| Objective:Utilizing hydrogen peroxide(H2O2)to induce injury in PC 12 cell models,simulating the pathological process of oxidative stress after ischemic stroke,and investigating the protective effect and possible mechanism of Mongolian medicine Zhachong Shisanwei Pill(ZSP)against oxidative stress injury in neurons after ischemic stroke by detecting cell viability,oxidative stress indicators,cell apoptosis,and key proteins in the MAPK pathway,providing experimental evidence for elucidating the scientific connotation of this prescription.Methods:(1)Preparation of oxidative injury model in PC 12 cells with H2O2.The CCK-8 method was used to detect the effects of different concentrations of H2O2(100 μM,200 μM,300 μM,400 μM,500 μM,600 μM,800 μM)on PC 12 cell viability,and an appropriate concentration of H2O2 was selected to establish an in vitro cell model for oxidative stress after ischemic stroke.(2)Preparation of ZSP-containing serum.Twenty male SD rats were randomly divided into a blank group and low,medium,and high dose ZSP groups.The medium dose ZSP group was gavaged with an equivalent dose for adults(70 kg),the low dose ZSP group with half the equivalent dose,and the high dose group with twice the equivalent dose.The blank group was given the same volume of purified water,and ZSP-containing serum was prepared after 7 consecutive days of administration.(3)Optimal concentration screening of ZSP-containing serum.Serum from the blank group and low,medium,and high dose ZSP groups were prepared at different concentrations(2%,5%,8%,and 10%)and applied to PC 12 cells for 24 hours.Cell proliferation toxicity was detected by the CCK8 method,and the most suitable drug concentration was selected.(4)The protective and antioxidant effects of ZSP-containing serum pretreatment on H2O2-induced injury in PC 12 cells were investigated.PC 12 cells were divided into control group(Control),model group(Model),low dose ZSP-containing serum group(L-ZSP),medium dose ZSP-containing serum group(M-ZSP),and high dose ZSP-containing serum group(H-ZSP).After 12 hours of intervention with ZSP-containing serum(5%concentration),H2O2(400μM)was used to induce injury for 1 hour.Cell morphology was observed under a microscope,cell survival rate was detected by the CCK-8 method,lactate dehydrogenase(LDH)activity was detected by colorimetry,reactive oxygen species(ROS)content was detected by flow cytometry,superoxide dismutase(SOD)activity was detected by the WST-1 method,malondialdehyde(MDA)content was detected by the TBA method,and catalase(CAT)activity was detected by the ammonium molybdate method.(5)The effects of ZSP-containing serum on H2O2-induced PC 12 cell apoptosis and the MAPK signaling pathway were investigated.Cell grouping was the same as in(4).Cell apoptosis was detected by Hoechst 33258 fluorescence staining and Annexin V-FITC/PI double staining,mitochondrial membrane potential changes were detected by JC-1 fluorescence probe,and the expression levels of Bcl-2,Bax,Caspase-9,Caspase-3,PARP,and key proteins in the MAPK pathway were detected by the WES automatic protein blot analysis system.Results:(1)Compared with the control group,the survival rate of PC 12 cells treated with different concentrations of H2O2 showed a dose-dependent decrease,with the 400 μM H2O2 concentration demonstrating a significant decrease in cell survival rate.Therefore,400μM H2O2 was selected to establish the oxidative stress injury model in PC 12 cells.(2)No significant cytotoxic effects were observed in PC 12 cells treated with 2%,5%,or 8%serum from the blank group and low,medium,and high dose ZSP groups.However,the 10%serum concentration showed cytotoxic effects,so a 5%serum concentration was selected for subsequent experiments.(3)Morphological observations showed that ZSP-containing serum pretreatment effectively alleviated the morphological changes of H2O2-induced injury in PC 12 cells,and the CCK-8 results showed that ZSP-containing serum significantly increased the survival rate of H2O2-injured PC 12 cells.(4)ZSP-containing serum pretreatment significantly reduced LDH release,ROS production,and MDA content while increasing SOD and CAT activities in H2O2-injured PC 12 cells,indicating that ZSP-containing serum has a protective effect against oxidative stress injury.(5)ZSP-containing serum pretreatment significantly reduced H2O2-induced apoptosis and mitochondrial membrane potential loss in PC 12 cells,and the expression of Bcl-2 increased,while the expression of Bax,Caspase-9,Caspase-3,and PARP decreased.Additionally,ZSP-containing serum pretreatment significantly inhibited the phosphorylation of MAPK pathway-related proteins(p3 8,JNK,and ERK)induced by H2O2,suggesting that ZSP-containing serum may alleviate oxidative stress injury and cell apoptosis by regulating the MAPK signaling pathway.Conclusion:ZSP can effectively protect PC 12 cells from hydrogen peroxide-induced oxidative stress injury and apoptosis by inhibiting the MAPK signaling pathway,suggesting that ZSP may have potential therapeutic effects in the treatment of ischemic stroke. |
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