Evaluating the anti-inflammatory potential of JN-KI3: the therapeutic role of PI3Kγ- selective inhibitors in asthma treatment

Evaluating the anti-inflammatory potential of JN-KI3: the therapeutic role of PI3Kγ- selective inhibitors in asthma treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluating the anti-inflammatory potential of JN-KI3: the therapeutic role of PI3Kγ- selective inhibitors in asthma treatment Lei Jia, Mengyun Ma, Wendian Xiong, Jingyu Zhu, Yanfei Cai, Yun Chen, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3856128/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction Asthma is a chronic airway inflammatory disease of the airways characterized by the involvement of numerous inflammatory cells and factors. Therefore, targeting airway inflammation is one of the crucial strategies for developing novel drugs in the treatment of asthma. Phosphoinositide 3-kinase gamma (PI3Kγ) has been demonstrated to have a significant impact on inflammation and immune responses, thus emerging as a promising therapeutic target for airway inflammatory disease, including asthma. Objective and method There are few studies reporting on the therapeutic effects of PI3Kγ-selective inhibitors in asthma disease. In this study, we investigated the anti-inflammatory and therapeutic effects of PI3Kγ-selective inhibitor JN-KI3 for treating asthma by utilizing both in vivo and in vitro approaches, thereby proving that PI3Kγ-selective inhibitors could be valuable in the treatment of asthma. Results In RAW264.7 macrophages, JN-KI3 effectively suppressed C5a-induced Akt phosphorylation in a concentration-dependent manner, with no discernible toxicity observed in RAW264.7 cells. Furthermore, JN-KI3 can inhibit the PI3K/Akt signaling pathway in lipopolysaccharide-induced RAW264.7 cells, leading to the suppression of transcription and expression of the classical inflammatory cytokines in a concentration-dependent manner. Finally, an ovalbumin-induced murine asthma model was constructed to evaluate the initial therapeutic effect of JN-KI3 for treating asthma. Oral administration of JN-KI3 inhibited the infiltration of inflammatory cells and the expression of T-helper type 2 cytokines in bronchoalveolar lavage fluid, which was associated with the suppression of the PI3K signaling pathway. Lung tissue and immunohistochemical studies demonstrated that JN-KI3 inhibited the accumulation of inflammatory cells around the bronchus and blood vessels, as well as the secretion of mucus and excessive deposition of collagen around the airway. In addition, it reduced the infiltration of white blood cells into the lungs. Conclusion JN-KI3 shows promise as a candidate for the treatment of asthma. Our study also suggests that the inhibitory effects of PI3Kγ on inflammation could offer an additional therapeutic strategy for pulmonary inflammatory diseases. PI3Kγ-selective inhibitor JN-KI3 anti-inflammation airway inflammatory disease asthma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Phosphoinositide 3-kinase (PI3K) is a lipid kinase involved in signal transduction. It controls various biological processes, including cellular growth, proliferation, and differentiation [ 1 , 2 ]. PI3Ks can be categorized into class I, II, and III according to their substrates [ 3 ]. Class I PI3Ks, which have attracted significant attention, are further divided into class IA (α, β, and δ) and IB (γ) based on their regulatory proteins and tissue specificity [ 4 , 5 ]. Class IA PI3Ks are triggered by a cascade of receptor tyrosine kinases (RTK), while class IB PI3K is activated by G protein-coupled receptors (GPCR) such as C5a, initiating downstream signaling pathways [ 6 ]. In contrast to the wide distribution of PI3Kα and β, PI3Kγ and PI3Kδ are predominantly expressed in the immune system, specifically the leukocytes [ 7 , 8 ]. Their selective expression of γ and δ closely ties them to human immune function, with the two subtypes fulfilling non-redundant roles [ 9 ]. Accumulated research has found that mutations in the genes encoding both PI3Kγ and δ result in primary immunodeficiency diseases [ 10 ]. As the sole representative of class IB, PI3Kγ plays a key role in regulating leukocyte migration, including neutrophils and eosinophils, and is involved in mediating the recruitment of certain immune cells to inflammation sites [ 11 , 12 ]. As a result, PI3Kγ has been implicated in various inflammatory conditions (such as asthma, allergies, rheumatoid arthritis, pulmonary fibrosis, etc.), immunodeficiency diseases, cardiovascular disease, and cancer-related inflammation. Consequently, there