The Effect Of CSEsiRNA On Hydrogen Sulifde-mediated EDHF Relaxation In Rat Middle Cerebral Artery

The endothelium consists of a single layer of cells lining the inner wall of thevasculature. It serves as a barrier between blood and tissues and actively participates inthe regulation of vascular function. Vascular homeostasis and relaxation are controlledby the endothelium via the synthesis and release of a number of endothelial-derivedrelaxing and constricting substances.NO,PGI₂and endothelial derived hyperpolarizingfactor （EDHF） are the substances which regulate the vascular of relaxing andconstricting. However, EDHF is the third activing factor independing on NO and PGI₂,as it is conventional that the mechanism and chemical character is unknown, presentlythe research on EDHF in the cerebral vascular remains little acknowledgement.Hydrogen sulfide （H₂S） is an important gas transmitter molecular in the body,which serves a vital physiological fuction as NO,CO gas molecular. H₂S is generatedthrough dissolving the L-cystein acid by cystathionine beta synthase （CBS） orcystathionine gamma lyase （CSE）, and CSE and CBS is tissue specific. CBS mainleexists in the brain, as CSE mainly exists in the cardiovascular system. RNAi is atechnology of gene silencing recently, which may carry out research on specific fuctionin specific tissue. This article further studies the effect on EDHF in rat middle cerebralarteries of H₂S.Object:The siRNA technique was used to explore whether H₂S mediated the EDHF dilationin rat MCA or not. Methods:1. Design and chemically synthesis three specified and one nonspecified CSE siRNA,which are CSEsiRNA1, CSEsiRNA2, CSEsiRNA3, negative control CSEsiRNA.Applying Lipofectamine2000to transfect EAhy926, observe transfection efficiencywith flourence microscope, test the CSE expression with Westernblot, and collectthe cells to detect the content of H₂S.2. The relaxant effects of ACh on isolated and perfused MCAs were detected byvasomotoricity experiment in vitro. The effects of non-NO-non-PGI₂relaxation wasinduced by ACh in the presence of Nω-nitro-L–arginine-methyl-ester (L-NAME,3×10^-5mol/L) and indomethacin(Indo,10^-5mol/L), The effects of the combinationapplication of apamin （100nmol/l） and charbdoxin（0.5nmol/l） orDL-propargylglycine （PPG,1×10-4mol/L） on the non-NO-non-PGI₂relaxationwere also observed. EAhy926cells with or without transfection of CSEsiRNA wasadded to mimick the EDHF-mediated dilation in the endothelium-denuded ratMCA.Results:1. The transfection efficiency is about60%, the expression of CSE protein decreasesfrom153.41±23.43mol/L/105in the normal group and150.22±17.56mol/l/105in theNeg-CSEsiRNA group to59.43±21.81mol/L/105in the CSEsiRNA3group, as well asthe content of H₂S decreases from0.96±0.05mol/L/105in the normal group and0.95±0.06mol/L/105in the Neg-CSEsiRNA group to0.54±0.08mol/L/105in theCSEsiRNA3group.2.In rat MCA preconstricted by30mmol/L KCl,ACh produced concentration-dependentrelaxation in the presense and absence of L-NAME (3×10^-5mol/L) and Indo(10^-5mol/L).ACh-induced vasorelaxation in the presense of L-NAME and Indo was markly inhibited by combination application of apamin （100nmol/L） and charbdoxin（0.5nmol/L） or by10^-4mol/L PPG （an inhibitor of CSE）.3. In the endothelium-denuded rat MCA, normal EAhy926cells, but not EAhy926cellswith transfection of CSEsiRNA could mimick the EDHF-mediated relaxation with anEmax of66.09±1.63%.Conclusions1. In the rat MCA, ACh-induced non-NO-non-PGI₂relaxation, the so-called EDHFdilation might be mediated by H₂S.2. In the endothelium-denuded rat MCA, the transfection of CSEsiRNA could abolishthe EDHF-mediated dilation mimicked by EAhy926cells...