The Relationship Between NO And Apoptosis Of Brain Cell In The Rat With Insulin Resistance

Background and ObjectivesWith the progress of medical technology and the decline in the birth rate, our country has been entering an aging society. The incidence of cerebrovascular disease and neurodegenerative disease appears an upward trend significantly. Cerebrovascular disease is the leading cause of death in China, and places a huge burden on the individual, family, and community. The population of metabolic syndrome (MS) including hypertension, insulin resistance (IR), obesity, lipid metabolism disorder, etc is growing, associated with reduced life span and quality of life. Its prevalence rate is more than 14% in China. MS is a term associated with the collection of a series of risk predictors that are associated with metabolic diseases including cardiovascular diseases, diabetes mellitus(DM) and nervous system disease.Several lines of evidence suggest that nerve apoptosis phenomenon exists in cerebrovascular diseases, neurodegenerative disorder, acute brain damage and epilepsy. There are many factors which can cause apoptosis, and studies both in vivo and in vitro are confirmed that excessive NO in tissue can induce cell apoptosis. Isosorbide mononitrate (ISMN), as an exogenous NO donor, is a commonly used drug in the patients with MS, which is clinically applied for the treatment of cardiovascular disease.Our study, by observing the apoptosis level of the mice brain with IR and with the treatment of ISMN, explore how ISMN lead to apoptosis in rat brain cells with IR. More functional and mechanistic studies are warranted to elucidate the possible mechanisms involved, verify the efficacy and safety of ISMN in the prolonged treatment of cardiovascular patients with MS, research new therapeutic targets, and provide a more effective therapeutic approach to the treatment of brain injury and disease via targeting of apoptosis.Materials and Methods1 Grouping and breeding methodFor this experiment,14-weeks-old male Wistar rats (weight 393.75±11.09 g) were purchased from Laboratory Animal Center of Henan Province, and the rat weights of each group had no statistical difference.The experiment was randomly divided into 4 groups with 10 rats in each group as follows:Control group (n=10) that received normal food for 12weeks; HFHG group that received High fat & High glucose diet (HFHG) food; ISMN group (n=10) that received isosorbide mononitrate (4.2mg/kg per d for 12 w,) and normal food for 12 weeks; UNITED group that received High fat & high glucose diet (HFHG) and isosorbide mononitrate. The groups above without the treatment of ISMN received an equivalent volume of saline for 12weeks.The formulation of HFHG was as follow: 79% normal diet,10% cane sugar,5% cholesterol and 1% lithocholic acid.2 Observing index2.1 After overnight fasting, blood samples were taken to assess serum levels of the fasting glucose (FBG, mmol/L) and fasting insulin (FINS, mIU/L).2.2 Levels of NO and iNOS were measured by Nitrate reduction method.2.3 The expression levels of Bcl-2, Bax mRNA were measured by quantitative real-time reverse transcriptase polymerase chain reaction. The expression levels of Bcl-2, Bax protein expressive were measured by western blot analysis.2.4 After blocking with 4% BSA, the cells were then incubated in TUNEL kit. To assess apoptosis, the numbers of TUNEL stained nuclei were counted by two investigators who worked blindly with regard to the treatments in five randomly selected microscopic fields at × 400 magnification per section.Results1 HOMA-IRHOMA-IR in the groups fed with HFHG and/or ISMN was higher than that of the control group. (P<0.05)2 Measurement of NO productionThe levels of NO in the groups fed with HFHG and/or ISMN were higher than that of the control group. (P<0.05)The levels of NO in the UNITED group were higher than that of in HFHG group and ISMN group. (P<0.05)3 Measurement of iNOS productionThe levels of iNOS in the groups fed with HFHG and/or ISMN were higher than that of the control group. (P<0.05)The levels of iNOS in the UNITED group were higher than that of in HFHG group and ISMN group. (P<0.05)4 Correlation analysis between HOMA-IR and NOThe NO level of the rat brain tissue was correlated with HOMA-IR in the HFHG group and the HOMA-IR was significantly correlated with NO level in the ISMN group.5 Assessment of apoptosis of brain cells and apoptotic indexThe number of brain cells apoptosis was higher in the groups fed with HFHG and/or ISMN than that of the control group at high magnification. (P<0.05) 6 Measurement of Bcl-2 mRNA, Bax mRNA and related proteinsCompared with the Control group, the level of Bcl-2 mRNA in HFHG group, ISMN group and UNITED group was decreased. (P<0.05)Compared with the Control group, the level of Bax mRNA in HFHG group, ISMN group and UNITED group was increased. (P<0.05)Compared with the Control group, the level of Bcl-2 protein expression in HFHG group, ISMN group and UNITED group was decreased. (P<0.05)Compared with the Control group, the level of Bax protein expression in HFHG group, ISMN group and UNITED group was increased. (P<0.05)Conclusion1 ISMN can induce rat brain cell apoptosis of all the groups with treatment of ISMN, and IR aggravates the apoptosis;2 There is a mutual induction between IR and NO in the process of apoptosis;3 Protective effect or toxic effect of NO is dependent on the generating ways and the quantity of its.