| Nowadays, the prevalence of diabetes mellitus is growing rapidly. It is thought that diabetes has become one of the three most important chronic non-communicable diseases, which is subsequent to cardiovascular diseases and cancer. Diabetes and its complications could cause multi-system damage, which result in high rate of disability and mortality, hence diabetes is not only a serious threat to human health but also a heavy economic burden to the whole society. Therefore, the prevention and treatment of diabetes have been the important and urgent public health issues, which are dependent on the intensive study about the pathogenesis of diabetes. It is widely accepted that pancreaticβ-cell dysfunction is one of the key factors in the onset and development of diabetes. So it is meaningful to do in-depth studies into the pathogeneses and mechanisms of pancreaticβ-cell dysfunction.Some past studies have indicated that long-term hyperglycemia led to pancreaticβ-cell dysfunction and apoptosis, which was termed "glucose toxicity". Further study suggested that oxidative stress was an important mechanism underlying pancreaticβ-cell "glucose toxicity". Generally, oxidative stress is such a phenomenon in which the production rate of the reactive oxygen species (ROS) exceeds the clearance rate of ROS, so ROS are over-cumulated and eventually do damage to cell structure and function. It has been confirmed that chronic hyperglycemia could induce higher levels of cellular ROS and markers of oxidative stress. Antioxidant drugs or gene transfer techniques could significantly enhance the protection ofβ-cell from oxidative stress. There are two patterns of manifestation for chronic hyperglycemia clinically—constant hyperglycemia and fluctuant hyperglycemia with the latter being observed more often. Fluctuant hyperglycemia is a state in which intermittent or paroxysmal high glucose could be observed, and clinically unreasonably controlled postprandial hyperglycemia is the main manifestation pattern of fluctuant hyperglycemia. Some major studies have suggested postprandial hyperglycemia is the independent risk factor for vascular complications of diabetes. Further research discovered that glucose fluctuations exhibited a more specific triggering effect on oxidative stress than chronic sustained hyperglycemia in patients with type 2 diabetes. A series of studies in vitro for human umbilical endothelial cells have also suggested fluctuant high glucose triggers more serious oxidative stress, and thus is more likely to promote the development of chronic complications of diabetes. Therefore, fluctuant high glucose may do more harm than constant high glucose. However, there are few reports on the oxidative injury of the fluctuant high glucose to the pancreaticβ-cell and it is meaningful to do research about this topic.Studies have shown that the decrease ofβ-cell mass caused by increased apoptosis is one of the important mechanisms underlying pancreatic islet dysfunction. So pancreaticβ-cell apoptosis may play an important part in the onset and development of diabetes. Among the factors inducing the increasing apoptosis ofβ-cell, high glucose has been under great attention. Previous researches indicated that constant high glucose promoted increasing apoptosis ofβ-cell. In the regulation ofβ-cell apoptosis, Bcl-2 gene family may play an important part. Under constant high glucose, the expressions of pro-apoptotic gene Bax inβ-cell increased significantly, whereas those of anti-apoptotic gene Bcl-2 were decreased, so that the expression ratio of Bcl-2 to Bax was decreased. The result showed that expression changes of Bax and Bcl-2 may participate in the regulation ofβ-cell apoptosis induced by high glucose. It has found that oxidative stress is closely related to cell apoptosis in recent years. Previous studies suggested that high glucose could aggravate endothelial cell apoptosis by inducing oxidative stress. A number of studies in vitro or vivo also showed that oxidative stress could induceβ-cell apoptosis and that cell apoptosis was inhibited with the expressions of pro-apoptotic protein Bax decreasing and those of anti-apoptotic protein Bcl-2 increasing after antioxidant treatment. Therefore, constant high glucose could promote the increase ofβ-cell apoptosis by enhancing oxidative stress and down-regulate the expression ratio of Bcl-2 to Bax.As mentioned before, all the cells includingβ-cell may be damaged by oxidative stress and many pro-apoptotic factors, but there exist related defense systems to antagonize such damage in organism. Heme oxygenase-1(HO-1) is a kind of cytoprotection protein which has been under