is a growing interest in the identification of PI3Kγ-selective inhibitors for the treatment of inflammatory diseases [ 13 ]. Asthma, one of the most common chronic airway inflammatory diseases in the world, has caused significant harm to the health of patients and it can even threaten the lives of patients if certain treatment measures are not taken [ 14 ]. Allergic asthma, being the predominant form of asthma, is distinguished by airway hyperresponsiveness and remodeling. Research indicates a critical involvement of T-helper type 2 (Th2) cells in the pathogenesis of asthma [ 15 ]. Upon encountering allergens or antigens presented by antigen-presenting cells (e.g., dendritic cells), Th2 cells initiate allergic reactions and produce cytokines, including interleukin-4 (IL-4), IL-5, and IL-13 [ 16 ]. This process leads to the infiltration of eosinophils into the airways, contributing to the development of allergic asthma. PI3Kγ, as a key factor influencing leukocyte migration, may also be involved in modulating the behavior of eosinophils, which are a pivotal element in the pathogenesis of allergic asthma [ 17 , 18 ]. Recently, some PI3Kγ inhibitors have been reported to be useful for the treatment of asthma [ 19 – 23 ]. Therefore, PI3Kγ is anticipated to serve as a potential therapeutic target for asthma. However, there are limited preclinical studies using PI3Kγ inhibitors for asthma, highlighting the urgent need to develop newer and more efficient PI3Kγ inhibitors for the treatment of asthma [ 24 ]. Recently, we identified a novel PI3Kγ inhibitors, JN-KI3, utilizing a machine learning-based multi-conformational structure virtual screening approach. The kinase test demonstrated that JN-KI3 remarkably inhibited PI3Kγ outside the cellular matrix. Additionally, JN-KI3 specifically inhibited the PI3K/Akt signaling pathway in a time- and concentration-dependent manner [ 25 ]. Therefore, the article comprehensively investigates the anti-inflammatory and anti-asthma effects of this lead compound in in vitro and in vivo models. The study flow is outlined in Fig. 1 . Materials and Methods Reagents The positive compounds, IPI-549 and dexamethasone (Dexa), were purchased from Top-science (TargetMol, USA) and Chenxin (Shandong, China). JN-KI3 was obtained from ChemDiv Inc. (San Diego, USA). Thiazolyl Blue Tetrazolium Bromide (MTT) was purchased from Solarbio (Beijing, China). Complement component 5a (C5a) was obtained from R&D Systems (California, USA). Lipopolysaccharide (LPS) and ovalbumin (OVA) were purchased from Sigma (St Louis, USA). A PrimeScrip™ RT reagent kit and QuantiNova™ SYBR® Green PCR Master Mix were obtained from TaKaRa Bio (Tokyo, Japan) and QIAGEN (Germany). Tumor necrosis factor-α (TNF-α), IL-6, and IL-1β ELISA kits were purchased from Cusabio biotech (Wuhan, China). The primary antibodies against phosphorylated Akt serine 473 (pAkt473) and total Akt were purchased from Cell Signaling Technology (Beverly, USA). The HRP-conjugated secondary antibody was purchased from Proteintech Group, Inc. (Wuhan, China). Cell culture RAW264.7 cells were sourced from the Cell Bank of the Shanghai Institute of Cell Biology and Biochemistry, Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma, USA), supplemented with 10% fetal bovine serum (Gibco, USA), and 100 µg/mL penicillin-streptomycin in 95% air and 5% CO 2 at 37°C. Cell viability assay RAW264.7 cells were plated at a density of 8 × 10 3 cells per well in a 96-well plate and incubated overnight. The cytotoxicity experiments were divided into different groups: the control group was treated with an equal volume of PBS, while the test group was exposed to JN-KI3 at various concentrations (0.16, 0.32, 0.64, 1.25, 2.5, 5, and 10 µM) for 72 h at 37°C. Then, 10 µL of 5 mg/mL MTT was added to each well, followed by a 4 h incubation. Subsequently, 100 µL MTT buffer was added to each well. The absorbance was measured at 550 nm with spectrophotometer. Western blot assay RAW264.7 cells were seeded in 6-well plates at a density of 1 × 10 5 cells per well and treated with different concentrations of JN-KI3 (0, 1, 2.5, 5, and 10 µM), in addition to the positive compound IPI-549 (1 µM). After incubation for 1 h, 12.5 µg/ml of C5a was added for 10 min. The cells were washed three times with PBS and then added 100 µL RIPA lysis buffer (Thermo Scientific, USA), which contained protease and phosphatase inhibitors. After full lysis on ice, the supernatant was collected by 12,000 rpm centrifugation for 20 min, and the protein concentration was determined by BCA protein assay kit (Sangon Biotech, China). Loading buffer was added