| BcakgroundAlzheimer's disease(AD)is a common age-dependent neurodegenerative disease.With the aging of society is more and more serious,the incidence of AD are higher.The pathogenesis of AD is not clear at present.Though the oxidative stress induced by amyloid beta protein(A)is one of the putative virulence factors.Because of the lack of AD treatment drugs in clinical,developing a new AD therapeutic drugs(such as three seven total saponins,PNS)become imperative.Objective1 To investigate the effect and mechanism of A ? 25-35-induced PC12 cells which were treatment by PNS.2 To investigate the effects of PNS on autophagy of APP/PS1 gene stably transfected PC12 cells and its mechanism.Methods1 Protective of PNS of Nrf2 signaling pathway on apoptosis of PC12 cells induced by A ? 25-351.1 Effect of PNS on the cell viability of PC12 cells cells were divided into 3.125,6.25,12.5,25,50and 100mg · L-1 PNS six different concentrations.The effect of PNS on the cell viability of PC12cells was tested at 4,8,12,16,24,48hsix different time points by MTT assay.1.2 Establishment of A? 25-35 cell damage model A?25-35 was divided into 5,10,20and 40 ?mol · L-1four different concentrations.The effect of A? 25-35 on the cell viability of PC12cells was tested at 6,12,24,48h four different time points by MTT assay.1.3 Determination protection condition of PNS cells were divided into control(PC12 cells).model(20 ? mol · L-1A? 25-35)?6.25(6.25 mg·L-1PNS)?12.5(12.5mg·L-1PNS)?25(25 mg · L-1PNS)?50(50 mg · L-1PNS)six different groups.The effect of PNS on the cell viability of PC12cells was tested at0,4,8,12,16,24,48h seven different time points by MTT assay.1.4 Effect of PNS on apoptosis,oxidative stress and cell viability The cells were divided into control group,control treatment group(PC12 cells+25mg · L-1PNS),model group and model treatment group(20 ? mol · L-1A? 25-35+25mg · L-1PNS)· Detect the changes of LDH,MDA,SOD,the leakage rate of CAT and GSH-Px In cell culture medium.TUNEL staining was used to detectcell apoptosis;ROS staining was used to detectcell oxidative stress;JC-1 was used to detect mitochondrial membrane potential;caspase-3 was detected by Elisa.1.5 Effect of PNS on Nrf2/HO-1 signaling pathway Grouping the same1.4.The Nuclear Nrf2?Total Nrf2?HO-1 and Lamin B was detected by western blotting.1.6 Effect of PNS on Nrf2/HO-1 signaling pathway with HO-1 inhibitor The cells were divided into control group,control treatment group,model group,model treatment group and HO-1 inhibitor group.HO-1 was detected by Elisa.2 Study on the effect of PNS on autophagy of APP/PS1 gene stably transfected PC12 cells(APPCs)and its mechanism2.1 Effect of PNS on the cell viability of APPCs PNS was divided into 3.125,6.25,12.5,25,50and 100mg ·L-1 six different concentrations.The effect of PNS on the cell viability of APP/PS1 transgenic bovine PC12cells was tested at 4,8,12,16,24,48hsix different time points by MTT assay.2.2 Effect of PNS on ROS in APPCs PNS was divided into 3.125,6.25,12.5 and 25mg · L-1 four different concentrations.The effect of PNS on ROS in APPCs was tested at 4,8,12,16,24,48h six different time points by ROS staining.2.3 Effect of PNS on mitochondrial membrane potential of APPCs The cells were divided into control group(PC12 cells),control treatment group(PC12cells+12.5mg · L-1 PNS),model group(APPCs)and model treatment group(APPCs+12.5mg · L-1 PNS).Mitochondrial membrane potentialwas detected by JC-lstaining.2.4 Effect of PNS on autophagy of APPCs Grouping the same 2.3.LC3 was detected by immunofluorescence method.LAMP?Beclin-1 and p62 were detected bywestern blotting.2.5 Effect of PNS on autophagy of APPCs with autophagy interference agent The cells were divided into control group,model group,model treatment group and interference agent group(APPCs+12.5mg · L-1PNS+interference agent).p62 was detected by western blotting.2.6 Effect of PNS on AMPK/mTOR and PI3K/mTOR signaling pathway Grouping the same 2.3.AMPK?p-AMPK?PI3K?p-PI3K?Akt?p-Akt?mTOR and p-mTOR were detected by western blotting.2.7 Effect of PNS on AMPK/mTOR and PI3K/mTOR signaling pathway with inhibitor Grouping the same 2.5.p-AMPK and p-PI3K were detected by western blotting.Results1 Protective of PNS of Nrf2 signaling pathway on apoptosis of PC12 cells induced by A ?25-351.1 Results of cell viability:PNS had no significant influence in the concentration of 3:125?6.25?12.5?25 and 50mg · L-1,and PNS can significantly reduced the activity of PC12 cells in the concentration of 100mg·L-1(P<0.01).1.2 Establishment of A ? 