| Objective:To observe the protective effect of bezoar and its effective components on cerebral ne urovascular units in vitro and to clarify the internal biological mechanism of bezoar a nd its effective components in the treatment of ischemic stroke.Methods:1.Establishment of cerebral neurovascular unit model and hypoxia/reoxygenation model in vitro(1)In vitro primary cell culture technology was used to extract cerebral cortical neuro ns,astrocytes and endothelial cells for cell identification;(2)The three kinds of cells were co-cultured by Transwell to construct the model of brain neurovascular unit in vitro.The success of the model was determined by TEER value;(3)The OGD/R model was constructed by hypoxia for 1h and reoxygenation for 24h.The success of the model was determined by TEER value,LDH and y-GT.2.Toxicity of bezoar and its active components to three kinds of cells Different concentrations of bezoar,taurine,TUDCA and UDCA were used to act on n eurons,astrocytes and endothelial cells for 24h and 48h,respectively.Cell viability wa s detected by CCK8,and cell toxicity was detected by different concentrations of each drug.3.Protective effects of bezoar and its active components on cerebral neurovascular uni ts(1)The OGD/R model was used to detect the effects of different doses of bezoar,tau rine,TUDCA and UDCA on cells,and CCK8 method was used to detect cell viabilit y and determine the optimal dose of each drug;(2)The optimal dose of drugs was used to intervene by detecting the BBB permeabili ty:TEER value,luciferin sodium permeability coefficient and ?-GT;Inflammatory cytok ines TNF-?,IL-6,IL-1?;Oxidative stress indexes SOD,NO,MDA and neuronal apopt osis rate;(3)Study on the protective mechanism of bezoar on cerebral neurovascular units The inhibitor LY294002 was added to detect that the intervention of bezoar on cells was realized through PI3K/Akt pathway.Apoptosis related proteins Bax,Bcl-2,caspase-3,metalloproteinases MMP2,MMP9,tight junction proteins ZO-1,Occludin,claudin-5,HIF-1?,VEGF,PI3K,Akt,p-Akt were detected.4.Protective effects of different compatibility components on cerebral neurovascular un its(1)The OGD/R model was used to screen the optimal dose of T+T and T+U;(2)The optimal dose of drugs was used to intervene by detecting the BBB permeabili ty:TEER value,luciferin sodium permeability coefficient and ?-GT;Inflammatory cytok ines TNF-?,IL-6,IL-1?;Oxidative stress indexes SOD,NO,MDA and neuronal apopt osis rate.5.Study on the action mechanism of the compatible components taurine(T)and urso deoxycholic acid(U)on the cerebral neurovascular unitsThe optimal dose of T+U was used to model OGD/R,and the apoptosis-related protei ns Bax,Bcl-2,Caspase-3,metalloproteinases MMP2,MMP9,tight junction proteins Z O-1,Occludin,claudin-5 and NF-K Bp65,p-p65,IKB? were detected.Expression of p-IKBa protein.6.Mechanism of action of the compatible components taurine(T)and ursodeoxycholic acid(T)on cerebral neurovascular unitsThe optimal dose of T+T was used to model OGD/R,and the apoptosis-related proteins Bax,Bcl-2,caspase-3,metalloproteinases MMP2,MMP9,tight junction proteins ZO-1,Occludin,Claudin-5,p38MAPK and p-p38MAPK were detected.Results1.Neurons,astrocytes and endothelial cells with high purity were obtained(1)After cultured for 7 days,the neurons matured,and the purity was greater than 95%by MAP2 immunofluorescence identification;(2)Astrocytes were cultured for 7 days and matured.The purity was greater than 95%after GFAP immunofluorescence identification;(3)After cultured for 7 days,the endothelial cells matured,and the purity was more than 95%after immunofluorescence identification by VIII factor.2.The neurovascular unit model of the brain was established by Transwell(1)The resistance value of this model tends to be stable in 3-5 days;(2)The state of the three cells was better than that of single cells in three-cell co-culture.3.The OGD/R model was successfully copiedAfter hypoxia for 1h and reoxygenation for 24h,the model was successfully built.Compared with the normal group,the resistance value