| Backgrounds:WHO claimed that obesity is one serious diseases in adults worldwide.Nonalcoholic fatty liver disease(NAFLD)is related to obesity,and the incidence has significantly increased in recent years.It has become a concern for multi-disciplinary discussions on nutrition,endocrinology,digestion,genetics,and cardiovascular.The pathogenesis of NAFLD has not been clearly studied.Many studies have found that the important factor that promotes the occurrence of NAFLD is the accumulation of fatty acids.Palmitic acid and oleic acid are the two fatty acids with the highest content in blood and diet.NAFLD is accompanied by inflammation.It is meaningful to conduct early intervention and targeted therapy of obesity by studying the relationship between inflammatory mediators and obesity.Objective:The purpose of the study was to study the lipid metabolism characteristics and model screening of L02 hepatocytes and HepG2 cells.The best model was selected to explore the relationship between IL-6 and lipid metabolism,clarify the role of IL-6 in L02 hepatocyte steatosis,and explore the possible mechanism in vitro.Methods:Part 1,model screening and characteristics of lipid metabolism in L02 hepatocytes and HepG2 cells.The growth curves of L02 hepatocytes were drawn,and then the appropriate cell inoculation concentration was selected.L02 hepatocytes or HepG2 cells were respectively incubated with sodium oleate or sodium palmitate at different concentrations(1200,600,300,150,75,37.5,0 ?mol/L)for 24h.Then IL-6 in the supernatant and the cell viability were detected.L02 hepatocytes or HepG2 hepatocytes were incubated with 300,150,75 and 0 ?mol/L sodium oleate for 24 h;and HepG2 hepatocytes were incubated with 300,150,75 and 0?mol/L sodium palmitate for 24 h;L02 hepatocytes were incubated with 150,75,37.5 and 0?mol/L sodium palmitate for 24 hours.Oil Red O staining was used to observed cell lipid accumulation,and the triglyceride in the cells was detected.Western blot was used to detect the expression of silent information regulator 1(SIRT1)and nuclear factor-?B(NF-?B),and Toll-like receptor 2(TLR2)and TLR4 on the cell surface were detected by flow cytometry.L02 hepatocytes were incubated with 300,75 and 0?mol/L sodium oleate diluted with complete medium and incomplete medium respectively,and after 24h incubation,the supernatant was taken to detect IL-6 by ELISA and FFA by gas chromatography-mass spectrometry,and the L02 hepatocytes were stained with the Oil Red O staining kit.Part 2,the relationship of IL-6 and lipid metabolism in L02 hepatocytes.L02 hepatocytes were incubated with 1200,600,300,150,75,37.5,0?mol/L sodium oleate for 24h,and the supernate was collected to detect IL-6.L02 hepatocytes were incubated with 300?mol/L,150?mol/L,75?mol/L or 0 sodium oleate on 0,0.5,1,2,4,6,8,10,12,16,18,20,22,24h.The supernate was collected and centrifuged for IL-6 and FFA detection.L02 hepatocytes were stained by BODIPY 493/503 for fluorescence microscope observation of lipid accumulation.L02 hepatocytes were stained by Oil Red O after treatment with 300?mol/L sodium oleate on 0,0.5,1,2,4,6,8,10,12,14,16,20,24h,and lipid accumalation was evaluated.Part 3,the role of IL-6 on L02 hepatocyte steatosis.The concentration of IL-6 diluted in complete medium was 400,100,25,6.250,1.563,0.391,0.098,0.024,0.006pg/mL or 0pg/mL IL-6,and then they were added to L02 hepatocytes for 24h incubation.12 parallel wells were designed for each concentration,half wells for each concentration were used for cell viability detection using CCK-8 solution after taking the supernatant.After 2h incubation,the absorbance at 450nm was detected by a microplate reader.Discard the supernatant of the remaining wells,and stain the bottom cells with Oil Red O.The concentration of IL-6 diluted in 10%BSA medium was 100,33.3,8.325,2.081,0.520,0.130,0.033,0.008,0.002pg/mL or Opg/mL IL-6,and then they were added to L02 hepatocytes for 24h incubation.Parallel wells were designed for each concentration,Half wells of each concentration were used for cell viability detection using CCK-8 solution after taking the supernatant.After 2h incubation,the