Observations Of Cultured Cortical Neurons Injury Induced By Streptozotocin And The Protections Of Pioglitazone And HN In Rats

Recent researches show that insulin is an important regulator of neuron survival and metabolism in the central nervous system (CNS), insulin can promote protein synthesis, synapse formation and maintain neurons growth, survival and differentiation. Studies have shown that insulin in central neural system is partly from the periphery, in addition, neuron can produce insulin themselves. Insulin from neuron is involved in the signal transduction, cell metabolism and modulation. Insulin receptors (IR) are densely distributed in the axons of hippocampus CA1 region pyramidal cells, the terminal of hypothalamus adrenergic neurons and the membrane surface of the tree-tree synaptosome in the olfactory bulb, meanwhiles the IRs is distributed in other cortex closely related to cognitive functions. Insulin combined with insulin receptors (IR) on the cell membrane, trigger the signal transduction and induce the cell-specific physiological and biochemical reactions.Alzheimer's disease (AD) is the most common neurodegenerative disease in central nervous system, characterized by the progressive deterioration of cognition and memory in association with the presence of senile plaques, neurofibrillary tangles, and massive loss of neurons, primarily in the cerebral cortex and hippocampus. Studies have confirmed that glucose metabolism disorders and the pathogenesis of AD are closely linked, especially in late-onset AD, glucose metabolism disorder may be an important trigger. Multiple evidences suggested that central neural system glucose metabolism disorders can induce cognitive impairment, followed the cerebral glucose metabolism disorder, the central cholinergic system which is closely related with memory decline, the central cholinergic system is also an important target of AD pathological damage. In addition, AD characteristic with cognitive impairment and dysfunction is often accompanied with cerebral glucose metabolism disorder. There is a very close relationship between the generation and secretion of Aβwhich is the key pathogenesis factor in AD with glucose metabolism disorder, they can co-promote each other. Because glucose metabolism disorders (diabetes) and AD has very close relationship, probably they have the same etiological factors and pathological processe. Someone proposed that AD may be a brain-specific neuro-endocrine disease, or known as "Type 3 Diabetes".STZ is an alkylate, which was injected into abdominal cavity can induce diabetes through destructing of pancreaticβcells. So it is used to analyze the pathological process of diabetes. In the STZ-induced diabetes model, neuron injury and AD-like pathological changes have been observed, such as AP formation, Tau protein hyperphosphorylation and cognitive barriers. Further studies found that STZ intraventricular injection could lead to emergence of AD-like pathology and degenerative changes in normal rats, including mitochondrial dysfunction, neuronal death, and cognitive dyfunction. The mechanism may be the impairment or destroy of the insulin signal transduction, the destruction of insulin receptor autophosphorylation and intrinsic tyrosine kinase activity, the increase of phosphorylated tyrosine phosphatase activity. They lead to the disturbance of insulin signal transduction, result in long-lasting brain glucose metabolism disorder, energy production dysfunction, hippocampal choline acetyl transferase activity decrease and oxidative stress response increase, eventually decline the learning,memory and cognition funtions. STZ can interfere insulin signal transduction system, cause disorder of glucose metabolism and form AD-like degenerative diseases. In cultured neuron, whether STZ could induce the same injury?Pioglitazone, one thiazolidinedione (TZD) group insulin sensitizing agent, can bind to and activate the peroxisome proliferator-activated receptor y (peroxisome p roliferatorsactivated recep tors, PPARs), and enhance the cell sensitivity to insulin. In STZ lateral cerebral ventricle injected rats pioglitazone have a protective effect on the damage of spatial memory, hippocampal synaptic structure. While rosiglitazone, another type of thiazolidine dione insulin-sensitizing agent, can obviously improve the learning and memory function of AD rats. De la Monte and his colleagues have found that thiazolidine dione group insulin sensitizing agent may be preventive to AD. Has insulin sensitizing agent pioglitazone also a protective effect on STZ-injuried cortical cells?HN, which was found in the brains of AD patients and composed of 24 amino acid linear peptide, can effectively inhibit a variety of FAD (Family Alzheimer's disease) gene mutations and Aβderivatives-induced neurotoxicity, and is considered as AD-specific neuroprotective peptide. HN can inhibit APP, PS1, PS2 mutation induced neuronal death, antagonize the complete Aβpeptide and Aβfragments induced neurotoxicity, but the mechanism is unclear. With the deeply studing, our laboratory found that HN can antagonize NMDA-induced excitotoxicity and hypoxia-induced cortical neuronal injury, suggested that HN had a broad neuroprotective effect, whether HN has a protective effect on