Study On The Effect Of Cyclosporine A On Prevention Of Injuries To Pancreatic β Cells In STZ-induced Diabetic Rats

Partâ… : The effects of cyclosporine A on dysfunction of pancreaticβcells inSTZ-induced diabetic ratsObjective: To establish the diabetic rats model induced by streptozotocin (STZ),and to investigate whether the STZ-induced dysfunction of pancreaticβcells could berecovered by the combined treatment with cyclosporine A (CsA) and insulin.Furthermore, the side-effects of CsA were also investigated in the present study.Methods: The diabetic rats model was established by intravenous administrationof STZ (45mg/kg). Forty male Sprague-Dawleys (SD) rats were randomly dividedinto five groups: control group (CON), diabetic control group (DM), insulin treatedgroup (INS), CsA treated group (CsA) and the combined- treated group (CpI) withCsA (1mg.kg.d) and insulin (8 U/d) simutaneously. All the rats were followed up toassess the weight and plasma glucose each week. The level of plasma glucose and thefunction of pancreaticβcells were evaluated by OGTT (oral glucose tolerance test)and insulin provocative test. Both hepatic and renal functions were monitored,including the urinary albumin for 24 hours, while calcium and phosphate in the serumwere also investigated. The morphological change in pancreaticβcells was studied byHE and AZON stain of paraffin sections, and the ultrastructures ofβcell wereobserved by transmission electron microscopy. The expression of insulin in islet wasdetected by immunohistochemistry.Results: thirty-two SD rats treated by STZ were all demonstrated the clinicalmanifestations of diabetes mellitus and were diagnosed by twice elevatory plasmaglucose (≥16 mmol/l). Random plasma glucose of the forth week was lowest in group CpI among diabetic rats, while the level of group INS was also lower than that ofgroup DM. The area of islet and the expression of insulin in rats of group CpI wereboth elevated than those of group DM and CsA, and tended to be higher than those ofgroup INS (the value of P was 0.056 and 0.057, respectively).As compared with that of group DM, the plasma glucose in group CpI and INSwas much more lower, while the insulin level in the serum in these two groups wassignificantly elevated in OGTT. The mean level of the AUC of glucose in group DM,INS, CpI and CON was 70.21±514.47, 50.21±8.79, 38.72±3.98 and 22.55±1.25,respectively. The differences between those groups were statistically significant. Thelower level of plasma glucose and the higher level of insulin in the serum were foundin CpI group as compared to that in INS group at the time of thirty and sixty minutesin OGTT. The significant differences in InsE₆₀ either etween group CpI and group DMor between CpI group and INS group were seen. Moreover, it was found that the indexof Homaβin CpI group was much better than that of DM group.By Light microscopy and electron microscopy, it was showed that remarkablemelioration of morphological features and less injury toβcells in CpI group than thatin INS group. The rats in CpI group had lower level of total bilirubin, serum creatinine,blood urea nitrogen and urine albumin than that in DM group.Conclusions: The model of STZ-induced diabetic rat was established. The trialshowed that CsA given 1mg/(kg. d) could protect the function of pancreaticβcells,but this protective effect needed the help of insulin. The protective effect of CsA oninjurues to kidney in STZ-induced diabetic rats seemed not barely lie on the bettercontrol of plasma glucose. The data obtained suggest that CsA could not injure thehepatic and renal function in STZ-induced diabetic rats at the dose of 1mg/(kg. d). Partâ…¡: The effects of cyclosporine A on immune functionin STZ-induced diabetic ratsObjective: The purpose of this part was to explore effects of CsA on immunefunction of pancreas and correlative lymph node in STZ-induced diabetic rats.Methods: The modeling and grouping of the experimental animals weredescribed as above. The expression of CD1αprotein, a specific surface marker ofdendritic cells, in pancreas was detected by western blotting, and the data of it wasadjusted by the expression ofβ-actin protein. Two rats of each diabetic group wereselected randomly to undertake the lymphocyte transformation test, whose pancreaticdraining lymph