| BackgroundThe incidence of diabetes and its complications are increased year by year with gradually expanding harm. Progressive injury of islet β-cell function and mass of is a common feature of type1and type2diabetes.Because pancreatic β cell secretory function active, making it long-term in vivo exposure to various stress injuries, such as glucose toxicity and lipid toxicity and oxidative stress. Diabetes, high blood sugar circulation will induce a state of oxidative stress, and chronic oxidative stress can cause pancreatic β cell damage and death. Compared with other cells in vivo, β cell antioxidant enzymes such as catalase and glutathione content is very low, which makes the β cells more sensitive to oxidative stress. More and more evidence as nrf2greatest oxidative stress in vivo system, capable of inducing a protective phase II detoxifying enzymes and antioxidant protein synthesis, and therefore it is possible to prompt nrf2through inhibition of oxidative stress in vivo and other regulatory energy metabolism and receptor interaction of transcription factors to slow or prevent the onset of diabetes. Dihydro-CDDO-Trifluoroethyl Amide (dh404) are triterpenoid derivatives of the compounds which can be modified cysteine Keapl section151, nrf2protein stability, promoting nuclear translocation nrf2to activate transcription nrf2function.Nrf2activation due to the magnitude of the state in which the cell-associated, ob leptin deficient/ob mice induced gene transcription after nrf2exhibit insulin resistance and fat synthesis dysfunction. However, given the leptin receptor deficient db/db mice orally nrf2inducer CDDO-Im, nrf2gene expression is activated β cell mass can significantly increase the volume of preventing the onset of diabetes in mice. Although the specific mechanisms of these phenomena is not yet clear, but recently obtained β-cell-specific nrf2/deletion experiments confirmed the nrf2to protect islet β cells oxidative stress injury. However, other potential independent mechanism nrf2fix β cell volume group yet to be confirmed.Ability giant autophagy (commonly referred to as autophagy) degradation cytoplasmic components is a highly conserved cellular mechanism, not only played an important role in maintaining the normal structure of the islets and the diabetic state of insulin resistance and β cell adaptive response to oxidative stress mechanism is essential. Autophagy autophagy began forming, which is a cytosolic contents of which include the dual-mode structure, and then forming the fusion with lysosomes digested autophagy lysosomal contents. Autophagy and the ubiquitin-proteasome system (UPS) is considered abnormal intracellular protein degradation product of the most important/clear pathway, UPS mainly through the ubiquitin pathway clear soluble misfolded or damaged proteins, but mainly clear soluble autophagic somewhat less toxic or non-ubiquitinated proteins soluble polymer. Importantly, autophagy to clear toxic ubiquitinated proteins islet β cells appears to be protective against oxidative stress caused by a key mechanism of diabetes, more and more evidence that nrf2-mediated autophagy clear ubiquitination inhibition of protein aggregation after the formation of reactive oxygen species. Since reactive oxygen species are important to activate autophagy induction medium, and nrf2various cells endogenous formation of reactive oxygen species inhibitory factor, so we have reason to believe that an independent mechanism nrf2through the formation of reactive oxygen species in easily of autophagic clearance. However, this supposed to be further explored, the islet nrf2activation diabetic state is able to promote autophagy cleared to protect β cells from oxidative stress-induced cell damage is not yet clear. AimThe role of oxidative stress in rat pancreatic islets of this study was to study islet β cells in vitro and in vitro and nrf2-mediated survival and function by inducing autophagy of ubiquitinated protein aggregation islet clear protective mechanism, while Through the diabetic mice after islet transplantation were nrf2agonist intervention to further confirm the protective effect nrf2islet, thus providing new ideas and strategies for the treatment of diabetes.MethodPart I mechanisms of the protective effect of nrf2agonist on isolated rat pancreatic islets in an oxidative stress setting1.For new patients and