| ObjectiveStudies have shown that hippocampus are damaged and secreted a large a mount of glucocorticoid in long-term chronic stress by HPA hypertrophy.Aut ophagy that is a self-protection mechanism under external stimulation is c rucial to damaged neuronal cells,then overactivation of autophagy will lead to internal environment disorder,impairment of its function and even deat h.Sinisan that comes from Treatise on Febrile Diseaseand has the effect of reducing depression and is composed of chaihu,zhishi.baishao,zhigancao.Pre-studies in our group has proved that Sinisan can improve depression-like behavior in chronic stress rats.Therefore,The primary hippocampal neuro ns were cultured and were used to establish chronic stress cells model by 200μM corticosterone.Modern experimental techniques such as Western-blot,PCR and immunofluorescence were used to detect proteins or gene to discuss the mechanism of chronic stress,the mechanism of Sinisan to provide a new theore tical basis for the treatment of chronic stress by traditional Chinese medi cine.Methods1.According to the relevant papers,the primary hippocampus neurons were cultured and observed under the microscope in 4h,24h,48h,5-7d on the 7th day,the primary hippocampus neurons were identified by immunofluorescence.2.CCK-8 was used to detect survival rates of hippocampal neuron cells at different concentrations of corticosterone(0μM,5μM,10μM,50μM,100μM,200μM,300μM,400μM,600μM,800μM)and at different time periods(6h,12h,24h,48h;6 groups were divided according to different concentrations of Corticosteo ne:0μM,10μM,50μM,100μM,200μM,400μM.Western-blot was used to detect the ex-pression of LC3-Ⅱ/LC3-I,Beclinl,ULK1,PI3K,p-Akt,p-mTOR,p-4E-BP1,p-70s pro teins to screen out optimal concentration of corticosterone.3.In order to screen out optimal serum concentrations,Western-blot and Real-time PCR was used to detect the expression of LC3-Ⅱ/LC3-Ⅰ,Beclin 1,ULK1,PI3K proteins and genes in the control group,the model group,10%con trol-containing serum group,10%sinisan-containing serum group,20%control-containing serum group,20%sinisan-containing serum group,30%control-con-taining serum group,30%sinisan-containing serum group.Immunofluorescence was used to detect the expression of LC3 protein in the above group s.Wester n-blot was used to detect the expression of LC3-Ⅱ/LC3-Ⅰ,Beclinl,ULK1 prot eins in 20%control-containing serum group,20%sinisan containing serum gro up,20%control-containing serum group+3-ma,20%sinisan-containing serum gro up+3-ma。4.Western-blot and Real-time PCR was used to detect the expression of PI3K,p-Akt,p-mTOR,p-4E-BP1,p-70s in the control group,the model group,10%control-containing serum group,10%sinisan-containing serum group,20%con-trol-contai ning serum group,20%sinisan-containing serum group,30%con-trol-containing serum group,30%sinisan-containing serum group.Western-bl lot was used to detect the expression of p-mTOR,p-4E-BP1,p-70s in 20%cont rol-containing serum group,20%sinisan-containing serum group,20%control-containing serum group+rapamycin,20%sinisan-containing serum group+rapam ycin and 20%control-containing serum group,20%sinisan-containing serum group,20%control-containing serum group+siRNA-NC,20%control-containing serum group+siRNA-mTOR,20%sinisan-containing serum group+siRNA-mTOR.CCK-8 was used to detect survival rates of hippocampal neuron cells in 20%control-containing serum group,20%sinisan-containing serum group,20%control-containing serum group+3-ma,20%sinisan-containing serum group+3-ma,20%control-containing serum+rapamycin,20%sinisan-containing serum+rapamycin.Results1.the results of morphological observation of primary hippocampal neu-ronal cells in newborn SD rats:After 4h of cultured cells,cells became roun d,elliptic,or spindle,grew small synapses and began to attach walls;After 24h of cultured cells,the hippocampus neuron cell were larger,and its morpho logy changed from circle to triangle,shuttle shape.The number and length o of synapses were increased;After 48h of cultured cells,cells are refractive and synapses grow further,synapses were connected to form a network.After 5-7d of cultured cells,the boundary of cells is clear,the refractive effect of cells was strong.Synaptic interconnection formed a dense network structu re.There were a certain aggregation of cells.The obvious nucleus were observ ed under high magnification mirror,the cytoplasm was uniform and consistent t,and the