ABSTRACT
Background Apoptosis is a primary clinical pathological mechanism of diabetic retinopathy (DR).Oxidative stress and high glucose can activate cell apoptosis pathway and thus leads to cellular damage.It is confirmed that tert-butyl hydroquinone (tBHQ) plays an antioxidation effect,however,whether it has a protective role on retinal cells in DR is still unelucidated.Objective This study was to investigate the effect of tBHQ on vascular endothelial growth factor (VEGF) and bcl-2 expressions in retina of type 2 diabetic rats and its possible mechanism via nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway.Methods Fifty clean healthy male SD rats were included in this experimental study.Ten rats were fed with normal diet as the normal control group,and other rats were fed with high fatty and high sugar food for 4 weeks.After 12 hours of fasting,streptozotoin (STZ) (30 mg/kg) was intraperitoneally injected to induce the type 2 diabetic models.The model rats were randomly divided into the diabetic control group and tBHQ group and 1％ tBHQ was added into the high fatty and sugar food 1 week after modeling in the tBHQ group.Fasting plasma glucose (FPG) level,blood total cholesterol (TC) level,blood triglyceride (TG) level,high density lipoprotein-cholesterol (HDL-C),low density lipoproteincholesterol (LDL-C) and fasting serum insulin (FINs) were detected 4 and 12 weeks after modeling,respectively,and radio immunoassay was used to detect the FIN levels of the rats.The relative expression of VEGF and bcl-2 in retinas of the rats were assayed by immunohistochemistry and fluorescence real-time quantitative PCR (qRT-PCR).The use of the animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals by State and Technology Commission.Results Type 2 diabetic models were successfully established in 35 rats with successful rate 92.1％.The FIN levels were significantly different among different groups and time points (Fgroup =22.480,P =0.000;Ftime =7.636,P =0.008).The FPG,TC,TG and LDL-C levels were significantly different among the groups (FPG:Fgroup =78.531,P =0.000;TC:Fgroup =28.049,P =0.000;TG:Fgroup =13.108,P =0.000;LDL-C:Fgroup =6.804,P＜0.05).Immunohistochemistry showed that VEGF and bcl-2 were mainly expressed in retinal ganglion cell layer,inner plexiform layer and outer plexiform layer.The expressions of VEGF and bcl-2 proteins were significantly different among different groups (VEGF:Fgroup =11.805,P =0.000;bcl-2:Fgroup =22.943,P =0.000);the expression level of bcl-2 protein was higher in 12 weeks after modeling than that in 4 weeks after modeling in the tBHQ group (P＜0.05).The expressions of VEGF and Bcl-2 mRNA in rat retinas were significantly different among different groups and time points (VEGF:Fgroup =79.220,P =0.000;Ftimo =6.090,P＜0.05;Bcl-2:Fgroup =105.000,P=0.000;Ftime =13.170,P=0.001).Four and eight weeks after modeling,the expressions of VEGF and Bcl-2 mRNA in the diabetic control group and tBHQ group were significantly higher than that in the normal control group,and the expressions of Bcl-2 mRNA in the tBHQ group were significantly higher than that in the model control group (all at P＜0.05);the expression of Bcl-2 mRNA was higher at 12 weeks after modeling than that at 4 weeks in the tBHQ group (P＜0.05).Conclusions tBHQ produces anti-oxidative-damage and anti-apoptosis effects on retinal cells by up-regulating VEGF expression and down-regulating bcl-2 expression in DR rats.In addition,tBHQ may have effects on lowering high blood sugar,regulating insulin and blood lipid levels.
ABSTRACT
Apoptosis or programmed cell death is a physiological process, essential for eliminating cells in excess or that are no longer necessary to the organism, acting on tissue homeostasis, although the phenomenon is also involved in pathological conditions. Apoptosis promotes activation of biochemical pathways inside cells called caspase pathway, of the proteins responsible for the cleavage of several cell substrates, leading to cell death. Antiapoptotic members of the Bcl-2 family (B cell CLL/lymphoma 2), that belong to the intrinsic route of the activation of caspases, such as Bcl-xL (extra-large B-cell lymphoma) and Bcl-w (Bcl-2-like 2), act predominantly to prevent that pro-apoptotic members, such as Bax (Bcl-2-associated X protein) and Bak (Bcl-2 relative bak) lead to cell death. Antiapoptotic molecules are considered potentially oncogenic. Murine models are known to be valuable systems for the experimental analysis of oncogenes in vivo, and for the identification of pharmacological targets for cancer and to assess antitumor therapies. Given the importance of tumorigenesis studies on the immune responses to cancer and the possibility of investigating the participation of antiapoptotic molecules in tumor progression in vivo, the development of new models may be platforms for studies on tumorigenesis, immune antitumor responses, investigation of the ectopic expression of antiapoptotic molecules and immunotherapies for tumors.
A apoptose, ou morte celular programada, é um processo fisiológico essencial para a eliminação de células em excesso ou que não são mais necessárias ao organismo, atuando na homeostase dos tecidos; entretanto, esse fenômeno também está envolvido em condições patológicas. A apoptose promove a ativação de vias bioquímicas dentro das células, denominada via das caspases, proteínas responsáveis pela clivagem de diversos substratos celulares, levando as células à morte. Membros antiapoptóticos da família Bcl-2 (B cell CLL/lymphoma 2), pertencentes à via intrínseca de ativação das caspases, como Bcl-xL (B-cell lymphoma-extra large) e Bcl-w (Bcl-2-like 2) atuam predominantemente prevenindo que os membros pró-apoptóticos, como Bax (Bcl-2-associated X protein) e Bak (Bcl-2 relative bak) ocasionem a morte celular. Moléculas antiapoptóticas são consideradas potencialmente oncogênicas. Sabe-se que os modelos murinos são sistemas valiosos para a análise experimental de oncogenes in vivo, bem como para a identificação de alvos farmacológicos do câncer e para avaliar terapias antitumorais. Em vista da importância dos estudos de tumorigênese e respostas imunes contra o câncer e da possibilidade de investigar a participação de moléculas antiapoptóticas na progressão tumoral in vivo, o desenvolvimento de novos modelos poderá servir como plataforma para estudos de tumorigênese, respostas imunes antitumorais, investigação de expressão ectópica de moléculas antiapoptóticas e imunoterapias contra tumores.