absorbance at 450nm was detected by a microplate reader.Discard the supernatant of the remaining 6 wells,and stain the bottom cells with Oil Red O.25pg/mL and 2.5pg/mL IL-6 diluted in complete medium,as well as complete medium solutions,were added to L02 cells incubated overnight,7 replicate wells per group.The supernatant was discarded and the bottom cells were stained with BODIPY 493/503 and DAPI reagent.ImageJ software was used to calculate the green and blue fluorescent area of the same view field,and then the ratio of green and blue fluorescent area was calculated,which represents the accumulation of lipids.The high and low concentrations of sodium oleate were respectively 300?mol/L and 75?mol/L,and the high and low concentrations of IL-6 were respectively 25pg/mL and 2.5pg/mL;there are 7 groups in the experiment,namely "high sodium oleate high IL-6 group","high sodium oleate low IL-6 group","high sodium oleate group","low sodium oleate high IL-6 group","low sodium oleate low IL-6 group","low sodium oleate group" and "control group".Then the corresponding IL-6 and sodium oleate were added to L02 hepatocytes for 24h incubation.Afterwards,flow cytometry was used to detect lipid deposition and CD36 expression in L02 hepatocytes.Part 4,effects of siRNA blocking IL-6 transcription on CD36 expression and lipid synthesis.IL-6 siRNA was used to transfect L02 hepatocytes,and RT-PCR was used to detect the transcription of IL-6 mRNA in the cells;Oil Red O staining was used to observe the effect of IL-6 siRNA on the lipid deposition of L02 hepatocytes induced by sodium oleate;In addition,flow cytometry was used to detect lipid accumulation and CD36 expression on the cell surface.Results:1.As the concentration of sodium oleate increased,cell viability decreased and cell growth inhibition rate increased.The IL-6 content in the supernatant of L02 cells incubated with 1200,600,300,150 ?mol/L sodium oleate was statistically significantly higher than that in the control group(P<0.001),the IL-6 content in the supernatant of L02 cells incubated with 75 ?mol/L sodium oleate was statistically significantly higher than that in the control group(P<0.01),the IL-6 content of the 1200?mol/L sodium oleate experimental group was statistically significantly higher than that of the 600?mol/L experimental group(P<0.001),and the IL-6 content of the 600?mol/L sodium oleate experimental group was statistically significantly higher than that of the 300?mol/L experimental group(P<0.01);The IL-6 content in the L02 cell supernatant of the 1200,600,300 ?mol/L sodium palmitate experimental groups was statistically significantly higher than that of the control group(P<0.001),the IL-6 content of the 1200?mol/L sodium palmitate experimental group was statistically significantly higher than that of the 600?mol/L experimental group(P<0.001),and the IL-6 content of the 600?mol/L sodium palmitate experimental group was statistically significantly higher than that of the 300?mol/L sodium palmitate experiment group(P<0.001).The IL-6 content of the 300?mol/L sodium palmitate experimental group was statistically significantly higher than that of the 150?mol/L sodium palmitate experimental group(P<0.001).300,150 and 75 ?mol/L sodium oleate induced L02 hepatocyte triglyceride synthesis,the content was statistically significantly higher than the control group(P<0.001,P<0.001 and P<0.01),300?mol/L sodium oleate statistically significantly increased HepG2 hepatocyte triglyceride content(P<0.01).The effect of 300?mol/L sodium palmitate on HepG2 cells was statistically significantly higher triglyceride than the control group(P<0.01).L02 hepatocytes and HepG2 cells were incubated with 300,150 and 75 ?mol/L sodium oleate for 24 h,HepG2 hepatocytes were incubated with 300,150 and 75 ?mol/L sodium palmitate for 24 h,L02 hepatocytes were incubated with 150,75 and 37.5?mol/L sodium palmitate for 24 hours;after the steps above,lipid droplets were observed to in the cells by Oil Red O staining,while no obvious lipid accumulation was observed in the control group through Oil Red O staining.The expression of TLR2 in L02 hepatocytes