STZ-induced neuron injury?Thus, this study include three parts, we observe:1) the damage of STZ on the cultured cortical neurons; 2) the protect effect of pioglitazone on STZ-induced injury in cultured cortical neurons; 3) the protect effect of HN on STZ-induced injury in cultured cortical neurons. PartⅠThe injury effects of STZ on cultured cortical neurons.We cultured the primary cortical neurons, on the 8th day, different concentrations of STZ solution (1μmol/L,10μmol/L, 100μmol/L, 1000μmol/L) and insulin (15 u/mL) were added into the cell medium.36 hours later, the vitality of cells, the number of living cells and the release of LDH was detected. The results showed:1) STZ significantly decreased cell viability detected by cck-8 assay. STZ(1μmol/L, 10μmol/L, 100μmol/L, lmmol/L) decreased the cell viability by18.88%,27.74%,37.09%,40.88% respectively (P<0.01, n=4), compared to the control; 2) STZ (1μmol/L, 10μmol/L, 100μmol/L, lmmol/L) reduced living cells detected by calcein-AM staining. (3) LDH (lactate dehydrogenase) assay showed that compared to the control group, STZ (1μmol/L, 10μmol/L, 100μmol/L, 1mmol/L) significantly increased the LDH release by 25.23%,35.66%,46.87%,50.52% respectively (P＜0.01, n=4).PartⅡThe protective effects of pioglitazone on STZ-induced injury in cultured cortical neuronsBased on the previous experiments, STZ (100μmol/L) were used to observe the protective role of pioglitazone and HN in the following experiments. In addition, insulin was conventional group because there is no statistical difference between insulin and control group. The exprimental results of pioglitazone group found that:(1) the test of CCK-8, when the nerve cell was cultured for 7 days, different concentrations of pioglitazone and insulin was added,24 hours later adding STZ (100μmol/L),36 hours later to observe the changes in nerve cell activity. Compared with the control group, cell viability of STZ group decreased significantly, and it decreased from(control group)100%to 63.93%(P<0.01, n=4); preincubated with pioglitazone (0.01μmol) for 24 hour, cell activity increased by 12.83%(P＜0.01, n= 4); pioglitazone (0.1μmol/L) cell activity increased by 6.06%(P<0.05, n=4); pioglitazone (1μmol/L and 10μmol/L) could not inhibit the cell viability decreased by STZ (P＞0.05). (2) Calcein-AM staining showed that STZ induced the number of living cells reduced significantly, accompanied by sharply reduction of the number of apophysis; adding pioglitazone (0.01μmol/L)in advance significantly inhibited the STZ induced reduction in the number of living cells and a marked increase in the number of apophysis. So did pioglitazone (0.1μmol/L), rather than pioglitazone (1μmol/L, 10μmol/L). (3) Compared with the control group, LDH release of STZ group were significantly increased, and it increased by 42.31%(P＜0.01, n= 4); pioglitazone (0.01μmol/L) can reduced cell injury induced by STZ, (P＜0.01, n= 4); pioglitazone (O.1μmol/L) can be reduced with STZ induced cell injury, and the release of LDH reduced by 10.04%(P＜0.05, n= 4); other than pioglitazone (1μmo/L and 10μmol/L).PartⅢThe protective effects of HN on STZ-induced injury in cultured cortical neuronsBased on the previous experiments, we observe whether HN played a protection role on STZ-induced injury in cultured cortical neurons (STZ 100μmol/L). The results showed that:(1) the test of CCK-8, when the nerve cell was cultured for 7 days, different concentrations of HN (0.1,1,10,100μmol/L) and insulin was added,16 hours later adding STZ (100μmol/L),36 hours later to observe the changes in nerve cell activity. Compared with the control group, cell viability of STZ group decreased significantly, and it decreased from 100%(control group) to 45.56%(P＜0.01, n=4); HN (0.1μmol) could not inhibit the reduction of cell viability induced by STZ (P＞0.05). The HN (0.1μmol) could not inhibit the reduction of cell viability induced by STZ (P>0.05), but HN (1μmol) can be antagonized cell injury induced by STZ, and cell viability was increased by 28.47%(P＜0.05, n=4), and also HN (10μmol, 100μmol) cell activity increased by 48.47%,53.04% respectively (P＜0.01, n=4). (2) Calcein-AM staining, adding HN (0.1μmol/L) in advance could not inhibit reduction in the number of living cells induced by STZ; but adding HN (1μmol/L) in advance can reduce the STZ induced reduction in the number of living cells and increasement of the number of synaptic. And the phenomenon of HN (10μmol/L, 100μmol/L) was more obviously. (3)Compared with the control group, LDH release of STZ group were significantly increased, and it increased by 52.75%(P<0.01, n= 4), HN (0.1μmol) did not inhibit cell injury induced by STZ, so that the release of LDH decrease by 9.36%(P> 0.05, n=4); HN (1μmol) can reduced cell injury induced by STZ, while the release of LDH reduced by 18.76%(P<0.05, n=4); HN (10μmol, 100μmol) can significantly reduce the cell damage induced by STZ, and also the release LDH reduce by 21.05%, and 23.65% respectively(P＜0.01, n= 4).Conclusion:1. STZ plays a role in nerve injury to nerve cells in vitro which was similar to intraventricular injection. 2. Pioglitazone antagonized STZ-induced cultured nerve cell injury and play a neuroprotective role.3. HN antagonized STZ-induced cultured nerve cell injury and play a neuroprotective role.