of the model group was reduced,and the next experimental study could be carried out.4.Screening drug toxicityDifferent concentrations of bezoar,taurine,ursodeoxycholic acid and ursodeoxycholic acid were used to intervene for 24h and 48h,respectively,to screen for different toxicity of the drugs.5.Screening the protective dose of drugsUsing non-toxic concentrations of various drugs,OGD/R modeling method was used to detect cell viability and screen out the best dose of protective effect.6.Protective effects of bezoar and its active components on brain neurovascular units in vitro(1)Bezoar and its active components decreased LDH,increased ?-GT,increased TEER value and decreased luciferin sodium permeability coefficient;(2)Bezoar and its active components reduced the levels of inflammatory cytokines TNF-?,IL-6 and IL-1?;(3)Bezoar and its active components decreased the levels of NO and MDA,and increased the level of SOD;(3)Bezoar and its active components inhibit neuronal apoptosis;(4)Bezoar decreased the protein expression of MMP2 and MMP9;(5)Bezoar can increase the expression of tight junction protein,and increase the protein expression of ZO-1,Occludin and Claudin-5;(6)Bezoar plays a protective role in NVU through HIF-?/VEGF and PI3K/Akt pathway.Bezoar reduces the protein expression levels of HIF-la and VEGF and increases the protein expression levels of PI3K and Akt.7.Protective effects of different compatibility components on cerebral neurovascular units(1)The optimal dose of the compatible components was selected,including 1 mM taurine and 1?M UDCA for T+T and 1 mM taurine and 1?M UDCA for T+U;(2)T+T and T+U effectively protect the blood-brain barrier,decrease TEER value,decrease luciferin sodium permeability coefficient,and increase y-GT;(3)T+T and T+U effectively alleviated cell damage and reduced LDH level;(4)T+T and T+U can effectively reduce inflammation and decrease the values of TNF-?,IL-6 and IL-1?;(5)T+T and T+U effectively inhibit the oxidation reaction,reduce the levels of NO and MDA,and increase the activity of SOD;(6)T+T and T+U inhibited the apoptosis rate of neurons.8.Mechanism of action of the compatible components taurine(T)and ursodeoxycholic acid(U)on cerebral neurovascular units(1)T+U inhibited neuronal apoptosis rate;(2)T+U increased the protein expression of ZO-1,Occludin and Claudin-5;(3)T+U decreased the protein expression of MMP2 and MMP9;(4)T+U decreased the protein expression of Bax and Caspase-3 and increased the protein expression of Bcl-2;(5)T+U decreased the protein expressions of MyD88,p-p65 and p-IKB?,and played a protective role in brain through MyD88/NF-?B signaling pathway.9.Mechanism of action of taurine and bezoar ursodeoxycholic acid on cerebral neurovascular units(1)T+T inhibited the apoptosis rate of neurons;(2)T+T increased the protein expression of ZO-1,Occludin and Claudin-5;(3)T+T decreased the protein expression of MMP2 and MMP9;(4)T+T decreased the protein expression of Bax and Caspase-3 and increased the protein expression of Bcl-2;(5)T+U decreased AQP4 and P38MAPK and increased the protein expression of CX43.Conclusion(1)Bezoar,taurine,ursodeoxycholic acid and ursodeoxycholic acid can reduce the inflammatory response,reduce the level of oxidative stress,inhibit the apoptosis of neurons to play a neuroprotective role;(2)Bezoar exerts protective effects through HIF-1?/VEGF and PI3K/Akt pathways;(3)Taurine and ursodeoxycholic acid,the combination of taurine and ursodeoxycholic acid,plays a synergistic role,which is better than the use of taurine alone;(4)The combination of taurine and ursodeoxycholic acid 1000:1 can inhibit the inflammatory response,reduce the level of oxidative stress,protect the blood-brain barrier,inhibit the apoptosis of neurons,and inhibit the MyD88/NF-?B pathway to play a protective role in the brain(5)The combination of taurine and ursodeoxycholic acid 1000:1 can inhibit the inflammatory response,reduce oxidative stress,protect the blood-brain barrier,inhibit the p38MAPK pathway,and play a brain protective role;... |
PDF Full Text Request