ARACHIDONIC ACID ENHANCES MITOPHAGY IN CYBRIDS

Mitochondria are involved in energy metabolism, regulation of immune responses, and apoptosis. To maintain their functional integrity, cells utilize mitophagy, a selective form of autophagy responsible for the removal of damaged mitochondria. The canonical mitophagy pathway is triggered by a loss of mitochondrial membrane potential, accompanied by stabilization of PINK1 and activation of Parkin. Disruptions in this cascade may reduce the efficiency of mitophagy. Therefore, alternative PINK1/Parkin-independent mechanisms are of particular interest.

Arachidonic acid is a polyunsaturated fatty acid involved in the regulation of inflammatory processes and the synthesis of inflammatory mediators. Currently, arachidonic acid is considered a potential modulator of autophagy-related processes. It has previously been shown to enhance autophagy in several cell types, including Sertoli cells, adipocytes, and monocytes. However, the molecular mechanisms underlying these effects remain insufficiently understood and require further clarification.

In the present study, we evaluated the role of arachidonic acid as a potential modulator of mitophagy in cells with impaired mitophagic function. Two cybrid cell cultures based on the THP-1 cell line were used: HSM2 (mitophagy was activated in response to FCCP treatment) and HSM1 (not activated). Cells were treated with FCCP (2 μM) or arachidonic acid (20 μM) for 2 hours. Mitophagy was assessed by quantifying the colocalization of mitochondria (MitoTracker Green) and lysosomes (LysoTracker Deep Red), calculated using Mander’s coefficient. Analysis was performed on images obtained by live-cell confocal microscopy of cells stained with mitochondrial and lysosomal fluorescent probes.

It was shown that FCCP activated mitophagy in HSM2 but not in HSM1. In contrast, arachidonic acid enhanced mitophagy in both HSM2 and HSM1. Additional gene expression analysis revealed that in HSM2, arachidonic acid and FCCP induced increased expression of PRKN, ATG7, ULK2, ATG4B, and TBK1, as well as LAMP2, DNM1L, and BNIP3, indicating enhanced autophagy. In HSM1, the transcriptional response to FCCP was attenuated, whereas arachidonic acid predominantly increased the expression of ATG7, TBK1, ULK2, and GABARAP, consistent with activation of alternative mitophagy pathways.

Thus, the obtained results demonstrated that arachidonic acid enhanced mitophagy even under conditions of impaired canonical PINK1/Parkin-dependent responses, likely through activation of alternative autophagy pathways.