Abstract
Objective: The aim of this study is to investigate the effects of a Fuzheng Paidu tablet on peripheral blood T lymphocytes, intestinal mucosa T lymphocytes, and immune organs in cyclophosphamide (CY)-induced immunosuppressed mice. Methods: The experimental mice (but not the control mice) were intraperitoneally injected with 80 mg/kg of CY solution every day for 3 consecutive days. Meanwhile, each mouse was administered with corresponding drugs for 7 continuous days. Then, 1 h after the last administration, each index was detected. Results: The Fuzheng Paidu tablet significantly increases the CD4+/CD8+ ratio (P < 0.01) and the number of CD3+ and CD4+ cells in immunosuppressed mice (P < 0.01). In addition, the tablet apparently enhances the CD3+, CD4+, and CD8+ levels in the intestinal mucosal immune system (P < 0.01) as well as reverses the reduction of spleen lymphoid nodules and lymphocytes (P < 0.01). It also significantly improves intestinal inflammation, thymic atrophy, and sparse thymocytes in immunosuppressed mice (P < 0.01). Discussion: The Fuzheng Paidu tablet greatly increases the levels of peripheral blood T lymphocytes and intestinal mucosal T lymphocytes as well as impoves atrophied thymuses and spleens in CY-induced immunosuppressed mice.
Introduction
The immune system is the defense function of the organism to resist the pathogenic microorganism, and to remove the self-stabilizing function of the body decay cells and the waste material, and to identify the monitoring function of malignant cells in body. The occurrence, development of many human diseases are associated with immune adjustment functions obstacle, such as colds, tumor and other diseases.Citation1
The Fuzheng Paidu tablet is a TCM receipt developed by the AIDS prevention and treatment experts at Henan University of Traditional Chinese Medicine based on their long-term clinical practice. The tablet is composed of 13 TCM materials, including ginseng and Astragalus. It can benefit, nourish Yin, and clear away heat and toxicity, and it is suitable for the clinical treatment of asymptomatic HIV patients with Qi and Yin deficiencies and who are inflicted with internal heat-toxicity.Citation2-5
In order to further investigate the features of the Fuzheng Paidu tablet, its effects on the peripheral blood T lymphocytes, intestinal mucosa T lymphocytes, and immune organs in cyclophosphamide (CY)-induced immunosuppressed mice were studied.
Results
Effects on peripheral blood T lymphocytes and intestinal mucosa T lymphocytes in cyclophosphamide-induced immunosuppressed mice.
The immunohistochemical method (SP method) was applied to determine the CD3+, CD4+, and CD8+ levels in the T lymphocyte subsets of the intestinal mucosa ().
As is shown in , the CD3+, CD4+, and CD4+/CD8+ levels were significantly reduced in the model group as compared to the blank control group (P < 0.01). These results indicated the successful establishment of the CY-induced immunosuppressed model. Furthermore, in comparison to the model group, the number of CD3+ and CD4+ cells and the CD4+/CD8+ ratio were obviously increased in the Lentinan group as well as in the high, middle, and low dose groups (P < 0.01). Finally, the effects of the Fuzheng Paidu high, middle, and low dose groups were similar to or better than the Lentinan group, meaning the CD3+ cell number was apparently enhanced (P < 0.01).
Table 1. Effects of Fuzheng Paidu Tablet on the peripheral blood CD3+, CD4+, CD8+, and CD4+/CD8+ levels in CY-induced immunosuppressed mice
As is shown in and , the CD3+, CD4+, and CD8+ subsets in the intestinal mucosal lamina propria were significantly reduced in the model as compared to the control groups (P < 0.01). This suggests that CY-induced immunosuppression not only affects the peripheral immune system but also strongly inhibits the intestinal mucosal immune system. Moreover, as compared to the model group, the Lentinan group and the Fuzheng Paidu high, middle, and low dose groups all had dramatically increased CD3+, CD4+, and CD8+ subsets in the intestinal mucosal immune system in the immunosuppressed mice (P < 0.01). Furthermore, the intestinal mucosal immune system's function was improved by lentinan as well as by the high dose of the Fuzheng Paidu Tablet. Compared to the normal control group, there was no significant difference in the CD3+ cell number (P > 0.05); however, there was an obvious increase in the number of CD4+ cells (P < 0.01).
