Inhibiting apolipoprotein C3 alleviates ferroptosis and cell damage induced by Pseudomonas aeruginosa

To explore the induction effect of Pseudomonas aeruginosa (PA) infection on ferroptosis and inflammatory response in human hepatoma cells (Hep3B) and the regulatory effect of apolipoprotein C3(APOC3) on ferroptosis sensitivity.

Hep3B hepatocytes (MOI=5, 10, 15) were infected with the wild standard strain of PA (PAO1) for 4 h, 8 h, and 12 h respectively, and the survival rate of cells under different infection concentrations and times was detected by CCK-8. The morphological changes of cells were observed under an inverted microscope to determine the infection conditions. After adding ferroptosis inhibitors or siRNA respectively to inhibit the expression of APOC3, lipid droplets were labeled using oil red O staining, and the formation of lipid droplets was observed under an inverted microscope. Reactive oxygen species (ROS) were detected using a fluorescent probe (DCFH-DA), and lipid peroxidation levels were detected using a microplate reader. The expression levels of Peroxisome Proliferator-Activated Receptor α (PPARα), APOC3 and related inflammatory factors were detected by RT-qPCR. The protein expression levels of APOC3 and the ferroptosis-related proteins SLC7A11 and ACSL4 were evaluated by Western blotting. The morphological changes of mitochondria in cells were observed by transmission electron microscopy. The content of Fe²⁺ in cells was detected using a divalent iron ion probe.

With the increase of infection concentration and the extension of time, the survival rate of Hep3B cells significantly decreased after PAO1 infection, and the cell morphology gradually changed from spindle-shaped to round and accompanied by fragmentation. The number of red lipid droplets within the cells increased with the increase of the bacterial load. The fluorescence intensity of ROS was enhanced. The expression of PPARα mRNA was down-regulated and the expression of APOC3 mRNA was up-regulated. The volume of mitochondria decreased, the membrane density increased, and the cristae were reduced or even disappeared. Western blotting further confirmed the changes in APOC3 protein expression and ferroptosis-related proteins, including SLC7A11 and ACSL4, following PAO1 infection and APOC3 knockdown. The intracellular lipid peroxidation level was significantly increased. Compared with the control group, the levels of IL-6 and TNF-α significantly increased after PA infection. After inhibiting the expression of APOC3, the expression level of ROS decreased, lipid peroxidation reduced, the number of lipid droplets decreased, the content of Fe²⁺ decreased, mitochondrial damage decreased, and the expression of ferroptosis-related factors decreased.

After the action of PA, cell activity significantly decreased, intracellular ROS generation increased, and intracellular lipid peroxidation was enhanced, leading to cell damage and death. Meanwhile, APOC3, as a key downstream target of PPARα, PA promotes the generation of APOC3 by inhibiting the expression of PPARα, and APOC3 reduces the hydrolysis of triglyceride-rich lipoproteins (TRL), thereby promoting lipid droplet formation. It causes the accumulation of lipids within cells. However, the continuous inhibitionof APOC3 can reverse this effect. That is, after PA infects cells, it can promote the increase of APOC3 expression, leading to lipid metabolism disorders, and APOC3 plays a promoting role in the process of PA promoting ferroptosis in cells.