| Part one The effect and mechanism of sodium hydrosulfide on mitochondrial dynamic in N2 a cellObjective: 1.To examine the effect of hydrogen sulfide on mitochondrial fission and fusion.2.To examine the effect of hydrogen sulfide on cell viability and mitochondrial ATP synthesis.3.To examine the effect of hydrogen sulfide on Drp1 m RNA and protein expression.4.To examine the effect of ERK1/2 signial pathway on hydrogen sulfide induecd mitochondrialfission inhibition.Methods: 1.Using TEM to analysis mitochondrial morphological changes of N2 a cell after treatment with different concentrations of Na HS at different durations.2.The MTT assay was used to evaluate the effect of Na HS on cell viability.3.ATP Bioluminescence Assay Kit was used to meansure ATP levels of N2 a cells treated with differern concentration of Na HS.4.Using QRT-PCR and Western blotting to examine the protein and m RNA expression levels of Drp1 in each group.5.After transfect Drp1 overexpressing lentiviral vector(LV-Drp1)to N2 a cells,Using TEM to analysis mitochondrial morphological changes in Na HS treated,Na HS + LV-Drp1 treated group.6.Using Western blot to meansure P-ERK1/2 and Drp1 proteinlevels in,Na HS treated,Na HS+ERK1/2 inhibitor group.Results: 1.The mitochondrial number per cell was significantly decreased(p<0.01 vs.control),elongated mitochondrial percent was significantly increased(p<0.01 vs.control)in 200,400μM Na HS group.The mitochondrial number per cell was significantly decreased(p<0.01 vs.control),elongated mitochondrial percent was significantly increased(p<0.01 vs.control)in 8h,16 h Na HS group.2.The cell viability was gradually increased in Na HS treatment group.There was a significant difference between 400,600μM Na HS group and the control group(p<0.05).3.The mitochondrial ATP levels was gradually increased in Na HS treatment group.There was a significant difference between 400,600μM Na HS group and the control group(p<0.05).4.The Drp1 protein levels was significantly decreased in 200,400μM Na HS group(p<0.05 vs.control),the Drp1 m RNA levels was significantly decreased in 400μM Na HS group(p<0.05 vs.control).5.Lentivirus can significantly increase the expression of Drp1(p<0.01 vs.control).The mitochondrial number per cell was significantly decreased(p<0.01 vs.control),elongated mitochondrial percent was significantly increased(p<0.01 vs.control)in 400μM Na HS treatment group.The mitochondrial number per cell was significantly decreased(p<0.01 vs.400μMNa HS),elongated mitochondrial percent was significantly increased(p<0.01 vs.400μM Na HS)in 400μM Na HS+LV-Drp1 group.6.400μM Na HS treatment increase the P-ERK1/2 expression levles(p<0.01 vs.control),ERK1/2 inhibitor PD98059 reverse this effect of Na HS(p<0.01 vs.400μM Na HS)in400μM Na HS+50μM PD98059 group.7.400μM Na HS treatment decreased Drp1 protein expression levles(p<0.01 vs.control),ERK1/2 inhibitor PD98059 reverse this effect of Na HS(p<0.01 vs.400μM Na HS)in400μM Na HS+50μM PD98059 group.Conclusion: 1.Na HS can inhibit the mitochondrial fission in an dose and time denpendent manner.2.Na HS can improve the cell viability and mitochondrial ATP generation.3.Na HS may inhibit mitochondrial fission via decrease the Drp1 expression.4.ERK1/2 signial pathway may involved in the Na HS-induced decrease of Drp1 expression and mitochondrial fission.Part two The effect of sodium hydrosulfide on mitochondrial morphology changes and cognitive impairmentof APP/PS1 transgenic miceObjective: 1.To explore whether supplementing hydrogen sulfide would improve cognitive function in APP/PS1 transgenic mice.2.To examine whether there is a corresponding mitochondrial morphology change in cognitive function improved transgenic mice.3.To examine whether there is a relationship between the improvement of cognitive function and the decrease of Drp1 expression.4.Whether Na HS is able to improve the Drp1 abnormal expression in transgenic mice.5.The role of ERK1/2 pathway in the effect of Na HS to Drp1 expression.Methods: 1.10,20,50μmol/kg Na HS was then administered intraperitoneally(i.p.)to 10 month old transgenic mice every 2 days for 2 