| Stroke is the second most common cause of death and disability in the world,and acute ischemic stroke is the most common type of stroke,which brings a heavy burden to families and society.It starts with sudden interruption of blood flow,oxygen,glucose and energy in the infarct area,which can lead to severe neurological functional disruption.Although subsequent reperfusion is critical for ischemic brain tissue to restore normal blood supply and function,it can paradoxically result in secondary damage,called I/R injury.I/R injury includes a series of pathologic cascading events,such as exitotoxicity,calcium influx,free radicals accumulation,inflammation response,BBB breakdown,brain edema,cell death and so on.Therefore,hunting for an effective therapies for cerebral I/R injury has been a main task in neuroscience for decades.Neuroprotection remains the central focus of cerebral I/R injury treatment.Despite the development of technologies and agents against stroke,few suitable neuroprotective agents for the prevention of cerebral I/R injury are available when translated into clinical application.Thus,what is responsible for these failures? Increasing scholars indicated the cause of these failures may be that the treatment focuses only on a single therapeutic target,ignoring the interaction between the different therapeutic targets.So there is urgent need of new research strategies to prevent cerebral I/R injury.In view of above-mentioned facts,scholars proposed a concept of Neurovascular Unit(NVU),which is regarded as a basic structural and functional unit of brain with cerebral function.It mainly consists of brain neurons,BMECs,astrocytes,even with basement membrane,pericytes,microglia and extracellular matrix.This concept emphasizes the complicated interactions among the above cells and their contribution to the pathophysiological mechanisms of brain.Thererfore,NVU has become an important model for studying multi-target,multi-level and combination therapy for stroke.Based on the this strategy,we have established a NVU model for in vitro study,which is a triple cells co-culture network model made up of three kinds of primary cells including SD rat BMECs,astrocytes and neurons.It can be used in brain research,potential drug targets screening and therapeutic drug discovery.Traditional Chinese herbal medicine with its multiple targets,high security features,has shown a unique advantage in the treatment of CIRI.Salvia miltiorrhiza(SM)is a traditional Chinese herb that has ?been? widely? ?used for treament of a variety of cardiovascular and cerebrovascular diseases and neurodegenerative disorders.Cryptotanshinone(CTs)is one of the major tanshinones isolated from the root of SM,which is lipophilic compound(Mw:296.35)and can crosses BBB.Modern pharmacological studies have shown that CTs not only has the effects of antioxidative,anti-inflammatory,antibacterial,anticancer,antiapoptosis,antiplatelet aggregation,antidiabetes and antiobesity,but also possesses the protential to prevent ischemia,atherosclerotic and Alzheimer’s disease.Recently,some scholars have focused their interests on researching the relationship between CTs and I/R injury.Some researches have demonstrated the protective effects of CTs on myocardial I/R injury and hepatic I/R injury.However,the protective effects and mechanisms of CTs on cerebral I/R injury remains unclear,especially at the cellular level.Clarification of the neuroprotective effects of CTs on cerebral I/R injury may provide new insight into the mechanism of neuroprotection.Oxygen glucose deprivation /reperfusion(OGD/R)is a commonly used method to simulate CIRI in vitro.The pathological changes of brain cells and BBB in this model are similar to those of CIRI in vivo.In the present study,we successfully established a OGD/R-damaged triple cells network model of SD rats to mimic the cerebral I/R injury in vitro,and explored the effects of CTs on cerebral ?I/R injury and possible mechanisms.Part I Establishment of a model of oxygen glucose deprivation/reperfusion injury in main brain cells network model of Sprague-Dawley ratsObjective: To establish a simple and reliable oxygen glucose deprivation/reperfusion(OGD/R)model of NVU at the cellular level in vitro through selecting the optimal treatment conditions of OGD/R,which can lay the foundation for research on the intervention effect of CTs on CIRI and its mechanism.Methods: Primary rat BMECs,astrocytes and neurons were obtained from cerebral cortex of SD rats,and cultured