Experimental Study Of The Immunomodulatory Effects Of Tupistra Chinensis Baker Polysaccharides

Polysaccharides, the macromolecules widely existing in variable organisms ranging from mammals, higher plants, bacteria, viruses, fungi and many other lower organisms, exhibit unusual biological properties. While many kinds of polysaccharides have been isolated and purified from all kinds of organisms, it has been thought that polysaccharides might be good material for the development of new therapeutic agents. The polysaccharides can be used as vaccines against infection of pathogenic bacteria and are also useful against diseases caused by aberrant and abnormal cell-cell interaction, such as cancer metastasis and inflammation. Polysaccharides immunomodulators were first discovered 60 years ago. Although very few has been rigorously studied, certain polysaccharides immunomodulators have been identified that have profound effects in the regulation of immune responses during the progression of infections diseases, and studies have begun to define structural aspects of these molecules that govern their function and interaction with cells of the host immune system.Tupistra chinensis Baker, also called niuweiqi, zhugenqi and so on in Chinese, is endemic in south-western regions of the People's Republic of China. As a Chinese folk medicine, this species has usually been used for treatment of rheumatic diseases, snake-bite and so on. Many steroidal saponins were isolated from rhizomes of Tupistra chinensis Baker, which was the main active component. It was found that Tupistra chinensis Baker had good anti-inflammatory activity and favorable exploitation value. And saponin is a very important substance for treatment of inflammation. The mechanism involves decreasing the quantity of mediators of inflammation like prostaglandin E2 and nitrogen monoxidum. Saponins of Tupistra chinensis Baker had significant cytotoxicity on HL-60, Caski and U251 tumor cells, strong inhibitory effect on S180 sarcoma cells in vivo and in vitro, and inhibitory effect on the platelets'aggregation and activation induced by TNBS. Also, it was reported that water extraction of Tupistra chinensis Baker could inhibit the early inflammatory exudation and edema, and had anti-inflammatory and analgesic effect. In the screening for immune active substances, we found that Tupistra chinensis Baker polysaccharides had immunological activity, which attracted our interest to study further. This paper reports the immunomodulatory effects of Tupistra chinensis Baker polysaccharidesⅡ.Objectives:1. To isolate the crude polysaccharides of Tupistra chinensis Baker and identify the active fraction.2. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the production of tumor necrosis factor a (TNF-α), interferon y (INF-y) and interleukin 2 (IL-2) by murine spleen cells, and the effect on spleen cells metabolic activity with methylthiazolyl tetrazolium(MTT) colorimetry assay. To investigate the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the production of tumor necrosis factor a (TNF-a) by murine peritoneal exudate cells and the effect on peritoneal exudate cells metabolic activity with methylthiazolyl tetrazolium (MTT) colorimetry assay. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the swelling degree in DTH and the level of serum hemolysin induced by RRBC immunization in normal mice in vivo.3. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the swelling degree in DTH, the phagocytic function of macrophages and the level of serum hemolysin induced by RRBC immunization in immunosuppressed mice induced by CTX in vivo.4. To study the effect of Tupistra chinensis Baker polysaccharides (TCBPⅡ) on the serum amount of tumor necrosis factorα(TNF-α) and interferonγ(INF-γ), the tumor growth inhibition rate, and the phagocytic function of macrophages in S180 tumor-bearing mice.Methods:1. Hot water extraction and ethanol precipitation were used to isolate the crude polysaccharides from the rhizomes of Tupistra chinensis, then Sevage method was adopted for deproteination. After that, further separation and purification was carried out by anion-exchange chromatography with DEAE-Sepharose fast flow.2. Splenocytes from normal mice were incubated at 37℃in the presence of Tupistra chinensis Baker polysaccharides (TCBPⅡ) for 48h. ELISA was employed to determine the amount of TNF-α, IFN-γ, and IL-2 in the culture supernatants. And MTT colorimetry was used for the assay of cell metabolic activity.3. Peritoneal exudate cells from normal mice were incubated at 37℃in the presence of Tupistra chinensis Baker polysaccharides (TCBPⅡ) for 10h. ELISA was employed to determine the amount of TNF-αin the culture supernatants. And MTT colorimetry was used for the assay of cell metabolic activity.4.50 normal mice were randomly divided into 5 groups, including normal saline group, positive control group and TCBPⅡ(50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. Mouse model for DTH was established by priming the mice with 1% dinitrofluorobenzene(DNFB) applied to the shaved abdomen skin and attacking on the right ear 7 days later. Twenty four hours after attack, the weight of double ears was weighed to evaluate the influence of TCBPⅡon the swelling of the ear tissues.5.50 normal mice were randomly divided into 5 groups, including normal saline group, positive control group and TCBPⅡ(50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. The next day after treatment, each mouse was then administered