Mechanism Study Of Serum L-glutamate Causing Coronary Artery Lesions In Kawasaki Disease

Objective This study aimed to investigate the effect of serum L-glutamate(L-glu)on coronary artery lesions in Kawasaki disease and its mechanism of inhibiting ribosomal autophagy(ribophagy)by inhibiting the cystine/glutamate reverse transporter(System XC~-).Methods Metabolites upregulated in children with Kawasaki disease were screened by LC-MS metabolomic profiling,and peripheral blood specimens were collected from eligible children with Kawasaki disease as well as normal children with healthy physical examination,and the expression of L-glu in peripheral serum was measured by enzyme-linked immunosorbent assay(ELISA).The CCK8 assay was used to clarify that serum L-glu triggers the death of umbilical vein endothelial cells(HUVECs).Single-cell sequencing data(GSE178765)of KD mice were downloaded from the Gene Expression Omnibus(GEO)database,and RNA sequencing data of vascular endothelial cells were extracted and analyzed to obtain the KD vascular HUVECs were verified to inhibit ribophagy by protein blotting(Western blot),quantitative real-time polymerase chain reaction(qRT-PCR),and immunofluorescence techniques.We verified that ribophagy was affected through the inhibition of the System XC~-pathway by measuring Western blot,reduced glutathione(GSH),and reactive oxygen species(ROS).Results Serum levels of L-glu were significantly higher in children with KD in the acute phase compared with the NC group(P<0.001),while serum levels of L-glu were significantly higher in children with KD complicated by CAL than in the group of children with KD(P<0.001);cell viability of HUVECs underwent a significant decrease in L-glu,KD,and L-glu+healthy children stimulated by serum compared with the control group(P<0.05 or P<0.01);L-glu-treated HUVECs showed no significant changes at low concentrations and for a short period of time,but cell viability of HUVECs showed a significant decrease with increasing concentration and time(P<0.001);KD vascular endothelial cell disorder was closely associated with ribosomes;The expression of LC3,ATG5,and ATG7 was significantly lower(P<0.05 or P<0.01)and rp S6 was significantly higher(P<0.001)in the L-glu group compared with the NC group;the expression of LC3,ATG5,and ATG7 was significantly higher in the L-glu+Rapamycin group compared with the L-glu group(P<0.05 or P<0.01 or P<0.001)and rp S6 was significantly decreased(P<0.001);the expression of System XC~-catalytic subunit(Slc7a11)was significantly decreased in HUVECs after L-glu stimulation(P<0.001),and the addition of Ferrostatin-1 significantly reversed this result(P<0.001);The intracellular GSH content was significantly lower in the L-glu group compared with the NC group(P<0.001),while the L-glu+Ferrostatin-1 group could increase the GSH content compared with the L-glu group(P<0.01);The ROS levels were significantly increased after L-glu stimulation of HUVECs(P<0.001);while ROS levels were significantly decreased in the L-glu+Ferrostatin-1 group compared with the L-glu group(P<0.05).Conclusions 1.Increased expression of L-glu in the serum of children with Kawasaki disease is a key factor contributing to coronary endothelial dysfunction;2.Serum L-glu promotes ROS and inhibits ribosomal autophagy in vascular endothelial cells by regulating System XC~-,leading to endothelial cell lesions in coronary arteries.