Figure 1. Effects of Fuzheng Paidu Tablet on the intestinal CD3+ cells in CY-induced immunosuppressed mice.
Figure 2. Effects of Fuzheng Paidu Tablet on the intestinal CD4+ cells in CY-induced immunosuppressed mice.
Figure 3. Effects of Fuzheng Paidu Tablet on the intestinal CD8+ cells in CY-induced immunosuppressed mice.
Table 2. Effects of Fuzheng Paidu Tablet on the intestine mucosa T cells in CY-induced immunosuppressed mice
As is shown in , the splenic lymphoid nodules and lymphocytes were relatively dense in the control mice spleens. The thymic lobule boundary was clear, and the cortex was clearly separated from the medulla. Moreover, the cortical lymphocytes were intensive. As compared to the control group, the model group had significantly small splenic lymphoid nodules, sparse lymphocytes, and thin thymus cortex, thereby implicating that the model was successfully constructed (P < 0.01). Furthermore, as compared to the model group, the Lentinan group and the Fuzheng Paidu high, middle, and low dose groups exhibited a smaller reduction in the splenic lymphoid nodules and lymphocytes, a thicker thymus cortex, and more intensive lymphocytes (P < 0.01). In addition, the low dose of the Fuzheng Paidu Tablet also obviously improved the phenomena (atrophic thymus and sparse lymphocytes) in the immunosuppressed mice (P < 0.01). These findings indicate that the Fuzheng Paidu Tablet regulates the immune function of CY-induced immunosuppressed mice.
Table 3. Effects of Fuzheng Paidu Tablet on the splenic lymphoid nodule size and lymphocyte number in the spleen of CY-induced immunosuppressed mice
Effects on immune organs in CY-induced immunosuppressed mice
The animals in each group were evaluated according to the small intestine semi-quantitative standard. Based on inflammation levels, the measurements were classified into one of 4 grades: ‘−’ represents normal epithelial cells, the lamina propria layer, submucosa, muscularis, and serosa; ‘+’ represents normal epithelial cells and mildly inflamed lamina propria layer and submucosa; ‘++’ represents normal epithelial cells and seriously inflamed lamina propria layer and submucosa; and ‘+++’ represents necrotic epithelial cells and seriously inflamed lamina propria layer and submucosa.
As shown in and , the control mice had normal epithelial cells, lamina propria layer, submucosa, muscularis, and serosa. In comparison to this control group, the model mice showed significantly necrotic epithelial cells and a seriously inflamed lamina propria layer and submucosa. The Ridit test indicated P < 0.01, which suggested that the model was successful. Furthermore, as compared to the model group, the Fuzheng Paidu high and middle dose groups exhibited obviously improved inflammatory symptoms (Ridit test, P < 0.01). On the other hand, the Fuzheng Paidu Tablet low dose group and the Lentinan group tended to improve intestinal mucosal injury. These findings suggest that the Fuzheng Paidu Tablet improves the immune function of CY-induced immunosuppressed mice.
Table 4. Effects of Fuzheng Paidu Tablet on the intestinal mucosal pathology in CY-induced immunosuppressed mice
Discussion
Immunosuppression model is often used in immune pharmacology studies, the Immunosuppression model currently mentioned has the radiation damage model, the animal model of thymus, immunosuppression induced inhibition model and gene knockout etc. Among them, the immunosuppressants model is simple and cheap, and it is widely used in the present immunological research.Citation6 In current immune toxicology, commonly used positive substances include cyclophosphamide (CY), hydrocortisone, dexamethasone, cyclosporin A, vincristine, and actinomycin.Citation7-8 In the present study, CY was used to construct an immunosuppressed mouse model because CY is a cytotoxic chemotherapy drug as well as an alkylating immune inhibitor. CY damages DNA structure and blocks replication, thus leading to cell death; furthermore, it has been reported that CY inhibits humoral and cellular immune responses in animals, causing immunosuppression.Citation9-10 Thus, CY is an ideal drug for establishing immunosuppressed models in the immune toxicology field.Citation11-12
The rationale behind this study is as follows. T cells play an essential role in the immune mechanism, and CD3+ is a marker for mature T cells.CD4+ and CD8+T cells are the central segment of immune response and immune regulation in organism, under the organism normal physiological conditions, CD4+/CD8+ to maintain a certain proportion, be in relative equilibrium and stability state, and between each other to induce and restrict, constitute T lymphocyte network system, maintain normal immune response in organism. When the proportional imbalance or function changes between the CD4+/CD8+ can lead to the disorder of immunity adjustment function, and cause a series of pathological changes in the body. While, a variety of immune cells and immune factors in the spleen can complete non-specific immune function by phagocytosis, and the specific immune function can also be played by the cell, cell-mediated cellular immune and humoral immunology. Lastly, the mucosal immune system plays an important role in immune defense as it is essential for clearing away any exogenous toxins or pathogenic microorganisms by physical, chemical, or immunological approaches.
