7.076 ± 0.462** | |
*p < 0.05, **p < 0.01 vs. Model group; ##p < 0.01 vs. Control group.
Twenty-four hours after the last administration, the proliferative capacity of splenic lymphocytes was investigated in vitro to determine the effect of KG. The SI of splenic lymphocytes that were stimulated by ConA in the untreated immunosuppressed group significantly decreased compared with the normal control group (p < 0.05). The SI of splenic lymphocytes in the AG group and KG-6 group was higher than in the untreated immunosuppressed group (p < 0.05). The SI in the KG-24 group was not significantly different from the untreated immunosuppressed group (p > 0.05;
Serum IL-2 levels in the untreated immunosuppressed group significantly decreased compared with the normal control group (p < 0.01). Serum IL-2 levels in the AG group and KG-6 group were higher than in the untreated immunosuppressed group (p < 0.01). Serum IL-2 levels in the KG-24 group were not significantly different from the untreated immunosuppressed group (p > 0.05;
The CD4+/CD8+ ratio in the untreated immunosuppressed group significantly decreased compared with the normal control group (p < 0.01). The CD4+/CD8+ ratio in the AG group, KG-24 group, and KG-6 group was higher than in the untreated immunosuppressed group (p < 0.01;
The number of peripheral blood leukocytes in the untreated immunosuppressed group significantly decreased compared with the normal control group (p < 0.01). The number of leukocytes in the AG group, KG-24 group, and KG-6 group was higher than in the untreated immunosuppressed group (p < 0.01 or p < 0.05;
The SI of splenic lymphocytes that were stimulated by LPS in the untreated immunosuppressed group significantly decreased compared with the normal control group (p < 0.01). The SI of splenic lymphocytes in the AG group and KG-6 group was higher than in the untreated immunosuppressed group (p < 0.05). The SI in the KG-24 group was not significantly different from the untreated immunosuppressed group (p > 0.05;
Serum IgG levels in the untreated immunosuppressed group significantly decreased compared with the normal control group (p < 0.01). Serum IgG levels in the AG group and KG-6 group were higher than in the untreated immunosuppressed group (p < 0.01). Serum IgG levels in the KG-24 group were not significantly different from the untreated immunosuppressed group (p > 0.05,
KG is often used to treat the cold virus. The overall course of a cold mainly consists of a conflict between the immune system and foreign pathogens. Another Chinese herbal compound, AG, is widely used today. Its main component is astragalus [Astragalus membranaceus (Fisch.) Bung., the dry root of Astragalus membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge]. AG is also included in the 2015 Chinese Pharmacopoeia. The immunomodulatory effect of AG has been reported [
Dexamethasone, a glucocorticoid, is widely used as immunosuppressive agent. In addition, dexamethasone is often used to induce immunosuppression as disease models in experimental studies [
Leukocytes are blood cells that play a primary role in the defense against foreign pathogens. Changes in the number of leukocytes are associated with some diseases [
leukocytes significantly increased after KG and AG administration, indicating that KG can regulate the number of leukocytes in immunosuppressed mice. T-lymphocytes play an important role in cellular immunity. They comprise a large and complex group that can be divided into several subsets. In these subsets, CD4+ cells are known as helper T-cells, and CD8+ cells are known as cytotoxic T-cells. The CD4+/CD8+ ratio can change when immunological diseases occur [
The spleen contains a large number of lymphocytes, including B-lymphocytes and T-lymphocytes, which play an important role in humoral and cellular immunity. We investigated the proliferative capacity of B- and T-lymphocytes that were stimulated by LPS and ConA to evaluate changes in the immune response [
IL-2 is produced by T-cells. It plays an important role in the immune system, including receptor activation, cytokine expression, and lymphocyte proliferation. Low levels of IL-2 reduce the immune response [
The results of KG-24 administration were not as promising as those of KG-6. The dose of KG in the the KG-24 group was likely much higher than the dose at which the drug reaches its maximal efficacy, whereas the dose of KG in the KG-6 group was likely slightly lower than the maximally effective dose. However, a more precise KG dose-response curve needs to be generated. To study the effect of KG on the immune system in mice, we developed a model of dexamethasone-induced immunosuppression. We then measured the phagocytic ability of mononuclear macrophages, organ weights, and the proliferative capacity of splenic lymphocytes, the number of peripheral blood leukocytes, the distribution of T-lymphocyte subsets, and serum IgG and IL-2 levels. The results of these analyses indicated that KG improved immunity and regulated immune function in immunosuppressed mice.
The present study showed that KG, which consists of Lonicerae japonicae Flos, Dryopteridis Crassirhizomatis Rhizoma, and Paeoniae Radix Rubra, can improve immune function in immunosuppressed mice, reflected by enhancements of nonspecific immunity, humoral immunity, and cellular immunity.
This work was supported in part by Targeting drug delivery system of Sichuan Province Youth Science and technology innovation team (2016TD0001).
The authors declare no conflicts of interest regarding the publication of this paper.
Gan, Y.M., Gu, M.X., Liu, D.L., Zhou, H.J., Zeng, C.Y., Yang, T.T., Li, H., Geng, F.N. and Du, J.R. (2019) Kanggan Granule Ameliorates Dexamethasone-Induced Immunosuppression in Mice. Journal of Biosciences and Medicines, 7, 80-91. https://doi.org/10.4236/jbm.2019.73008