The Influence To PI3K/Akt Signal Pathway And Hsp70 In Hippocampal Tissue Of Rats With Chronic Manganese Sulfate Exposure

Objective By establishing the rat model with chronic manganese sufate (MnS04) exposure, we observed the level of oxidation stress index and Hsp70 content in plasma, the expression quantity of Hsp70 and apoptosis related gene in the hippocampus tissue. We also observed the expression and phosphorylation quantity of PI3K/Akt signaling pathway related proteins and the Hsp70 protein expression to explore the mechanisms about inducing the nerve cells apoptosis and effects on PI3K/Akt signaling pathway. Meanwhile, we may discuss if Hsp70 could be redarded to be the biomarker during the chronic MnSO4 exposure.Methods Choose 40 healthy SPF male SD rats and divide into four groups at random named blank control group, low dose group, middle dose group and high dose group, which raising 10 in each group in cages with free drinking and feeding. The four groups are individually intraperitoneal injected with physiological saline and 5 mg/kg,10 mg/kg,20 mg/kg of MnSO4·H2O solution for 24 weeks with 5 times one week and no injection in weekend. After the 24 weeks exposure, we weight them and kille the rats with cervical dislocation method, collecte the samples and calculate the organ coefficient. Detect the plasma SOD, GSH-Px, CAT and MDA levels of the rats with the dedicated kits. The plasma Hsp70 levels are detected with the ELISA kit and all the kits are from Nanjing Jiancheng Bioengineering Institute. Separate the hippocampus tissue and test the expression levels of Caspase-3, Bcl-2, Box, Akt-1, FoxO3a, and Hsp70 mRNA with fluorogenic quantitative PCR. Test the expresstion levels of Hsp70, Akt and p-Akt protein with Western Blot.Results(1) With the increase of manganese sulfate infected dose, the rat heart, liver, spleen, lungs and kidney viscera coefficient is on the rise, testicular viscera coefficient decreased significantly (P<0.05). In high dose group, the heart, liver, spleen, lungs and kidney viscera coefficient are significantly higher than control group and low dose group (P<0.05), testicular viscera coefficient is significantly lower than the other three groups (P<0.05). In middle dose group and low dose group, the spleen viscera coefficient is significantly higher than control group (P<0.05).(2) After the chronic MnSO4 infection, the plasma SOD, GSH-Px and CAT levels of rats in high dose group are (135.46±35.41) U/ml, (845.81±26.06) U and (3.06±0.42) U/ml for respectively, all of them are significantly lower than control group (P<0.01) and the low dose group (P<0.05). In high dose group, the plasma MDA concentration is (3.05±0.37) nmol/L, which is significantly higher than (2.53±0.21) nmol/L in control group (P<0.01) and (2.61±0.35) nmol/L in low dose group (P<0.05). The plasma Hsp70 content is (1020.50±134.86) ng/ml in high dose group, which is significantly higher than (807.15±160.23) ng/ml in control group (P<0.05) and (830.64±161.02) ng/ml in low dose group (P<0.05).(3) The expression levels of Akt-1 and FoxO3α mRNA in hippocampus decreases with the increasing infected dose (P<0.05). The expression levels of Hsp70 mRNA in high dose group is significantly higher than the other groups (P<0.05). In high dose group, the expression levels of Caspase-3 and Bcl-2 mRNA is significantly lower than the low dose group (P<0.05) and Box mRNA is significantly higher than the control group and low dose group (P<0.05).(4) Results of Western Blot experiments show that, with the increase of dose of MnSO4 infection, the expression levels of Akt protein don’t have statistical differences in four groups and the levels of Akt (pS473) protein is significantly on the rise (P<0.05). The expression level of Hsp70 protein in high dose group is significantly higher than control group (P<0.05) and low dose group (P<0.05).(5) The results of Pearson correlation analysis show that the plasma Hsp70 levels were negatively associated with plasma SOD, CAT and GSH-Px levels, correlation coefficient are r1=-0.4439, r2=-0.4516, r3=-0.5226 respectively (P<0.05). The plasma Hsp70 levels were positively associated with plasma MDA levels (r4=0.4785, P<0.05). Plasma Hsp70 levels were positively associated with hippocampal Hsp70 levels (r5=7415, P<0.05).Conclusion(1) Chronic manganese sulfate exposure may cause the decreasing of in vivo antioxidant ability, elevated levels of lipid peroxidation, resulting in increased cell injury and apoptosis.(2) Chronic manganese sulfate exposure may activate PI3K/Akt pathway and regulate FoxO3a phosphorylation which is one of transcription factors and induce Hsp70 expression increased in hippocampal cells at the same time.(3) Chronic manganese sulfate exposure may increase the hippocampal Hsp70 protein levels which was positively associated with plasma Hsp70 levels. It suggestes that Hsp70 may be considered as one biological effective biomarker in chronic MnSO4 exposure.