Effects Of Proteasome Inhibitor MG132 On The Pathogenesis Of Early Period Diabetic Retinopathy

Abstract:Objective:Diabetic retinopathy (DR) is one of the most prevalent and serious microvascular complications of diabetes mellitus (DM). The exact pathogenesis of diabetic retinopathy is not clear. Oxidative stress is deemed to the common pathway in the development of diabetic retinopathy. Therefore, the mechanism of oxidative stress on in-depth study DR will be of great significance. There will be many factors leading to oxidative stress in diabetes, such as the activation of nuclear factor-KB (NF-κB), start downstream gene transcription, up-regulates a variety of DR-related cytokines, inflammatory factors and adhesion molecules expression, Ubiquitin-proteasome pathway (UPP) is an important way of the major intracellular degradation of specific proteins for the injury. In recent years, studies have shown that NF-κB signal pathway key elements-inhibitory factor-KB (IκB) is degradation of regulation by the UPP. However, the relationship between UPP and NF-κB/IκB signal pathway in diabetes retinopathy is unclear. In this research, we chose streptozotocin (STZ) to induce early DM rat model, and used ubiquitin proteasome inhibitor MG132 as the specific inhibitor of UPP intervention in DM rats, blocked UPP on the degradation of IκB ubiquitination, observed ubiquitin proteasome inhibitor on NF-κB and its inhibitory signaling protein IκB kinase regulation of the degradation in the early DR, and the retinal ganglion cells (RGCs) apoptosis effects to explore the mechanisms of the ubiquitin proteasome pathway in early DR in diabetic rats and the intervention of proteasome inhibitors on DR.Methods:1.Establishment of diabetes mellitus rats model and grouping: Fifty healthy male Wistar rats weight approximately 170 grams were conventionally fed a week, and 10 were randomly selected as normal control group (NC group),40 for the DM model group. The diabetic rat model was made by intraperitoneal injection STZ 60mg/kg in DM model group. After then, the model groups(30) were divided into:DM group, DM+G132 low-concentration intervention group, and DM+MG132 high concentration intervention group randomly. Since the third day from establishment DM model, In DM+MG132 low concentration intervention group, the rats were intraperitoneally injected with 0.05mg/ kg MG132 DMSO solution once daily, in DM+MG132 high concentration intervention group, the rats were intraperitoneally injected with 0.1mg/kg MG132 DMSO solution once daily In DM and NC group, the rats were intraperitoneally injected with the same dose of 0.5% Dimethyl sulfoxide (DMSO) buffer solution once daily.2. Generally observedthe rats, and 6 weeks and 8 weeks after drug administration, with 3% sodium pentobarbital anesthetized,5 rats in each group were sacrificed, heart blood for fasting blood glucose determination.3. The eyeballs were removed, and made into eye cup, embedded in paraffin, the retinal pathologies of rats were observed by the HE staining.4. The expression of NF-κB and IκB were studied by Streptavidin Peroxidase (SP) immunohistochemistry.5. Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method.6. Statistical Analysis:application SPSS 14.0 statistical softwere to analyze data, data were expressed as mean±standard deviation. Between the two groups using Student’s t test, multiple groups were analyzed using analysis of variance. P<0.05 indicated statistically significant. Results:1. General indicators of changes in rats:Compared with the NC group, there are obviously more drinking, urine, body weight decreased two days after STZ injection in model group, all these parameters is more obvious to 6 weeks and 8 weeks.2. The expression of NF-κB was up-regulated in DM group as compared with NC group, it’s expression was down-regulated in MG132 high concentration intervention group compared with DM group; But the expression of IκB was down-regulated in DM group as compared with NC group, it’s expression was up-regulated in MG132 high concentration intervention group compared with DR placebo group (P<0.05).3. The detection of TUNEL positive retinal ganglion cells was up-regulated in DM group as compared with NC group, the expression was down-regulated in DM+ high concentration intervention group compared with DR placebo group (P<0.05). On the contrary, NF-κB and IκB expression strength and apoptosis of RGCs in MG132 low concentration intervention group compared with DR placebo group were no significant difference (P> 0.05).Conclusion:1. Single intraperitoneal injection 60 mg/kg STZ can successfully induce diabetes in rat. With the modeling time, the indicators of blood glucose and body weight of rats change in line with changes in diabetic mellitus.2. NF-κB signal pathway activation, and NF-κB activation and IKBa ubiquitin degradation increase in the course of diabetic retinopathy suggest UPP involved in NF-κB signal pathway regulation in diabetic retinopathy.3. Proteasome inhibitor MG132 can inhibit IKBa ubiquitination degradation, block the translocation of NF-κB activation, inhibit of apoptosis of RGCs, and improve the early DR lesions in diabetic retinopathy, indicating that proteasome inhibitor MG132 may play an early protective role in diabetic retinopathy.