Berberine Alleviates Palmitic Acid-induced Podocyte Apoptosis By Reducing Reactive Oxygen Species-endoplasmic Reticulum Stress

BackgroundAs terminal differentiated cell,the podocyte is a crucial part of the glomerular filtration barrier,and crucial to maintain the normal function of glomerular filtration.Abnormal Lipid accumulation in podocyte can lead to the cellular lipotoxicity,injury and apoptosis,which is a key factor in progression of chronic kidney disease(CKD).Berberine(BBR),an isoquinoline alkaloid extracted from the medicinal plants such as coptis chinensis,used as a clinical favorable medicine to treat intestinal infectious disease,has been acknowledged for its lipid-lowering effect and prevention of progression of CKD in recent years.However,the role of BBR on palmitic acid(PA)-induced podocyte damage remains unknown.ObjectiveThis study intends to investigate the effect of berberine on PA–induced podocyte apoptosis,further to clarify whether its mechanism is related to its suppression of ROS-endoplasmic reticulum stress inpodocytes,and to provide a basic evidence for the protective effect of berberine on lipid nephrotoxicity.MethodsMouse podocytes were treated with PA,an ER stress inhibitor4-phenyl butyric acid(4-PBA),BBR or a potent antioxidant N-acetylcysteine(NAC).Cell Counting Kit 8(CCK-8)colorimetric assay was used to detect cell death.Cell apoptosis rate was counted using flow cytometry assay.The expression of endoplasmic reticulum stress(ER stress)-related proteins including Heavy-chain Binding protein(BIP),protein kinase RNA-like endoplasmic reticulum kinase(PERK),activating transcription factor4(ATF4),C?EBP homologous protein(CHOP),activating transcription factor6(ATF6),inositol requiring enzyme 1α(IRE1α)and Cleaved-caspase12 and apoptosis-related proteins Cleaved-caspase3 were detected by western blot assay.Protein expression of BIP was also measured by immunofluorescence.And DCFH-DA fluorescence staining was used to evaluate reactive oxygen species(ROS)with fluorescence microscopy.Results(1)Compared with the control group,PA can increase podocyte apoptosis and the expression of apoptosis-related protein Cleaved-caspase3.Berberine can reduce PA-induced podocyte apoptosis.(2)PA can increase the expression of endoplasmic reticulumstress-related proteins BIP,PERK,ATF4,CHOP,ATF6,IRE1α in a dose-dependent manner in podocytes.Moreover,PA can cause a mass of accumulation of BIP immunofluorescent particles in podocytes.(3)Endoplasmic reticulum stress inhibitor 4-PBA can alleviate PA-induced podocyte apoptosis and downregulate the expression of Cleaved-caspase3 and endoplasmic reticulum stress related proteins BIP,PERK,ATF4,CHOP,ATF6,IRE1α and Cleaved-caspase12.In addition,it can reduce the fluorescence intensity of BIP in podocytes induced by PA.(4)PA can increase ROS levels in podocytes in a dose-dependent manner,and antioxidant reagent NAC can reduce ROS production in podocytes.Besides,NAC can reduce PA-induced podocyte apoptosis and the expression of Cleaved-caspase3 and endoplasmic reticulum stress-related proteins BIP,PERK,ATF4,CHOP,ATF6,IRE1α,Cleaved-caspase12.(5)Berberin can reduce the expression of endoplasmic reticulum stress-related proteins BIP,PERK,ATF4,CHOP,ATF6,IRE1α and Cleaved-caspase12 and ROS production induced by PA in podocytes.Meanwhile,berberine can reduce PA-induced BIP fluorescence intensity.Conclusion(1)PA can induce podocyte apoptosis,and its mechanism may be associated with the elevation of ER stress and ROS production.(2)BBR can protect against the PA-induced podocyte apoptosis viasuppressing ROS-dependent ER stress.