months,the 10 month old wild type mice as blank control,the 10 month old transgenic mice treated with equal dose of physiologic saline as 14 n egative control group.When the mice were 12 months old,The Morris Water Maze was used to measure the indicators of cognitive function.2.Using TEM to analysis mitochondrial morphological changes in each group of mice.3.Using immunohistochemistry analysis to measure Drp1 expression in the hippocampus of each group.4.Using QRT-PCR and Western blotting to examine the protein and m RNA expression levels of Drp1 in each group of mice.5.Using Western blot to measure P-ERK1/2 and Drp1 protein expression levels after treatment with Na HS and ERK1/2 inhibitor for two month.Results:1.In The Morris Water Maze,there was no significant difference in swimming speed between blank and negative control group;different concentration of Na HS-treated group and the negative control group(p> 0.05).In the place navigation test(PNT),the escape latencies of the negativecontrol group was significantly increased(Days3 p<0.05,Days4 p<0.001,Days 5 p<0.001 vs.blank control group);The swimming distance was significantly increased(Days3 p<0.05,Days4 p<0.01,Days 5 p<0.01 vs.blank control group).50μmol/kg Na HS treatment significantly reduced the escap latencies(Days3 p<0.05,Days4 p<0.001,Days 5 p<0.001 vs.negative control group)and swimming distance(Days3 p<0.05,Days4 p<0.01,Days 5 p<0.01 vs.negative control group).20μmol/kg Na HS treatment also significantly reduced the escap latencies(Days 5 p<0.01 vs.negative control group)and swimming distance(Days4 p<0.05,Days 5 p<0.05 vs.negative control group).In spatial probe test,the negative control group shows a lower percentage of time spent in the target quadrant and less crossing times to the blank control group(p<0.01 vs.blank control group).50μmol/kg Na HS treatment significantly increase the percentage of time spent in the target quadrant and shows less crossing times(p<0.01 vs.negative control group).2.The TEM shows that the number of mitochondria in negative control group was significantly increased(p <0.05 vs.blank control group),and the proportion of elongated mitochondria was significantly reduced(p <0.05 vs.blank control group).The number of mitochondria in 50μmol/kg Na HS treated group was significantly reduced(p<0.01 vs.negative control group),the proportion of elongated mitochondria was significantly increased(p <0.01 vs.negative control group).3.Immunohistochemistry analysis shows increased Drp1 expression in the hoppocampus of negative control group,Na HS treated shows decreased Drp1 expression.4.Negative control group Drp1 m RNA and protein levels were significantly increased(p<0.01 vs.the control group),but in the Na HS treated groups they were significantly decreased,in which 50μmol/kg Na HS treatment group decreased most significantly(p<0.01 vs.the negative control group).5.After 5mg/kg ERK1/2 pathway inhibitor PD98059 lateral cerebral ventricle and 50μmol/kg Na HS intraperitoneal injection for two months,the ERK1/2 phosphorylation level of the negative control group was significantly decreased(p<0.01 vs.the blank control group),Drp1 expression was significantly increased(p <0.01 vs.the blank control group),after 50μmol/kg Na HS treatment,the phosphorylation levels of ERK1/2 was significantly increased(p<0.05 vs.negative control group),Drp1 expression was significantly decreased(p<0.05 vs.negative control group),while giving the PD98059 and Na HS treatment,the phosphorylation levels of ERK1/2 was significantly decreased(p<0.05 vs.50μmol/kg Na HS),Drp1 expression was significantly increased(p<0.05 vs.50μmol/kg Na HS).Conclusion: 1.Na HS can improve the cognitive impairment of 12-month-old transgenic mice.2.There are abnormal increased mitochondrial fission in the transgenic mice,Na HS can suppresses this abnormal changes.3.The expression of Drp1 in transgenic mice was abnormal increased,Na HS inhibit the overexpression of Drp1.4.ERK1/2 signal pathway may involved in the Na HS-induced decrease of Drp1 expression and mitochondrial fission. |
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