| Background and Objective:Cerebral stroke is a group of diseases characterized by cerebral ischemia or hemorrhagic injury as clinical manifestation,also known as stroke or cerebrovascular accident.There are two main types of stroke,including hemorrhagic stroke(cerebral hemorrhage or subarachnoid hemorrhage)and ischemic stroke(cerebral infarction and cerebral thrombosis),of which ischemic stroke is the most common.Stroke is the second leading fatal diseases in the world,with acute onset and high fatality rate.Under physiological conditions,the normal cerebral blood flow(CBF)was maintained at about50-60 m L/100 g/min.When cerebral ischemia occurred,CBF decreased sharply and the damage spread from center to periphery.The CBF in the ischemic core area was about less than 7 m L/100 g/min,in which irreversible death of neurons occurred within minutes to hours of ischemia.Outside of the ischemic core,there were some low perfusion brain tissues,and CBF was maintained at 7~17 m L/100g/min,which was ischemic penumbra(IP).Owing to the collateral circulation of the brain,the irreversible death of neurons in IP has not yet occurred.Therefore,neurons still can survive and recover after reperfusion to restore cerebral blood flow.Therefore,in most cases,prompt thrombolysis is essential for the treatment of acute ischemic stroke.The guidelines for the diagnosis and treatment of acute ischemic stroke in China clearly states that for patients with ischemic stroke,thrombolytic therapy should be given together with neuroprotective drugs to improve neurological function.Therefore,revealing important signaling molecules and their related signaling pathways,so as to protect neurons in the ischemic penumbra,is conducive to the formulation of neuroprotective therapeutic strategies.Cerebral ischemia activates many complex cellular signal cascades that are critical to cell survival and damage induced by multiple stimuli.Mitogen activated protein kinase(MAPK)is a kind of serine / threonine protein kinase which widely exists in eukaryotic cells.It can transfer extracellular signals to cells.These enzymes enable cells to respond to ischemia and hypoxia.Among them,p38 MAPK pathway is one of the main pathways.In cerebral ischemia-reperfusion injury,p38 MAPK signal transduction pathway isactivated,which transmits extracellular stimulation signal to cells,which is an important way to transfer cellular information that mediates cell response.Rhizoma coptidis is one of traditional Chinese medicine,first published in the herbs of the Tang Dynasty,cold in property,bitter in flavour.The previous study of our research group showed that the treatment with picroside Ⅱ in rats following acute cerebral ischemia injury could obviously reduce the ultrastructure damage of cortical neurons,decrease neuronal apoptosis,alleviate cerebral edema,and improve the neurological dysfunction.The results suggest that picroside Ⅱ may have the neuroprotective effect of anti-inflammation,anti-oxidation and inhibition of neuronal apoptosis.However,the cellular and molecular mechanism involved in these regulatory effects remains unclear.Therefore,we tried to establish middle cerebral artery occlusion/reperfusion(MCAO/R)model in rats and oxygen glucose deprivation/Reoxygen(OGD/R)model in SH-SY5 Y cells to imitate cerebral ischemia reperfusion injury.To investigate the neuroprotective effect of picroside Ⅱ on cerebral ischemia reperfusion injury and its relationship with p38 signaling pathway.Methods:1.In vivo animal experiment: Total of 200 adult healthy male Wistar rats,SPF grade,6 months old,weighing 230 g-250 g.In the natural environment(day / night12h/12h),routine adaptive feeding for 1 week.According to the principle of randomized control,all animals were randomly divided into sham group,model group,picroside(Picr)group,Anisomycin(p38 agonist,An)group,An+Picr group,and SB203580(p38inhibitor,SB)group and SB+Picr group.Middle cerebral artery occlusion / reperfusion(MCAO/R)models were established via the insertion of a monofilament suture from internal-external carotid artery into the middle cerebral artery.Modified neurological severity score(mNSS)and transcranial Doppler detecting regional cerebral blood flow(r CBF)were used as the criteria for successful modeling.The cerebral infarct volumes were measured with tetrazolium chloride(TTC)staining.The structure of neuron was observed using hematoxylin-eosin staining(HE)staining and transmission electron microscopy(TEM).Apoptosis were counted by terminal deoxynucleotidyl transferase d UTP nick end labeling assay(TUNEL).The expression of phosphorylated p38(p-p38)in the cortex of ischemic region was assessed using the immunohistochemistry(IHC).And the expressions of p-p38,phosphorylated p53(p-p53),interleukin-6(IL-6)and tumor necrotic factorα(TNFα)in the cortex of ischemic region were determined by Western Blot(WB).2.In vitro cell culture: The oxygen glucose deprivation / reoxygenation(OGD/R)model was established by SH-SY5 Y cells,and