Study Of Immunosuppressive Activity And Apoptosis-inducing Effect Of Total Triterpenoids From Poria Cocos On Human Colorectal Carcinoma RKO Cells

Background and objective:As a commonly used traditional Chinese medicine, Poria cocos has a high application value in daily diet and clinical use. Current studies show that the pharmacological effect of Poria cocos is mainly determined by the component of triterpenoids and polysaccharides. In this paper, our research focuses on the pharmacological effects of Total Triterpenoids from Poria cocos （TTP） and discussion of the prospect of its clinical application, hoping to provide some useful data for the development of TTP. The present study includes two parts, the first part determines whether TTP has immunosuppressive activity, and study the mechanism involved in the immunosuppressive effect in mice in vitro and in vivo, and the second part concerns the inhibitory effect of TTP on human colorectal carcinoma RKO cells, the apoptosis-inducing mechanism, and the potential application in cancer chemotherapy.Method:1) Preparation of total triterpenoids from Poria cocos （TTP）:Small cubes of Poria cocos were ground to powder and transferred into a flask with addition of a certain amount of 75% ethanol. Then reflux extraction was performed for 3 times, 2h for each time. After that, the extraction mixture was filtered while it was hot. The supernatant was collected and concentrated by low-temperature vacuum drying method to get the ethanol extract. The ethanol extract was then dispersed with some water and extracted by ethyl acetate for 5 times. The fraction of ethyl acetate was collected and dried by low-temperature vacuum dehydration, which was used as TTP.2) The effect of TTP on the function of mouse splenocytes in vitro:MTT method was used to determine the effect of TTP on the function of mouse splenocytes in vitro, including on proliferation of mouse splenocytes induced by Con A and LPS as well as the lymphocyte transformation induced by MLC （mixed lymphocyte culture response）. These studies were designed to understand the immune modulatory mechanism of the triterpenoids. The effect of TTP on T lymphocyte subsets of mice induced by Con A was assessed by flow cytometry. ELISA method was used to determine the effects of TTP on the production of cytokines （IL-2 and IFN-y） from splenocytes induced by Con A and the production of antibodies induced by LPS.3) The effect of TTP on the immune function in vivo. Spectrophotometric method was used to determine the change of serum hemolysin in mice immunized by rabbit red blood cells and the serum IL-4 was measured by ELISA method. The change of ear swelling degree, spleen index and thymus index in mouse model of delayed-type hypersensitivity （DTH） induced by DNFB was exploited to study the effect of TTP on cellular immune function. Parameters such as arthritis index, paw edema and body mass in adjuvant-induced arthritis rats （AA rats） model were used to evaluate the therapeutic effect of TTP on autoimmune diseases.4) The study of TTP on human colorectal carcinoma RKO cells:RKO cells were plated in 96 well plates and treated with TTP, and then the proliferative activity was determined by MTT method. After that, the RKO cells were seeded in 6 well plates and treated with TTP. Morphologic changes of the cell nuclei were examined after stained with DAPI. Mitochondrial transmembrane potential was evaluated by JC-1 staining. Bax, Bcl-2 and mitochondrial cytochrome C （Cyt C） were detected by western blotting. And the activity of caspase 9 or caspase 3 was determined by enzymatic colorimetric assay.Results:1) An average of 3.23g alcohol extract and 1.27g ethyl acetate extract were obtained by extraction of 100g Poria cocos powder, with average yields of 3.23% and 1.27%, respectively.2) TTP with concentrations above 10μg/mL significantly inhibited the mixed lymphocyte culture response in a concentration-dependent manner. Compared with the model group, the difference was statistically significant.3) Con A significantly promoted the proliferation of mouse spleen cells. TTP at a concentration of 10μg/mL exhibited inhibition on the proliferation of mouse spleen cells induced by Con A, but there was no statistical significance. The rest concentrations of TTP （20,40,and 80μg/mL） significantly inhibited the proliferation of mouse spleen cells induced by Con A in a concentration-dependent manner.4) Flow cytometry was used to study the effect of TTP on mouse T lymphocyte subsets induced by Con A. The results showed that Con A promoted the proliferation of CD₃⁺, CD₃⁺CD₄⁺ and CD₃⁺CD₈⁺ cells. After treatment, it was observed that all concentrations of TTP remarkablly inhibited the proliferation of CD₃⁺ and CD₃⁺ CD₄⁺ cells induced by Con A, but exerted only a little effect on CD₃⁺ CD₈⁺ cells.5) Based on the results of ELISA measurement, Con A significantly promoted mouse spleen cells to secrete IL-2 and IFN-γ. It was observed that TTP at all preseted concentrations （80,40,20, and 10μg/mL） significantly inhibited the secretion of IL-2 and IFN-γ induced by Con A.6) The result of MTT measurement showed that LPS significantly promoted the proliferation or metabolic activity of mouse spleen cells and TTP at all preseted concentrations （80,40,20, and 10μg/mL） significantly inhibited the LPS-induced proliferation in a concentration-dependent manner.7) The result of ELISA measurement showed that TTP at all preseted concentrations （80,40,20, and 10μg/mL） inhibited the secretion of IgG and IgM from mouse spleen cells induced by LPS with statistical significance.8) The data of serum hemolysin and IL-4 demonstrated that TTP in doses studied （400,200, and 100 mg/kg.b.w） decreased the level of serum hemolysin and IL-4. Compared with the vehicle control group, the difference was statistically significant.9) By examining the effect of TTP on mouse delayed-type hypersensitivity （DTH）, It was observed that the high and middle doses of TTP （400 and 