Abietic acid attenuates LPS-induced acute lung injury by restoring Th17/Treg balance

Purpose: To investigate the mechanism of action and effects of abietic acid (AA) in a mouse acute lung injury (ALI) model. Methods: A mouse ALI model was established by lipopolysaccharide (LPS) induction. Lung tissues were examined for histological alterations and scored based on the degree of injury. Myeloperoxidase (MPO), IL-6, IL-1β, and TNF-α levels were measured by enzyme-linked immunosorbent assay (ELISA) while the numbers of Th17 and Treg cells were assessed by immunofluorescence. Protein expression levels of p-STAT3, p-STAT5, RORrt, and FOXP3 were analyzed by immunoblot assay. Expression of peroxisome proliferator-activated receptor γ (PPARγ) was assessed by immunoblot. Results: AA ameliorated LPS-induced lung injury in mice. Furthermore, AA ameliorated LPS-induced pneumonia in mice (p < 0.05) and restored Th17/Treg balance and Th17/Treg transcription factor expression that was altered by LPS induction. AA also activated PPARγ expression to restore Th17/Treg balance (p < 0.05). Conclusion: The results indicate that AA attenuates LPS-induced ALI in mice by restoring Th17/Treg balance. Thus, AA is a potential drug for the management of ALI; however, AA must first be evaluated in clinical studies.


INTRODUCTION
Acute lung injury (ALI) is a leading cause of death in patients with respiratory failure [1]. ALI may occur as a result of pneumonia, inhalation of harmful substances, or indirect injuries such as ischemia-reperfusion. ALI induces damaging hypoxic and ischemic stress and the release of lipopolysaccharide (LPS), a bacterial endotoxin.
LPS stimulates the release of severe inflammatory signals, leading to lung injury by inflammatory leukocyte infiltration [2]. Therefore, it is important to identify drugs that suppress the inflammatory response in ALI.
Immune homeostasis in vivo depends on a balance among T-helper lymphocyte subsets, including Th1, Th2, Th17, and Treg cells. Th17 cells secrete IL-17, which induces the production of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, thereby exacerbating the inflammatory response [3]. In contrast, Tregs secrete anti-inflammatory mediators such as IL-10 and TGF-β to mediate immune homeostasis. Imbalance between Th17 cells and Treg cells contributes to the development of various diseases [4]. Mediation of the balance of Th17 cells and Treg cells can improve sepsis-induced ALI. Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear hormone receptor family and one of the PPAR isotypes [5]. PPARγ activation plays anti-inflammatory and anti-apoptotic roles in several inflammatory diseases, including ALI. There is growing evidence that nuclear receptors, especially PPARγ, play crucial roles in regulating the Th17/Treg balance.
Abietic acid (AA) is an abietic diterpene compound, mainly derived from Pimenta racemosa var grissea [6], that has been reported to have anti-allergy, anti-inflammation, antiobesity, anti-convulsion, and anti-tumor effects. Rosinic acid inhibits IL-1β-induced inflammation in human osteoarthritis chondrocytes by activating PPAR-γ and has a protective effect on allergic asthma in mice induced by OVA [7]. In this study, the effect of AA on the treatment of ALI was investigated.

Animals and treatments
Male C57BL/6 mice (~8 weeks old) were obtained from SLAC (Shanghai, China) and kept in a sterile environment. All experimental procedures in this study were approved by the Ethics Committee of Ankang Central Hospital (approval no. 2021-021) and conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines [8]. The mice were anesthetized by injection of 4 % chloral hydrate (CIHy, 0.1 ml/10 g) and instilled with sterile PBS (control group) or 3 mg/kg LPS (Sigma, Escherichia coli 0111:B4) dissolved in 50 μL PBS. AA (10, 20, or 40 mg/kg) was administered by oral gavage 1 h after LPS treatment. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected, and pathological changes such as cytokine production, total cells, and neutrophils were assessed.

Histological examination
Lung tissues were fixed with 5 % PFA, embedded in paraffin, and sectioned. The sections were counterstained with hematoxylin and eosin (H&E).

Lung injury scores
Lung injuries were scored based on hemorrhage, alveolar wall thickness, inflammatory infiltration, alveolar congestion, and hyaline membrane formation. A score of 0 represents no damage, 1 represents mild damage, 2 represents moderate damage, 3 represents severe damage, and 4 represents very severe histological alterations.

Evaluation of wet-to-dry (W/D) lung weight ratio
Mice were sacrificed by neck breakage, and their lungs were removed and weighed immediately. The lungs were dried in an oven until they reached a constant weight. The wet/dry weight ratios of the lungs were calculated.

Enzyme-linked immunosorbent assay (ELISA)
IL-1β, TNF-α, and IL-6 levels in BALF samples were assessed using ELISA kits (Beyotime, Beijing, China) according to the manufacturer's instructions. Briefly, samples were aspirated into wells and incubated with biotin-conjugated primary antibodies. Then, avidin-conjugated horseradish peroxidase (HRP) was added to each well. Determination was performed by the addition of substrate, and absorbance was measured at 350 nm using a microplate reader.