the concentration gradient was set to explore the optimal protective dose of picroside Ⅱ against SH-SY5 Y cell model induced by OGD/R.Then they were divided into control group,model group,picroside(Picr)group,Anisomycin(p38 agonist,An)group,An + Picr group,SB203580(p38 inhibitor,SB)group and SB+Picr group according to the drug treatment.Cell morphology was observed under light microscopy.Cellular viability was detected by CCK8.Apoptosis were assessed by flow cytometry(FCM)and Cleaved caspase-3 protein expression.Expressions of p-p38 and p-p53 were detected using immuno-fluorescence(IF)and Western Blot(WB).IL-6 and TNFα were detected using immuno-fluorescence(IF)and Western Blot(WB)and Elisa.Results:1.The results of MCAO/R experiment showed as follows:(1)Neurobehavioral function change: m NSS scores showed that the scores of rats in model group,An group and An+Picr group were higher than those in the control group(P<0.01,n=20).Compared with the model group,treatment with picroside Ⅱ,SB203580 and the combination of them could decrease the scores significantly(P< 0.01,n = 20).(2)Changes of cerebral infarction volumes: TTC test showed that large white infarct appeared in rats of model group,An group and An+Picr group.After treatment with picroside Ⅱ,SB203580 or SB2035820 combined with picroside Ⅱ,the cerebral infarct volume decreased significantly compared with the model group(P<0.01,n=5).(3)Morphological and structural changes of neurons: HE stainning showed that the cortical neurons in the sham group were neatly arranged and the structure of the neurons was intact.After MCAO/R,the neurons in the model,An and An+Picr groups were disarranged,the cells were condensed and stained deeply especially in the An group.However,the neurons in the Picr group,SB group and SB+Picr group were less damaged,the structure of neurons was intact,some cells were irregular in shape and the nuclear membrane was not uniform.(4)Ultrastructural changes of the neurons: TEM showed that the cortical neurons in the sham group were intact,and there were various organelles in the cytoplasm.After MCAO/R,the bilayer nuclear membrane of the neurons in the model,An and An+Picr groups was not clear.Cytoplasmic organelles were dissolved and disappeared.In Picr,SB and SB+Picr groups,the neuronal damage was mild,the ultrastructure was intact and the nuclear membrane was unclear.(5)Changes of apoptosis: TUNEL assay showed that apoptotic cells in the model group and An group were significantly increased compared with that in the sham group(P<0.01,n=5).However,apoptotic cells in Picr group,An+Picr group,SB group and SB+Picr group were significantly decreased compared with that in the model group(P<0.05,n=5).(6)Changes of p-p38 protein expression: WB and IHC showed that a small amount of p-p38 positive cells existed in the sham group.After MCAO/R,the expressions of p-p38 in the model group,An group,and An+Picr group were significantly higher than that in the sham group,with a strong positive expression(P<0.01,n=5).Compared with the model group,p-p38 protein expressions of Picr group,An+Picr group,SB group and SB+ Picr group were significantly decreased and weakly positive(P<0.05,n=5).(7)Changes of p-p53 protein expression: WB test showed that p-p53 protein expression was at a low lovel in the sham group.After MCAO/R,the p-p53 expression in the model group and An group was significantly higher than sham group(P<0.01,n=5).P-p53 protein levels in Picr group,An+Picr group were significantly lower than those in the model group(P<0.05,n=5).Compared with Picr group,those in SB group and SB+Picr group were further decreased,but no difference was found(P >0.05,n=5).(8)Changes of IL-6 protein expression: WB test showed that following MCAO/R,IL-6 protein expression was increased in the model group,An group and An+ Picr group,significantly higher than that in the sham group(P<0.01,n=5).Compared with the model group,the expression of IL-6 protein in Picr group,SB group and SB+ Picr group was significantly lower(P<0.05,n=5),which was close to that in the sham group(P>0.01,n=5).(9)Changes of TNFα protein expression: WB showed that the expressions of TNFαprotein in model group and An group were significantly higher than that in sham group(P<0.01,n=5)after MCAO/R.Compared with the model group,the expressions of TNFαprotein in Picr group and An+Picr group decreased,but there was no statistical difference(P>0.05,n=5).2.The results of SH-SY5 Y cells after OGD/R experiment showed as follows:(1)Establishment of OGD/R model: according to the detection results of CCK8,the damage of cells was relatively mild when hypoxia was 4 ~ 6 h,and the cells could repair themselves.However,when the hypoxia time reached 12 h,the cells were severely damaged and only a few cells survived.After 8 h of hypoxia,about 50% ~ 60% of the cells survived.According to the cellular viability,we finally selected hypoxia for 8 h andreoxygenation for 4h for downstream experiments.