Study On Immunomodulatory Activity And Mechanism Of Periplaneta Americana Glycoprotein On Immunocompromised Mice

Objective:To optimize the extraction process of the glycoprotein from Periplaneta Americana,isolate and purify the glycoprotein,and to screen its immunoactivity in vitro.The immunosuppression mouse model was established to study the activity of the selected active components in vivo.The immunoregulatory activity of Periplanella American glycoprotein and its influence on mRNA expression of various related factors of NF-κB signaling pathway were evaluated,and the mechanism of its immunoregulation was preliminarily discussed.Methods:1.PAG was extracted from Periplaneta Americana by water extraction,alcohol precipitation and Sevage method to take out of Free protein.The extraction process of PAG was optimized by response surface method.2.PAG was isolated and purified by macroporous resin and DEAE-FF,and the effects of each component on the proliferation ability of RAW264.7 were studied by MTT method.3.The contents of polysaccharides and peptides in PAG and PAGW were determined by UV-Vis method,and the composition of monosaccharides was determined by HPLC.The scavenging rates of DPPH free radical,total reduction rate,hydroxyl free radical and ABTS free radical of PAG and PAGW were studied by antioxidant activity in vitro.4.Taking immunocompromised mice as the research object,the contents of RBC,WBC,HGB and PLT in mice were determined by automatic animal blood cell analyzer,and the effects of PAG and PAGW on the blood indexes of immunocompromised mice were studied.MTT assay was used to determine the effects of PAG and PAGW on the proliferation of spleen lymphocytes in mice.The effects of PAG and PAGW on CD₃⁺,CD₄⁺and CD₈⁺in spleen cells were determined by flow cytometry.The contents of Ig G,Ig A,Ig M,IL-1β,IL-6 and TNF-αin serum were determined by ELISA.The mRNA expression of factors related to NF-κB signaling pathway in mouse spleen cells was detected by RT-q PCR.Results:1.The optimal extraction conditions of PAG were as follows:solid-liquid ratio of1∶9,extraction temperature at 90℃,extraction time of 2.25 h,extraction times of 4times.The extraction yield of glycoprotein was 5.10%,and the relative error was 2.62%with the predicted value of the model.2.PAG was eluted by macroporous resin to get PAGW,The extraction yield of PAGW was 2.26%.PAGW was separated and purified to get PAGW-1,PAGW-2,PAGW-3,PAGW-4,PAGW-5 and PAGW-6 components.The concentration of the drug was 800μg/m L,the proliferation ability of PAGW and PAG on RAW264.7 were greater than 100%,and the other components had almost no proliferation ability.3.The polysaccharide content of PAG and PAGW were 20.64%and 21.24%,respectively;The Protein tcontents of PAG and PAGW were 65.99%and 76.35%,respectively.The monosaccharide composition of PAG and PAGW were mannose,glucosamine hydrochloride,rhamnose,glucose,galactose,xylose,arabose and fucose,and the molar ratio was 17.0∶13.2∶9.6∶53.4∶21.8∶1.0∶4.6∶1.3.The molar ratio of PAGW was 21.3∶16.7∶12.3∶73.4∶31.8∶1.0∶5.2∶1.5.The IC₅₀values of PAG and PAGW on DPPH free radical scavenging were 0.028 mg/m L and 0.025mg/m L.When the concentration of PAG and PAGW were 0.16 mg/m L,the absorbances were 0.531 and 0.593,it was proved that PAG and PAGW had a certain reducing ability.The IC₅₀values of PAG and PAGW on hydroxyl radical scavenging were 0.78 mg/m L and 0.748 mg/m L,respectively.The IC₅₀values of ABTS free radical scavenging ability were 0.091 mg/m L and 0.075 mg/m L,respectively.4.Under the administration dose,the indicators change as follows:（1）Orginal index:PAG and PAGW could increase the spleen and thymus index of immunocompromised mice,and the spleen and thymus index of PAG-H,PAGW-L,PAGW-M and PAGW-H groups were significantly increased compared with the model group（P<0.05,P<0.01）.（2）Whole blood RBC,WBC,HGB and PLT:PAG and PAGW could improve the blood indexes of immunocompromised mice.The contents of WBC and RBC in the administration group were increased,with statistical significance（P<0.05,P<0.01）;the contents of HGB and PLT in the PAGW group were significantly increased,with statistical significance（P<0.05）.（3）Proliferation of splenic lymphocytes:PAG and PAGW could promote the proliferation of splenic lymphocytes;Compared with model group,the proliferation ability of B lymphocytes was significantly increased by PAG-M,PAG-H,PAGW-L,PAGW-M and PAGW-H（P<0.05,P<0.01）;Compared with model group,the proliferation ability of T lymphocytes in PAG and PAGW low-medium-high-dose groups was significantly increased（P<0.05,P<0.01）;（4）PAG and PAGW could regulate the number of CD₃⁺,CD₄⁺and CD₈⁺cells,and make the disordered organism recover gradually;Compared with the model group,The number of CD₃⁺cells was increased,the number of CD₄⁺cells was decreased,and the number of CD₈⁺cells was basically unchanged in the administration group.Compared with the model group,CD₄⁺/CD₈⁺was significantly decreased in PAG-H and PAGW groups,with statistical significance（P<0.01）.（5）PAG and PAGW could increase the contents of Ig G,Ig A,Ig M,IL-1β,IL-6 and TNF-αin serum of immunocompromised mice.Compared with model group,the differences among other groups except PAG-L group were statistically significant（P<0.05,P<0.01）.（6）CTX can increase the mRNA expression of TLR4,My D88,TRAF-6,NF-κB p65,p-IκBαand p-NF-κB p65 in the NF-κB pathway,compared with the model group.The mRNA expressions of TLR4,My D88,TRAF-6,NF-κB p65,p-IκBαand p-NF-κB p65 in the administration group were significantly decreased（P<0.01）,indicating that PAG and PAGW can regulate the immune function of the body through the NF-κB signaling pathway.Conclusion:1.The optimal extraction conditions of glycoprotein PAG from Periplaneta Americana were as follows:solid-liquid ratio of 1∶9,extraction temperature at 90℃,extraction time of 2.25 h,extraction times of 4 times.2.Both PAG and PAGW had better immunomodulatory activity in vitro,but the immunomodulatory activity of PAGW was higher than that of PAG.Six components of PAGW were further isolated,and their activities were all lower than those of PAG and PAGW.PAGW had the best immunomodulatory activity in Periplaneta Americana.3.The protein content of PAG and PAGW was higher than that of polysaccharide content.PAG and PAGW had scavenging ability of DPPH free radical,hydroxyl free radical,ABTS free radical and total reducing ability.4.PAG and PAGW can restore the body weight,organ index,blood index,immunoglobulin,serum of cytokines and spleen tissue of immunocompromised mice,increase the splenic lymphocytes and accelerate the proliferative ability.PAG and PAGW can down-regulate the expression of related factors that are highly expressed in CTX-induced on the NF-κB signaling pathway,it suggested that PAG and PAGW have better immunoregulatory activities,The regulatory mechanism may be through NF-κB signaling pathway.The immune activity of PAGW group on mice was better than PAG group.