The Immunosuppressive And Protective Ability Of Glucose Regulated Proteins 78 For Improvement Of Type 1 Diabetes In Streptozotocin-induced Diabetic Mice

Objective: Endoplasmic reticulum(ER) stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cells, and contributes to the development of type 1 diabetes. The chaperone molecule, glucose regulated proteins 78(GRP78), is required to maintain ER function during toxic insults. In this study, we investigated the changes of GRP78 expression in different phase of streptozotocin(STZ)-affected beta-cells, to explore the relationship between GRP78 and the response of beta-cell to ER stress; we investigated the effect of GRP78 on the beta-cell apoptosis; and we investigated the effect of GRP78 on the development of STZ-induced type I diabetes mice.Methods:①The expression of GRP78 in different phase of STZ-affected beta-cell A insulinoma cell line(NIT-1) treated with STZ for different time course, and STZ-induced diabetic Balb/C mices at different time points were used as the model system. The level of GRP78 and C/EBP homologous protein (CHOP) mRNA were detected by real-time PCR and their protein by immunoblot, apoptosis and necrosis were measured by flow cytometry, and morphological changes of apoptotic cells by fluorescence microscope. In addition, the changes of GRP78 protein in STZ-treated non-diabetic mices was also detected by immunoblot;②The effect of GRP78 on the beta-cell apoptosis induced by STZ, cytokines and CTLNIT-1 cells transfected with GRP78 was established, named NIT-GRP78, and used to study apoptosis, which was induced by streptozotocin, inflammatory cytokines or cytotoxic T lymphocyte(CTL). Apoptosis of NIT-1 or NIT-GRP78 cells was detected by flow cytometry, by monitoring the transcription of CHOP with real-time PCR, the concentration of nitric oxide and the activity of superoxide dismutase with colorimetric method; ③The effect of GRP78 on the development of STZ-induced type I diabetes mice NIT-1 cells transfected with GRP78 were used to study the immunosuppressive and protective ability of GRP78, which was evaluated by extended CTL killing assay measured with flow cytometry through target cells dyed with CFSE cultured with effector cells and finally stained with PI, by measurement of concentrations of IL-4 and IFN-γdetected with ELISA, by transplantation of these cells into STZ-induced diabetic Balb/c mices.Results:①The expression of GRP78 in different phase of STZ-affected beta-cell GRP78 expression significantly increased in early phase but decreased in later phase of affected beta-cell, while along with the decrease of GRP78, CHOP was induced and apoptosis occured. Interestingly, the GRP78 protein of STZ-treated non-diabetic mices increased stably compared with control;②The effect of GRP78 on the development of STZ-induced type I diabetes mice In comparison to NIT-1 cells, NIT-GRP78 cells responded to the streptozotocin or cytokines treatments with decreased concentration of nitric oxide, but increased activity of superoxide dismutase. In addition, the level of CHOP was also decreased in the NIT-GRP78 cells, which may mediate the resistance of the GRP78 overexpressed NIT-1 cells from apoptosis. NIT-GRP78 cells were also more resistant than NIT-1 cells to CTL specific killing;③The effect of GRP78 on the beta-cell apoptosis induced by STZ, cytokines and CTLCultured with NIT-GFP-primed lymphocytes, NIT-GRP78 cells had moderate necrosis("+ +", P<0.05) compared with the severe necrosis of NIT-GFP cells("+ + +"). While cultured with NIT-GRP78-primed and expanded lymphocytes, NIT-GRP78 cells had mild necrosis("+", P<0.05), NIT-GFP cells had moderate necrosis("+ +", P<0.05). A increase of IL-4(P<0.01) secretion from beta-cell-primed splenocytes when GRP78 presences was observed. Diabetic mice reached normoglycemia promptly and gained weight after transplantation of NIT-GRP78. More importantly, the survival time for recipients transplanted with NIT-GRP78 cells was significantly longer than that with NIT-GFP cells(P < 0.01). In addition, we observed a significant increase of insulin concentration after glucose stimulation for diabetic mice received NIT-GRP78 cells at day 7 of post-transplantation.Conclusions: From the results we can conclude that there is a close relationship between GRP78 and the response of beta-cells to ER stress; Modulating GRP78 expression could be useful in preventing pancreatic beta-cell from the immunological destruction in type 1 diabetes individuals; GRP78 could have a dual functions of protecting NIT-1 cells from CTL mediated lysis and of stimulating a population of T cells with cytokine profile of IL-4 which may be part of a mechanism to down-regulate an immune response, should be paid more attention to improvement of immunorejection in beta-cell transplantation.