increasing attention in recent years. HO-1 distributes extensively in the microsomes of many types of mammalian cells. The expressions of HO-1 are low under physiological conditions, but under the induction of such factors as hemoglobin, heavy metals, peroxides, oxidized low-density lipoprotein and so on, its expressions and activity could be significantly enhanced. It is well accepted that HO-1 is one of the important anti-oxidant protective factors in the body, and the increased expressions of HO-1 under the induction of many factors could be considered as the adaptive protection response of cells to antagonize oxidative stress. In recent years, studies have also found that the increased expressions of HO-1 could reflect the state of oxidative stress under the condition of high glucose to some extent.Researches in recent years have shown that the up-regulation of HO-1 expressions plays an important part in terms of opposing oxidative stress, which is a heated topic in such fields as organ transplantation, ischemia-reperfusion injury, cardiovascular and cerebrovascular diseases and so on. High glucose could enhance dramatically the oxidative stress of vascular endothelial cells, and up-regulating the expression of HO-1 could be an effective way to antagonize the oxidative stress induced by high glucose, on the contrary, down-regulating the expression of HO-1 may make oxidative stress more severe. Researches on pancreaticβ-cell have shown that induction of the increased expression of HO-1 could significantly diminish the levels of ROS in INS-1 cells and effectively improve their insulin-secreting functions under the condition of constant high glucose. These results indicated that HO-1 may protectβ-cell by acting against oxidative stress under the condition of high glucose. Current studies have suggested that the mechanisms on antagonizing oxidative stress of HO-1 may include the following aspects:1) the antioxidant function of its metabolic products; 2) weakening the activities of the enzymes related to the generation of ROS; 3) induction of the expressions of other antioxidant enzymes.Up-regulating the expression of HO-1 may play an anti-apoptotic role in many types of cells. Transfection with HO-1 gene could obviously alleviate the growth arrest and apoptosis of microvascular endothelial cells. Increased expression of HO-1 induced by hemin and curcuma could effectively inhibit the apoptosis of renal proximal tubular epithelial cells induced by angiotensinⅡ, whereas down-regulating the expression of HO-1 could aggravate the apoptosis. The anti-apoptotic role of HO-1 in the field of protectingβ-cell has been a heated topic in recent years. Researches showed that up-regulating the expression of HO-1 could be an effective way to alleviate the apoptosis levels ofβ-cell lines and significantly improve the islet function. Nowadays, the anti-apoptotic mechanisms of HO-1 have not been clarified fully, but the following factors may be related to its anti-apoptotic role:1) up-regulating the expression of p21; 2) the participation of NF-κB and MAPK signaling pathways; 3) regulating genes relating to apoptosis Bax and Bcl-2.As mentioned before, constant high glucose could induce the increased expression of HO-1 by enhancing the oxidative stress, then how about the expression of HO-1 in pancreaticβ-cell under the condition of fluctuant high glucose? In terms of the effects on the oxidative stress ofβ-cell, is there any difference between fluctuation high glucose and constant high glucose? Does HO-1 could offer protection toβ-cells by bringing its anti-oxidant and anti-apoptotic roles into full play? There are few studies concerning these aspects, and further studies are needed to clarify these questions. In this study, the rat insulinoma-derived cell line INS-1 is used as theβ-cell model, and the expression of HO-1 protein, ROS levels and the expression of proteins related to apoptosis are detected under the condition of fluctuant high glucose and the changes of the above-mentioned indices are also studied after regulating the expression of HO-1, finally our purpose is to investigate the protective effect and related mechanisms of HO-1 on pancreaticβ-cell under the condition of fluctuant high glucose, thus provide new ideas for studies on the protection ofβ-cell.1. To observ the oxidative stress changes of INS-1 cells the condition of fluctuant high glucose. 