three-dimensional sense was strong;2.immunefluorescence identification of the primary hippocampal neurons in newborn SD rats:Hippocampal neurons were plump and synapses were connect ed to form a network,which was consistent with morphological images.Their cell purity is more than 90%;3.The effect of different concentrations of corticosterone on the sur-+vival rates of hippocampal neurons:When hippocampal neurons were cultured by corticosterone for 6h,compared with 0μM group,the cell survival rates o f hippocampal neurons were descresed in 100μM,200μM,300μM,400μM,600μM,800μM group(P<0.01);When hippocampal neurons were cultured by cortices terone for 12h,compared with 0μM group,the cell survival rates of hippocampal neur ons were descresed in 800μM group(P<0.01);When hippocampal neurons were cu ltured by corticosterone for 24h,compared with 0μM group,the cell survival rates of hippocampal neurons were incresed in 5μM,10μM group(P<0.01);the cell survival rates of hippocampal neurons were descresed in100μM,200μM,300μM,400μM,600μM,800μM group(P<0.01);When hippocampal neurons were cultured by corticosterone for 48h,compared with 0μM group,the cell su rvival rates of hippocampal neurons were incresed in 5μM group(P<0.01);the cell survival rates of hippocampal neurons were descresed in 50μM,100μM,200μM,300μM,400μM,600μM,800μM group(P<0.01);4.The effects of different concentrations of corticosterone on autoph-agy proteins in hippocampus neurons:Compared with 0μM group,the expression levels of Beclinl,LC3-II/LC3-I,ULK1 protein were increased in 10μM,50μM,100μM,200μM,400μM group(P<0.01);5.The effects of different concentrations of corticosterone on the PI3K/Akt/mTOR pathway proteins in hippocampal neurons:Compared with 0μM group,the expression levels of PI3K,p-Akt,p-70s,p-4E-BP1,p-mTOR were increased in 10μM group(P<0.01;P<0.01;P<0.01;P<0.01;P<0.05);The expression levels of PI3K,p-Akt,p-70s,p-mTOR(p<0.01;P<0.05;P<0.01;P<0.05)were increased and p-4E-BP1(P<0.01)were decreased in 50μM group.The expression levels of PI3K,p-Akt,p-4E-BPl,p-mTOR were decreased in 100μM group(P<0.01;P<0.01;P<0.01;P<0.05);The expression levels of PI3K,p-Akt,p-4E-BP1,p-mTO R,p-70s were dec reased in 200μM and 400μM group(P<0.01);6.The effect of different concentrations of sinisan-containing serum on the survival rates of hippocampal neurons in 200μM corticosterone:Compa red with the control group,the survival rates of hippocampal neurons were decreased in the model group(P<0.01);Compared with the corresponding con-centration of control-containing serum groups,the survival rates of hippo-campal neurons were increased in sinisan-containing serum(10%,20%,30%)gro ups(P<0.05;P<0.01;P<0.05);7.The effect of different concentrations of sinisan-containing serum on the autophagy proteins of hippocampus neurons in 200μM corticosterone:Co mpared with the control group,the expression levels of Beclinl,LC3-Ⅱ/LC3-Ⅰ,ULK1 protein were increased in the model group(P<0.01);Compared with the corresponding concentration of control-containing serum groups,the expres-sion levels of Beclinl,LC3-Ⅱ/LC3-Ⅰ,ULK1 protein were decreased in sinisan-containing serum(10%,20%,30%)groups(P<0.01);8.The effect of different concentrations of sinisan-containing serum on the autophagy mRNA of hippocampus neurons in 200μM corticosterone:Com-pared with the control group,the expression levels of Beclinl,LC3,ULK1 mRNA were increased in the model group(P<0.05;P<0.01;P<0.05);Compared with 10%control-containing serum group,the expression levels of Beclinl mRNA were decreased in(P<0.01);Compared with 20%control-containing serum group,the expression levels of LC3,Beclinl mRNA were decreased in 20%sinisan-containing serum group(P<0.01);Compared with 30%control-containing serum group,the expression levels of Beclinl mRNA were decreased in 30%sinisan-containing serum group(P<0.01);9.The effect of different concentrations of sinisan-containing serum on the density of LC3 spots of hippocampus neurons in 200μM corticosterone:Compared with the control group,the density of LC3 spots was increased in the model group(P<0.01);Compared with 20%,30%control-containing serum grou ps,the density of LC3 spots was decreased in 20%,30%sinisan-containing ser um groups(P<0.01);10.The effect of 3-ma mediated 20%sinisan-containing serum on the auto phagy protein of hippocampus neurons in 200μM corticosterone:Compared with 20%control-containing serum group,the expression levels