Study On Protective Effect And Mechanism Of Artemisia Annua And Dihydroartemisinin On Cerebrovascular In A Septic Mouse Model

Objective: To study the protective effect and mechanism of Artemisia annua and its active component dihydroartemisinin(DHA)on blood-brain barrier(BBB)damage in sepsis mice.To clarify the molecular mechanism of DHA protect BBB permeability during sepsis by inhibiting snail family transcriptional repressor 1(SNAI1)then promoting the expression of tight junction protein occludin(OCLN).Methods:(1)Cecal ligation and puncture(CLP)sepsis mice model was constructed and gavaged with Artemisia annua.The effects of Artemisia annua on S100β levels in serum,Evans blue exudation in brain tissues,brain water content and TNF-α level were detected by ELISA,Evans blue dye and dry-wet weight experiments respectively.These were used to investigate the protective effect of Artemisia annua on early brain injury,BBB injury,brain water content and TNF-α level in septic mice.(2)Establishment of CLP sepsis mice model and gavaged with DHA.The effects of DHA on early brain injury,BBB permeability,brain water content and TNF-α level was detected by ELISA,Evans blue,dry/wet weight experiments.The effects of DHA on m RNA and protein expression of occludin(OCLN)and claudin 5(CLDN5)were detected by RT-q PCR,Western blot,immunohistochemistry and immunofluorescence.The in vitro cell model was established by TNF-α induced human microvascular endothelial cells(h CMEC)/D3(TNF-α-h CMEC/D3)and pretreated with DHA.The effects of DHA on cell viability and cell permeability were investigated by CCK-8 assay and FITC-Dextran assay.The effects of DHA on the m RNA and protein expression of OCLN and CLDN5 were examined by RT-q PCR,Western blot,immunohistochemistry and immunofluorescence assay respectively.These were used to explore the protective effect of DHA on brain injury in CLP sepsis mice from the perspective of BBB permeability,and to explore its possible mechanism from the perspective of regulating tight junction protein expression.(3)The in vivo CLP mouse model and in vitro TNF-α h CMEC/D3 cell model were established to investigate the m RNA and protein levels of SNAI1 in cerebral microvasculars by using RT-q PCR,Western blot and immunofluorescence experiments.h CMEC/D3 were transfected with SNAI1 small interfering RNA(si RNA)and SNAI1 overexpressed plasmid.Small interfering RNA(si RNA)was used to inhibited SNAI1 expression in h CMEC/D3 cells,and the regulation of SNAI1 on OCLN m RNA and protein expression was detected by RT-q PCR and Western blot.SNAI1 was overexpressed in TNF-α-h CMEC/D3 cells,and the effects of DHA on OCLN m RNA and protein levels and cell permeability were detected by RT-q PCR,Western blot and FITC-Dextran assay.This study explored the mechanism of DHA reducing permeability of cerebrovascular endothelial cells under TNF-α condition from the perspective of SNAI1 regulating OCLN.Results:(1)Artemisia annua could significantly reduce the levels of S100β in serum,Evans blue exudation in brain tissue,brain water content and TNF-α in CLP sepsis model mice.(2)DHA significantly reduced S100β level in serum,Evans blue exudation,brain water content and TNF-α level in CLP sepsis model mice;DHA increased the m RNA and protein levels of tight junction proteins OCLN and CLDN5 in cerebral blood vessels of CLP sepsis mice.50 and 100 ng/m L DHA had no significant effect on the cell activity of TNF-α-h CMEC/D3,while 250 and 500 ng/m L DHA significantly reduced the cell activity of TNF-α-h CMEC/D3.50 and 100 ng/m L DHA significantly increased the expression of OCLN in TNF-α-h CMEC/D3,but had no significant effect on the protein expression of CLDN5,suggesting that the protective effect of DHA on cerebrovascular endothelial permeability may be through influencing OCLN rather than CLDN5.DHA could significantly inhibited the permeability of TNF-α-h CMEC/D3,but had no significant effect on the permeability of h CMEC/D3 cells without TNF-α treatment.DHA significantly up-regulated the m RNA and protein level of OCLN in TNF-α-h CMEC/D3,but had no significant effect on OCLN expression in cells without TNF-α treatment.(3)DHA significantly reduced the m RNA and protein level of SNAI1 in brain microvessels of CLP model mice;DHA decreased the m RNA and protein levels of SNAI1 in TNF-α-h CMEC/D3;SNAI1 si RNA up-regulates the m RNA and protein expression of OCLN in h CMEC/D3.SNAI1 overexpressed reduced OCLN expression in TNF-α-h CMEC/D3,but DHA added after SNAI1 overexpression had no significant effect on OCLN expression.Conclusins:(1)Artemisia annua pretreatment had a protective effect on BBB damage in CLP sepsis mice,significantly reduced BBB permeability and alleviated early brain injury and edema in CLP sepsis mice.(2)DHA,one of the effective components of Artemisia annua,also had protective effects on BBB injury in CLP sepsis mice,significantly reducing BBB permeability,alleviating early brain injury and brain edema,decreasing the level of TNF-α in brain tissue,and increasing the expression of tight junction proteins OCLN and CLDN5.(3)DHA up-regulated the expression of OCLN and decreased the permeability of TNF-α induced cells in vitro cerebrovascular endothelial cell culture model.(4)DHA upregulated the expression of OCLN by inhibiting the level of SNAI1 and affecting cell permeability under TNF-α condition.In in vitro cerebral vascular endothelial model,DHA reduce the permeability of cells induced by TNF-α,and raised the expression of OCLN inhibited by TNF-α.(4)DHA up-regulated the expression of OCLN and decreased cell permeability by inhibiting the level of SNAI1 in cerebrovascular endothelial cells in condition of TNF-α.