Effect Of PKCβ/p66shc Of Oxidative Stress Signal Pathway In Injury Of The Central Nervous System Induced By Experiment Fluorosis

Objective To investigate the expression of protein kinase Cβ(PKCβ),peptidyl-prolyl cis-trans isomerase(Pin1), adaptor protein p66 shc and phosphop66 shc in of PKCβ/p66 shc oxidative stress signaling pathway in the brain of rats with chronic fluorosis and SH-SY5 Y cells exposed to excessive amount of fluoride,and to reveal the possible mechanism in nerve injury induced by fluorosis. Methods The animal model with chronic fluorosis was successfully estabolished. Thirty SD rats were divided randomly into three groups of 10 each(a half females and a half males), e.g, the normal control group(less than 0.5 mg/L of fluoride) in drinking water, low fluoride exposure group(10 mg/L fluoride of prepared with NaF), high fluoride exposure group(50 mg/L fluoride). The experiment period was 6 months.The fluoride content in urine was detected by Fluoride-ion selective electrode. The ability of learning and memory of rats was examined by Morris water maze test. The histopathologic observation for the cortices was performed under light microscope.The expression of neurons(employing neuron-specific nuclear antigen, NeuN) in the cortices was detected by immunohistochemistry(IHC). The protein levels of PKCβ,Pin1, p66 shc and phospho-p66 shc of brain tissues were detected by Western blotting.The cellular model of fluorosis was estabolished by exposure of fluoride to SH-SY5 Y cells. The experiments were divided into three groups, e.g. the control group without treatment with fluoride, the fluoride group treated with 500 ?mol/L NaF, and the PKCβ inhibition group with 500 ? mol/L NaF and 0.2 ? mol/L LY333531(PKCβ inhibitor). Cell viability was tested by the assay of CCK-8. The protein levels of PKCβ, Pin1, p66 shc and phospho-p66 shc in SH-SY5 Y cells weredetected by Western blotting. The activity of SOD and the content of MDA were measured by biochemistry method. The level of reactive oxygen species(ROS) were detected by the ROS assay kit. Mitochondrial membrane potential was detected by the mitochondrial membrane potential assay kit. The apoptosis of SH-SY5 Y cells was determined by flow cytometry. Results The results obtained from animal investigation showed thatin the rats exposed to low or high fluoride, the fluorine contents in urine were significantly higher than controls. The escape latency of the rats with chronic fluorosis was siginificantly delayedas compared with that controls.Under the light microscope, no obviously histopathological changes in the cortical regions of the rat brains with fluorosis were observed under the light microscope. In rat brains with low- and high-fluoride exposure as compared with controls, the decreased numbers of neurons stained by NeuN, and the raised expressions of PKCβ,Pin1 and phospho-p66 shc detected by Western blotting were found. The results obtained from cultured SH-SY5 Y cells showed that the cell viability of SH-SY5 Y cells detected by CCK-8 test were significantly decreased at the concentrations of500 μmol/L fluoride and above for 48 hr of incubation with a manner of dose dependence. In the SH-SY5 Y cells exposed to excessive fluoride as compared with the control group, the declined survival rates were detected, while obviously prevented by the treatment of PKCβ inhibitor. In addition, in the group treated with fluoride as compared with controls, the protein expressions of PKCβ, Pin1 and phospho-p66 shc were sigfinicantly increased, which was obviously attenuated by the treatment with PKCβ inhibitor. The SOD activity was significantly lower and MDA content higher in the group exposed by fluoride than those in the control group.Whereas, the changed of SOD and MDA induced by fluoride were prevented by the treatment of PKCβ inhibitor. On the other hand, the levels of ROS and apoptosis rate were significantly increased, while mitochondrial membrane potential was decreased in the cells exposed with fluoride. Interestingly, these changes indicated above were significantly attenuated by the treatment of PKCβ inhibitor. Conclusion The PKCβ/p66 shc oxidative stress signaling pathway is activated in the brains of rats with chronic fluorosis and the cultured neurons exposed to excessive fluoride andleads to cytotoxicity, which can be attenuated by the treatment of PKCβ inhibitor.PKCβ/p66 shc oxidative stress pathway may play an important role in the mechanisms of nerve injury induced by chronic fluorosis.