Effect Of Hydrogen Sulfide On Pathogenesis Of The Central Presbycusis And Its Correlation With The PI3K/AKT And CaMKKβ/AMPK Signal Pathways

Part I Effects of H2S on the pathogenesis of central presbycusis in D-gal induced mimetic aging ratsObjective:To study the effect of hydrogen sulfide on the pathogenesis of central presbycusis on the basis of D-gal induced mimetic aging rats.Methods:SD rats of 1 month old were grouped and treated as follows:①mimetic aging group:D-gal was injected subcutaneously for 8 weeks(500mg/kg/day)。Three subgroups were divided after the mimetic aging rats were established,one injected intraperitoneally with NaHS(dissolved in normal saline)for 10 days as the schedule of 1.4mg/kg/day(group of mimetic aging +NaHS in 3 months old);one injected with normal saline as the same schedule(group of mimetic aging in 3 months old);one keep on feeding for another 6 months followed by divided into another two subgroups:ⅰ)intraperitoneally injected with NaHS for continuous 10 days as the schedule of 1.4mg/kg/day(group of mimetic aging+NaHS in 9 months old);ⅱ)intraperitoneally injected with NaHS as the same schedule(group of mimetic aging in 9 months old).②Control group:injected with normal saline subcutaneously as the same schedule as the mimetic aging group.Eight weeks later,divided into 2 subgroups:one injected intraperitoneally with normal saline for 10 days(group of control in 3 months old);the other group keep on feeding for 6 months followed by injected intraperitoneally with normal saline(group of control in 9 months old).Utilizing western blot to determine the phosphorylation level of PI3K/AKT and AMPK in auditory cortex;determine the expression level of GRP78、lamin A/C、lamin B1 in auditory cortex,which is closely related with oxidative stress;determine the antioxidants of PRDX-2 and TXNRD1;determine the mitochondrial base excise repair enzymes of DNA POLG and OGG-1;determine the oxphos complexes and hsp90a in auditory cortex.Colorimetry was used to determine the content of GSH and MDA,the activity of SOD.By utilizing PCR to determine the occurrence of CD.Using IHC to determine the expression of Caspase-3 and GFAP.By using Nissl staining to counting the number of neurons in auditory cortex.Results:As suggested by western blot,the PI3K/AKT and AMPK pathway in 3-month-old mimetic aging group showed an increased level of phosphorylation compared with in the corresponding controls.However,these changes were not statistically significant in the 9-month-old rats.In the NaHS treatment group,phosphorylation level of PI3K/AKT and AMPK pathway increased significantly.The mimetic aging rats of 3-month-old and 9-month-old showed an increased level of GRP78、laminA/C and laminB1 compared with the corresponding controls and NaHS reversed these changes.Expression of PRDX-2 and TXNRD1 were declined in the mimetic aging rats of 3-month-old and 9-month-old and were revrsed by NaHS treatment.DNA POLG,OGG-1 in 3 months expressed in mild higher level than the mimetic aging group but with no statistical difference.In the 9-month-old,the mimetic aging group exhibited a decreased level of DNA POLG and OGG-1 than the corresponding control group,the differences were statistically significant.Expression of mitochondrial respiratory chains is increased in the 3-month-old mimetic aging group as compared with the control rats and was declined by NaHS treatment,especially in the complex Ⅰ and complex Ⅱ.In the mimetic aging group of 9-month-old rats,expression of mitochondrial respiratory chains is decreased slightly and not showed a statistically significance.Expression of HSP90a is upregulated in the mimetic aging group of 3-month-old rats while downregulated in the 9-month-old mimetic aging group,NaHS increased the expression of HSP90a in both 3-month-old mimetic aging rats and 9-month-old mimetic aging rats.The contents of GSH and activity of SOD in mimetic aging were decreased as compared with the corresponding controls and were reversed by NaHS treatment.The contents of MDA in mimetic aging group of 3-month-old and 9-month-old were increased as compared with the control group,NaHS treatment decreased the change significantly in 3-month-old rats and slightly in 9-month-old rats.The occurrence of CD in mimetic aging rats of 3-month-old and 9-month-old were increased as compared with the corresponding control groups,NaHS reversed this change in 3-month-old rats but not in 9-month-old rats.Expression of caspase-3 and GFAP were increased in the mimetic aging group of 3-month-old and 9-month-old and decreased by the treatment of NaHS.As indicated by Nissl staining,the neuron number in auditory cortex in mimetic