Modern pharmacological studies have shown that the American ginseng in the Fuzheng Paidu Tablet contains saponins, polysaccharides, amino acids, trace elements, vitamins, and other components. From among these components, active polysaccharides activate macrophages and T lymphocytes by regulating the reticuloendothelial system and complement system. Furthermore, they improve a person's immunity by inducing a variety of immune factors that can impact human immunity, the specific immune system, cellular immunity, and humoral immunity. On the other hand, Astragalus can promote T helper cell proliferation and enhance its function; it can increase the CD4/CD8 ratio and protect against CD4 depletion; and it significantly enhances the production of virus-induced interferon. Moreover, Glycyrrhiza not only mediates interferon production and increases the activity of natural killer cells, but this also inhibits HIV proliferation and activates the immune system. Lastly, both Hedyotis diffusa and Forsythia act to inhibit HIV activity.Citation13
In the present study, the findings suggest that the CD3+, CD4+, and CD4+/CD8+ levels were significantly reduced in the model group as compared to the blank control group (P < 0.01). These results indicated the successful establishment of the CY-induced immunosuppressed model. Furthermore, in comparison to the model group, the number of CD3+ and CD4+ cells and the CD4+/CD8+ ratio were obviously increased in the Lentinan group as well as in the high, middle, and low dose groups (P < 0.01). As compared to the model group, the Lentinan group and the Fuzheng Paidu high, middle, and low dose groups all had dramatically increased CD3+, CD4+, and CD8+ subsets in the intestinal mucosal immune system in the immunosuppressed mice (P < 0.01);And exhibited a smaller reduction in the splenic lymphoid nodules and lymphocytes, a thicker thymus cortex, and more intensive lymphocytes (P < 0.01) and the response of inflammation in the small intestine. The results showed that Fuzheng Paidu was significantly enhanced the immune function of CY-induced immunosuppressed mice. This study provides an experimental basis for the clinical application of the Fuzheng Paidu Tablet.
Methods and Materials
Methods
Effects on peripheral blood T lymphocytes and intestinal mucosa T lymphocytes in cyclophosphamide-induced immunosuppressed mice.
First, 30 female and 30 male mice (18 ∼ 21 g) were randomly divided into 6 groups. Five of the groups were used to construct the CY-induced immunosuppressed mouse models, and 80 mg/kg of cyclophosphamide solution (4 mg/ml; 0.2 ml/10 g) was injected into these mice once-a-day for 3 days. On the first day of modeling, the 5 experimental groups received high, middle, and low doses of the Fuzheng Paidu Tablet suspension (0.14 g/ml, 0.07 g/ml, and 0.035 g/ml; 0.2 ml/10 g), the Lentinan Tablet suspension (0.01 g/ml; 0.2 ml/10 g), and the same volume of normal saline (0.2 ml/10 g), respectively. The remaining group was used as a blank control and received the same volume of saline (0.2 ml/10 g). The drugs were administrated once-a-day for 7 continuous days. Then, 1 h after the last administration on Day 7, their eyeballs were extracted, and their blood was drawn, while EDTA was applied as an anticoagulant afterwards. The prepared whole blood was used to detect the peripheral blood CD3+, CD4+, and CD8+ levels using flow cytometry. The control and experimental tubes were labeled, and 2 ul of anti-CD3-PC5 monoclonal antibody, 1 μl of anti-CD4-FITC monoclonal antibody, and 1 μl of anti-CD25-PE monoclonal antibody were added to the experimental tubes, whereas 2 μl of anti-IgG-FITC/IgG-PE/ IgG-PC5 antibody was added to the corresponding control tubes. In addition, 2 ul of anti-CD3-PC5 monoclonal antibody and 1 μl of anti-CD8-FITC monoclonal antibody were added to the other experimental tubes, and 2 μl of anti-IgG-FITC/IgG-PE/ IgG-PC5 antibody was added to the corresponding control tubes. Afterwards, 40 μl of whole blood samples were added to each tube. The tubes were then well-mixed and placed into the dark for 20 min. Next, 200 μl of opticyle C hemolysin was added to each tube, and the tubes were then placed in the dark for 10 min. Next, 500 μl of PBS was added to each tube, and the tubes were again placed in the dark for 10 min. The results are shown in .
