The Mechanism Of Apoptosis Through Endoplasmic Reticulum Stress By Fatty Acid-Binding Protein 4 In HMC

Objective: Diabetic nephropathy(DN),as the most common complication in diabetes,is becoming the leading cause of end-stage renal disease due to the increasing morbidity and mortality worldwide.The histological hallmarks of DN first include thickening of the glomerular basement membrane(GBM),diffuse expansion of glomerular mesangial matrices,accumulated extracellular matrix,and then segmental nodular glomerular sclerosis,glomerular filtration rate decreased and finally lead to renal failure.The apoptosis of mesangial cells and the accumulation of extracellular matrix play important roles in the progression of DN.It is widely accepted that hyperglycemia and glycation end products aggravate diabetic nephropathy.As research continues,accumulation of lipids in non-adipose tissues can contribute to cellular dysfunction and cell death,a phenomenon that is called lipotoxicity attracts people’s attention.But how it works is still unclear.Fatty acid binding protein 4(FABP4)—also called aP2,is a member of FABPs.It is a carrier protein for fatty acids and other lipophilic substances and exerts diverse effects on modulation of inflammation,insulin resistance,and other metabolic pathways.Endoplasmic reticulum(ER)is the central site for lipid synthesis,calcium storage,protein folding and mature in eukaryotic cell,It takes part in the intrinsic pathway of apoptosis.However,when the homeostasis of ER is disrupted,endoplasmic reticulum stress(ERS)begins.Prolonged ER stress causes cell apoptosis.ER acts as an organelle sensor of lipid-related stress.High level of FFA-induced apoptosis,known as lipoapoptosis,is mediated by ERS in several types of cells including pancreatic β cells and hepatocytes.But the mechanisms underlying the apoptosis of β cells and hepatocytes remain incompletely understood.Our previous research confirm for the first time that the normal HMC express FABP4 and the expression of FABP4、ERS related proteins as well as apoptosis related proteins are up-regulated by high glucose or FFAs.We assumed that FABP4 might be involved in ERS and its related apoptosis induced by FFAs.The present study was designed basing on our previous research.We explored the role of FABP4 in ERS and apoptosis induced by high glucose and FFAs on the cellular level.Two parts were involved in this experiment: 1 RNA interference to knock down FABP4 in HMC,comparing the changes on the expression of ERS related proteins,apoptosis related proteins and their mRNA levels before and after transfection,exploring the effect of FABP4,FFA,ERS on apoptosis.2 Comparing the changes on the expression of FABP4,ERS related proteins,apoptosis related proteins and their mRNA levels before and after the interference of pioglitazone(PIO)induced by high glucose or FFA,to explicit the role of FABP4,FFA,ERS on apoptosis.Method:1 RNA interference to knock down FABP4,observing the changes on the expression of ERS related proteins and apoptosis related proteins in HMC before and after transfection induced by high-glucose or high FFAs.(1)Grouping: HMC cells were randomly divided into three groups which include high oleic acid(OA),high glucose(HG)and high palmitic acid(PA).Each stimulation contains control group(Control,Ctrl),empty plasmid group(nontargeting control,NCsi),plasmid silencing group(FABP4 siRNA,FABP4si),stimulation plus plasmid silencing group(Stimuli + FABP4 siRNA),stimulation(Stimuli)group.(2)Observing the expression of FABP4,ERS related proteins and apoptosis related proteins in HMC induced by OA,HG and PA detected by Western Blot and Real Time PCR;(3)RNA interference to knock down FABP4 in HMC.Observing the changes on the expression of FABP4,ERS related proteins and apoptosis related proteins induced by OA,HG and PA detected by Real-TimePCR and WesternBlot.2 Using pioglitazone to intervene HMC,observing the expression of FABP4,ERS related proteins and apoptosis related proteins stimulated with high glucose or high FFAs.