25-35 model:the vitality of PC12 cells decreased to 55.4437%of the normal control group,when the model making after 24h with A? 25-35 in the concentration of 20 ? mol · L-1.1.3 Determination of the best protective effect of PNS:The maximum protective effect of PNS was in the concentration of 25 mg · L-1and Pretreatment time 24h.1.4 Results of apoptosis and cellular oxidative stress?Compared with the control group,the cell rate of LDH,the number of TUNEL positive cells and the positive cell rate and the expression ofcaspase-3,ROS and MDA were significantly increased(P<0.01),mitochondrial membrane potential,SOD,CAT,GSH-Px were significantly decreased(P<0.01);compared with the model group,the cell rate of LDH,the number of TUNEL positive cells and the positive cell rate and the expression ofcaspase-3,ROS and MDA in model treatment group were significantlydecreased(P<0.01),mitochondrial membrane potential,SOD,CAT and GSH-Px were significantly increased.1.5 Results of Nrf2/HO-1 signal pathway:Compared with control group,the expression of lamin B and total Nrf2 in each group had no effect(P>0.05),the expression of HO-1 and Nucear Nrf2 in PNS+control group and model treatment group were significantly increased(P<0.01).The results adding HO-1 inhibitor showed that compared withmodel treatment group,the expression of HO-1 in HO-1 inhibitor group was significantly decreased(P<0.01).2 Study on the effect of PNS on autophagy of APP/PS1 gene stably transfected PC12 cells(APPCs)and its mechanism2.1 Results of cell viability:PNS had no significant influence in the concentration of 3.125?6.25?12.5?25mg · L-1 PNS can significantly reduced the activity of PC12 cells in the concentration of 100g · L-1(P<0.01)and in the concentration of 50mg · L-1 Incubatting 24h(P<0.01).2.2 Results of ROS,mitochondrial membrane potential and autophagy related protein:Compared with the control group,the fluorescence intensity of ROS of APPCs,the expression of LC3?Beclin-1 and p62 were significantly increased(P<0.01),the mitochondria membrane potential and the expression of LAMP were significantly decreased(P<0.01);compared with model group,adding PNS with different concentration and time can reduce the fluorescence intensity of ROS(P<0.05,R<0.01),the expression of LC3.Beclin-1 and p62 were significantly decreased(P<0.01),the mitochondria membrane potential and the expression of LAMP were significantly increased(P<0.01).Adding the 3-MA,compared withmodel treatment group,the expression of p62 in 3-MA group was significantly decreased(P<0.01).Adding the rapamycin,compared withmodel treatment group,the expression of p62 in rapamycin group had no obviously change(P>0.05).2.3 Results of AMPK/mTOR and PI3K/mTOR signaling pathway:Compared with the control group,the expression of AMPK?PI3K?Akt and mTOR in each group had no obviously change(P>0.05),the expression of p-AMPK?p-PI3K?p-Akt and p-mTOR had no obviously change(P>0.05).Compared with the model group,the expression of p-AMPK in model treatment group was significantly increased(P<0.01),the expression of p-PI3K?p-Akt and p-mTOR were significantly decreased(P<0.01).After add PI3K inhibitor,Compared with the model treatment group,the expression of p-PI3K in PI3K inhibitor was significantly increased(P<0.01).After add AMPK inhibitor,Compared with the model treatment group,the expression of p-AMPK in AMPK inhibitor had no no obviously change(P>0.05).Conclusion1 The maximum non-toxic dose of PNS to PC12 cells was 50 mg ·L-1.2 PNS pre-incubation can significantly inhibit the A&25-35-induced apoptosis and oxidative stress in PC12 cells.3 PNS pre-incubation can significantly inhibit the A ? 25-35-induced apoptosis and oxidative stress in PC12 cells through Nrf2/HO-1 signaling pathway.4 The maximum non-toxic dose of PNS to APPCs was 25 mg· L-1.5 PNS pre-incubation can significantly inhibit the Mitochondrial membrane potential depolarization in APPCs and increase autophagy.6 PNS pre-incubation can enhance cell autophagy.7 PNS could enhance the autophagy of APPCs through AMPK/mTOR and PI3K/mTOR signaling pathway.In conclusion,PNS has protective effect on injured nerve cells.PNS could adjust the apoptosis and autophagy of nerve cells induced by A ? 25-35-induced oxidative stress,which may through regulatory signaling pathway.PNS may apply to treating AD on clinical. |
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