Role Of Acid-sensing Ion Channels (ASICs) In Acid-induced Vascular Endothelial Cell Injury In Children With Henoch-sch(?)nlein Purpura

Objective Investigate the expression of acid sensing ion channels(ASICs)on human microvascular endothelial cells(HDMECs)and its role in regulation in vascular endothelial inflammatory injury in children with HSP through the observation of the effect of HSP serum a Ig A1 and extracellular acid on ASICs expression on HDMECs and its influence on the secretion of inflammatory cytokines and expression of cytoskeletal proteins,and the protection of acid sensitive ion channel blocker in endothelial cell injury.Methods1.Specimen Collection and the isolation and purification of a Ig A1 Ten children with acute HSP and ten normal healthy children were enrolled in the Study.5ml Serum samples were obtained from peripheral vein of all subjects in the morning.Serum a Ig A1 was purified by jacalin affinity chromatography and protein purification apparatus.2.Experimental grouping HDMECs cells can be divided into 10 groups,1 group as the blank control group,2group of p H6.5 group,3 group of normal Ig Al,4 group of normal Ig Al+ p H6.5 group,5group of normal Ig Al+p H6.5+amiloride group,6 group of normal Ig Al+p H6.5+Pc TX1,7 group of HSP Ig Al,8 group of HSP Ig Al+p H6.5,9 group of HSP Ig Al+p H6.5+amiloride,10 group of HSP Ig Al+p H6.5+Pc TX1,each group of three holes;taken Ig A1 from healthy controls or patients with HSP in six orifice,Ig A1 fromhealthy controls was added to 2-6 groups,Ig A1 from patients with HSP was added to 7to 10 groups,and adjust the medium Ig A1 concentration 250?g/ml,refuse culture supernatant after 6h;5,9 group to join amiloride 100 umol/L,6,10 group to join Pc TX1 20nmol/L,incubation and a half hours;After half an hour,adding acid to2,4,5,6,8,9,10,adjust the medium p H value of 6.5,incubation 4 hours.3.IL-8,NO and TM concentration the concentration levels of IL-8,NO and TM were detected by enzyme-linked immunosorbent assay(ELISA).All operations are executed strictly according to kit instructions.4.Analysis ASICs,destrin and SM-a m RNA and protein expression Use real-time quantitative polymerase chain reaction(q PCR)and Western blot(WB)method to analysis ASIC1 a,ASIC2a,ASIC3,destrin and SM-a m RNA and protein expression,All operations are executed strictly according to kit instructions.Result1.In the pre experiment,the expression of ASIC1 a,ASIC2a,ASIC3 on HDMECs in p H6.5 group was significantly higher than that in the other three groups,the difference was statistically significant(P <0.05).2.Extracellular acidification and the a Ig A1 separad from HSP children can raise HDMECs acid sensitive ion channels(ASIC1a ASIC2 a ASIC3)expression,the ASICs expression capacity of p H6.5 and HSP Ig Al group are more than blank control group and normal Ig Al group,and HSP Ig Al+p H6.5 group is more than HSP Ig Al group,the differences were statistically significant(P<0.05).3.Extracellular acidification and the a Ig A1 separad from HSP children can stimulate HDMECs releasing NO,TM and IL-8.NO,TM and IL-8 level of p H6.5 and HSP Ig Al group are more than blank control group and normal Ig Al group,and HSP Ig Al+p H6.5group is more than HSP Ig Al group.Amiloride and Pc TX1 can restrain the excessiverelease of inflammatory cytokines,NO,TM and IL-8 level of HSP Ig Al+p H6.5+amiloride group and HSP Ig Al+p H6.5+Pc TX1 are lessthan HSP Ig Al+p H6.5 group.In addition,in group HSP Ig Al+ p H6.5,IL-8,TM,NO levels were positively correlated with the expression level of ASIC1 a m RNA(R=0.947,0.864,0.829);IL-8,TM,NO levels were positively correlated with the expression level of ASIC2 a m RNA(R=0.833,0.827,0.874);IL-8,TM,NO levels were positively correlated with the expression level of ASIC3 m RNA(R=0.777,0.851,0.693),the difference statistically significant(P<0.05).4.Extracellular acidification and the a Ig A1 separad from HSP children can reduce HDMECs cytoskeleton proteins(destrin,SM-a)expression.The destrin and SM-a expression capacity of p H6.5 and HSP Ig Al group are less than blank control group and normal Ig Al group,and HSP Ig Al+p H6.5 group is less than HSP Ig Al group.Amiloride and Pc TX1 can inhibit reaction of reduced expressions of destrin and SM-a,The destrin and SM-a expression capacity of HSP Ig Al+p H6.5+amiloride group and HSP Ig Al+p H6.5+Pc TX1 are more than HSP Ig Al+p H6.5 group.In addition,in group HSP Ig Al+ p H6.5,the destrin m RNA level and the SM-? m RNA level arenegatively related to the ASIC1 a m RNA level(destrin,R=-0.713;SM-?,R=-0.867).The destrin m RNA level and the SM-? m RNA level are negatively related to the ASIC2 a m RNA level(destrin,R=-0.842;SM-?,R=-0.798).The destrin m RNA level and the SM-? m RNA level are negatively related to the ASIC3 m RNA level(destrin,R=-0.671;SM-?,R=-0.742),the difference statistically significant(P<0.05).Conclusions Extracellular acidification and a Ig A1 of HSP could induce ASICs expression on HDMECs and cause endothelial cell damage by releasing a large number of inflammatory cytokines and reducing cytoskeletal proteins,and the extracellularacidification effect even more,while the ASICs blocker can inhibit these effects,protec vascular endothelial cell,also demonstrat that ASICs may be involved in the inflammation process of vascular endothelial cells from the other side.