to all protein samples prepared for western blot assays and boiled at 100°C for 5 min. The protein samples were resolved using 10% SDS-PAGE and subsequently transferred onto nitrocellulose filter (NC) membranes. The membranes were blocked with 5% skim milk diluted in 1 x TBST with 0.3% Tween 20 for 2 h. After that, the membranes were incubated overnight at 4°C with primary antibodies (phosphorylated Akt serine 473, 1:2000; total Akt, 1:1000). After washing the membrane three times with TBST, all the membranes were incubated with the corresponding secondary antibody at room temperature for 2 h. The blots were visualized using ECL solution after the membranes had been washed three times. Quantitative real-time polymerase chain reaction (qRT-PCR) RAW264.7 cells (5 × 10 4 cells/well) were treated with JN-KI3 at concentrations of 0, 1, 2.5, 5, and10 µM for 1h, followed by incubation with 500 ng/mL LPS for 16 h. The total RNA was then extracted using Trizol Reagent (Thermo Scientific, USA), followed by reverse transcription into cDNA and amplification by qRT-PCR. The thermocycling program consisted of holding at 95°C for 2 min, followed by 40 cycles of 5 s at 95°C and 10 s at 60°C. The primer sequences are listed in Table 1 . Table 1 Primer sequences used for RT-PCR Genes Primer sequence (5′ to 3′) TNF-α F: 5′-GCCCACGTCGTAGCAAACCA-3′ R: 5′-GCAGGGGCTCTTGACGGCAG-3′ IL-6 F: 5′-TAGTCCTTCCTACCCCAATTTCC-3′ R: 5′-TTGGTCCTTAGCCACTCCTTC-3′ IL-1β F: 5′-TGTGCAAGTGTCTGAAGCAGC-3′ R: 5′-TGGAAGCAGCCCTTCATCTT-3′ GAPDH F: 5′-AGCCTCGTCCCGTAGACAA-3′ R: 5′-AATCTCCACTTTGCCACTGC-3′ ELISA assay RAW264.7 cells (5 × 10 4 cells/well) were treated with various concentrations of JN-KI3 (0, 1, 2.5, 5, and 10 µM) for 1 h. Following a 1 h incubation, cells were stimulated with LPS at a concentration of 500ng/mL for 16 h. The supernatant was taken out to measure the levels of TNF-α, IL-6, and IL-1β by using an ELISA kit according to the manufacturer’s instructions. A450 nm were determined on a Tecan microplate reader. Animal Female BALB/c mice (6–8 weeks, 19–21 g) were acquired from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and housed under normal conditions as follows: 23 ± 2°C, 55 ± 5% humidity, and 12 h day/night cycle. All mice were given plenty of water and food. The protocol of animal experiments was approved by the Animal Ethics Committee of Jiangnan University (JN.No20220315b0360530[087]). Induction of an asthma mice model The mice were randomly divided into 6 groups (6 mice/group): (1) control; (2) OVA-treated; (3) Dexa-treated (5 mg/kg); (4) JN-KI3-treated (20 mg/kg); (5) JN-KI3 (40 mg/kg), and (6) JN-KI3 (80 mg/kg) groups. To induce asthma, each mice was intraperitoneally injected with 100 µl OVA mixture (20 µg OVA and 2 mg aluminum hydroxide adjuvant in 0.1 mL PBS) in both the model and administration groups. The mice in the control group received the same dose of aluminum hydroxide adjuvant on days 1 and 14 for sensitization. Subsequently, the OVA-challenged mice were exposed to ultrasonic atomization of 5% OVA for 20 min from day 22 to day 28 (Fig. 1 ) . The mice were sacrificed 24 h after the final challenge. Bronchoalveolar lavage fluid (BALF) collection and leukocyte counts The bronchial tubes of the mice were ligated on the right side, and the left lung was washed with 0.5 mL of PBS and repeated three times to collect BALF. The collected BALF was separated at 800 g for 10 min at 4°C. The supernatant was stored at -80°C for subsequent ELISA analysis, and the cell precipitation was resuspended with 100 µl PBS for cell counting. Western blot analysis Lung tissue samples were collected for western blot analysis to measure Akt phosphorylation levels. After protein extraction using RIPA, equal amounts were loaded into the wells of an SDS-PAGE gel and subsequently transferred to NC membranes. Immunoblotting analyses were then performed to assess the levels of pAkt473 and total Akt protein. Measurement of IL-5, IL-13 and OVA-IgE levels The concentration of IL-5 and IL-13 in the BALF, as well as OVA-specific IgE in the serum, were measured by ELISA according to the manufacturer’s protocol. Histological analysis of lung tissue The right lung was excised and fixed with 4% paraformaldehyde. After dehydration, the lung tissue was embedded in paraffin, sliced into 3 µm sections, and subjected to staining with hematoxylin-eosin (HE), Masson’s Trichrome (Masson), and periodic acid Schiff (PAS). HE staining was used to evaluate lung inflammation, and then a blind method was utilized to assess the level of inflammation using the following criteria: 0, normal; 1, occasional