200mg/kg.b.w） inhibited the ear swelling and spleen index. Compared with the vehicle control group, the difference was statistically significant（P= 0.001, P= 0.014）. However, the effect of low dose （100mg/kg.b.w） was not significant, and there was no statistical significance （P=0.089）.10) The study of TTP acting on adjuvant-induced arthritis rats （AA rats） showed that the treatment started to work on the 20th day. And TTP in all doses showed improvement on arthritis index. From d20 to d28, the arthritis index was improved progressively. The difference was statistically significant. Based on the result of paw edema, the arthritis presentation of the rats became obviously apparent on the 12th day, and the treatment started to work on the 20th day. Compared with the model group, TTP in the high and middle doses significantly inhibited the paw edema. The difference was statistically significant. However, the effect of low dose of TTP was not significant, and there was no statistical significance. This trend lasted continuously until the 28th day. As for the change of body mass, all treatments did not show any significant effect.11) The MTT result of TTP acting on RKO cancer cells for 24,48, and 72 h showed that except for the concentration of 10μg/mL in which there was no statistically significant effect observed after treatment for 24h and 48h compared with the corresponding vehicle control, the rest concentrations associated with treatment durations significantly inhibited the proliferation of RKO cells （P<0.05）. And the inhibition showed time- and concentration-dependent manner. The IC50 values of treatment for 24,48 and 72h were 97.98,42.99 and 34.14μg/mL, respectively. All the data demonstrate that TTP can inhibit the proliferation of RKO cells.12) After DAPI staining, the cells of vehicle control group were examined under the fluorescence microscope. It was clearly observed that the nuclei were stained evenly, intact and with no fragmentation. Only a very few cells with pyknotic nucleus were also observed. There were a few karyopyknotic cells observed in 20 μg/mL TTP-treated group. Treatment of the cells with 40μg/mL TTP caused more karyopyknotic and karyorrhexic cells. As we know, karyopyknosis and nuclear fragmentation are important characteristics of cell apoptosis. Therefore, the above data suggest that TTP induce apoptosis of RKO cells.13) After JC-1 staining, the cells of vehicle control group were examined under the fluorescence microscope. It was observed that most cells appeared in orange fluorescence, which indicated that the mitochondrial membrane potential was high. After treatment with 20μg/mL TTP, some cells showed in green fluorescence, which suggested that the membrane potential was low. And in the 40μg/mL TTP-treated group, green fluorescent cells were increased. The results suggest that TTP induce RKO cell apoptosis via mitochondrial pathway.14) The result of Western blotting showed that after treatment with TTP （20 and 40 μg/mL） for 48 h, the content of Bax in the cells increased with the rise in TTP concentration （the gray density and area of Western blot increased）. Furthermore, the content of Bcl-2 and mitochondrial Cyt C decreased, （the gray density and area of Western blot decreased）. The results again suggest that TTP induce RKO cell apoptosis via mitochondrial pathway15) Detection of the activity of caspase 3 and caspase 9 demonstrated that after treatment of RKO with TTP （20,40 and 80μg/mL） for 24h, the activity of caspase 9 increased significantly. Compared with the vehicle control group, the activity increased by 63.5%,96.3% and 156.9%. After 48h, the activity of caspase-3 also rose remarkably by 47.6%,78.1% and 112.4%. All the data above further suggest that TTP can induce RKO cell apoptosis via mitochondrial pathway.Conclusion:1) TTP inhibits lymphocyte proliferation in mixed lymphocyte culture response, proliferation of mouse spleen cells induced by Con A, and production of IL-2 and IFN-γ by mouse spleen cells induced by Con A, suggesting it may exert a direct inhibition on proliferation of T lymphocytes and T cell function. The underlying mechanism may involve inhibition of proliferation and diffrenciation of CD4+ T cell subset, as evidenced by the inhibition of cellular immune response model in vivo, i.e. mouse delayed-type hypersensitivity （DTH）.2) TTP inhibits proliferation of mouse spleen cells and production of IgG and IgM by mouse spleen cells induced by LPS, suggesting it may exert a direct inhibition on proliferation of B lymphocytes and diffrenciation of B lymphocytes to antibody-producing cells, as evidenced by the inhibition of humoral immune response model, i.e. induction of serum hemolysin in mice immunized by rabbit red blood cells in vivo. The underlying mechanism may involve inhibition of production of IL-4.3) The concentration of TTP used to inhibit proliferation of mouse spleen cells induced by LPS is a little bit lower than that by Con A, and the minimum effective dose of TTP used to inhibit production of serum hemolysin is a bit lower than that used to inhibit delayed-type hypersensitivity, suggesting that TTP is more effective in suppressing humoral immune response than in suppressing cellular immune response.4) TTP decreases pleen index but has no significant effect on thymus index, indicating TTP suppresses immune response via affectting the lymphocytes in peripheral immune organ （spleen） in mice, but not the central immune organ （thymus）.5) TTP reduces arthritis index and paw swelling in Freund’s adjuvant arthritis model and does not significantly affect the body mass of rats, indicating the potential in the treatment of human rheumatoid arthritis and low toxicity of TTP.6) TTP inhibits proliferation of RKO cells, which may be related to induction of cell apoptosis and mitochondrial apoptotic pathway may be involved in the process.This apoptotic effect of TTP on RKO cells reveals its potential in the treatment of human colorectal carcinoma.