(1)Grouping: HMC cells were randomly divided into three groups that include high oleic acid(OA),high glucose(HG)and high palmitic acid(PA).Each stimulation contains control group(Control,Ctrl),0.5% DMSO group,stimulation plus pioglitazone treated group(Stimuli + Pi),stimulation group(Stimuli).(2)Observing the expression of FABP4,ERS related proteins and apoptosis related proteins in HMC induced by OA,HG and PA detected by Western Blot and Real Time-PCR;(3)Intervened by pioglitazone in HMC,Observing the expression of FABP4,endoplasmic reticulum stress related proteins and apoptosis related proteins stimulated with OA,HG and PA detected by Real Time PCR and Western Blot before.Results: 1 The first part(1)The expression and RNA level of FABP4 stimulated with OA,HG PA : The expression of FABP4 is dramatically up-regulated by OA,HG,PA detected by Real Time-PCR and Western Blot;(2)The effect of OA,HG and PA on ERS related proteins and apoptosis related proteins in HMC: The expressions of p-IRE1 a,p-PERK,ATF6,GRP78,CHOP,Cleaved Caspase3,Cleaved Caspase12 were induced by OA,HG,PA detected by Real Time-PCR and Western Blot;(3)The effect of RNA interference on FABP4: FABP4 expression was remarkably decreased by siRNA compared to the Control group;(4)The changes of ERS related proteins and apoptosis related proteins before and after RNA interference: The expressions of p-IRE1 a,p-PERK,ATF6,GRP78,CHOP,Cleaved Caspase3,Cleaved Caspase12 were much lower than that in stimulation group after knocking down FABP4 by RNA interference;(5)The effect of empty vector plasmid on the expressions of FABP4,ERS related proteins and apoptosis related proteins in HMC: The NC has no significant difference on the expression of FABP4,ERS related proteins and apoptosis related proteins in HMC detected by Real Time-PCR and Western Blot compared to the Control group.2 The second part(1)Observing the changes on the expression of FABP4 stimulated with OA,HG,PA before and after pioglitazone intervention: Pioglitazone remarkably decreased the expression of FABP4 induced by OA/HG on mRNA and protein levels whereas it has no effect on FABP4 expression in PA-stimulated HMCs;(2)The influence of pioglitazone treatment on the expression of ERS related proteins and apoptosis related proteins stimulated with OA、HG、PA detected by Western Blot: Treated by pioglitazone,the expressions of p-IRE1 a,p-PERK,ATF6,GRP78,CHOP,Cleaved Caspase3 were significantly declined in HMCs stimulated by OA/HG whereas it has no effect on PA-treated cells;(3)The influence of pioglitazone treatment on the RNA level of ERS related proteins and apoptosis related proteins stimulated with OA、HG、PA detected by Real Time-PCR: Treated with pioglitazone,the RNA level of IRE1 a,PERK,ATF6,GRP78,CHOP is significantly declined induced by OA/HG.But it has no effect on PA-treated cells.Conclusion:1 The expression and RNA level of FABP4,ERS related proteins and apoptosis related proteins is induced by OA/HG/PA in HMC;This proves that high glucose and high FFAs induce expression of FABP4 and initiate ERS and ERS related apoptosis pathway.2 FABP4 knocked down by RNA interference reduces the expression and RNA level of ERS related proteins and apoptosis related proteins induced by OA/HG/PA in HMC;This proves that FABP4 mediates apoptosis via endoplasmic reticulum stress induced by high glucose and high FFAs.FABP4 is in the upstream of ERS;3 PPARγ agonist Pioglitazone downregulates the expression of FABP4 induded by OA/HG,then relieve the ERS and the apoptosis.Pioglitazone does not decrease the expression of FABP4 induded by PA,thus it dose not relieve the ERS and the apoptosis.This further proves that high glucose and OA induce apoptosis via ERS by FABP4.The relationship among PPARγ、FABP4 and saturated fatty acid still need further study.