Activation of NRF2-PGC1α-TFAM Signaling by Bacosine Promotes Mitochondrial Biogenesis Through Midzone Fission to Prevent Oral Fibrosis

Abstract

Oral submucous fibrosis (OSF) is characterized by chronic oxidative stress-induced mitochondrial dysfunction, fibroblast activation, and collagen accumulation. Arecoline exposure disrupts mitochondrial homeostasis by elevating ROS levels, impairing mitochondrial membrane potential, triggering mitochondrial hyperfusion and mitochondrial DNA damage. Our study aims to suppress the arecoline induced submucosal fibrosis and identified bacosine, a flavonoid derived from Bacopa monnieri, to maintain mitochondrial biogenesis. Mitochondrial biogenesis is the process by which cells create new mitochondria to meet increased energy demands or to replace damaged ones. Bacosine treatment increased the mitochondrial biogenesis marker PGC1-α, mitochondrial transcription factor A (TFAM) and mitochondrial residing protein COXIV. Since mitochondrial biogenesis also involves replication of mitochondrial DNA (mtDNA), we have checked the mitochondrial copy number by qRT-PCR and found that bacosine treatment increased the mitochondrial copy number. Consistently bacosine treatment enhanced the newly synthesized mitochondria as evidenced by increased green:red ratio in mitotimer transfected cells. Then to demonstrate the underlying mechanism responsible for mitochondrial biogenesis, inhibition of NRF2 (via pharmacological inhibitor ML-385 or shRNA) or suppression of PGC1-α/TFAM blocked these effects, confirming that bacosine acts through the NRF2-PGC1α-TFAM axis. From these studies we have suggested that bacosine promotes mitochondrial biogenes via NRF2-PGC1 α-TFAM axis. Interestingly we found enhanced mitochondrial biogenesis also promotes mitochondrial midzone fission as evidence by bacosine increased the expression of fission mediators DRP1, MFF, PPA2, and MTP18, while reducing MFN1 and FIS1. Moreover, TOM20-based mitochondrial skeleton imaging further confirmed increased mitochondrial fragmentation. Moreover bacosine treatment also increased the mitotracker green (mitochondrial mass) and mitotracker red (functional mitochondria) staining in flow cytometry. Notably, bacosine reversed arecoline-induced mitochondrial hyperfusion and dysfunction. Finally, inhibition of either the NRF2-PGC1α-TFAM axis or midzone fission prevented bacosine from suppressing OSF markers αSMA, COL1A, and CTGF, indicating that both mitochondrial biogenesis and mitochondrial midzone fission are essential for its anti-fibrotic effect. Collectively, our results demonstrate that bacosine inhibits arecoline-induced OSF by restoring mitochondrial health through NRF2-PGC1α-TFAM-mediated mitochondrial biogenesis and midzone fission, highlighting a mitochondria-centered therapeutic strategy for oral fibrosis.