| ObjectiveHypoxia induced brain damage(BD)is mainly caused by hippocampal neuron injury,with high incidence rate and limited treatment methods,which cannot effectively reduce the risk of death and disability.Astragaloside Ⅳ(ASⅣ),the main active substance of Chinese traditional medicine Astragalus,has a certain protective effect on brain damage,but the mechanism is still unclear.Calpain-1 participates in mediating the damage of hippocampal neurons in brain tissue.Therefore,this experiment mainly explores the protective effect of ASⅣ on hypoxia induced hippocampal neurons in mice and its mechanism of action related to calpain-1.MethodsIn vivo level,C57BL/6 mice and calpain-1 knockout mice were modeled hypoxia brain damage(HBD)using a hypoxia chamber.In this experiment,six groups including 40 C57BL/6 mice were randomly assigned to four treatment groups: blank control(Con group),hypoxia brain damage(HBD group),astragaloside Ⅳ 60 mg/kg(ASⅣ 60 group),astragaloside Ⅳ 120 mg/kg(ASⅣ120 group);twenty knockout mice were randomly divided into two groups,normoxic calpain-1 knockout group(CK group),hypoxic calpain-1 knockout group(MK group).Mice were administered ASⅣ(60 and 120 mg/kg)by oral gavage once daily for 28 days.Detection of oxygen saturation(Sp O2)in mice by oxygen saturation meter.ELISA was used to detect the contents of blood urea nitrogen(BUN),lactate dehydrogenase(LDH),lactate(LD),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione reductase(GSH-px)in the serum of mice.The ability of learning and memory of mice was examined by Morris water maze.Observation of Morphological Changes in the CA1 Region of the Hippocampus by Hematoxylin Eosin(HE)Staining.The degree of neuronal apoptosis in brain tissues was observed by TUNEL staining.ROS content in hippocampal CA1 region was detected by DHE probe.Immunohistochemistry was used to evaluate the expression of caspase-3 and calpain-1 in the hippocampal CA1 region of the mouse brain.The protein levels of caspase-3,Bax,Bcl-2,HIF-1α and calpain-1 in brain tissues were determined by Western blot analysis.In vitro cell experiments,mouse hippocampal neurons(HT22)were cultured in a 3% O2 hypoxia chamber for 24 hours to establish a cell hypoxia damage model.The experiment was divided into 8 groups: blank control group,hypoxia model group(HMG group),MDL-28170 group(MDL group),HMG+MDL group,HMG+ASⅣ 50 μmol/L group,HMG+ASⅣ 100 μmol/L group,p LV-NC group,HMG+ASⅣ+p LV-CAPN1 group.The activity of HT22 cells in each group was detected by CCK-8 method.The content of ROS in HT22 cells was detected by DHE staining.Apoptosis of HT22 cells was measured by flow cytometry.Determination of MDA,SOD and GSH-px in HT22 cells by ELISA method.The changes of mitochondrial membrane potential in HT22 cells were detected by JC-1.The expression of caspase-3 and calpain-1 in HT22 cells was measured by immunofluorescence chemistry.The expression of Bcl-2,Bax,HIF-1α,caspase-3 and calpain-1 in HT22 cells was detected by Western blot.ResultsIn vivo level: compared with the blank control group,the hippocampal neurons in the HBD group of mice were damaged,characterized by a decrease in related biochemical indicators Sp O2,an increase in LDH,BUN,and LD(P<0.05),as well as blurred cell boundaries and disordered arrangement in the CA1 area of the mouse hippocampus;the spatial learning and memory abilities of HBD group mice decreased,manifested as an increase in escape latency and path length,and a decrease in the number of times they crossed the target quadrant(P<0.01);the HBD group mice experienced oxidative stress,characterized by a significant decrease in SOD activity and GSH-px content,a significant increase in MDA content(P<0.01),and an increase in ROS production in the hippocampal CA1 region;the HBD group mice showed apoptosis,while the calpain-1/HIF-1α/caspase-3 pathway was upregulated,manifested by increased expression of caspase-3,HIF-1α,Bax,and calpain-1proteins,decreased expression of Bcl-2 protein(P<0.01),increased number of TUNEL positive cells,and increased expression of caspase-3 in the hippocampal CA1 region.Compared with the HBD group,the damage of hippocampal neurons in the MK and ASⅣ groups of mice was improved,manifested by an increase in related biochemical indicators Sp O2,a decrease in LDH,BUN,and LD(P<0.05),and a clear and relatively neat cell boundary and arrangement in the CA1 area of the mouse hippocampus;the spatial learning and memory abilities of the MK and ASⅣ groups of mice were enhanced,manifested by a decrease in their escape latency and path length,and an increase in the number of times they crossed the target quadrant(P<0.01);the oxidative stress of the MK and ASⅣ groups of mice was improved,manifested by an increase in SOD activity and GSH-px content,a significant decrease in MDA content(P<0.01),and a decrease in ROS production in the hippocampal CA1 region;apoptosis was improved in the MK and ASⅣ groups of mice,while the calpain-1/HIF-1α/caspase-3 pathway was downregulated,characterized by decreased expression of caspase-3,HIF-1α,Bax,and calpain-1 proteins,increased expression of Bcl-2 protein(P<0.01),decreased number of TUNEL positive cells,and decreased expression of caspase-3 in the hippocampal CA1 region.In vitro,compared with the blank control group,HT22 cells in the HMG group were damaged,accompanied by oxidative stress and apoptosis,which were manifested as decreased cell viability,decreased SOD and GSH-px activities,increased MDA content(P<0.01),significantly increased ROS level,decreased mitochondrial membrane potential,increased number of positive apoptotic cells and apoptosis rate in the HMG group,increased expression of caspase-3,Bax,HIF-1α and calpain-1 proteins,and decreased expression of Bcl-2 protein(P<0.01),the fluorescence expression of caspase-3 and calpain-1increased.Compared with the HMG group,the damage of HT22 cells in the HMG+MDL group and the ASⅣ group was improved,accompanied by oxidative stress and improved apoptosis,which were manifested as increased cell viability,increased SOD and GSH-px activities,decreased MDA content(P<0.01),significantly reduced ROS level,significantly improved mitochondrial membrane potential abnormality,decreased the number of positive apoptotic cells and apoptosis rate,decreased caspase-3,Bax,HIF-1α and calpain-1protein expression,and increased Bcl-2 protein expression(P<0.01),the fluorescence expression of caspase-3 and calpain-1 decreased.In CAPN1 lentivirus vector transfection experiment: compared with the HMG group,the HMG+ASⅣ group showed improved cell damage,accompanied by improved oxidative stress and apoptosis,manifested by increased activity of HT22 cells,decreased MDA and increased SOD levels,and down regulated the expression levels of caspase-3 and calpain-1 proteins in HT22 cells(P<0.01).Compared with the HMG+ASⅣ group,the HMG+ASⅣ+p LV-CAPN1 group showed a decrease in HT22 cell viability,an increase in MDA content,a decrease in SOD levels,and an increase in caspase-3 and calpain-1 protein expression(P<0.01).ConclusionASⅣ has protective effect on hypoxia-induced hippocampal neurons in mice,and can improve mice’s learning and memory ability,and lessen oxidative stress and cell apoptosis,which is regulated through calpain-1/HIF-1α/caspase-3 protein pathway. |
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