STZ-induced diabetic mouse islets new-onset diabetes immunofluorescence staining and RT-PCR studies nrf2to determine the expression of the state of new onset diabetes islet β cells nrf2through.2. And then carried nrF2agonist dh404pretreatment intervention by H2O2in vitro stimulation of isolated rat islets as oxidative stress model, by detecting glucose-stimulated insulin secretion (GSIS), LDH cytotoxicity detection kit islet mortality and TUNEL staining, islet β cell apoptosis, the protective effect of dh404death induced by oxidative stress and function of rat islets.3.Mechanism for further research, through in vitro isolated rat islets of nrf2, NQO1,4HNE, LC3, p62and ubiquitinated proteins by immunohistochemistry and Western blot, clear nrf2agonist dh404against H2O2-induced oxidation of rat islets stress, nrf2pathway, autophagy activity and protein ubiquitination clear role and mechanism.Part II Nrf2activation by agonists protects islet p-cell function by enhancing oxidative stress-induced protein ubiquitination autophagic clearance1.H2O2treated INS-1cells as models of oxidative stress dh404intervention expression through interference nrf2nrf2RNAi, for LDH cytotoxicity detection, to determine the effect of oxidative stress induced dh404β on cell viability.2.Further Western blot analysis of ubiquitinated proteins accumulate and autophagy-related proteins LC3-II, changes in protein expression levels of p62to verify dh404islet β cells to promote autophagy clearance of ubiquitinated proteins are polymers is pathway by activating nrf2accomplished.3.This article uses autophagy and lysosomal fusion inhibitor chloroquine (CQ) inhibited autophagy activity, nrf2RNAi be nrf2knockout, Western blot analysis performed by the ubiquitin-soluble and non-soluble proteins extracted ingredients INS-1cells protein polymer accumulation and LC3-II, changes in p62protein levels, to confirm the β cell nrf2and dh404-induced autophagy between ubiquitinated proteins polymers clear there is a direct link.Part III Research on the role of Nrf2agonist effect after islet transplantation in diabetic mice1.On cultured islet microvascular endothelial cells MS-1cells were pretreated and high glucose dh40448h, carried LDH cytotoxic cell death detection, detection of oxidative stress and inflammatory cytokines and Western blot and RT-PCR assay nrf2The expression levels of NQO1, AT1, VEGF-A and eNOS, and to verify dh404activated nrf2on pancreatic β cells, oxidative stress and inflammation and microvascular endothelial cells to islet function.2.Concentric necrotic area of mouse islets were cultured in vitro dh404pretreatment and high glucose48h, eVOS observed islets, GSIS function and Western blot and RT-PCR assay nrf2, the expression levels of NQO1, AT1, VEGF-A, and to clarify the impact dh404high glucose-induced pancreatic microcirculation and endothelial function islet death and injury.3.In vivo studies using STZ-induced diabetic mice as a model receptor portal vein islet transplantation performed islet transplantation and dh404(10mg/kg body weight) orally for15days, at15and30days of intraperitoneal injection of glucose tolerance test (IPGTT) observe the effects of islet transplantation and dh404intervention on blood glucose and insulin secretory function spectrum, and to verify the impact dh404after islet transplantation inflammatory response in mice serum levels of inflammatory mediators. ResultPart I mechanisms of the protective effect of nrf2agonist on isolated rat pancreatic islets in an oxidative stress setting1.Confocal immunofluorescence microscopy results of new cases of pancreatic specimens STZ-induced diabetes and new-onset diabetes in mice, indicating diabetic islets smaller size, structural disorder, insulin-colored cells decreased, while nrf2expression increased, while normal blood glucose islets, islet bulky islet overall color depth, nrf2expression less than diabetes, and low fluorescence intensity compared to the surrounding tissue, suggesting that the early onset of diabetes nrf2is active. Meanwhile gene RT-PCR assay STZ-induced diabetic mice islets emerging nrf2and NQO1were detected mRNA expression levels of new-onset diabetic mice islets nrf2and NQO1upregulation.2.After H2O2stimulation of rat pancreatic islets in vitro model of oxidative stress as nrf2agonist dh404pretreatment intervention GSIS in rat islets improved islet β cell