| Hyperglycemia is a common complication of renal transplantation. On the one hand, we use insulin to keep the normal glycemia; on the other hand it is possible to reduce the incidence of posttransplantation diabetes mellitus by using basal insulin, so whether it is from a preventive point of view or from a therapeutic point of view that insulin is recommended for transplant patients. As we all know that it is very important for the patient that the clinical efficacy and the sensitivity to the immunosuppressive drugs are closely related. But Professor Hirano sensei’s team coming from Tokyo University of Pharmacy and Life Sciences found that the use of exogenous insulin can reduce the sensitivity to the immunosuppressive drugsafter renal transplantation, while the specific mechanism is still unclear. We hypothesized that insulin may increase the function of immune cells, enhance the immune response, resulting in reduced sensitivity to immunosuppressive drugs, and ultimately increase the dosage of immunosuppressive drugs. So we constructed a system to verify the suppose by following experiments:We first investigated the effects of insulin on responses of T-cell mitogen-activated human peripheral-blood mononuclear cells (PBMC) to several immunosuppressive drugs in our present study. For example, antiproliferative efficacies of prednisolone(PSL), hydrocortisone(HC), cyclosporine(CyA), tacrolimus(FK506), and mycophenolic acid(MPA)against concanavalin A-stimulated PBMC were evaluated in the presence of physiological (5μunits/mL) and super physiological (50μunits/mL) concentrations of insulin.What we have found in the experiment is that insulinhad no effects on the mitogen-induced proliferation of PBMC. The IC50 values of prednisolone, tacrolimus, and mycophenolic acid against the mitogen-activated PBMC in the presence of 5 or 50μunits/mL insulin were significantly higher than those of these drugs without insulin (p<0.05-0.001).The IC50 values of hydrocortisone and cyclosporine significantly increased by 50μunits/mL insulin(p<0.05),while the insulin effects on the pharmacodynamics of these two drugs were not significant at 5μunits/mL. In a word, insulin at physiological or superphysiological concentrations attenuates suppressive efficacies of prednisolone, hydrocortisone, cyclosporine, tacrolimus and mycophenolic acid against T-cell mitogen-activated PBMC of healthy subjects. And it indicates that insulin has antagonistic effect on immunosuppressive agents or can improve immune system function.Supposing that insulin can increase the cellular immune function, then, insulin has directly effect on the immune cells toactivate the immune response or there hasother mechanisms related to immune response? Anyway, there is a close relation between insulin effect and theinsulin receptor expression on cell-surface.So we detect the insulin receptor in PBMC by flow cytometry. Insulin receptor expression was detected on CD4 and CD 14 positive monocytes in PBMC in the present study. The major of PBMC is lymphocyte, and there is a little monocyte, and we stimulated PBMC with a T cell mitogen named concanavalin A. Concanavalin A stimulates all of T cells and monocytes in PBMC. In the present study, we could not detect insulin receptor expression on CD4+T cells or CD8+T cells. On the other hand, we detected the insulin receptor expression on CD4+(monocytes) and CD14+cells (monocytes) after stimulation by concanavalin A. These observations suggest that insulin binds to the insulin receptors on monocytes, which subsequently stimulate monocytes to secrete monocyte derived-cytokines and modulate T cell immunity. Thus, our data suggest that the attenuating effects of insulin on the immunosuppressive drug pharmacodynamics are mediated by monocytes through insulin receptors on these cells. In addition, the effect of insulin attenuating cyclosporine immunosuppressive efficacy against the mitogen-activated proliferation of PBMC was recovered byinsulin receptor antagonist S961 (at 100nM).These observations also support that attenuation of immunosuppressive efficacy of cyclosporine by insulinis mediated through the insulin receptors on monocytes.There are three kinds of rejection after organ transplantationincluding hyperacute rejection, acute rejection and chronic rejection.No matter which type of rejection, the adhesion of immune cells and endothelial cells has an important role in the whole process. Suppressing theadhesion of immune cells and endothelial cell is the central to anti-rejection reactions. So if insulin can boost the adhsion of immune cells and endothelial cell or not? To this end, we use HUVEC (human umbilical vein endothelial cells) to observe insulin-induced adhesion of PBMC and HUVEC process. The results from fluorescence microscopeshow thatinsulin can effectively induce fluorescence labeled probe PBMC and HUVEC adhesion, and has a dose-dependent manner; at the same time, sICAM-1 (intercellular adhesion molecule 1) and E-selectin (E-selectin), these two important adhesion chemokine, upregulated (P<0.05), suggesting that insulin mayenhance the immune response by increasing the adhesion between PBMC and HUVEC.Tanshinone IIA is an active ingredient coming from Chinese Herbs Salvia which has anti-inflammatory, anti-oxidation, anti-tumor, regulate blood sugar, and its mechanism most related to improvethe endothelial functioneffectively. However, there is a lack of the study about how Tanshinone IIA regulate the immune system function. In this study, we choose Tan IIA as the main object, observe the adhsion between PBMC and HUVEC at the same time when we study the insulin. The results show that Tanshinone IIA at 1μM and 10μM, can significantly promote adhesion between the two types of cells (P<0.05, P<0.01), and