The Effects And Mechanisms Of Advanced Glycation End Products-induced Calcium Dyshomeostasis In Intervertebral Disc Degeneration

Objective:Previous studies identified advanced glycation end product（AGE）accumulation in the intervertebral disc（IVD）as an essential risk factor associated with IVD degeneration via accelerated cell apoptosis and impeded extracellular-matrix metabolism;however,the underlying mechanisms have not been fully elucidated.Herein,we aimed to evaluate the effects of AGEs on endoplasmic reticulum（ER）stress,apoptosis,and subcellular calcium ion(Ca²⁺)redistribution.Methods:Human nucleus pulposus（NP）cells were incubated with various concentration of AGEs,including PBS,AGEs 100μg/ml and AGEs 200μg/ml,for 0 h,12 h and 24 h in the presence of calcium antagonists U73122,xestospongin C and Ryanodine or not.Calcium sensitive probe Fluo-4AM/Mag-Fluo-4AM were used to detect cytosolic/ER luminal calcium concentration(([Ca²⁺]c/[Ca²⁺]er).RT-PCR and western blot were used to explore the expression levels of ER-resident pumps and channels（SERCA and IP3R,RyR）,intracellular IP3 levels were detected by ELISA,cell apoptosis was observed with flow cytometry using AnnexinV-APC/7-AAD.Results:AGEs induced sustained[Ca²⁺]c elevation and[Ca²⁺]er depletion in a concentration-and time-dependent manner in NP cells.Furthermore,we observed significant increases and decreases in levels of the ER-resident Ca²⁺-release channels inositol 1,4,5-triphosphate receptor and ryanodine receptor and ER Ca2+-reuptake pumps sarco/endoplasmic reticulum Ca²⁺-ATPase,respectively.Pharmacologically blocking ER Ca²⁺release using Ca²⁺antagonists significantly ameliorated Ca²⁺dyshomeostasis,ER stress,and subsequent apoptosis in NP cells.Conclusions:Impaired Ca²⁺homeostasis plays an essential role in AGE-mediated ER stress and subsequent apoptosis in NP cells,with blockade of ER Ca²⁺release partially ameliorating subcellular Ca²⁺redistribution,ER stress,and apoptosis.Our findings provide novel mechanistic insight into the role of AGEs in the pathogenesis of IVD degeneration and a potential therapeutic strategy...