2. To investigate into the effects of HO-1 on the oxidative stress of INS-1 cells underthe condition of fluctuant high glucose.1. Cell culture:The rat insulinoma-derived INS-1 cells, a widely usedβ-cell surrogate, were cultured in 5% CO2 at 37℃in RPMI-1640 complete medium containing 10% fetal bovine serum(FBS),11.1 mmol/L D-glucose,1.0 mmol/L sodium pyruvate,50μmol/L 2-mercaptoethanol,100 U penicillin/mL and 100μg streptomycin/mL.2. There were five groups according to different concentrations of glucose and intervention reagents:(1) The normal control group (Ctrl group, cells were cultured in RPMI-1640 complete medium containing 5.5 mmol/L glucose);(2) The constant high glucose group (CHG group, cells were cultured in RPMI-1640 complete medium containing 16.7 mmol/L glucose);(3) The fluctuant high glucose group (FHG group, cells were cultured in RPMI-1640 complete medium containing 16.7 mmol/L glucose for two hours and then in RPMI-1640 complete medium containing 5.5 mmol/L glucose for three hours, and such fluctuant exposures were repeated three times and finally cells were cultured in RPMI-1640 complete medium containing 5.5 mmol/L glucose for nine hours at night);(4) CoPP (20μmol/L)+fluctuant high glucose group (CoPP+FHG group, cells were pretreated by HO-1 inducer CoPP for 24 hours and then exposed in fluctuant high glucose);(5) ZnPP (20μmol/L)+fluctuant high glucose group (ZnPP+FHG group, cells were pretreated by HO-1 inhibitor CoPP for 24 hours and then exposed in fluctuant high glucose). The INS-1 cells of these five groups were cultured in basal medium for 24 hours before intervention to keep synchronized growth of cells. Each group was officially intervened for 72 hours.3.The expressions of HO-1 protein were detected by western blot. The total protein was extracted from the INS-1 cells and then the concentrations of the protein were determined by Bradford assay. The protein was fractionated by SDS-polyacrylamide gel electrophoresis and then electroblotted to a polyvinylidene fluoride (PVDF) membrane. Nonspecific binding sites were blocked by 5% nonfat milk for one hour at room temperature. The blots were then incubated with the polyclonal rabbit antibody against HO-1 and this was followed by a one-hour incubation period with the peroxidase-labeled anti-rabbit IgG. For quantification, the protein bands were analyzed by Image-Pro Plus 6.0 software. The gray ratio of HO-1 protein band to theβ-actin protein band was regarded as the expression levels of HO-1 protein.4. Determination of ROS levels:Cells were resuspended in medium containing 10μmol/L DCFH-DA (molecular probes) and then incubated at 37℃for 40min. The mean fluorescence intensity (MFI), which was regarded as the ROS level, was detected by flow cytometric analysis using FACScan (Becton-Dickinson, Mountain View, CA) at the excitation wavelength of 488nm and the emission wavelength of 525nm.5. Cell viabilities were evaluated by MTT assay:The optical density (OD) value was read at 490nm in a plate reader. The reduction in optical density was used as a measurement of cell viability, normalized to cells incubated in the normal control group, which were considered 100% viable.6. Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. The data was expressed as the mean±S.D. Statistical significance of differences among groups was evaluated by one-way ANOVA and multiple comparisons were carried out using the Least-significant Difference (LSD) method. P<0.05 was considered as significant.1. The expression of HO-1 protein:The expression levels of HO-1 protein in the Ctrl group, CHG group, FHG group, CoPP+FHG group and ZnPP+FHG group were 0.28±0.02,0.41±0.01,0.49±0.01,0.55±0.01 and 0.39±0.01 respectively. In comparison with the Ctrl group, the HO-1 protein expressions in CHG group and FHG group were increased significantly (P<0.01, respectively) with the FHG group higher than the CHG group (P<0.01). In contrast with the FHG group, the HO-1 protein expressions of CoPP+FHG group were increased significantly (P<0.01) but those of ZnPP+FHG group were decreased significantly (P<0.01).2. ROS levels:The ROS levels of the Ctrl group, CHG group, FHG group, CoPP+FHG group and ZnPP+FHG group were 45.13±2.83,75.70±14.00,197.80±7.08,106.75±12.81 and 247.39±15.44 respectively. In comparison with the Ctrl group, the ROS levels in CHG group and FHG group were increased significantly (P<0.01, respectively) with the FHG group higher than the CHG group (P<0.01). Comparing to the FHG group, the ROS levels of CoPP+FHG group were decreased significantly (P<0.01) but those of ZnPP+FHG group were increased significantly (P<0.01).3. Cell viabilities:The OD values of the Ctrl group, CHG group, FHG group, CoPP+FHG group and ZnPP+FHG group were 0.97±0.01,0.85±0.02,0.66±0.01,0.78±0.03 and 0.25±0.05 respectively. In contrast with the Ctrl group, the cell viabilities of the CHG group and FHG group were decreased by 