Study On The Role Of HO-1 In Neuronal Oxidative Stress Induced By Palmitic Acid

Objective: Alzheimer’s disease(AD) is a chronic neurodegenerative disease featuring progressive impairments in memory and cognitive. The levels of medium-, long- and very long chain fatty acids have been found to be increased in the brains of AD patients. Oxidative stress is an important pathogenic mechanism of AD. Saturated fatty acids including palmitic acid(C16) promote the generation of reactive oxygen species(ROS) and induce oxidative stress through a variety of mechanisms that plays an important role in the occurrence and development of AD. So, reducing the oxidative damage by antioxidants may become an important target for the prevention and treatment of AD.Heme oxygenase-1(HO-1) is one of the antioxidases in the brain. It can alleviate cellular oxidative damage by the removement of its metabolites(bilirubin) on active oxygen. The upstream of HO-1 gene has antioxidant response element(ARE). Nuclear factor erythroid-derived 2-like 2(NRF2) is the activating factor of ARE. Under normal circumstances, NRF2 is located in the cytoplasm. When oxidative stress occurs in the cells, NRF2 is phosphorylated by several kinases, such as ERK and transferred into the nucleus to turn on the transcription of HO-1. The expression of HO-1 was increased in the brain of AD patients. These informations pointed out that HO-1 may enhance the antioxidant ability of the brain to resist the pathological process of AD by a compensatory increase.This study intends to use palmitic acid C16 as a stimulus and neuroblastoma BE(2)-M17 cells as the research object to study the up-regulatory mechanism and anti-oxidative role of HO-1 at the cellular level in the state of neuronal oxidative stress induced by saturated fatty acids, in order to provide theoretical and experimental basis for the target research of the prevention of diabetic AD.Methods:1 Cell cultureNeuroblastoma BE(2)- M17 cells were routinely cultured.2 Determination of cellular ROS contentDHE staining or chemiluminescence was performed to detect the content of ROS in cells.3 The detection of m RNA expression of target genes by real-time Quantitative RT-PCRPromega total RNA extraction kit was used to extract total RNA from neurons. 2 μg of total RNA was taken to perform reverse transcription. The relative expression of HO-1 and NRF2 m RNA was carried out by quantitative RT-PCR with 18 s r RNA as internal control4 The detection of protein expression of target genes by western blotTotal protein extracted from neurons was used to determine the protein expression of HO-1, NRF2, ERK and its phosphorylation level5 The detection of localization and protein levels of target genes by cell immunofluorescence stainingImmunofluorescence staining was performed to observe the protein expression of HO-1 and NRF2 and the nucleic localization of NRF2.6 Statistical analysisAll statistical analyses were conducted using SPSS 17.0 software. The data are expressed as means ± SD. Data were evaluated using One-way ANOVA for multiple group comparison. Statistical significance was set at P < 0.05.Results:1 Palmitic acid(C16) induced neuronal oxidative stress.Different dosages of C16 stimulated cells for 2 h. The levels of cellular ROS were detected by DHE staining. Results showed that compared with control group, the content of ROS increased in a dose-dependent manner after C16 treatment and the highest increase was at the dosage of 200 μM, indicating that palmitic acid induces neuronal oxidative stress.2 The expression of HO-1 was compensatory increased under the state of oxidative stress.Different dosages of C16 stimulated cells for 2 h. Quantitative RT-PCR was performed to detect HO-1 m RNA expression, and western blot and cell immunofluorescence staining detecte HO-1 protein expression. The Results showed that compared with control group, the m RNA and protein expression of HO-1 were both increased in a dose-dependent manner after C16 treatment, indicating that HO-1 expression was compensatory increased under the state of oxidative stress.3 The up-regulation of HO-1 alleviated neuronal oxidative stress induced by palmitic acid.1) ERK kinase was activated under the state of oxidative stress.Different dosages of C16 stimulated cells for 2 h. Western blot was performed to detect the expression of ERK kinase and its phosphorylation level. Results showed that there was no obvious change in the expression of ERK, but the level of p-ERK was dose-dependently increased. These results showed that ERK kinase was activated under the state of oxidative stress.In order to make clear the relationship between the activation of ERK and the expression of HO-1, the cells was divided into four groups: control group, C16 group, ERK inhibitor group and ERK inhibitor + C16 group. Western blot was performed to detect HO-1 expression, ERK expression and its phosphorylation level. The results showed that HO-1expression and p-ERK level were significantly increased in C16 group, compared with control group. However, HO-1expression and p-ERK level were significantly decreased in ERK inhibitor + C16 group, compared with C16 group. Those results showed that HO-1 expression may be related to the activation of ERK signaling pathway.2) Nuclear transcription factor NRF2 was activated under the state of oxidative stress.Different dosages of C16 stimulated cells for 2 h. Cellular immunofluorescence staining was performed to detect the activity of NRF2, a substrate of ERK and an upstream regulatory factor of HO-1, namely the transfer of NRF2 into the nuclear. The results found that the level of NRF2 in nucleus was significantly increased after C16 treatment. At the same time, quantitative RT-PCR and western blot were performed to detect the expression of NRF2. The results showed that compared with control group, NRF2 m RNA and protein expression were significantly increased after C16 treatment, suggesting that the up-regulation of HO-1 may be associated with NRF2 activation3) The up-regulation of HO-1 alleviated oxidative stress induced by palmitic acid.Cells were divided into four groups: control group, NRF2 agonist group, C16 group and NRF2 agonist + C16 group. The results of western blot showed that compared with control group, the expression of HO-1 was increased after C16 or NRF2 agonist treatment. At the same time, compared with C16 or NRF2 agonist groups, the expression of HO-1 was obviously increased after C16 and NRF2 agonist treatment. Chemiluminescence was used to detect the levels of intracellular ROS, and the results showed that compared with control group, the levels of ROS increased obviously after C16 treatment; at the same time, compared with C16 treatment, the levels of ROS was obviously decreased after C16 and NRF2 agonist treatment. These results indicated that the increase of HO-1 can alleviate oxidative stress induced by palmitic acid in neurons.Conclusions:1 That palmitic acid induced neuronal oxidative stress is one of pathogenic mechanisms of AD.2 The compensatory increase of HO-1 was associated with the activation of ERK which actives NRF2 further under the state of neuronal oxidative stress induced by palmitic acid.3 The up-regulation of HO-1 alleviated neuronal oxidative stress induced by palmitic acid.