Docosahexaenoic acid alleviates LPS-induced cytotoxicity in HL-1 cardiac cells via improving stress-induced mitochondrial fragmentation

Figures

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  Fig. 1. Effects of DHA on cell death in the LPS-induced HL-1 cardiac cells. A) Cell viability was scored by methyl thiazolyl tetrazolium (MTT) assay. B) LDH content in the cell supernatant was utilized as a surrogate for cell death and quantified using Cytotoxicity Detection Kit. C) Caspase 3 activity was used to analyze apoptosis. D) The expression level of Cleaved caspase-3 was assayed by Western blot. n = 3 independent experiments. *P<0.05, **P<0.01, compared with the control group; #P<0.05, ##P<0.01compared with the LPS group.

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  Fig. 2. Effects of DHA on ROS production in the LPS-induced HL-1 cardiac cells. A) DCFH-DA was used to detect cytoplasmic H2O2 by fluorescence microscopy. B) DHA mitigated LPS-induced ROS production. n=6 independent experiments. C) DHA increased the level of SOD activity in LPS-induced HL-1 cells. n=3 independent experiments. **P<0.01, compared with the control group; #P<0.05, ##P<0.01compared with the LPS group.

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  Fig. 3. Effects of DHA on myocardial mitochondria in the LPS-induced HL-1 cardiac cells. A) Representative TEM images of HL-1 cells in control, DHA-treated, LPS-treated, and DHA + LPS-treated cells (scale bar = 1 μm). Black arrows indicate normal mitochondria, red arrows indicate abnormal mitochondria. B) Representative fluorescence images of JC-1 staining show the change in high mitochondrial membrane potential (red) and low mitochondrial membrane potential (green), which is used to evaluate mitochondrial damage. LPS treatment increased green fluorescence, whereas DHA decreased green fluorescence (scale bar = 20 μm).

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  Fig. 4. Effects of DHA on mitochondrial fission in the LPS-induced HL-1 cardiac cells. A) Representative confocal microscopic images showing mitochondrial morphology stained by MitoTracker Red (scale bar = 10 μm). B) Percentage of HL-1 cells with fragmented mitochondria. n=6 independent experiments. ∗∗P<0.01, compared with the control group; ##P<0.01, compared with the LPS group.

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  Fig. 5. Effects of DHA on the expression of mitochondrial respiratory chain-associated proteins in LPS-induced HL-1 cells. A-D) Representative Western blot images showing the effect of DHA on the expressions of NDUFA9, SDHB, UQCRC2, and COX IV. Quantitative analysis of above proteins among all groups n = 3 independent experiments. **P < 0.01, compared with the control group; #P<0.05, ##P<0.01, compared with the LPS group.

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  Fig. 6. Effects of DHA on the expression of mitochondrial dynamics related proteins in LPS-induced HL-1 cells. A) Representative Western blot images showing the effect of DHA on the expressions of Mfn2 and Opa1. B) Representative Western blot images showing the effect of DHA on the expressions of Drp1, p-Drp1(Ser616), p-Drp1(Ser637) in LPS-induced HL-1 cells. C–F) Quantitative analysis of above proteins among all groups. n = 3 independent experiments. **P < 0.01, compared with the control group; ##P<0.01, compared with the LPS group.