After the blood had been drawn, the mice were sacrificed by cervical dislocation, and the thymuses, spleen, and small intestine were taken and fixed in 10% formalin. These results are shown in and .
The small intestine was carefully removed, fixed in 10% formalin, and used to make paraffin-embedded sections.
Materials
Drugs
The Fuzheng Paidu Tablet was provided by the Henan Onitl Pharmaceutical Company and contains ginseng, Astragalus, Atractylodes, Radix Saposhnikoviae, Ligustrum lucidum, Cornus officinalis, Radix Adenophorae, lithospermum, weeping forsythia, Hedyotis diffusa willd, glycyrrhiza, and so on. The main components of the Lentinan Tablet, purchased from Zhejiang Apeloa Bio-Pharmaceutical Co., Ltd., are mushroom polysaccharides. Finally, the CY injection solution was obtained from Jiangsu Hengrui Medicine Co. Ltd.
Instrument
EDTA anticoagulant tubes and a JY601 electronic balance were both manufactured by Shanghai Minqiao Medical Instrument Co. Ltd. A TDL-40B centrifuge was obtained from the Shanghai Anting Scientific Instrument Factory, and an FA(N)/JA(N) electronic balance was purchased from Shanghai Minqiao Precision Instrument Co., Ltd. Furthermore, an adjustable liquid shifter was purchased from Shanghai Leibo Analysis Instrument Co. Ltd., and a Flow cytometry (EPICS-XL) was obtained from Beckman Coulter, USA. In addition, a Zeiss-HM440E tissue slicer was purchased from Zeiss, German, and a ZMN-20802 autostainer was obtained from the Changzhou Huali Company. Finally, a KD-TS3 automatic tissue hydroextractor was purchased from the Zhejiang Kedi Company, and an OLYMPUS BX-60 biological microscope was purchased from Olympus, Japan.
Reagents
Physiological saline (Lot# 070620021) was purchased from Zhengzhou Chemical Pharmaceutical Factory, and formaldehyde (Lot# 031208) was obtained from the Shanghai Purchase and Supply Station. Monoclonal anti-CD3+, CD4+, and CD8 antibodies were produced by eBioscience Co., USA. Anti-IgG-FITC/IgG-PE antibodies and opticyle C hemolysin were produced by Beckman Coulter, USA and were provided by Zhengzhou Biocell Biotechnology Co., Ltd.
The reagents used for the immunohitochemical SP method included anti-CD3 (RMA-0543), CD4(MAB-0251), and CD8(RMA-0514) antibodies (Fuzhou Maixin Biotechnology Co., Ltd). All of these antibodies were suitable for use when purchased. Goat-anti-rabbit and rabbit-anti-goat secondary antibodies were purchased from Pierce, USA. The three-antibody system was produced by Beijing Zhongshan Biotechnology Co., Ltd, including biotin-labeled goat-anti-rabbit antibodies, rabbit-anti-goat antibodies, and HRP avidin.
Animals
Kunming mice were purchased from the Wuhan Institute of Biological Products Co., Ltd. (Animal quality certificate No: 0036893) and then were housed in a barrier facility. These mice were 18 ∼ 21 g in weight and were 6 weeks-old. Additionally, there were equal numbers of male and females. The laboratory temperature was 20 ∼ 25°C, and the humidity was 50% ∼ 60%. The animals had free access to food and water.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Funding
Deep processing of genuine medicinal materials (GMM), the Engineering Laboratory of Henan (2012-1431-10); Henan Science and Technology Innovation Team (2012IRTSTHN011); Zhengzhou Science and Technology Innovation Team(131PCXTD612); The Cooperation Center of TCM development and GMM deep processing at Henan University of Traditional Chinese Medicine [2012]188-2.
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