Tanshinone ⅡA Inhibits Alveolar Macrophage Polarization, Inflammation and Mitochondrial Damage by Regulating the PAPR-1 Signaling Pathway

Poly (ADP-ribose) polymerase-1 (PARP-1) is the most extensively studied member of the PARP family. It is characterized by a zinc-finger DNA-binding domain, an auto-modification domain, and a catalytic domain responsible for adding branched ADP-ribose moieties to target proteins (1). In recent years, numerous studies have shown that PARP-1 is involved in regulating inflammatory damage by modulating the expression of various cytokines, chemokines, and adhesion molecules, thereby playing a key role in the inflammation cycle (2, 3). Our previous studies have also demonstrated that inhibiting PARP-1 expression can significantly alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI) (4).

The PARP-1 regulates the inflammatory response in various cell types, including innate immune cells (macrophages, neutrophils), adaptive immune cells (lymphocytes), and non-immune cells (such as fibroblasts and endothelial cells) (5, 6). Modulation of PARP-1 expression, alterations in its enzymatic activity, and post-translational modifications can significantly influence the inflammatory activation process of macrophages. In macrophages, PARP-1 plays a crucial role in the regulation of transcription, signaling, inflammasome activity, metabolism, and redox balance. This regulation supports the polarization of macrophages toward the pro-inflammatory M1 phenotype, thereby contributing to excessive inflammatory responses and subsequent tissue damage (7, 8).

Alveolar macrophages (AMs), a critical subset of innate immune cells, are essential for the initiation, resolution, and tissue repair processes associated with lung inflammation (9). Based on activation status, function, and secreted factors, AMs can be categorized into two types: Classically activated M1 macrophages and alternatively activated M2 macrophages. Typically, M1 macrophages are activated by interferon-γ and LPS. They predominantly secrete interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and other inflammatory mediators, which play important roles in the early stages of inflammation (10-12). Furthermore, activated M1 macrophages can recruit monocytes from peripheral blood into the alveoli and promote their polarization into the M1 phenotype, further amplifying the inflammatory response. Therefore, effective regulation of macrophage polarization and inhibition of the subsequent cascade expansion of the inflammatory response are key challenges in the treatment of ALI.

Tanshinone IIA is the principal active ingredient in the Chinese herb Salvia miltiorrhiza, renowned for its potent anti-inflammatory activity (13). In the treatment of pulmonary inflammatory diseases, tanshinone IIA has demonstrated significant therapeutic efficacy in conditions such as asthma, ALI, and chronic obstructive pulmonary disease (COPD) (14, 15). It can markedly inhibit inflammatory damage and oxidative stress in lung tissue, reduce pulmonary edema, and suppress the adhesion and migration of neutrophils, macrophages, and other immune cells into the lung tissue. Research has also shown that tanshinone IIA can promote the cleavage of PARP-1 and reduce its expression (16, 17).

Consequently, we hypothesize that the anti-inflammatory activity of tanshinone IIA may be associated with its ability to regulate macrophage polarization, mitigate mitochondrial damage, and modulate the expression of inflammatory factors via the PARP-1 signaling pathway. Therefore, we investigated the regulatory mechanism of tanshinone IIA on macrophage polarization, mitochondrial damage, and inflammatory response using a LPS-induced AM model.

The AMs, a major component of the innate immune system, reside in the alveoli and perform critical functions including phagocytosis, both pro-inflammatory and anti-inflammatory activities, and the activation of T helper 1 (Th1) and T helper 2 (Th2) responses (24). The AMs are essential for maintaining pulmonary immune homeostasis, and disturbances in their inflammatory response, oxidative balance, and polarization status contribute significantly to the onset and progression of acute pulmonary inflammatory diseases such as ALI. It is well established that LPS induction promotes M1 polarization in AMs, subsequently stimulating the secretion of pro-inflammatory mediators such as IL-1β, IL-6, TNF-α (12). The release of these pro-inflammatory factors further amplifies inflammation, leading to increased lung damage. Moreover, excessive inflammation reciprocally promotes oxidative and mitochondrial damage.