12% (P<0.01),31% (P<0.01) respectively with the FHG group lower than CHG group(P<0.01). In comparison with the FHG group, the cell viabilities of CoPP+FHG group were increased significantly (P<0.01) but those of ZnPP+FHG group were decreased significantly (P<0.01).1. High glucose could induce significant oxidative stress of INS-1 cells. In comparison with constant high glucose, fluctuant high glucose may trigger more severe oxidative stress and thus pose greater damage to INS-1 cells.2. Compensatory increase of HO-1 protein expressions could not counteract efficiently the oxidative stress of INS-1 cells under the condition of fluctuant high glucose.3. Under the condition of fluctuant high glucose, the inducible increase of HO-1 protein expressions could make ROS levels decrease and cell viabilities improve, and hence it was suggested that the inducible increase of HO-1 expressions may protect the INS-1 cells by acting against oxidative stress; inversely, the inhibition of HO-1 expressions may aggravate oxidative stress and thus does damage to cells. Therefore, HO-1 may be one of the important antioxidant factors in INS-1 cells.To investigate into the protective effect of HO-1 on INS-1 cells by observing the expression changes of Bax and Bcl-2 after regulating the expressions of HO-1 protein under the condition of fluctuant high glucose.1. Cell culture and experimental grouping were just the same as those of the chapter 1.2. The expressions of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were determined by immunocytochemical method. The positive parts ranging from light yellow to dark brown yellow were located in cytoplasm or cell membrane under the microscope. Five complete and non-overlapping high-power fields were selected randomly from the slides of each group. The average optical density (OD) value of the positive parts in each field was determined by Image-Pro Plus 6.0 image analysis software. The expression levels of related proteins were shown by the OD values.3. Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. The data was expressed as the mean±S.D. If equal variances were achieved, one-way ANOVA was performed to evaluate the statistical significance of differences among groups and multiple comparisons were carried out using LSD method; If not, Welch approximate analysis of variance was performed to analyze the statistical significance of differences among groups and multiple comparisons were carried out using Tamhane's T2 method. P<0.05 was considered as significant.1. The expressions of pro-apoptotic protein Bax:The OD values of pro-apoptotic protein Bax in the Ctrl group, CHG group, FHG group, CoPP+FHG group and ZnPP+FHG group were 0.11±0.02,0.22±0.03,0.19±0.03,0.07±0.02 and 0.26±0.02 respectively. In comparison with the Ctrl group, the Bax protein expressions in CHG group and FHG group were increased significantly (P<0.01, respectively), but there was no significant difference between FHG group and CHG group (P=0.091). Comparing to the FHG group, the expression levels of Bax protein in CoPP+FHG group were decreased significantly (P<0.01) but those in ZnPP+FHG group were increased significantly (P<0.01).2. The expressions of anti-apoptotic protein Bcl-2:The OD values of anti-apoptotic protein Bcl-2 in the Ctrl group, CHG group, FHG group, CoPP+FHG group and ZnPP+FHG group were 0.26±0.01,0.17±0.01,0.19±0.01,0.27±0.03 and 0.16±0.01 respectively. In contrast with the Ctrl group, the Bcl-2 protein expressions in the CHG group and the FHG group were decreased significantly (P<0.01, respectively) but there was no significant difference between the FHG group and the CHG group (P=0.410). Comparing to the FHG group, the expression levels of Bcl-2 protein in CoPP+FHG group were increased significantly (P<0.01) but those in ZnPP+FHG group were decreased significantly (P<0.01).1. In comparison with the normal control group, the expressions of the pro-apoptotic protein Bax in constant high glucose and fluctuant high glucose were increased significantly and those of the anti-apoptotic protein Bcl-2 in these two states of high glucose were decreased significantly, therefore it was suggested high glucose may aggravate the apoptosis of INS-1 cells by up-regualting the expressions of Bax and inhibiting those of Bcl-2.2. Under the condition of fluctuant high glucose, the inducible increase of HO-1 may counteract the apoptosis of INS-1 cells by promoting the expressions of Bcl-2 and inhibiting those of Bax; the inhibition of HO-1 expressions may promote apoptosis by down-regulating the expressions of Bcl-2 and up-regulating those of Bax.
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