FNDC5/PPARa Pathway Alleviates THP-1-derived Macrophage Pyroptosis and Its Mechanism

Atherosclerosis (AS) is a chronic inflammatory disease. Pyroptosis is a newly discovered, pro-inflammatory cell death that can trigger and amplify the occurrence and progression of AS. Researchers are still uncertain about the anti-atherosclerotic mechanism of "fibronectin type III domain-containing protein 5" (FNDC5).

The study aimed to investigate the ability of FNDC5-mediated, "peroxisome proliferator activated receptor alpha" (PPARa) to inhibit oxidized low-density lipoprotein (ox-LDL)-induced, THP-1-derived macrophage pyroptosis and to determine a potential molecular mechanism at the cellular level.

The research team performed a laboratory study.

The study took place in the Department of Cardiovascular Medicine at the Affiliated Hospital of Guzhou Medical University at the Medical Research Institute at Guizhou Medical University in Guiyang, Guizhou, China.

The research team: (1) constructed and stably transfected FNDC5 gene-overexpressing and FNDC5 gene-silencing lentiviral vectors into THP-1 cells; (2) observed the cell morphology under an inverted fluorescence microscope and screened the stably transfected THP-1 cells with puromycin; (3) verified the transfection efficiency using quantitative real-time polymerase chain reaction (qRT-PCR) and Western Blot; (4) used phorbol to induce THP-1 cells into macrophages; (5) cultured the THP-1-derived macrophages with different concentrations of ox-LDL-25, 50, 75, and 100 µg/ml-for 24 h; (6) performed Hoechst 33342/ propidium iodide (PI) double staining and examined lactate dehydrogenase (LDH) and interleukin-1 beta (IL-1ß) activity to determine the effects of ox-LDL on THP-1-derived macrophage pyroptosis; (7) selected the optimal ox-LDL concentration; (8) divided the THP-1-derived macrophages into seven groups: NC group (no ox-LDL intervention), ox-LDL group, PBS group, Mock1 group, Ad-FNDC5 group, Mock2 group, and Sh-FNDC5 group; (9) examined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and "nuclear factor kappa-light chain enhancer of activated B cells P65" (NF-κB P65), and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining, respectively; (10) treated the THP-1-derived macrophages with FNDC5 expression with GW6471, a specific PPARα antagonist; (11) determined the expressions of functional proteins and the pyroptosis of THP-1-derived macrophages, including FNDC5, PPARa, and NF-κB P65, and those related to the pyroptosis pathway, using Western Blot and Hoechst 33342/PI double staining and detection of the LDH and IL-1ß activity, respectively.

With the stably transfected THP-1 cells with FNDC5 overexpression or silencing the ox-LDL-induced, THP-1-derived, macrophage pyroptosis occurred in a concentration-dependent manner. Compared with the ox-LDL, phosphate buffered saline (PBS), Mock1, and Mock2 groups, the Ad-FNDC5 group had a significant increase in expression of FNDC5 and of peroxisome proliferator activated receptor alpha (PPARa) proteins (P < .05). The "nuclear factor kappa-light chain enhancer of activated B cells P65: (NF-κB P65), NOD-like receptor thermal protein domain associated protein 3, (NLRP3), Caspase-1, gasdermin D (GSDMD, IL-1ß and IL-18 protein expressions, percentage of PI-positive cells, LDH activity, and IL-1ß activity decreased significantly (P < .05); the results in the Sh-FNDC5 group were opposite to those in the Ad-FNDC5 group. 3. Intervention with GW6471 (PPARa antagonist) in the stably transfected THP-1-derived macrophages with FNDC5 overexpression abolished the protective effect of FNDC5 against ox-LDL-induced THP-1-derived macrophage pyroptosis.

Irisin/PPARa inhibited THP-1-derived macrophage pyroptosis and inflammation and delayed AS by inhibiting the NF-κB/NLRP3 pathway.