(2)Discussion on the concentration of picroside Ⅱ: according to the detection results of CCK8 and observation of cell morphology under light microscope,the cellular viability of SH-SY5 Y cells was significantly improved after treatment with 20,50 and100 μg/m L picroside Ⅱ,and the cellular viability with different concentration gradient was not significantly different betweent each other(P>0.05),so 20 μg/m L of picorside Ⅱ was selected for the downstream experiment.(3)Cellular viability: CCK8 detection and observation of cell morphology showed that after OGD/R,in model group,An group and An+Picr group SH-SY5 Y cells was impaired,the protuberance disappeared and the cellular viability decreased obviously compared with control group(P<0.01,n=6).Treatment with picroside Ⅱ,SB2035820 or combination with each other,the damage of SH-SY5 Y cells was recovered,and the cellular viability was increased significantly in the model group(P<0.01,n=6).(4)Changes of apoptosis: FCM detection showed that following OGD/R,a large number of apoptosis occurred in the model group,and the apoptotic cells were further increased after the administration of Anisomycin,but there was no statistical difference between the two groups(P>0.05,n=5).Compared with the model group,apoptotic cells were significantly reduced(P<0.05,n=5)after treatment with picroside Ⅱ,SB2035820 or combined treatment.(5)Changes of Cleaved caspase-3 protein expression: WB test showed that the expression of Cleaved caspase-3 protein was weak in the control group.After OGD/R,Cleaved Caspase-3 protein expressions in the model and An groups was significantly higher than that in the control group(P <0.01,n=6),while Cleaved Caspase-3 protein expression in the Picr group,An+Picr group,SB group and SB+Picr group was significantly lower than that in the model group(P<0.01,n=6).(6)Changes of p-p38 protein expression: WB and IF tests showed that p-p38 was slightly expressed in the control group.After OGD/R,the expression of p-p38 in the model group and An+Picr group was significantly higher than that in the control group(P<0.01,n=6),and that in the Anisomycin group was further increased than that in the model group(P<0.01,n=6).Compared with the model group,in Picr group,SB group and SB+Picr group p-p38 expressions were significantly decreased and positive cells were little and weak.(P<0.01,n=6).(7)Changes of p-p53 protein expression: WB and IF tests showed that p-p53 was slightly expressed in the control group.After OGD/R,the expressions of p-p53 in themodel group,An group,and the An+Picr group were significantly higher than that in the control group(P<0.01,n=6).Compared with the model group,the p-p53 expressions of SH-SY5 Y cells in the Picr,SB and SB+Picr groups were significantly decreased and weakly positive(P<0.01,n=6).(8)Changes of IL-6 protein expression: WB and IF tests showed that after OGD/R,the expressions of IL-6 in the model group,An group and An+Picr group were significantly higher than that in the control group(P <0.01,n=6).Compared with the model group,the IL-6 protein expression of SH-SY5 Y cells in the Picr,SB and SB+ Picr groups was significantly decreased and weakly positive(P <0.01,n=6).ELISA showed that IL-6 protein was secreted less in the control group.After OGD/R,the secretion of IL-6 protein in the model group,An group and An+Picr group was significantly higher than that in the control group(P<0.01,n=6),while the secretion of IL-6 protein in the Picr group,SB group and SB+Picr group was significantly lower than that in the model group(P<0.01,n=6).(9)Changes of TNFα protein expression: WB test showed that after OGD/R,TNFαin the model group was significantly increased compared with the control group(P <0.05,n=6),while TNFα expression was not significantly decreased after picroside Ⅱ administration(P>0.05,n=6).TNFα expression was further increased in the An group and An+Picr group compared to the model group(P<0.01,n=6),while the increased TNFα expression was completely reversed in the SB group and SB+ Picr group,close to the control group(P >0.05,n=6).ELISA showed that TNFα protein was secreted less in the control group.After OGD/R,TNFα protein secretion in the model group,An group and An+Picr group was significantly higher than that in the control group(P<0.01,n=6);TNFα protein secretion in the Picr group was much lower than that in the model group,but there was no statistical difference(P>0.05,n=6).TNFα protein secretion in SB group and SB+Picr group was significantly lower than that in the model group(P<0.01,n=6).Conclusion:In vivo animal experiments and in vitro cell cultures confirmed that cerebral ischemia / reperfusion(oxygen glucose deprivation / reoxygenation)injury could activate p38 signaling pathway to mediate neuronal apoptosis and inflammatory response.Picroside Ⅱ could inhibit the inflammatory response after cerebral ischemia / reperfusion injury by inhibiting the p38-p53 signaling pathway,so as to play a neuroprotective role. |
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