| Background: Stroke is one of the most common causes of death,and is associated with high rates of morbidity,disability and multi-complications,which is a severe threat to health worldwidely.Stroke can cause serious health and economic effects,which greatly consume medical resources.Specifically,cerebral ischemic stroke contributes up to 80% of total stroke.Ischemic stroke is induced by thrombosis,embolism or systemic hypoperfusion,which can lead to a restriction of blood flow to the brain,resulting in insufficient supply of oxygen and glucose to support cellular homeostasis.Meanwhile,reperfusion plays an important role in ischemic injury.It also contributes to delayed secondary brain tissue damage and additional complications.Cerebral ischemia/reperfusion injury induces a multifactor,multi-mechanism and malignant cascade reaction,such as energy metabolism dysregulation,reactive oxygen species production,excitotoxicity,calcium overload,blood-brain barrier injury,inflammation,apoptosis gene activation and so on.In addition,autophagy has recently been proposed as an important mechanism in cerebral ischemia/reperfusion injury.Autophagy not only have functions as a defense mechanism by removing damaged organelles and metabolites from the cytoplasm,but also triggers the cell death program to induce cell death.These mechanisms are interrelated and overlaped,and reinforced each other,which creats a vicious cycle and eventually leads to neuronal cell death.Nowadays,therapies of cerebral ischemia/reperfusion injury include acute thrombolysis,intravascular therapies and cerebrovascular bypass surgery,but the therapeutic effects remain limited.Therefore,it is necessary to search for new therapeutic targets.The fruit of Lycium barbarum is an important traditional Chinese herbal medicine with multiple biological activities and pharmacological functions.Lycium barbarum polysaccharide(LBP)is the major active ingredient of Lycium barbarum.Previous studies have shown that LBP can modulate immunity,have anti-tumer effects,and protect against the effects of aging and oxidation.Moreover,a recent research has reported that LBP also protects neurons.However,it is still unclear how LBP plays the protective role.The PI3K/Akt/m TOR signaling pathway plays a central role in controlling cell proliferation,cell growth,cell metabolism and survival through various underlying mechanisms.The neuroprotective effects of this signaling pathway in cerebral ischemia have been widely studied.Previous studies have indicated that the PI3K/Akt/m TOR signaling pathway can alleviate autophagy when it is activated.The pathway also inhibits apoptosis,promotes the cell cycle,and inhibits autophagy.Therefore,the PI3K/Akt/m TOR signaling pathway is highly related to apoptosis and autophagy.Based on the above research background,the aims of this study are to explore the mechanisms through which the PI3K/Akt/m TOR signaling pathway regulates apoptosis or autophagy in LBP treated neurons.Objectives: In the study,oxygen glucose deprivation and reoxygenation(OGD/R)injured model in primary cultured hippocampal neurons was used to observe and investigate:(1)the protective effect of LBP pretreatment on neurons,(2)the effect of LBP pretreatment on neuronal apoptosis,(3)the effect of LBP pretreatment on neuronal autophagy,(4)weather LBP pretreatment is related to the PI3K/Akt/m TOR signaling pathway.Methods: 1.Cultured primary hippocampal neurons which were exposed to oxygen-glucose deprivation for 3h followed by a 24 h reperfusion(OGD/R)were used as an in vitro model of cerebral ischemia-reperfusion injury.2.Cells were randomly divided into 5 groups: control group,OGD/R group,low,middle and high concentration LBP(15,30,60?g/ml)groups.LBP was added at the start of the reoxygenation.3.The survival rate of hippocampal neurons was evaluated by MTT assay.The neuronal injury was measured by detecting the release of LDH.4.ROS formation was measured by the fluorescent probe DCFH-DA,and the levels of MDA were measured using the MDA commercial kit.5.The neuronal apoptosis was evaluated by using terminal d UTP nick end labeling TUNEL assay.6.Immunofluorescence staining was used to detect the expression of cleaved Caspase-3,LC3 and Beclin 1.7.The numbers of autophagosome and autolysosome were visualized using transmission electron microscope.8.Western blotting assay was used to evaluate protein expression of cleaved-Caspase 3,Caspase 3,Bax,Bcl-2,LC3-II,LC3-I,Beclin 1,p62,p-m TOR,m TOR,p-Akt,and AktResults: Part one: Compared to the control group,OGD/R treatment significantly decreased cell viability and increased LDH leakage.Compared with the OGD/R group,LBP(15,30,60?g/ml)treatment significantly inhibited LDH leakage and increased cell viability.In addition,OGD/R treatment induced a significant increase in ROS formation and MDA levels,compared to the control group.However,LBP(15,30,60?g/ml)treatment significantly suppressed ROS formation and MDA levels after OGD/R.Part two: TUNEL-positive cells increased in the OGD/R group compared with the control group.In the LBP(15,30,60?g/ml)treatment groups,fewer TUNEL-positive cells were detected compared with the OGD/R group.Furthermore,we measured essential apoptosis-related proteins and found OGD/R treatment increase the ratio of cleaved Caspase-3/Caspase-3 and decrease Bcl-2/Bax protein ratio.LBP(15,30,60?g/ml)treatment increased Bcl-2/Bax protein ratio and decreased Caspase-3/Caspase-3 protein ratio.Meanwhile,the immunofluorescence staining results of cleaved Caspase-3 was consistent with WB results.Part three: After OGD/R injury,Beclin 1 expression and the LC3II/LC3 I ratio were increased,and p62 expression was decreased,compared to the control group.LBP(15,30,60?g/ml)treatment reduced Beclin 1 expression and the LC3II/LC3 I ratio,and increased p62 expression.Besides,the immunofluorescence staining results of LC3 and Beclin 1 were consistent with WB results.The electron microscopy reveled autophagosome or autophagolysosomal numbers were increased in the OGD/R group compared with control group.LBP 60?g/ml treatment observed fewer autophagosome or autophagolysosomal numbers.Part four: After OGD/R injury,p-Akt and p-m TOR expression were decreased,compared with the control group.However,LBP(15,30,60?g/ml)treatment enhanced p-Akt and p-m TOR expression.Furthermore,we used the PI3K-specific inhibitor LY294002 to clarify the relationship between LBP and PI3K/Akt/m TOR signaling pathway.LBP 60?g/ml enhanced p-Akt and p-m TOR expression,and reduced LC3-II and cleaved Caspase-3 expression.Conclusion: 1.LBP treatment has protective effects on OGD/R-induced cell damages in primary hippocampal neurons.2.LBP treatment ameliorates OGD/R-induced cell apoptosis in primary hippocampal neurons.3.LBP treatment inhibits OGD/R-induced autophagic cell death in primary hippocampal neurons.4.Neuroprotective effects of LBP on OGD/R-induced hippocampal neuron damages by activating the PI3K/Akt/m TOR signaling pathway. |
PDF Full Text Request