The Activation And Function Of NLRP3-related Inflammatory Factors IL-1βand IL-18 In Primary Brain Microvascular Endothelial Cells

Objective Using lipopolysaccharide(LPS)and adenosine triphosphate(ATP)as stimuli to investigate the activation and function of NLRP3-related inflammatory factors IL-1β and IL-18 in primary brain microvascular endothelial cells(BMECs),to further explore the correlation between cerebral small vessel disease(CSVD),and to provide experimental and theoretical basis for finding new diagnostic and therapeutic markers and develop new CSVD drugs of CSVD,which are based on immune inflammation.Method Obtaining BMECs with high purity and observing the growth by cell morphology.Using the immunofluorescence staining to detect the expression of factor VIII related antigen(vWF)and glial fibrillary acidic protein(GFAP)in BMECs.The experiment was divided into normal control group,LPS group,LPS and ATP co-stimulation group.Using ELISA to detect the expression of NLRP3-related inflammatory factors IL-1β and IL-18,and using MTT to detect the cell survival rate in each experimental group.Results(1)Cell morphology showed typical "beads" or "branching" after 48 hours of isolating and culturing the brain microvascular segments,On the 4th day of culture,BMECs were found to creep out around the microvascular segments of the brain,which were typical "short fusiform".On the fourth day of culture,endothelial cells appeared typical "short spindle".On the 8th day of culture,the cell confluence could reach more than 80%,showing typical "paving stone" or "whirlpool shape".Immunofluorescence staining results showed the expression of v WF in BMECs was positive,while the expression of GFAP was negative.The purity of endothelial cells(the expression rate of v WF positive cells)was over 95%.(2)Compared with the normal control group,ELISA results showed that the expression of IL-1β and IL-18 had no significant effect in LPS stimulation group(P>0.05),but it increased significantly in LPS and ATP co-stimulation group(P<0.05).(3)Compared with the normal control group,MTT results showed that the survival rate of cells in LPS alone stimulation group was decreased,but there was no significant difference(P>0.05);while the survival rate of cells was significantly decreased in LPS and ATP co-stimulation group,and there were almost no surviving cells,the difference was statistically significant(P<0.05).Conclusions(1)LPS and ATP can stimulate the production of the NLRP3-related inflammatory factors IL-1β and IL-18 in primary BMECs.(2)LPS and ATP can significantly reduce the survival rate of primary BMECs.(3)It may be related to the pathogenesis of CSVD that the NLRP3-related inflammatory factors IL-1β and IL-18.