The Function And Mechanism Of Sp And Cmp On Inhibition Of PCV2Infection

Pachymaran is extracted from Poria cocos (Schw.) Wolf and has strong antitumor and immunomodulatory activities and cell-mediated immune activities.Pachymaran is not easily applied in the clinic because of its low water-solubility. Sulfation pachymaran (SP) is obtained from artificially sulfated modification and Carboxymethylpachymaran (CMP) is obtained from artificially carboxymethylation.The two polysaccharides are water-soluble and exhibit good immune regulation and antitumor activities. Porcine circovirus type2(PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), a worldwide disease that debilitates nursery and fattening pigs with systemic lymphadenopathy and interstitial pneumonia.Microscopic lesions in the lymphoid tissues of pigs severely affected with PMWS exhibits lymphocyte depletion with loss of lymphoid follicles (mainly in the B cell-dependent areas) and reduced cellularity in the parafollicular T cell-dependent parts.So swine infected with PCV2always exhibit immunosuppression in a long-term.The objective of this study is to investigate the function of SP and CMP on porcine immunocytes inoculated with PCV2in vitro and immunological regulation of CMP in mice after infected with PCV2in vivo.1Effects of SP or CMP on proliferation of splenic lymphocytes and cytokine after inoculated with PCV2In order to investigate the immunomodulatory mechanism of SP or CMP on the porcine splenic lymphocytes after the cells were inoculated with PCV2.The splenic lymphocytes from pigs which were negative for both antigens and antibodies of PRRSV or PCV2were treated with SP or CMP after inoculation with PCV2for2h.Lymphocyte proliferation was determined using the MTT method. The levels of IL-1β, IL-2, IL-8, IL-10and TNF-a were detected by ELISA methods.The results showed that the two polysaccharides were able to promote the proliferation activities of splenic lymphocytes with or without inoculation of PCV2.The SP at concentrations from50μg/mL to400μg/mL or CMP at concentrations from200μg/mL to400μg/mL was able to stimulate proliferation of splenic B lymphocytes in vitro.The level of IL-1β in splenic lymphocytes inoculated with PCV2decreased after splenic lymphocytes were treated with SP at different concentrations from50μg/mL to200μg/mL.The level of IL-2in splenic lymphocytes inoculated with PCV2increased after splenic lymphocytes were treated with SP or CMP.The level of IL-10in splenic lymphocytes inoculated with PCV2decreased when splenic lymphocytes were treated CMP at with100μg/mL,while the level of TNF-α increased.These results showed that SP and CMP were able to stimulate proliferation of swine splenic T and B lymphocytes with or without inoculation of PCV2in vitro.2Effects of SP or CMP on activities and phagocytosis immune function of porcine alveolar macrophage after inoculated with PCV2The effects of SP or CMP on immunological function of swine alveolar macrophage inoculated with porcine circovirus type2(PCV2) were observed.The swine alveolar macrophages were separeated from pigs which were negative for both antigens and antibodies of PRRSV or PCV2and treated with SP or CMP after inoculation of PCV2.The phagocytosis of swine alveolar macrophage was observed by neutral red method,the nitric oxide (NO) level was determined by Griess method and the cytokine levels of IL-1β、IL-8、 TNF-a and MCP-1were measured by ELISA method.The results showed that SP and CMP could stimulate proliferation of swine alveolar macrophages.The content of NO decreased after swine alveolar macrophages were treated with PCV2and SP for4h,8h and24h, while the levels of NO decreased after swine alveolar macrophages were treated with PCV2and SP for4h and8h.The levels of TNF-α increased while the IL-8decreased after swine alveolar macrophages treated with PCV2and SP or CMP.These results suggests that SP and CMP might play an important role in regulating immunological functions by improving phagocytotic activity and promoting secretion of cytokines of swine alveolar macrophages.3Effect of SP or CMP on oxidative stress in the swine splenic lymphocytes after inocubated with PCV2The effect of SP or CMP on oxidative stress in the swine splenic lymphocytes inocubated withPCV2were observed.The splenic lymphocytes from pigs which were negative for both antigens and antibodies of PRRSV or PCV2were treated with SP or CMP at different concentrations from25μg/mL to200μg/mL post-inoculation of PCV2. The reactive oxygen species (ROS) level,total superoxidase dismutase (T-SOD) activity and total antioxidant capacity (TAOC) were measured. The results showed that SP and CMP were able to decrease the level of ROS in the swine splenic lymphocytes after inoculated with PCV2,and also could increased the activities of T-SOD and TAOC.Thesr results showed that SP and CMP could enhance the anitoxidantive activities of lymphocytes so as to protect the lymphocyte infection.4Effect of CMP on immune function of mice after infected with PCV2To investigate the effect of CMP on the immunological function of mice infected with PCV2,100of Kunming inbred mice were randomly divided into five groups (20mice each).Four groups were inoculated (intranasal and intraperitoneal injection at a dose of0.2mL) four times with PCV2(once daily on Dayl, Day3, Day5and Day7at1000TCID50of PCV2) and labeled the mPCV2group.The mice in the another group were sham-inoculated with a sterile cell culture medium using the same experimental conditions and functioned as the normal control group.Three groups of mPCV2mice were then given intraperitoneal injections of50,100,or200mg/kg body weight CMP once daily on Day8,Day9and Day10post-inoculation (dpi).At the same time,another group of mPCV2mice and the normal control group received the same volume of distilled water under the same conditions.On Day11dpi,the spleen and thymus indices were calculated.Lymphocyte proliferation in vitro was determined using the MTT method.Determination of cytokine levels in murine serum was carried out by using ELISA method.The activities of T-SOD and TAOC and GSH in the spleen and thymus were determined.The results showed that CMP administration significantly improved the spleen or thymus index,promoted the proliferation activities of T or B lymphocytes,and increased the production of glutathione, the superoxidase dismutase capacity and the total antioxidant capacity in the spleen or thymus of PCV2-infected mice.The administration of different CMP doses to PVC2-inoculated mice resulted in the upregulation in the levels of IL-2,IL-6and IFN-a,otherwise the downregulation of IL-10levels in the serum.These findings suggest that CMP has potential application in regulating immune functions to overcome the immunosuppresion caused by PCV2infection in mice.The findings may also prove useful in designing effective therapies against PCV2infection.