in vitro.The rate of cell purity was detected by immunofluorescence staining.Then we established main brain cell network model of SD rats in vitro by transwell insert.These models were randomly divided into 2 groups:Control group and OGD/R group,and each group was further divided into R 0h,0.5h,3h,6h,12 h,24h and 72 h were 7 different reperfusion time groups(each group was established three holes).We set up OGD/R model by beginning reperfusion after OGD2 hours.Cell viability and the activity of lactate dehydrogenase(LDH)in supernatants were detected by the corresponding assay kit.TEERs were detected by ERS-2 System in brain main cell network model of SD rats.Results:1.After primary cultured for 7 d,BMECs had spindle shape and arranged into whirlpool form.The cell purity was >95% by immunofluorescence staining of VIII antibody.2.After 10 days of primary culture,the astrocytes were digested and passage.After cultured for 9 d,the second generation cells were flat irregular polygon,weak refraction and confluent.The cell purity was >90% by immunofluorescence staining of GFAP.3.After primary cultured for 7 d,neurons appeared ovoid,spindle and multipolar,and had abundant cytoplasm and a large number of neurites formed extensive networks.The cell purity was >90% by immunofluorescence staining of MAP2.4.BMECs,astrocytes and neurons were co-cultured by transwell insert.TEERs became stable from the third day to the fifth day of co-culture(P>0.05),which marked that the successful establishment of main brain cells network model of SD rats.5.After OGD2 h treatment,these triple cell network models of SD rats were confirmed reperfusion treatment.The cell viability of neurons began to decline at R 3h(P<0.01),and cell viability of neurons at R 24 h and R 72 h were more lower than that at R 3h(P<0.01).But survival rates of BMECs and astrocytes began to decrease at R 6h(P<0.05),and survival rates of BMECs and astrocytes were more lower than that at R6h(P<0.01).LDH activity in supernatants began also to increase at R 6h(P<0.05),and LDH activity in supernatants at R 24 h and R 72 h were more higher than that at R3h(P<0.01).TEER value began also to decline at R 6h(P<0.05),and TEER value at R12 h,R 24 h and R 72 h were more lower than that at R 6h(P<0.05,<0.01 and <0.01respectively).Conclusion:1.After OGD 2h/R 24 h treatment,brain triple cell network model of SD rats was successfully established as OGD/R damage model.2.The decrease of TEERs can be used as a criterion to judge the successful establishment of OGD/R injury model in brain triple cell network model of SD rats.Part II Intervention effects of Cryptotanshinone on oxygen and glucose deprivation/reperfusion injury in main brain cells network model of Sprague-Dawley RatsObjective: To observe the effect of CTs on the OGD/R damage in main brain cells network model of SD rats,and verify the neuroprotective effect of CTs on CIRI.Methods:(1)We established main brain cells network models of SD rats,then these models were randomly divided into 9 different dosage groups of CTs(0.2,0.5,1.0,2.5,5,10,20,50 and 100μM,each group was established three holes).After 24 h treated by CTs,the cell viability of neurons were tested by CCK-8 in order to select the appropriate concentration of CTs.(2)The main brain cells network models of SD rats were randomly divided into pretreatment group and immediate treatment group,and each group was further divided into Control group and the appropriate concentration of CTs groups(each group was established three holes).Every group was treated with OGD2h/R 24 h except Control group.The severity of cells damage were tested by LDH detection kit in order to determine the best mode of administration and effective dose CTs for further experiments.(3)The main brain cells network models of SD rats were randomly divided into 4 groups: Control group,OGD2h/R 24 h group,CTs 2.5μM group and CTs 5.0μM group(each group was established three holes).Every group was treated with OGD2h/R 24 h except Control group.The cellular morphology were observed by inverted phase contrast microscope.The cell viability and the severity of cells damage were tested by corresponding detection kits.Results:1.After 24 h treated by CTs,0.2,0.5,1.0,5.0,and 10 μM CTs had no significant effect on the cell viability of neurons in the brain triple cell network model of SD rats(P>0.05).2.In pre-treatment group: compared with group OGD2h/R24 h,the leakage rate of LDH were significantly decreased in CTs1.0,2.5,5.0 and 10 M groups(P<0.05,0.01,0.01 