node (PDLN), mesentery lymph node (SLN) and the left half ofspleen were taken to use. The activation of proliferation of T lymphocytes wasassayed by method of MTT, and the concentration of interleukin-2 in supematant wasmeasured with a commercially available enzyme-linked immunosor-bent assay(ELISA) kit.Results: The mean level of the expression of CD1αprotein in group CON, DM,INS, CsA and CpI was 0.046±0.009, 1.895±0.268, 1.565±0.261, 0.583±0.125 and0.292±0.098, respectively. The differences were statistically significant.The stimulatory index (SI) of T lymphocytes deriving from PDLN and SLN ofDM group and PDLN of INS group elevated remarkably, whereas the index of the restT lymphocytes remained stable.Basing on the results of SI, we just measured the concentration of interleukin-2in supernatant of cultured T lymphocytes deriving from PDLN. The level ofinterleukin-2 in supernatant was highest in group DM, higher in group INS, andlowest in group CsA and CpI. There was no significant difference of the level ofinterleukin-2 between the last two groups.Conclusions: These observations suggested that insulin may have the sameeffect as CsA on preventing the pancreatic islet from infiltration of dendritic cells. But,in all probability, it could do less even nothing on immune function of PDLN. Partâ…¢: The effects of cyclosporine A on apoptosis of pancreaticβcells andexpression of pro-inflammatory cytokines in STZ-induced diabetic ratsObjective: The aim of this part was to investigate the effects of CsA onapoptosis of pancreaticβcells in STZ-induced diabetic rats and to evaluate theinfluence of pro-inflammatory cytokines.Methods: The modeling and grouping of the experimental animals weredescribed as part one. Using the TDT mediated dUTP nick end labeling (TUNEL)technique to identify the apoptotic cells, and the immunohistochemical technique wasused to confirm the apoptotic cells asβcell. The expression of TNFα, IL-1βandMCP-1 protein in islet were detected by immunohistochemistry, and the data of it wasadjusted by the area of islet. The level of mRNA of TNFα, MCP-1 and apoptoticprotein caspase-3 were detected by real-time quantitative-polymerase chain reaction(real-time Q-PCR), and the data of it was adjusted by the level of mRNA ofβ-actin.Results: Expression of TNFαprotein was higher in group DM and INS than thatof group CsA, CpI and the control group. There was no significant difference betweenthe first two groups or among the last three groups. Results of expression of TNFαmRNA were in conformity with those of TNFαprotein, except that there was nosignificant difference between group CsA and any other groups.The level of MCP-1 mRNA was consistent with that of MCP-1 proteincompletely. The expression of MCP-1 elevated greatly in diabetic rats, especially inDM and INS group, as compared to that in control.The expression of IL-1βprotein was highest in group DM, higher in group CsA,and lowest in group INS and CpI. No significant difference was found between thelast two groups. β-cell apoptosis was a key event contributing to the pathogenesis of type 1diabetes mellitus, and there was a synergistic effect between the treatment of CsA andinsulin on reducing the frequencies ofβ-cell apoptosis. Caspase-3 was the majoreffector caspase involved in apoptotic pathways, and the studying of it also revealedthe best efficacy of combined treatment of CsA and insulin.Multivariate analysis revealed that expression of caspase-3 mRNA, MCP-1mRNA, IL-1βprotein and 4-week random plasma glucose were positively correlatedwith the frequencies ofβ-cell apoptosis, while only 4-week weight showed negativelycorrelated with it. The analysis of multiple linear regulation showed that theexpression of caspase-3 and IL-1βmay contribute to the apoptosis ofβcell.Conclusions: The CsA treatment (1mg. kg. d) in STZ-induced diabetic ratspartially restoredβ-cell function through acting as a preventer ofβ-cell apoptosis. Indiabetic rats, the expression of pro-inflammatory cytokines was elevated remarkably.This high expression of pro-inflammatory cytokines in the pancreatic islet of diabeticrats could be restrained by the combined effect of CsA and insulin. In addition, CsAmay do more on the expression of TNFαand MCP-1, whereas the expression of IL-1βcould be suppressed mainly by using of insulin.