intraperitoneally with 20% RRBCs in a total volume of 0.2 ml.The sixth day after the immunization, the serum samples were collected for haemolysin assay.6. The immunosuppressed mice were induced by cyclophosphamide (CTX). Cellular immunity was assessed with the mouse delayed type hypersensitivity (DTH) model induced by DNFB, Phagocytosis of mononuclear macrophage was determined by the method of carbon particle clearance test, and humoral immunity was evaluated with the production of specific antibody (haemolysin) against rabbit red blood cells.7. Mouse models bearing S180 solid tumor were established. The control group was then administered intraperitoneally with sterile physiological saline solution at 0.1ml/10g body weight. The administered volume of other groups was the same as that of control. The positive control group was administered with Ganoderma lucidum polysaccharides(GLP), at 100 mg/kg intraperitoneally and the low, median and high dose groups of Tupistra chinensis Baker polysaccharides (TCBPⅡ)were administered at 50,100, and 200mg/kg intraperitoneally. After 7 days, the serum samples were collected. Then TNF-αand IFN-γlevels in the samples were measured by using enzyme-linked immunosorbent assay (ELISA). Then the implanted tumors and spleens were removed and weighed, and the tumor growth inhibition rate.8.50 normal mice were randomly divided into 5 groups, including normal saline group, Ganoderma lucidum polysaccharides(GLP100mg/kg) and TCBPⅡ (50,100, and 200mg/kg) groups, and the treatment was given once daily for 7 consecutive days. Phagocytosis of macrophages in S180 tumor-bearing mice was evaluated with percentage of phagocytized chicken red blood cells(CRBC).Results:1. With DEAE-Sepharose fast flow method, three fractions were obtained, designated TCBPⅠ,TCBPⅡ,and TCBPⅢ. The highest content of the fractions was observed for TCBPⅠ, followed by TCBPⅡ, and finally TCBPⅢ. TCBPⅠ(50,100,200μg/ml) had no effect on splenocyte metabolic activity(P=0.597, P=0.398,P=0.073). TCBPⅢat concentrations of 50 and 100μg/ml did not enhance the splenocyte metabolic activity (P=0.497,P=0.398), however, significantly enhancing activity was found at the concentration of 200μg/ml (P=0.034). TCBPⅡwith the highest activity was subjet to further study.2. Compared with control group, TCBPⅡ(50,100,200μg/ml) significantly enhanced the splenocyte metabolic activity (P=0.048,P=0.001,P=0.000) and the production of TNF-αcytokine (P=0.016,P=0.000,P=0.000) in a dose-dependent manner in a certain concentration range, but it could not enhance IFN-γand IL-2 in normal mice in vitro. The TCBPⅡ(50,100,200μg/ml) markedly promoted the metabolic activity of mouse peritoneal exudate cells (P=0.004,P=0.000, P=0.000) and production of TNF-αby the cells in normal mice in vitro (P=0.044,P=0.001,P=0.000). TCBPⅡ(50,100,200 mg/kg) could not affect the swelling degree in DTH and the level of serum hemolysin induced by HRBC immunization in normal mice.3. TCBPⅡ(100,200 mg/kg) remarkably increased the swelling degree in DTH in immunosuppressed mice induced by CTX in vivo (P=0.014,P=0.006) and enhanced celluar immunity function. TCBPⅡ(100,200 mg/kg) remarkably promoted the serum clearance of carbon particles in immunosuppressed mice induced by CTX in vivo (P=0.017,P=0.012), indicating an increase in phagocytic function of macrophages. TCBPⅡ(50,100,200 mg/kg) increased the level of serum hemolysin induced by HRBC immunization in immunosuppressed mice (P=0.011,P=0.001, P=0.000), indicating an enhance in humoral immunity.4. TCBPⅡ(50,100,200 mg/kg) could inhibit the growth of S180 solid tumor (P =0.000,P=0.000,P=0.000), with the inhibition rate of 26.7%,32.5%, and 40.7%. And TCBPⅡ(100,200 mg/kg) increased the serum levels of TNF-αin S180 solid tumor mice (P=0.039,P=0.001). The effects were dose-dependent in a certain concentration range. TCBPⅡ(100,200 mg/kg) remarkably potentiated the rate and index of phagocytosis of CRBC by peritoneal macrophages in the tumor-bearing mice in vivo respectively (P=0.005, P=0.000), (P=0.011, P=0.003)Conclusion:1. The fraction TCBPⅡ(Tupistra chinensis Baker polysaccharidesⅡ) is the highest in activity and with considerable content.2. TCBPⅡcan significantly enhance immunocyte metabolic activity and promote secretion of TNF-αby immunocytes in normal mice in vitro, but can not affect IFN-γand IL-2. TCBPⅡcan not enhance the swelling degree in DTH and the level of serum hemolysin in normal mice.3. The experimental model of immunosuppressed mice induced by cyclophosphamide (CTX) was established successfully.4. TCBPⅡhas the capacity of enhancement of immune function in immunosuppressed mice induced by CTX in vivo. TCBPⅡcan markedly potentiate DTH, elevate the corrected phagocytic index (α), promote the phagocytic function of macrophages, and increase the level of serum hemolysin in immunosuppressed mice. It is suggested that TCBPⅡis able to enhance the function of cellular and humoral immunity in immunosuppressed mice induced by CTX in vivo. 5. The experimental model of S180 solid tumor-bearing mice was established successfully.6. TCBPⅡcan inhibit the growth of S180 solid tumor, increase the serum level of TNF-a, and potentiate the rate and index of phagocytosis of CRBC by peritoneal macrophages in the tumor-bearing mice in vivo.7. The preliminary experimental results demonstrate that TCBPⅡis a sort of effective immunomodulator, and the underling mechanism may be related with its potentiation of the macrophages phagocytosis and the production of TNF-a in mice.