sICAM-1 and E-selectinupregulated (P<0.05); this indicate that Tanshinone IIA is similar to insulin on the effects of enhancing the immune response.When we use insulin to prevent and treat the complications after organ transplantation, it candecrease the sensitivity of immunosuppressive agents which related to improve the immune response; can give a suggestion for the treatment in the clinical who use immunosuppressive agents as well as insulin after transplantation.The use of immunosuppressive agentsanti-rejection after transplantation can also suppress the immune function, resulting in manyclinical bottleneck, such asposttranplantation diabetes mellitus and secondary infections.Our study shows that insulin at even physiological concentrationattenuatessuppressive efficacies of severalimmunosuppressive drugs against mitogen-activated proliferation of human PBMC, maybe partially via insulin receptors on monocytes but not on lymphocyte.And it is one of the possible mechanisms that immunopotentiation of insulinis by increasing the adhesion of PBMC-HUVEC.Tanshinone IIA has a similar way to regulate the immune system as insulinby increasing the PBMC-HUVEC.However, the pros and cons of insulin in organ transplantation application and tanshinone IIA clinical value in organ transplantation needs to be further evaluated.The study include three parts as following:Study I:Insulin effects on responses of mitogen-activated human peripheral-blood mononuclear cells (PBMC) to several immunosuppressive drugs, such as prednisolone, hydrocortisone, cyclosporine, tacrolimus and mycophenolic acid.Objective:To investigate the effects of insulin on responses of mitogen-activated human peripheral-blood mononuclear cells (PBMC) to several immunosuppressive drugs, such as prednisolone, hydrocortisone, cyclosporine, tacrolimus and mycophenolic acid.Methods:Antiproliferative efficacies of prednisolone, hydrocortisone, cyclosporine, tacrolimus, and mycophenolic acid against concanavalin A-stimulated PBMC were evaluated in the presence of physiological (5μunits/mL) and super physiological (50μunits/mL) concentrations of insulin.Results:1. Physiological and super physiological concentrations of insulinhad no effects on the mitogen-induced proliferation of PBMC.2. The IC50 values of prednisolone, tacrolimus, and mycophenolic acid against the mitogen-activated PBMC in the presence of 5 or 50μunits/mL insulin were significantly higher than those of these drugs without insulin (p<0.05-0.001).3.The IC50 values of hydrocortisone and cyclosporine significantly increased by 50μunits/mL insulin(p<0.05),while the insulin effects on the pharmacodynamics of these drugs were not significantly changed by 5μunits/mL insulin.Conclusions:These results indicate that insulin at even physiological concentrationattenuates suppressive efficacies of severalimmunosuppressive drugs against mitogen-activated proliferation of human PBMC.Study II:Study the mechanism of insulin decreasing the responses of mitogen-activated human peripheral-blood mononuclear cells (PBMC) to several immunosuppressive drugs.Objective:To study the mechanism of insulin decreasing the responses of mitogen-activated human peripheral-blood mononuclear cells (PBMC) to several immunosuppressive drugs.Methods:The surface expression of insulin receptor on PBMC was detected with a PE-conjugated mouse anti-human CD220 antibody, PE conjugated anti-mouse IgG1κ antibody was used as an isotype control by flow cytometry. And using MTT method to detect insulin receptor antagonist S961 effect on the decreased immunosuppressive efficacy of cyclosporine by insulin.Results:1. Insulin receptors were detected on the mitogen-activated CD4+/CD14+ monocytes in PBMC.2.Insulin receptor antagonist had no effects on the mitogen-induced proliferation of PBMC.3. Insulin receptor antagonist S961 can partially recover the decreased immunosuppressive-efficacy of cyclosporine by insulin.Conclusions:These results indicate that insulin at even physiological concentrationattenuates suppressive efficacies of severalimmunosuppressive drugs against mitogen-activated proliferation of human PBMC, maybe partially via insulin receptors on monocytes but not on lymphocyte.Study III:Effects of insulin and Tanshinone IIAon PBMC-HUVEC adhesion.Objective:To investigate the effects of insulin and Tanshinone on PBMC-HUVEC adhesion.Methods:Primary human umbilical vein endothelial cells (HUVEC) were isolated from human umbilical veins with enzyme digestion. PBMC were isolated from human peripheral vein blood. HUVEC were divided into blank control group,5μunits/mL insulin group,50μunits/mL insulin group, 100μunits/mL insulin group group. After treated for 24h, PBMC which marked by BCECF-AM were added and then the number of adherent cell was observed by fluorescence microscope. Tanshinone ⅡA use the same method as Insulin. And we also detect the contentof sICAM-1 and E-selectin.Results:Compared with blank control group, the number of adherent cell was increased by insulin, and there were significantly higher than those control group(p<0.05) when the finnal concentration of insulin is 50μunits/mL and 100μunits/mL.Also the number of adherent cell was increased by Tanshinone ⅡA, and there were significantly higher than those control group(p<0.05-0.001) when the finnal concentration of Tanshinone ⅡA is 1μM and 10μM.And insulin can increase the content of sICAM-1 and E-selectin. Tanshinone ⅡA can also increasethe content of sICAM-1 and E-selectin.Conclusions:It is one of the possible mechanisms that immunopotentiation of insulinis by increasing the adhesion of PBMC-HUVEC. AndTanshinone ⅡA has a similar way to regulate the immune system as insulinby increasing the PBMC-HUVEC. |
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