and 0.05 respectively).In immediate treatment group: compared with OGD2h/R24 h group,the leakage rate of LDH had no significant difference in all of CTs groups(P>0.05).3.In CTs 2.5μM and CTs 5.0μM groups,the OGD/R-induced pathological changes of BMECs,astrocytes and neurons were significantly restored.Compared with OGD2h/R24 h group,the cell viability of three kinds of cells were significantly increased(P<0.01),the leakage rate of LDH of cells were decreased significantly in CTs 2.5μM group and CTs 5.0μM group(P<0.05 and <0.01 respectively).Conclusion:1.The best way of administration of CTs for OGD/R injury of main brain cells network model of SD rats were pre-treatment,and the effective doses of CTs were 2.5and 5.0 μM.2.CTs pre-treatment was able to restore the cell shrinkage,reduce cell shedding,increase cell activity and reduce cell damage induced by OGD/R.CTs has protective effect on OGD/R damage in main brain cells network model of SD rats.Part III The mechanism of intervention effects of Cryptotanshinone on oxygen and glucose deprivation/reperfusion injury in main brain cells network model of Sprague-Dawley RatsObjective: To study the intervention mechanism of CTs on the OGD/R damage in the brain triple cell network model of SD rats,and provide a new way for the research of brain protection mechanism and drug screening.Methods: The main brain cells network models of SD rats were randomly divided into 4 groups: Control group,OGD2h/R 24 h group,CTs 2.5μM group and CTs 5.0μM group(each group was established three holes).Every group was treated with OGD2h/R24 h except Control group.The level of intracellular ROS in neurons,and the level of MDA and NO and the activity of SOD in supernatants were measured using the commercial assay kits.The contents of IL-1β,TNF-α and IL-6 of the supernatant were detected by Quantikine? ELISA kits according to the manufacture’s instructions,respectively.To identify apoptotic neurons,TUNEL staining were performed using an in situ cell death detection kit.TEERs were detected by ERS-2 System in brain triple cell network model of SD rats.The permeability of BMECs to Na F after OGD/R damage was detected by fluorescence chemiluminescence detector.The proteins levels of Caspase-3,cleaved-Caspase-3,PARP,cleaved-PARP,Bax,Bcl-2 and MAPKs in neurons and the proteins levels of ZO-1,Occludin,Claudin-5,MMP-2 and MMP-9 in BMECs were determined by Western blot analysis.Results:1.CTs pre-treatment could reduce the level of intracellular ROS in neurons and the levels of MDA and NO in supernatants(P<0.01),but could increase the SOD activity(P<0.01)in supernatants of main brain cells network model of SD rats after OGD/R.2.CTs pre-treatment could decrease the levels of pro-inflammatory cytokines IL-1β,TNF-α and IL-6(P<0.01)in supernatants of main brain cells network model of SD rats after OGD/R.3.CTs pre-treatment could decrease the apoptosis rate of neurons and the expression levels of cleaved-Caspase-3 and cleaved-PARP(P<0.01)in neurons of main brain cells network model of SD rats after OGD/R.4.CTs pre-treatment could also increase the expression level of Bcl-2(P<0.01),while decrease the expression level of Bax(P<0.01)in neurons of main brain cells network model of SD rats after OGD/R,thereby increasing the ratio of Bcl-2/Bax.5.CTs pre-treatment could reduce the expression levels of p-JNK and p-p38MAPK(P<0.01)in neurons of main brain cells network model of SD rats after OGD/R.6.CTs pre-treatment could increase the TEER value(P<0.01),while reduce the permeability of BMECs to Na F(P<0.01),and could increase the expression of ZO-1,Occludin and Claudin-5(P<0.01),while decrease the expression of MMP-9(P<0.01)in BMECs of main brain cells network model of SD rats after OGD/R.Conclusion:1.CTs exerted intervention effects on OGD/R injury by attenuation of oxidative stress,inflammation,neuronal apoptosis and blood-brain barrier disruption in main brain cells network model of SD rats.2.The mechanisms of CTs inhibiting neuronal apoptosis might be related with up-regulating the ratio of Bcl-2/bax and inhibiting JNK and p38 MAPK signaling pathways in main brain cells network model of SD rats.3.The mechanisms of CTs improving the barrier function of BBB might be to up-regulate the expression of tight junction proteins ZO-1,Occludin and Claudin-5,and reduce the degradation of extracellular matrix by inhibiting the expression of MMP-9 in main brain cells network model of SD rats. |
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