Effect Of Oxidized Low-density Lipoprotein On Proliferation And Calcium Signal In Lens Capsule Epithelial Cells

BackgroundSerum lipid levels,especially oxidized low-density lipoprotein levels,are related to the occurrence of cataracts,but the mechanism of oxidized low-density lipoproteins in cataract development is unclear.Numerous literatures support that calcium(Ca2+)overload in the lens is involved in the opacity of the lens.Store-operated Ca2+entry(SOCE)controlled by the Ca2+pool is a vital way to mediate Ca2+exchange in lens capsule epithelial cells.Orai family proteins(forming a SOCE channel)are located on the cell membrane in response to the depletion of Ca2+storage in the endoplasmic reticulum and control the influx of extracellular Ca2+.In our study,we cultured human lens capsule epithelial cells(HLEpiCs)in a medium containing oxidized low-density lipoprotein to simulate a high-fat environment in vitro,and explored the impact mechanism of oxidative low-density lipoprotein on cell proliferation and calcium signals of HLEpiCs.This study provides a theoretical basis for drug development for the treatment of obesity or hyperlipidemia-related cataracts.Objective1.To explore the role of oxidized low-density lipoprotein in proliferation of HLEpiCs;2.To explore the role of oxidized low-density lipoprotein in calcium signals of HLEpiCs.Methods1.RNA extraction,sequencing and analysisRNA from HLEpiCs treated with oxidized low-density lipoprotein and controls was extracted.The RNA samples were sequenced using the Illumina platform and raw data was obtained.The data that have passed the quality inspection will be subjected to difference analysis,cluster analysis and various biological analyses.2.Western Blot experimentWestern Blot was used to detect Orai1,Orai2,and Orai3 protein expression levels in HLEpiCs treated with oxidized low-density lipoprotein and control.3.Cell proliferation experimentHLEpiCs with different processing factors were seeded in 96-well plates(103-104 cells/well),100 ?L of culture medium per well,and cultured for 24 h.10 ?L of CCK-8 solution was added to each well,and the cells were incubated in an incubator for about 2 h.The cell culture plate was placed in a microplate reader,and the absorbance of HLEpiCs with various processing factors at a wavelength of 450 nm was measured.4.Ca2+ imaging experimentHLEpiCs with different treatment factors were inoculated on coverslips,cultured for 24 hours,and then added with fluorescent dye(Fluo-8)and incubated for 30 min.Adenosine triphosphate(ATP)and calcium chloride(CaCl2)were added to detect Ca2+release and SOCE.5.Identification of knockout ratsFirst,DNA was extracted from rat toe tissue,and rat DNA was amplified by PCR amplification technology.Knockout rats were determined based on the results of agarose gel electrophoresis.6.Statistical analysisAll data are expressed as mean±standard error(mean±S.E.M.).Graphpad Prism 8.0 software was used for Student's unpaired t test.When the results showed that P<0.05,the difference between the two groups was considered statistically significant.Results(1)The results of cluster analysis showed that oxidized low-density lipoprotein enhanced the expression of Orai3 in HLEpiCs,but the expression of Orail and Orai2 was not significantly changed.(2)Oxidized low-density lipoprotein promoted the proliferation of HLEpiCs,which is dependent on the concentration.(3)Oxidized low-density lipoprotein significantly increased ATP-induced SOCE in HLEpiCs,but there was no significant effect on Ca2+ release.(4)The results of Western Blot showed that oxidized low-density lipoprotein enhanced the expression of Orai3 in HLEpiCs,but the expression of Orail and Orai2 was not significantly changed.(5)Knockdown of Orai3 significantly reduced cell proliferation and ATP-induced SOCE in HLEpiCs.Conclusion(1)Oxidized low-density lipoprotein enhanced the expression of Orai3 in HLEpiCs,but the expression of Orai1 and Orai2 was not significantly changed.(2)Oxidized low-density lipoprotein promoted the proliferation of HLEpiCs,and Orai3 siRNA inhibited the enhanced proliferation caused by oxidized low-density lipoprotein.(3)Oxidized LDL increased ATP-induced SOCE in HLEpiCs,and Orai3 siRNA inhibited the increase in SOCE levels caused by oxidized low-density lipoprotein.