treated with 300 and 150 ?mol/L sodium oleate was statistically significantly higher than that in the control group(P<0.001 and P<0.05);the expression of TLR2 in L02 cells treated with 300 and 150 ?mol/L sodium palmitate was significantly higher than that in the control group(P<0.05);TLR4 expression did not increase significantly.The expression of SIRT1 protein in L02 hepatocytes of 300,150,75?mol/L sodium oleate and 150 ?mol/L sodium palmitate was lower than that of control group.NF-?B p65 expression in L02 hepatocytes and HepG2 hepatocytes treated with 300,150,75?mol/L sodium oleate and 300,150?mol/L sodium palmitate was higher than that of the control group.The IL-6 content of L02 cells incubated with complete medium to dissolve sodium oleate decreased with the decrease of sodium oleate content,while the dose-response effect of IL-6 and sodium oleate wasn't observed in L02 hepatocytes incubated with 10%BSA MEM medium to dissolve sodium oleate.Treated with sodium oleate in 10%BSA MEM medium,the lipid accumulation of L02 hepatocytes was lower than that of the complete medium group.Treated with 10%BSA medium,the cell growth state under microscope was worse than that of the MEM complete medium group.2.With the incubation time extention of sodium oleate,the concentration of IL-6 increased in the supernate of L02 hepatocytes.After 24h incubation of L02 hepatocytes,IL-6 increased as the concentration of sodium oleate increased.FFA in the supernate decreased as sodium oleate incubation time extended.Lipid accumulation increased as the sodium oleate increased and incubation time extended.Oil Red O staining deepened as incubation time extended from 2 hours on.3.When IL-6 was dissolved in 10%BSA incomplete medium,the activity of L02 cells gradually decreased as the IL-6 concentration increased from the concentration of 0.033pg/mL on.100pg/mL IL-6 had no inhibitory effect on L02 cells in complete medium.IL-6 was dissolved in 10%BSA incomplete medium or complete medium,and then was incubated with L02 cells for 24h.Lipid accumulation of L02 cells was observed through Oil Red O staining.Using BODIPY 493/503 and DAPI staining to observe the lipid accumulation of L02 cells caused by IL-6,it was found that the lipid accumulation of cells in the 25pg/mL IL-6 treatment group was statistically significantly higher than that of the control group(P<0.01);the lipid deposition of cells in the 25pg/mL IL-6 treatment group was statistically significantly higher than that in the 2.5pg/mL IL-6 treatment group(P<0.05).2.5pg/mL IL-6 significantly increased the level of cellular lipid accumulation induced by 300?mol/L sodium oleate and 75?mol/L sodium oleate(P<0.05,P<0.01).4.The results of qPCR showed that IL-6 siRNA inhibited the transcription of IL-6 in L02 hepatocytes,and IL-6 mRNA was not detected.After IL-6 siRNA was added,the IL-6 content in the supernatant decreased,but no significant difference was observed.After IL-6 transcription was inhibited,CD36 expression was inhibited,and lipid synthesis was inhibited.Conclusion:1.L02 hepatocytes treated with sodium oleate as a lipid deposition and inflammation model were screened and suitable for cell IL-6 and lipid metabolism observation.Sodium oleate induced L02 hepatocytes to secrete IL-6 through the TLR2/NF-?B pathway;sodium oleate induced L02 lipid accumulation,which was related to IL-6/PPAR?.2.IL-6 production and lipid accumulation in L02 hepatacytes were dose-and time-dependent with sodium oleate.3.IL-6 could promote the lipid deposit of L02 cells and also promote the lipid deposit of L02 cells induced by sodium oleate.4.This experiment suggested that the lipid synthesis induced by sodium oleate might be related to IL-6/CD36,and there was also a relationship between IL-6 and CD36.5.Based on the above research results,the pathway of sodium oleate-induced IL-6 production may be TLR2/NF-?B/IL-6,and the mechanism of IL-6-induced lipid synthesis may be IL-6/CD36/PPARy.Therefore,sodium oleate induced L02 hepatocyte lipid synthesis,which might be related to the TLR2/NF-?B/IL-6/CD36/PPAR? pathway. |
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