presence of inflammatory cells; 2, presence of inflammatory cells surrounding the bronchi or blood vessels; 3, 2–4 layers of inflammatory cells surrounding the bronchi or blood vessels; 4, presence of more than 4 layers of inflammatory cells around most bronchi or blood vessels [ 26 ]. Masson trichrome was employed to assess collagen deposition and airway remodeling. The area occupied by collagen was quantified using ImageJ software to calculate the percentage of collagen fibers [ 27 ]. In addition, PAS staining was used to observe airway mucus production. Goblet cell proliferation was quantified using a five-point scoring system as follows: 0, 75%. A minimum of 8 bronchi and parenchyma regions were counted per lung tissue sample [ 26 ]. Statistical analysis The data were expressed as the mean ± standard error of the mean (SEM) and analyzed using Graphpad prism 9.0 software (GraphPad Software Inc., San Diego, CA, United States). One-way ANOVA analysis was conducted to assess the differences between groups. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the control group. Results Impact of JN-KI3 on PI3Kγ-dependent cellular functions The structure of JN-KI3 is illustrated in Fig. 1 . The enzyme inhibition assay revealed that JN-KI3 exhibits greater selectivity towards PI3Kγ in comparison to the other three isoforms. In order to eliminate chemical toxic interference, the MTT assay was performed to determine the cytotoxic effect of JN-KI3 on RAW264.7 cells following 24, 48, and 72 h of treatment. As shown in Fig. 2 A-C, at a high concentration of 10 µM, JN-KI3 did not induce toxicity in RAW264.7 cells. To further validate the selectivity of JN-KI3 among class I PI3Ks within a cellular context, we examined the PI3Kγ-dependent cellular potencies in C5a-stimulated RAW264.7 cells. C5a can stimulate GPCR signals via PI3Kγ. As shown in Fig. 2 D, compared to the unstimulated condition, C5a induced significant phosphorylation of Akt at Ser473 (pAkt473). IPI-549, a well-established selective PI3Kγ inhibitor, served as the positive control for inhibiting Akt phosphorylation. Notably, IPI-549 considerably suppressed Akt phosphorylation, and JN-KI3 also showed concentration-dependent inhibition of Akt phosphorylation while not affecting total Akt expression. Effect of JN-KI3 on PI3K/Akt signaling pathways in LPS-induced RAW264.7 cells The LPS-induced RAW264.7 macrophage inflammatory model is a widely utilized model for inflammation. Studies have shown that LPS binds to Toll-like receptors (TLR) secreted by macrophages, leading to the activation of the PI3K/Akt signaling pathway, subsequently resulting in the release of a variety of inflammatory factors that mediate signaling [ 19 , 28 – 30 ]. As shown in Fig. 3 , the level of pAkt473 was significantly increased by LPS compared to the control, suggesting activation of the PI3K signaling pathways by LPS, whereas Akt phosphorylation was significantly suppressed by JN-KI3 in a concentration- and time- dependent manner. Effect of JN-KI3 on expression of TNF-α, IL-6 and IL-1β in LPS-induced RAW264.7 macrophages. As an important link in the immune system, macrophages play a crucial role in the host's response to pathogen infection and inflammation [ 31 ]. LPS induces classical activation of macrophages (M1), leading to the destruction of pathogens and the production of pro-inflammatory mediators such as TNF-α, IL-6, and IL-1β [ 32 , 33 ]. As pro-inflammatory cytokines play a key role in the inflammatory process, we investigated the inhibition of classical pro-inflammatory cytokines by JN-KI3. As shown in Fig. 4 , the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β significantly increased in RAW264.7 cells following LPS treatment. After treatment of varied concentrations of JN-KI3, the mRNA expression of these three pro-inflammatory factors decreased by varying degrees compared to the control group, exhibiting concentration-dependence behavior. Then, an ELISA assay was performed to measure the protein levels of these three pro-inflammatory factors in LPS-induced RAW264.7 cells. As shown in Fig. 5 , LPS significantly enhanced the protein expression of these pro-inflammatory factors, whereas the levels of IL-1β and IL-6 in the JN-KI3-treated group exhibited concentration-dependent reduction compared to those in the control group. Surprisingly, while JN-KI3 exhibited concentration-dependent inhibition of TNF-α expression at low concentrations, it seemed to lose its inhibitory effect at high concentrations. By establishing an extracellular inflammatory cell model with RAW264.7 cells, we preliminarily confirmed that JN-KI3 