Study On Lipid Metabolism Of Cadmium On HepG2 Cells Based On LC-MS

Backgrounds:Cadmium（Cd）is a non-essential trace element,which has a half-life of 10-30 years in the human body.The research on its pollution status and toxicity mechanism has attracted more attention.Current research reports that chronic low-dose Cd exposure could cause health risks to the general population.Many inflammatory biomarkers are released after cadmium exposure,which are related to different diseases,such as osteoporosis,hypertension,atherosclerosis,and cardiovascular disease,obesity and type 2 diabetes.In recent years,lipidomics have gradually become hotspots,but the lipid metabolome after cadmium exposure has not been systematically studied.At the same time,many studies have shown that there is an association between cadmium and obesity,diabetes and metabolic syndrome,but it is not clear.Therefore,this project intends to study different doses of Cd Cl₂exposured to HepG2 cells at different time points.The effect of cadmium chloride on the HepG2 cell viability was measured by CCK-8 kits,calculated IC₅₀,and chose the exposure dose and time of Cd Cl₂.Using liquid-mass spectrometry technology to study The lipidomics of HepG2 cells exposed to Cd Cl₂was measured by LC-MS technology,and analyzed potential differential metabolites.Method:1.HepG2 cells were exposured to six different doses（1μM,2μM,4μM,6μM,8μM,10μM）of Cd Cl₂for 24 h and 48 h,respectively.The kits were used to mearsure the concentration of ACC,MDH,TC and TG in the HepG2 cells.2.HepG2 cells were measured via high-resolution mass spectrometry technology,and conducted non-targeted lipidomics research.Potential differential metabolites and related pathways of HepG2 cells exposed Cd Cl₂ were screened through multivariate statistical analysis.3.Potential differential metabolites and related pathways of HepG2 cells exposed Cd Cl₂ were identified via sMRM detection method and targeted lipid analysis method.Results:1.Cell biochemical indicators and oil red O staining results showed that HepG2 cells were exposured to three doses（2μM,4μM and 8μM）Cd Cl₂ for 24 h.The contents of TC and TG in the HepG2 cells were significantly increased,and significant accumulation of lipids in cells.The accumulation is significant.This indicated that cadmium had a lipid accumulation effect on HepG2 cells.2.HepG2 cells were measured via high-resolution mass spectrometry technology,and conducted non-targeted lipidomics research.86 major potential differential lipid metabolites were obtained under the conditions of VIP>1 and P<0.05 through QI software and multivariate statistical analysis.Pathway analysis of potential differential lipid metabolites obtained two metabolic pathways.One is sphingolipid metabolism pathway,and it main enriched lipid metabolites were Ceramides（Cer）and Sphingolipids（SM）.The other is glycerophospholipid metabolism pathway,and it main enriched lipid metabolites were PC and PE.The results indicated that Cd could increase or decrease the content of some sphingolipids and phospholipids metabolites in HepG2 cells.3.HepG2 cells were measured via high-throughput and broadly targeted lipidomics.Data was processed and normalized through R and multivariate statistical analysis.9 main potential differential lipid metabolites were obtained under the condition that VIP was greater than 1 and P value was less than 0.05,and FC was greater than 1.5 and less than 0.5.Based on the research results of our research team,we found that the liver of ICR mice,serum and liver of SD rats and HepG2 cells were screened nine common potential differential metabolites by high-resolution LC/MS technology for non-targeted lipidomics.We further screened out two common potential differential metabolites（Ceramide（d18:1/24:0）and PI（18:0/20:4））through the existing mature high-throughput and broadly targeted lipidomics research in our laboratory.We found that Ceramide（d18:1/24:0）levels in the liver of ICR mice,serum and liver of SD rats and HepG2 cells were up-regulated,and PI（d18:0/20:4）levels were down-regulated by using targeted lipid analysis method for verification.Conclusion:Combining in vivo and in vitro non-targeted lipidomics and high-throughput and broadly targeted lipidomics methods,two common potential differential metabolites Ceramide（d18:1/24:0）and PI（d18:0/20:4）)were screened.They were verified by targeted lipid analysis method.We found that the contents of Ceramide（d18:1/24:0）in the liver of ICR mice,serum and liver of SD rats and HepG2 cells were up-regulated.The contents of PI（d18:0/20:4）were down-regulated.