The PARP-1 is implicated in regulating numerous inflammatory processes (6), closely associated with the activation of immune cells — including macrophages, neutrophils, dendritic cells, and lymphocytes — as well as the inflammatory responses of non-immune cells such as fibroblasts and endothelial cells. Among these, the polarization and activation of AMs play a pivotal role in the acute phase of pulmonary inflammation (11). Research has shown that PARP-1 is crucial in regulating LPS-induced macrophage polarization, enhancing polarization towards the M1 phenotype by affecting both transcriptional and post-transcriptional processes. The PARP-1 facilitates the expression and mRNA stability of pro-inflammatory factors. In response to LPS, PARP-1 is phosphorylated at the conserved Y829 site by the c-Abl tyrosine kinase, which in turn activates NF-κB (7). The NF-κB is a key transcription factor governing the expression of inflammation-related genes and orchestrating innate immune responses. The activation of NF-κB in macrophages initiates an inflammatory cascade and promotes their recruitment to sites of infection, where activated M1 macrophages secrete TNF-α, IL-1β, IL-6, and other cytokines to mobilize neutrophils and initiate the host response (25).

The activity of NF-κB is regulated in part by PARP-1. The inhibitor of kappa B (IκB) retains NF-κB in the cytoplasm in resting cells, preventing its nuclear localization and transcriptional activity (26). The PARP-1 activates and phosphorylates inhibitor of kappa B kinase (IKK), which phosphorylates IκB, promoting its degradation and the release of NF-κB, thereby enabling its nuclear translocation and activation. The p65 subunit of NF-κB (p-p65) is phosphorylated and translocates to the nucleus to drive transcription of target genes (27).

Tanshinone IIA is widely recognized for its potent anti-inflammatory properties and exhibits significant therapeutic efficacy in various pulmonary inflammatory diseases, including ALI (28). Previous studies have explored the mechanisms by which tanshinone IIA treats pulmonary inflammation, demonstrating its ability to inhibit NF-κB (29), suppress NLRP3 inflammasome activation (30), and regulate macrophage polarization (15). However, the regulatory mechanism of tanshinone IIA on pulmonary inflammation-related signaling pathways via PARP-1 has not been systematically studied.

Our findings demonstrate that the anti-inflammatory effects of tanshinone IIA are mediated by its suppression of inflammatory factor expression and M1 polarization of AMs through the PARP-1 pathway. Molecular docking and molecular dynamics simulation studies revealed a strong binding affinity of tanshinone IIA to PARP-1. Western blot analysis further confirmed that tanshinone IIA inhibits PARP-1 and its associated signaling pathway, resulting in the suppression of IκB-α phosphorylation. This, in turn, inhibits the phosphorylation and activation of NF-κB p65, suppresses NLRP3 inflammasome activation, and ultimately reduces the expression of pro-inflammatory factors.

Mitochondria are critical organelles involved in cellular energy metabolism, signal transduction, and apoptosis. Studies have shown that mitochondrial damage and cellular inflammatory responses are mutually reinforcing (31). Inflammatory responses can induce mitochondrial damage, and inflammatory cytokines can significantly alter mitochondrial dynamics and compromise mitochondrial energy metabolism, ultimately leading to apoptosis (32). Damaged mitochondria can further amplify inflammation by overproducing mitochondrial ROS and releasing damage-associated molecular patterns (DAMPs) (33). Additionally, alterations in mitochondrial energy metabolism can influence macrophage polarization (34).

The PARP-1 hyperactivity can indirectly affect metabolic homeostasis. Excessive activation of PARP-1 leads to depletion of nicotinamide adenine dinucleotide (NAD⁺), resulting in reduced glycolysis, impaired electron transport chain function, and decreased adenosine triphosphate (ATP) production — ultimately compromising mitochondrial and cellular function (35). Furthermore, PARP-1 is involved in the regulation of mitochondrial fission and fusion protein expression, and its inhibition can enhance mitochondrial stability (36, 37). Our results indicate that tanshinone IIA may directly prevent mitochondrial damage caused by NAD⁺ depletion through the inhibition of PARP-1 and may also regulate mitochondrial fission/fusion balance by inhibiting PARP-1. Additionally, tanshinone IIA reduces cellular inflammatory injury via the PARP-1 pathway, thereby mitigating mitochondrial damage.

The development of therapeutics targeting the PARP-1 pathway for regulation of inflammatory responses is gaining increasing attention. Our study supports the hypothesis that the therapeutic effects of tanshinone IIA in pulmonary inflammatory diseases are attributable to its interaction with the PARP-1 signaling pathway, a finding with significant implications for the future application and exploration of tanshinone IIA. Nevertheless, our research is currently limited to in vitro mechanistic studies. Further in vivo investigations and comprehensive mechanistic analyses will be required to validate these findings.