of Beclinl,LC3-Ⅱ/LC3-Ⅰ,ULK1 protein were decreased in 20%sinisan-containing serum group(P<0.01)and 20%control-containing serum group+3-ma(P<0.01;P<0.05;P<0.01);Compared with 20%control-containing serum group+3-ma,the expression levels of ULK1 protein were decreased in 20%sinisa control-containing serum group+3-ma(P<0.01);11.The effect of different concentrations of sinisan-containing serum on the PI3K/Akt/mTOR pathways protein of hippocampus neurons in 200μM cor-ticosterone:Compared with the control group,the expression levels of PI3K,p-Akt,p-70s,p-4E-BP1 protein were decreased in the model group(P<0.01);Comp ared with 10%control-containing serum group,the expression levels of PI3K,p-Akt,p-mTOR,p-70s,p-4E-BP1 protein were increased in 10%sinisan-conaining serum group(P<0.01);Compared with 20%control-containing serum gro up,the expression levels of PI3K,p-Akt,p-mTOR,p-70s,p-4E-BP1 protein were increase d in 20%sinisan-containing serum group(P<0.01);Compared with 30%control-containing serum group,the expression levels of p-mTOR,p-70s,p-4E-BP1 pro-tein were increased in 30%sinisan-containing serum group(P<0.05;P<0.01;P<0.01);12.The effect of different concentrations of sinisan-containing serum on the P3IK/Akt/mTOR pathways mRNA of hippocampus neurons in 200μM cortico sterone:Compared with the control group,the expression levels of Akt,mTOR,70s,4E-BP1 mRNA were decreased in the model group(P<0.01);Compared with 10%control-containing serum group,the expression levels of mTOR mRNA were incr eased in 10%sinisan-containing serum group(P<0.01);Compared with 20%con-trol-containing serum group,the expression levels of Akt,mTOR,4E-BP1 mRNA were increased in 20%sinisan-containing serum group(P<0.01);Compared with 30%control-containing serum group,the expression levels of mTOR,70s,4E-BP1,Akt mRNA were increased in 30%sinisan-containing serum group(P<0.01;P<0.01;P<0.01:P<0.05);13.The effect of rapamycin mediated 20%sinisan-containing serum on the P3IK/Akt/mTOR pathways protein of hippocampus neurons in 200μM corticoster ne:Compared with 20%control-containing serum group,the expression levels of p-mTOR,p-70s,p-4E-BP1 protein were increased in 20%sinisan-containing serum group(P<0.01)and decresed in 20%control-containing serum+rapamy-cin(P<0.01);Compared with 20%sinisan-containing serum group,the expression levels of p-mTOR,p-70s,p-4E-BP1 protein were decreased in 20%sinisan-conta ining serum group+rapamycin(P<0.01);14.The effect of siRNA-mTOR mediated 20%sinisan-containing serum on the P3IK/Akt/mTOR pathways protein of hippocampus neurons in 200μM cortico sterone:Compared with 20%control-containing serum group,the expression lev els of p-mTOR,p-70s,p-4E-BP1 protein were increased in 20%sinisan-contain ing serum group(P<0.01)and decresed in 20%control-containing serum group+siRNA-mTOR(P<0.01);Compared with 20%sinisan-containing serum group,the ex-pression levels of p-mTOR,p-70s,p-4E-BP1 protein were decreased in 20%sini san-containing serum group+siRNA-mTOR(P<0.01);15.The effect of 3-ma/rapamycin mediated 20%sinisan-containing serum on the survival rates of hippocampus neurons in 200μM corticosterone:Compa red with 20%control-containing serum group,the survival rates of hippocam pus neurons were increased in 20%sinisan-containing serum group(P<0.05);Compared with 20%sinisan-containing serum group,the survival rates of hipp ocampus neurons were decreased 20%sinisan-containing serum group+rapamy-cin(P<0.05);Compared with 20%control-containing serum group+3-ma,the sur-vival rates of hippocampus neurons were increased in 20%sinisan-containing serum group+3-ma(P<0.01);Compared with 20%control-containing serum group+rapamycin,the survival rates of hippocampus neurons were increased in 20%sinisan-containing serum group+rapamycin(P<0.01).Conclusion1.The survival rates of hippocampal neurons were increased by corti-costerone less than 50μM,and decreased between 50-800μM with the increase of time and concentration of corticosterone;2.200μM corticosterone inhibits the P3IK/Akt/mTOR signal pathway,acti-vat es autophagy on the primary hippocampus neurons;3.The sinisan-containing serum increases the survival rates of hippo-campal neurons;4.The sinisan-containing serum inhibits the excessive autophagy of hip-pocampal neurons;5.The sinisan-containing serum activates the P3IK/Akt/mTOR signal path-ways of hippocampal neurons by 200μM corticosterone. |
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