| 1.BackgroundThe blood brain barrier?BBB?is a complex consists of multiple cells,which is composed of unimpeded brain microvascular endothelial cells?BMECs?,tight junctions?TJ?,pericytes,astrocyte foot processes,basement membrane,neurons,Microglia and very small cell gap,and the most important component of BBB is BMECs,while the TJ of BMECs is the important structure to ensure the barrier function.LPS is a glycoprotein on the outer membrane of Gram-negative bacilli,which can cause strong inflammatory reaction in the body,directly or indirectly damage BMECs and turn on the TJ to further cause changes in BBB permeability and to accelerate the development of cerebrovascular inflammation.Therefore,how to repair the structure and function of tight junctions is the key to the treatment of cerebrovascular inflammation diseases after the occurrence of cerebrovascular inflammation.Catalpol is the main active ingredient of Rehmannia glutinosa,which has anti-inflammatory,inhibition of capillary permeability,anti-oxidation,anti-apoptotic,hypoglycemic and other effects.Butthe protective effect of catalpol on the inflammatory injury of tight junctions of BMECs is lack of research.RhoA/ROCK is a major signaling pathway that regulates BBB permeability.A variety of inflammatory factors activate the RhoA/ROCK signaling pathway,which will increase BBB permeability and damage the brain.In this study,we explored the protective mechanism of catalpol on LPS-induced the injury of BMECs,which would provide experimental evidence for the treatment of cerebrovascular inflammation.2.ObjectivesTo study the protective effect and the mechanism of catalpol on LPS-induced the destruction of BMECs,and to provide experimental data for the development of catalpol for the treatment of cerebrovascular inflammation diseases.3.Methods 3.1 Establishment of LPS induced damage model of BMECsBMECs was primary cultured and identified.The effect of LPS on the survival rate of BMECs was detected by MTT assay.The median lethal concentration and the time were determined by LD50 to screen out the optimal concentration and time of LPS.3.2 Effects of catalpol on functional damage in BMECs induced by LPSTransendothelial resistance?TEER?was detected by transmembrane resistance meter.Permeability was detected by fluorescein sodium.The content of Endothelin-1?ET-1?was measured by enzyme-linked immunosorbent assay?ELISA?.To observe the protective effects of catalpol?0.3?M,3.0?M,30.0?M?on cell barrier and secretory functional of BMECs induced by LPS.3.3 Morphological effects of catalpol on damage of BBB induced by LPS3.3.1 In vivo: The effect of catalpol on vascular permeability of brain cortex induced by LPS was detected by EB assay.3.3.2 In vitro: BMECs was primary cultured.Immunofluorescence double staining was used to detect Claudin-5,cytoplasmic protein ZO-1 and cytoskeletal proteinin.And the effect of catalpol on the tight junction structure of BMECs was detected by transmission electron microscopy.3.4 The effect on RhoA/ROCK gene and protein expression of catalpol on the destruction of BMECs caused by LPSqPCR was used to detect the expression of Rho,RhoA,ROCK2,Occlaudin,Claudin-5,ZO-1,ZO-2 and ZO-3 mRNA.Western blot was used to detect the expression of RhoA,ROCK2 and tight junction Protein Claudin-5,ZO-1 protein expression.At the same time,the protective mechanism of catalpol on the destruction of BMECs caused by LPS was studied by using RhoA/ROCK signal inhibitor Fasudil.4 Results 4.1 Establishment of BMECs model induced by LPSAccording to the principle of LD50,the optimal concentration and the time of LPS-induced model were screened for 10 ?g·mL-1 LPS for 24 h to construct BMECs injury model.4.2 Catalpol antagonizes barrier permeability and secretion of endothelin in BMECs by LPSIn the model of BMECs induced by LPS,catalpol increased transmembrane resistance of BMECs and the amount of sodium fluorescein penetration,and the secretion of ET-1 were also reduced.4.3 Morphological changes of BBB permeability and tight junction of protective effects from catalpol induced by LPS4.3.1 In vivo: The C57 mice of model group showed that the whole brain were severely stained with blue dye and the fluorescence signal of the whole cerebral cortex red fluorescence signal was stronger and there was a large amount of EB leakage in the brain tissue.After the catalpol treatment,the brain were lighter stained with blue dye and cortical red fluorescence signal was weakened.4.3.2 In vitro: The expression of Claudin-5 and ZO-1 in the model group was significantly decreased,the F-actin cytoskeleton was rearranged and the nucleus was significantly reduced.The expression of Claudin-5 and ZO-1 was increased in each dose of catalpol group,and the F-actin cytoskeleton rearrangement was reversed by LPS.The results of transmission electron microscopy showed that catalpol significantly antagonized the shorter of Fir-like structure from TJ induced by LPS.4.4 Catalpol protects tight junctions of BMECs induced by LPS on RhoA/ROCK signal pathwayThe expression of Occludin,Claudin-5,ZO-1,ZO-2 and ZO-3 mRNA in the model group induced by LPS was significantly lower than that in the normal group?P <0.01?,Rho,RhoA and ROCK2 mRNA were significantly increased?P <0.01?.Compared with the model group injured by LPS,the levels of Occlaudin,Claudin-5,ZO-1,ZO-2 and ZO-3 mRNA were significantly increased in each dose of catalpol group and Fasudil?P <0.05?.The expression of Rho,RhoA and ROCK2 mRNA was significantly decreased?P <0.01?.The expression of Claudin-5 and ZO-1 in the model group induced by LPS was significantly lower than that in normal group?P <0.01?.The expression of RhoA,ROCK2 were significantly increased?P <0.01?.Compared with the group of model,the expression of Claudin-5 and ZO-1 in each dose of catalpol group and Fasudil were significantly increased?P <0.05?,RhoA,ROCK2 expression was significantly decreased?P <0.05?.5 ConclusionCatalpol has a protective effect on the destruction of tight junction of BMECs induced by LPS,and its protective mechanism may be closely related to RhoA/ROCK pathway. |
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