mortality.3.1mmunohistochemistry and Western blot showed that rat islets after dh404intervention group compared with H2O2, nrf2and NQO1expression levels were significantly increased,4HNE expression decreased expression of LC3, p62and ubiquitinated protein polymer lower, indicating nrf2dh404agonists inhibited H2O2-induced oxidative stress in rat islets, activation of the nrf2pathway, autophagy promotes clearance of ubiquitinated proteins cleared.Part II Nrf2activation by agonists protects islet β-cell function by enhancing oxidative stress-induced protein ubiquitination autophagic clearance1.LDH cytotoxicity test results showed that after the dh404intervention, β cell survival rate was significantly higher than the pure H2O2-treated group on H2O2treated INS-1cells, and this protective effect was significantly inhibited in nrf2RNAi interference after being confirmed dh404effects on oxidative stress-induced β cell survival pathway is accomplished by nrf2.2.Western blot analysis showed that after nrf2RNAi interference, scavenging dh404-induced autophagy on the accumulation of ubiquitinated proteins significantly reduced, suggesting dh404islet β cells to promote autophagy of ubiquitinated protein polymers are cleared by nrf2pathway to complete the activation.3.Western blot analysis showed that in control cells dh404and H2O2-treated, CQ treatment caused a significant accumulation of soluble protein component of non-ubiquitinated protein polymers and LC3-II and p62protein levels were significantly increased, however, This effect nrf2significantly suppressed after knockout. Confirmed nrf2in dh404-induced autophagy of ubiquitinated proteins P cells of the polymer removal process is essentialPart III Research on the role of Nrf2agonist effect after islet transplantation in diabetic mice1.Dh404preconditioning induced by high glucose MS-1cells after48h, mortality, oxidative stress and inflammatory cytokine levels has been significantly improved. Western blot and RT-PCR showed nrf2and NQO1expression levels were significantly higher and lower levels of ATI expression, the expression levels of VEGF-A and eNOS upregulation. After activation nrf2tips dh404inhibits pancreatic microcirculation oxidative stress and endothelial inflammation, while improving endothelial function.2.0f mouse islets were cultured in vitro pretreatment and high glucose dh40448h, islets and get concentric necrotic area GSIS function significantly improved, Western blot and RT-PCR showed elevated nrf2and NQO1expression levels, ATI expression levels lower levels of VEGF-A expression upregulation. Confirmed dh404high glucose-induced pancreatic microcirculation endothelial function has a protective effect.3.Vivo study showed that after the intervention dh404islet transplantation in diabetic mice, IPGTT showed that islet transplantation group compared with pure glucose and insulin secretion dh404spectrum intervention group of mice has been significantly improved while also a significant inflammatory reaction suppression. ConclusionIn our study, the expression of the first to reveal the nrf2new onset diabetes patients and mouse islets are regulated, suggesting that human clinical relevance nrf2the pathogenesis of diabetes. After the high glucose-induced oxidative stress toxicity, ROS and toxic protein products generated by the function of islet β cell damage and accelerated cell death, nrf2as the body’s largest anti-oxidative stress system is activated, clear ROS inhibition of oxidative stress while the reaction is also able to prevent damage to the toxic protein β cells, protecting the β cell secretory function and reduced cell death, which is a compensatory mechanism in islet oxidative stress, but with oxidative stress This persistence of state compensatory mechanism soon decompensation, nrf2protective effect against oxidative stress injury ultimately insufficient to fully maintain the functional integrity of β cells, so we need to use drugs to further strengthen nrF2for β cytoprotective, in fact, the drug can inhibit β activation nrF2cellular oxidative stress state of death and injury. From the mechanism, our research shows that not only can dh404islets nrf2activation pathways directly inhibit oxidative stress, but also activates autophagy nrf2-mediated protein ubiquitination polymer removal process by an independent anti-oxidation mechanism. |
PDF Full Text Request