Effects Of The Naoshenkang Capsule On Oxidative Stress In The Hippocampus Of Diabetic Rats

Background:Diabetes has been considered as an increased risk factor for cognitive dysfunction. Recently, evidence has accumulated to suggest that oxidative stress plays a key role in the pathogenesis of diabetes-induced cognitive dysfunction though the mechanism underlying has not been understood. High glucose level can increase ROS production through the activation of protein kinase C (PKC) and other intracellular signaling pathways. When cellular excessive production of ROS overwhelms its antioxidant capacity, it can lead to damage of cellular macromolecules such as lipids, proteins and DNA. The transcriptional factor hypoxia inducible factor-1(HIF-1) is a key regulator in cellular responses to reduced oxygen concentrations and occupies a central position in regulating oxygen homeostasis. HIF-1is stabilized to enhance transcription of hypoxia-inducible genes such as erythropoietin and VEGF to achieve metabolic adaptation during hypoxia. However, sustained and prolonged activation of the HIF-1pathway would then rather induce cell death responses than initiate a neuroprotective adaptive response due to the activation of p53and other related genes. The hippocampus is closely associated with the regulation of short and long term memory and spatial navigation, but it is particularly vulnerable to hypoxia and oxidative stress. We have previously shown that HIF-1α expression was increased in the hippocampus of diabetic rats and may result in the development of diabetes-related cognitive dysfunction. Recent studies have demonstrated that the activation of hypoxia-inducible factor-1requires reactive oxygen species generated at mitochondrion by NADPH oxidase under hypoxic conditions. NADPH oxidase subunit4(Nox4) is a new target gene of HIF-1 involved in the response to hypoxia. NADPH oxidase, as a major source of ROS, can regulate normal physiological activities, but its overexpression possibly contributes to various disorders such as diabetes mellitus, Alzheimer’s and other neurodegenerative diseases. Some scholars have found that the intervention on overexpression of NOX4in renal and myocardial tissue of diabetic rats contributed to various protective effects, and demonstrated that the expression of NOX4was upregulated in neurons under ischemic conditions. Thus, anti-oxidation and intervention on over-expression of NOX4and HIF-1α may play an important role in the treatment of cognitive impairment in diabetes. Previous studies have reported that Naoshenkang capsule and its constitutive extracts possess plenty of obvious therapeutic effects such as antihyperglycemic activity, promoting the insulin sensitivity, resisting free radical, inhibiting hippocampal neuronal apoptosis, and thus improving clinical symptoms of diabetic cognitive function, but the molecular mechanisms underlying have not been delineated.Objective:This study aimed to gain greater insight into the effects of NOX4and HIF-1α on the pathogenesis of diabetes-induced cognitive dysfunction and the possible mechanisms of Naoshenkang capsule to ameliorate cognitive impairment in diabetic rats.Methods:Diabetic rat models were established by an intraperitoneal injection of streptozotocin (STZ). Twelve weeks later, all the rats were randomly divided into five groups:the normal group, the diabetic group, small-dose of Naoshenkang capsule group, high-dose of Naoshenkang capsule group and western medicine group. In this study, we assessed the cognitive function of streptozotocin-induced diabetic rats by Morris water maze test and BAEP measurement. The activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) in hippocampus were measured to evaluate the status of oxidative stress. Nissl staining was performed to analyze hippocampal CA1pyramidal damage histopathologically. The NOX4and HIF-1α rotein expressions in the hippocampus of all the rats were detected using immunohistochemistry. The expression of NOX4, HIF-1α, Bax and Bcl-2mRNA was measured by real-time RT-PCR. Results:Compared with the normal group, the escape latency, the waves Ⅱ, VPL, and Ⅰ-Ⅴ IPL of BAEP in the diabetic group were significantly longer than the normal group (P<0.01). The diabetic rats developed significant impairment in learning and memory behaviors and brainstem neuroelectrophysiological lesion as indicated by the deficits in water maze tests and BAEP measurement as compared to control rats, respectively. Furthermore, MDA level increased and SOD activity decreased in diabetic rats. However, treatment with Naoshenkang capsule significantly ameliorated learning and memory impairment and reversed the diabetes-induced changes of these parameters. Reduced level of MDA, obviously elevated activities of SOD were found in Naoshenkang-treated groups. What’s more, Naoshenkang capsule remarkably inhibited the expression of NOX4, HIF-1α at mRNA and protein levels, respectively. Besides, Naoshenkang capsule could also significantly suppress the expression of Bax and inhibit the expression of Bcl-2. These effects were paralleled with normalization of oxidative stress markers and expressions of NOX4and HIF-1α in hippocampus. These results suggested that oxidative stress is an exacerbating factor of cognitive dysfunction in diabetic rats and that antioxidant therapies are of value to diabetes-induced cognitive dysfunction.Conclusion:Our results showed that NOX4and HIF-1α signaling pathways might work in a coordinated fashion in the pathogenesis of diabetes-induced cognitive dysfunction. These findings indicate that the neuroprotective effect of Naoshenkang capsule appears to be closely associated with its anti-oxidative and anti-apoptotic properties that can regulate the expressions of NOX4, HIF-1α, Bax and Bcl-2. Inhibition of NOX4and HIF-1α may hopefully serve as an useful strategy to improve cognitive function.