Immunofluorescence
Lung tissues were fixed with 5 % PFA, embedded in OCT, and sectioned. Sections were incubated with primary antibodies to CD4, IL-10, and IL-17A at 4°C overnight. After rinsing with PBS, sections were incubated with the respective secondary antibodies and counterstained with DAPI. Images were captured using a fluorescence microscope.

Statistical analysis
Statistical analysis was performed using GraphPad 7.0. Data are presented as mean ± SEM. P < 0.05 was considered statistically significant.

AA relieved LPS-induced lung injury in mice
After induction of ALI by LPS, lung tissues were examined histologically. Mice in the LPS group displayed tissue injury characterized by irregular alveolar integrity and size, partial collapse of alveoli, enlarged spaces between alveoli, and inflammatory cell infiltration. However, these injuries were reversed after the administration of AA (Figures 1 A and B). In addition, the wet/dry ratio of lung tissue increased in the LPS group, but this increase was reversed upon AA treatment (Figure 1 C). These results suggested that AA may ameliorate lung injury.

AA inhibited inflammatory cytokine production in mice
Total cells and neutrophils in the BALF samples from each group were evaluated. Total cell and neutrophil numbers in the BALF samples from LPS-induced mice increased significantly compared with those in control samples, and AA treatment reversed these increases (Figure 2 A). MPO activity reflects neutrophil infiltration into damaged tissue. The MPO level increased in LPS-induced mice, and this increase was reversed by AA treatment (Figure 2 B). Levels of the inflammatory cytokines IL-6, IL-1β, and TNFα also increased in LPS-induced mice, and this increase was reversed by AA treatment (Figure 2 C).

AA mediated the Th17/Treg balance via the STAT pathway
To unravel the mechanism of action of AA, the effects of AA on molecules in the STAT pathway were investigated. AA treatment increased p-STAT5, RORrt, and FOXP3 levels and decreased the level of p-STAT3 in LPS-induced mice ( Figure 4). These data confirmed that AA restores the Th17/Treg balance by modulating the STAT pathway.

AA modulated Th17/Treg balance by activating PPARγ
To further understand the molecular mechanism of AA, the effect of AA on PPARg was examined. The level of PPARg decreased in LPS-induced mice, and this decrease was reversed with AA treatment (Figure 5 A). To verify the effect of AA on PPARg, the PPARg inhibitor GW9662 was used. As shown above, levels of p-STAT5 and FOXP3 increased and levels of p-STAT3 and RORrt decreased in LPS+AA mice, and these alterations were reversed with administration of GW9662 (Figure 5 B). Thus, AA modulated the Th17/Treg balance by activating PPARγ.

DISCUSSION
ALI is characterized by acute hypoxic respiratory failure caused by injury of alveolar epithelial cells [9]. ALI is caused by inflammation; however, understanding the pathogenesis of ALI requires further study [10]. Currently, there are no effective treatments for the severe stage of ALI (oxygenation index < 200). In this study, it was found that AA, an abietic diterpene compound, may be an effective treatment for ALI. H&E staining and lung injury scoring showed that AA ameliorated LPS-induced lung injury in mice. Furthermore, ELISA and FCM assays revealed that AA ameliorated LPS-induced pneumonia and restored LPS-induced Th17/Treg imbalance in mice. Immunoblot assays revealed that AA activated PPARγ and affected the expression of transcription factors that contribute to Th17 and Treg cell differentiation. It was previously shown that AA ameliorates psoriasis-like inflammation and modulates the gut microbiota in mice. In addition, AA suppressed non-small-cell lung cancer (NSCLC) cell growth by suppressing the IKKβ/NF-κB pathway. Similarly, AA ameliorated inflammation in our ALI mouse model. In this study, AA activated PPARγ and mediated the Th17/Treg balance, thus attenuating inflammation in the ALI model; however, the precise mechanism of AA requires further study.
AA modulated the Th17/Treg balance in the ALI mice model. Immune homeostasis depends on the balance between lymphocyte subsets and Treg cells [11]. Th17 cells secrete IL-17 and induce production of pro-inflammatory cytokines, thereby exacerbating the inflammatory response [12]. Tregs secrete anti-inflammatory mediators to maintain immune homeostasis [13]. Imbalances in Th and Treg cells contribute to the development of various diseases [14]. Restoration of the balance of Th17 cells and Treg cells has been shown to improve ALI [15]. Mechanistically, we suggest that AA attenuates ALI by modulating the Th17/Treg balance.
PPARγ is a key member of the PPAR family [16], and PPARγ activation plays anti-inflammatory and anti-apoptotic roles in many inflammatory disease models. PPARγ also plays a crucial role in regulating the Th17/Treg balance [17]. In this study, LPS induced a Th17/Treg imbalance, and AA treatment restored the Th17/Treg balance by promoting the expression of transcription factors. In addition, AA activated PPARγ to regulate the Th17/Treg balance. Hence, PPARγ may serve as a target for the treatment of ALI and other inflammatory diseases.

CONCLUSION
The findings of this study show that AA attenuates LPS-induced ALI by activating PPARγ and restoring Th17/Treg balance. Thus, AA is a potential drug for the treatment of ALI.