Myofibroblasts are clinically and biologically important pathological features of pulmonary fibrosis, and their numbers on surgical lung biopsy directly correlate with progressive physiological deterioration and shortened survival. Although resident fibroblasts activate myofibroblasts in response to injury, their origin in lung fibrosis remains controversial. AECs gradually lose their epithelial cell markers and polarity during human lung fibrogenesis. Simultaneously, these cells express mesenchymal markers and acquire single-cell motility. The epithelial-mesenchymal transition exists in lung biopsy samples from fibrotic patients.

However, scientists obtained conflicting results and did not detect double-positive cells for E-cadherin or surfactant-associated protein A, as well as α-SMA or vimentin, in lung tissues from patients with idiopathic pulmonary fibrosis non-specific interstitial pneumonia.

Furthermore, pericytes and two epithelial cell populations are excluded as the origin of myofibroblasts. Our current work revealed that myofibroblasts, which can originate from AECs-II, were detected in patients with pulmonary fibrosis. This phenomenon also existed in the lung tissues from bleomycin-induced lung fibrotic rats and cultured A549 and MRC-5 cells activated by TGF-β1.

Thus, using these models is significant to further investigate the action of astaxanthin against pulmonary fibrosis by promoting myofibroblast apoptosis, which is based on Drp1-mediated mitochondrial fission.

A more recent study found that pulmonary fibrosis exhibited several cancer-like pathogenic features; thus, pulmonary fibrosis is considered to be a nonproliferative disorder of the lung. Like cancer cells, myofibroblasts show epigenetic and genetic abnormalities and functional features such as uncontrolled proliferation, resistance to apoptosis, and high migration rates. Thus, the promotion of myofibroblast apoptosis is becoming a potential therapeutic strategy for fibrotic diseases.

The current results showed that astaxanthin strongly improved lung parenchymal injury reduced collagen deposition, and promoted myofibroblast apoptosis through the Drp1-mediated mitochondrial fission signaling pathway.

Several studies reported that astaxanthin effectively co-localized with mitochondria in many types of cells and tissues, such as leucocytes, mesangial cells, blastocysts, liver, and muscles, implicating a role of astaxanthin in the mitochondria. Studies have shown that astaxanthin can impair mitochondrial function and induce apoptosis in specific cancer cells through mitochondrion-dependent pathways. Mitochondrial fission is important to maintain cellular function and initiate intrinsic apoptosis. Few functional food agents were identified to influence mitochondrial morphology leading to apoptosis. The present results showed that astaxanthin strongly promoted mitochondrial fission in myofibroblasts.

Dynamin-related protein-1, a conserved evolution protein, is one of the proteins proposed to participate in the fission process. Dynamin-related protein-1 is a cytosolic protein with an N-terminal GTPase domain, a dynamin-like middle domain, and a C-terminal GTPase effector domain. It is considered the principal activator of mitochondrial fission in a GTP-dependent manner.

Mitochondrial fission may be controlled by regulating the expression and altering the activity of fission proteins, whereas Drp1 activity is often altered by mitochondrial recruitment, post-translational modification, and conversion into its oligomer state. Its localization to the mitochondrial surface is important for mitochondrial fission.

Overexpression of Drp1 was critical for cytoplasmic irradiation-induced mitochondrial fission. Consistent with the previous work, our observation revealed that astaxanthin significantly increased the Drp1 level and promoted the recruitment of Drp1 into mitochondria. Moreover, Drp1 deficiency by RNA interference and the use of a dominant-negative mutant protein (K38A) and a chemical inhibitor (mdivi-1) are known to delay the release of cytochrome c and subsequent apoptosis on the challenge with apoptotic stimuli in various types of cultured mammalian cells. Drp1 persistent accumulation in the mitochondria was associated with cell apoptosis or death. In agreement, Drp1 siRNA verified that Drp1 was essential for astaxanthin-induced mitochondrial fission leading to myofibroblast apoptosis.

Drp1 was transcriptionally up-regulated by p53, which conveyed the apoptotic signal of p53 by triggering mitochondrial fission in cardiomyocytes. The current data demonstrated that the regulation of astaxanthin on the Drp1 level was highly correlated with p53. PUMA is exclusively located in the mitochondria, which can facilitate the irreversible immobilization of Drp1 to the mitochondrial outer membrane through protein-protein interactions, which form multimeric complexes. In agreement, the present data showed that Drp1 was co-localized with PUMA during astaxanthin-induced mitochondrial fission, leading to myofibroblast apoptosis.

In summary, we clarified the effects of the pharmacological inhibition of astaxanthin on pulmonary fibrosis. We found that this action required induction of myofibroblast apoptosis by activating the Drp1-mediated mitochondrial fission pathway. This finding suggested that Drp1 exhibits potential benefits for developing astaxanthin as a novel drug for pulmonary fibrosis.