A handful of PI3K inhibitors have been explored for the treatment of pulmonary diseases. However, their systemic distributional toxicity, stemming from tissue distribution, restricts their therapeutic window. PI3K-selective inhibitors show promise due to the various toxicities produced by pan-PI3K inhibitors [37]. Therefore, PI3K-selective inhibitors are progressively demonstrating their advantages [38]. Recent studies have identified PI3Kγ as a promising target for treating inflammatory and autoimmune diseases owing to its predominant expression in leukocytes [39–41]. It has been discovered that PI3Kγ−/− neutrophils and macrophages demonstrate a certain level of chemotactic impairment when stimulated by GPCR agonists such as C5a [32, 42]. Neutrophils and macrophages serve as the prime defense barrier against bacterial and microbial invasions in the body, and their ability to facilitate healing of infection sites heavily relies on their chemotactic abilities, notably influenced by chemokines and diverse cytokines [18]. In addition, upon stimulation, neutrophils and macrophages can generate reactive oxygen species to exert antimicrobial effects. The process heavily relies on the production of PIP3 by PI3Kγ, the loss of which directly inhibits the respiratory burst of neutrophils [10, 43].
Inflammation represents a complex series of defense responses initiated by the body to shield against internal and external factors [44]. If the source of inflammation is not promptly eradicated, an excessive chronic inflammatory response can lead to cellular death, tissue necrosis, and possibly progress to conditions such as asthma, rheumatoid arthritis, diabetes, and even cancer. Asthma is a lung disease characterized by Th2 dominance, triggering airway hyperresponsiveness and airway remodeling, leading to significant physical harm for patients [14]. The primary treatment for asthma involves the use of corticosteroids; however, up to 10% of patients show resistance to these medications, making the development of novel therapeutic target for asthma a pivotal focus [45, 46]. On account of the tissue-specific expression of PI3Kγ kinase, characterized by abnormal activation of immune cells (primarily white blood cells) and lung fibroblasts, PI3Kγ has become a potential therapeutic target for asthma and other lung diseases [47]. Therefore, the development of novel PI3Kγ inhibitors for the treatment of asthma has promising prospects for application. Our laboratory has previously identified JN-KI3, a novel scaffold-based selective inhibitor of PI3Kγ, demonstrating a superior selectivity towards PI3Kγ over other PI3K isoforms [25]. Hence, the focus of this study was to assess the anti-inflammatory properties of JN-KI3 and confirm its preliminary therapeutic effects on asthma.
To assess the potential in vitro anti-inflammatory effects of JN-KI3, we used the murine macrophage RAW264.7 cell line. First, to assess the potential toxic interference of the compound itself, the MTT assay was performed to determine the cytotoxicity of JN-KI3 for RAW264.7 cells. The results showed that even at higher concentrations (10 µM), the cell viability remained above 95% after 72 h of treatment, demonstrating the absence of cytotoxicity associated with JN-KI3 on RAW264.7 cells. Subsequently, to further verify the effect of JN-KI3 on PI3Kγ signaling pathways, C5a-induced PI3Kγ signaling in RAW264.7 was employed. C5a, being a GPCR activator, can specifically stimulate the PI3Kγ signaling pathway [48, 49]. The western blot analysis revealed a significant increase in the expression of pAKT473 following C5a stimulation, and JN-KI3 displayed a concentration-dependent inhibition of cellular C5a-induced pAKT473. Then, the anti-inflammatory activity of JN-KI3 was investigated by inducing the production of inflammatory cytokines in RAW264.7 macrophage cells using LPS. LPS, the primary component of the outer membrane of Gram-negative bacteria, activates TLR4 and initiates a cascade of pro-inflammatory responses [31, 50]. Studies have found that macrophages, when stimulated in the inflammatory response, secrete TNF-α, IL-1β, and IL-6 [51, 52]. Our data demonstrated that RAW264.7 cells produced an inflammatory response and released TNF-α, IL-6, and IL-1β under LPS simulation. Moreover, JN-KI3 reduced LPS-activated Akt phosphorylation and downregulated the transcription and expression of TNF-α, IL-6, and IL-1β in a dose-dependent manner, suggesting that JN-KI3 can diminish the production of inflammatory factors by modulating the PI3K/Akt pathway. These in vitro results suggest that JN-KI3 may yield anti-inflammatory effects by specifically inhibiting the PI3Kγ signaling pathway.
Functional studies have substantiated the significant role of PI3Kγ in regulating inflammation, particularly in lung diseases [19]. Therefore, to examine the hypothesis that JN-KI3 can function as an inhibitor for pulmonary inflammation, we constructed a mouse asthma model induced by OVA. OVA sensitization and challenge can significantly cause the infiltration of inflammatory cells, thus increasing the number of inflammatory cells. Hence, the number of inflammatory cells in the BALF was initially determined. The inflammatory cell counts in the BALF showed abundant expression of inflammatory cells in the lungs of the OVA group. JN-KI3 significantly reduced the expression of inflammatory cells, particularly neutrophils, eosinophils, and macrophages, and restored them to normal levels. Subsequently, the western blot analysis of mouse lung tissues showed that JN-KI3 significantly inhibited Akt phosphorylation, suggesting its potential in suppressing the infiltration of lung inflammatory cells through the inhibition of PI3Kγ signaling pathway. Asthma is a lung disease characterized by a Th2 immune response, wherein cells facilitate inflammatory cell infiltration by releasing factors such as IL-4, IL-5 and IL-13, resulting in lung airway remodeling and increased airway hyperresponsiveness [53, 54]. The ELISA assay found that JN-KI3 significantly reduced the expression of IL-4, IL-5 and IL-13 in BALF. It also indicated that at high concentration, the expression levels returned to normal. These finding suggest that JN-KI3 can downregulate the production of Th2 cytokines by inhibiting the PI3K/Akt signaling pathway. Then, the histopathological analysis of lung by H&E, Masson trichrome staining and PAS staining showed that oral administration of JN-KI3 effectively inhibited the accumulation of inflammatory cells around the bronchus and blood vessels, reduced the overproduction of cupped cells and mucus, and mitigated the excessive deposition of collagen around the airways. These findings demonstrated that JN-KI3 effectively delayed the pathological changes occurring in the lung during asthma and exhibited a certain therapeutic effect. Ultimately, the immunohistochemical analysis of lung tissue revealed that JN-KI3 significantly inhibited leukocyte infiltration following OVA exposure. In conclusion, the results from these mouse models of asthma suggest that JN-KI3 has a preliminary therapeutic effect on OVA-induced asthma.