Docosahexaenoic Acid Alleviated LPS-induced Cardiomyocyte Damage And The Underlying Mechanism

Objective: To investigate whether docosahexaenoic acid(DHA)attenuates lipopolysaccharide(LPS)-induced cardiomyocyte damage in HL-1 cells and the possible mechanism.Methods:(1)The optimal time point was determined by accessing the effect of1μg/m L LPS on the cell viability of HL-1 cells at different time points(2h、4h、8h、16h、24h)on.(2)The optimal concentrations of DHA were selected by accessing the effect of different concentrations of DHA(50μM、100μM、200μM、250μM、500μM)on the cell viability of HL-1 cells.(3)The experimental groups were as follows:control group,DHA group,LPS group,and DHA+LPS group.To investigate the possible effect of DHA on LPS-induced cytotoxicity,the cell viability,the lactate dehydrogenase(LDH)activity,the caspase-3 activity,and cleaved caspase-3 protein were detected.Oxidative stress was assessed by the level of reactive oxygen species(ROS)and the activity of superoxide dismutase(SOD).Morphology and function of mitochondria were accessed by transmission electron microscopy,JC-1 kit,and mitochondrial respiratory chain-associated proteins including NDUFA9,SDHB,UQCRC2,and COX IV.The mitochondrial fragmentation was accessed by fluorescence microscopy and the mitochondrial fission-fusion related proteins including Opa1,Mfn2,Drp1,p-Drp1(ser 616),and p-Drp1(ser 637).Results:(1)After treated with LPS for 8 hours,the viability of HL-1 cells decreased significantly in a time-dependent manner(P<0.05).(2)Cell viability did not show marked changes under the DHA concentration ranged from 50-200 μM.However,it decreased notably when treated in the DHA concentration higher than 250 μM(P<0.001).(3)Compared with the control group,the LPS group showed a decreasing level of cell viability and SOD activity and an increasing level of LDH activity,the level of apoptosis,and ROS(p<0.01),while the DHA+LPS group showed no significant changes(p>0.05).Compared with the LPS group,cell viability and SOD activity were up-regulated,LDH activity,the level of apoptosis,and ROS were down-regulated in the DHA+LPS group(p<0.05).Compared with the control group,LPS induced mitochondrial damage,down-regulated the membrane potential and the expression of NDUFA9,SDHB,and COX IV(p<0.01),but did not affect the expression of UQCRC2(p>0.05).The DHA+LPS group showed no significant changes when comparing to the control group.Compared with the LPS group,the mitochondrial damage was attenuated,the membrane potential and the expression of NDUFA9,SDHB,and COX IV(p<0.05)were increased in the DHA+LPS group,but no significant difference were detected in UQCRC2(p>0.05).Compared with the control group,the LPS group had an increasing level of mitochondrial fragmentation and p-Drp1(ser 616)(p<0.01)but showed no changes in Opa1,Mfn2,and p-Drp1(ser 637)(p>0.05).Compared with the LPS group,the mitochondrial fragmentation and the expression of p-Drp1(ser 616)were reduced in the DHA+LPS group(p<0.01),but there was no significant difference in Opa1,Mfn2,and p-Drp1(ser 637)(p>0.05).Conclusion: DHA could attenuate LPS-induced cytotoxicity in HL-1 cells.The protective mechanism was associated with the downregulation of p-Drp1(ser 616),inhibition of mitochondrial division,improvement of mitochondrial dysfunction,and reduction of oxidative stress.