aging rats of 3-month-old and 9-month-old rats were decreased but not statistically changed by treatment of NaHS.Conclusion:The level of oxidative stress in D-gal induced mimetic aging rats is increased as aging develops.mitochonria function and repairment as well as the ability of antioxidation and protein homeostasis are declined in the mimetic aging group.These changes are reversed by NaHS treatment.Thus,NaHS may be a potential drugs in preventing the process of aging and age-related disease.Partn Ⅱ The role of H2S in preventing the process of central presbycusis in vitro and its correlation with PI3K/AKT and CaMKKβ/AMPK signal pathway.Objective:To explore the effects of hydrogen sulfide on the aging process of neurons in auditory cortex and its relationship with PI3K/AKT and CaMKKβ/AMPK pathway.Methods:Neurons of auditory cortex from SD rats within postnatal 2 days were primary cultured.Six groups were divided as follows:control group;senescent group,senescent cellular model induced by D-gal;NaHS group,senescent neurons treated with NaHS;LY group,the senescent cells were pretreated with LY294002 followed by treated with NaHS;CC group,the senescent cells were pretreated with Compound C followed by treated with NaHS;STO group,the senescent cells were pretreated with STO 609 followed by treated with NaHS.P-galactosidase staining was used to evaluate the senescence of neurons,PCR was used to determine the occurrence of CD.Western blot was used to detected the phosphorylation level of PI3K/AKT and AMPK,to detect the expression of GRP78,lamin A/C,lamin B1,PRDX2,TXNRD1,DNA POLG,OGG1,OXPHOS and HSP90a in cultured neurons.The contents of GSH,the activity of SOD and CAT were evaluated by colorimetric.ROS was detected by probe.Flow cytometry was utilized to determine the apoptosis and mitochondrial membrane potential.Chemiluminescence was used to evaluate the production of ATP.Results:As indicated by β-galactosidase staining,D-gal induced senescent cells showed more positive cells than the control and were increased as the D-gal concentration augmented.The CD were in a higher level in senescent group than the control but was reversed slightly in the NaHS treated group and not showed a statistical significant.Phosphorylation level of PI3K/AKT and AMPK is higher in D-gal induced senescent cells than the control and NaHS promoted the phosphorylation level in a further.After treated with LY294002,Compound C,STO 609,NaHS not promoted the phosphorylation level of PI3K/AKT and AMPK.GRP78,laminA/C and laminB1 as well as the production of ROS were arised in the senescent neurons as compared with the control and were reversed by NaHS.This reversion could not be observed in the groups inhibited by LY294002,Compound C,and STO 609.The expression of PRDX2 and TXNRD1 were declined in the senescent cells as compared with the control and were upregulated by NaHS treatment.This upregulation could not be observed in the groups inhibited by LY294002,Compound C,and STO 609.DNA POLG,OGG-1 expressed in a higher level in the D-gal treated group than the control group and NaHS increased the expression in a further.The increasement was not observed in the LY294002,Compound C,and STO 609 pretreated groups.The expression of OXPHOS is in a higher level in the senescent neurons and was decreased by NaHS,especially complex Ⅰ,Ⅱ and Ⅳ.This decreasement was inhibited in the groups pretreated with LY294002,Compound C,and STO 609,especially complex Ⅰ and Ⅱ.Expression of HSP90a is upregulated in the D-gal treated group and increased in a further in the group treated with NaHS.Likewise,in the groups preated with LY294002,Compound C,STO 609 the increasement was not observed.Contents of GSH and activity of SOD,CAT were decreased in the senescent neurons and reversed by NaHS.This reversion could not be observed in the groups inhibited by LY294002,Compound C,and STO 609.Abnormal mitochondria and apoptosis were increased in the senescent group and were decreased by NaHS treatment,this decrasement was precluded by PI3K,AMPK and CaMKKβ inhibition.ATP production was declined in the D-gal induced senescent group and was increased by NaHS treatment.Likely,in the groups preated with LY294002,Compound C,STO 609 the increasement was not observed.Conclusion:Extrogenous H2S is able to promote the capability of antioxidation,increase the expression of mitochondrial base excision enzymes,protect the mitochondrial biogenesis,decrease oxidative stress induced damage and attenuate neuronal apoptosis.Activation of PI3K/AKT and CaMKKβ/AMPK is involved in this effect.Thus,PI3K/AKT and CaMKKβ/AMPK signal pathway mediated the beneficial effect carried by NaHS.