can attenuate inflammatory responses by inhibiting the LPS-induced activation of PI3K/Akt pathways in RAW264.7 cells. Effect of JN-KI3 on the infiltration of inflammatory cells in BALF through the PI3K signaling pathway To evaluate the anti-inflammatory and anti-asthmatic effects, as well as the potential mechanism of action of JN-KI3, an in vivo study was conducted using an OVA-induced asthma mouse model (Fig. 5 A). After 24 h of the last-day OVA stimulation, BALF was collected from the mice to evaluate the impact of Dexa and JN-KI3 on the recruitment of inflammatory cells in the lungs. As shown in Fig. 5 B, compared with the control group, the inflammatory cells in the lungs of the OVA-stimulated group substantially increased, while the cell count exhibited a noticeable reduction in response to JN-KI3 in a dose-dependent manner. In addition, there was a significant increase in the counts of neutrophils, eosinophils, and macrophages in the OVA group of mice. Conversely, these inflammatory cells were significantly reduced in the JN-KI3-treated group, particularly under JN-KI3 administration at 80 mg/kg, displaying an effect similar to the positive control, Dexa. Therefore, JN-KI3 was able to exert an inhibitory effect on the infiltration of inflammatory cell in the lungs of asthmatic mice. Then, the inhibition of Akt phosphorylation in lung tissue homogenates of mice after treatment with JN-KI3 was analyzed using western blot analysis. Figure 5 C demonstrates a significant increase in Akt protein phosphorylation, while the total Akt level remained constant. The administration of JN-KI3 resulted in a concentration-dependent reduction in Akt phosphorylation, indicating that orally administered JN-KI3 accessed the lung tissues and interacted with the target protein, subsequently inhibiting PI3K/Akt signaling. Effect of JN-KI3 on cytokine levels in BALF OVA-induced asthma is primarily characterized by the release of pro-inflammatory Th2 cytokines, including IL4, IL-5, and IL-13 [ 34 , 35 ]. Consequently, the expression levels of these pro-inflammatory factors in BALF were initially assessed using the ELISA assay. As shown in Fig. 6 , the expression of all three cytokines was significantly higher in the OVA-treated group compared to the lower expression level in the control group. The positive control group Dexa demonstrated a clear inhibitory effect on pro-inflammatory factors. After oral administration of JN-KI3, the levels of these pro-inflammatory factors decreased to varying degrees in a dose-dependent manner. At a high concentration of JN-KI3 (80 mg/kg), the anti-inflammatory efficacy was equivalent to that of Dexa, the positive control group. These results suggest that JN-KI3 has the potential to reduce OVA-induced Th2 cytokine infiltration in lung tissues. Histopathology examination of the lungs To gain further insight into the pathological changes and the extent of lung inflammation, we performed lung section analysis on all 4 groups of mice. Upon H&E staining, it was observed that the OVA-stimulated group showed a substantial infiltration of inflammatory cells in the bronchial and perivascular connective tissues of the lungs compared to the control group, (Fig. 7 A). Whereas the accumulation of inflammatory cells was significantly reduced following treatment with Dexa and JN-KI3 treatment. Analysis of bronchial collagen distribution was conducted using Masson’s trichrome staining. As shown in Fig. 7 B, lung tissues exhibited excessive collagen deposition around the bronchi following OVA stimulation, with varying degrees of reduction in collagen accumulation around the trachea observed after treatment with Dexa or JN-KI3. To determine the potential of JN-KI3 in reducing mucus production in the lungs of OVA-treated mice, lung tissues were subjected to PAS staining (Fig. 7 C). After OVA stimulation, there was a substantial increase in goblet cell metaplasia and mucus secretion in the lungs of mice. In contrast, mucus production was significantly reduced after treatment with Dexa and JN-KI3. The analysis of the mice lung sections revealed that JN-KI3 exhibited an inhibitory effect on airway remodeling and demonstrated therapeutic potential for treating asthma in mice. Effects of JN-KI3 on OVA-induced white blood cell infiltration into lung tissue Asthma is an inflammatory pulmonary disease characterized by heightened leukocyte infiltration into the airways and diminished respiratory function [ 36 ]. To evaluate the effect of JN-KI3 on OVA-induced peribronchial leukocyte recruitment, mice lung tissue sections were subjected to immunohistochemical analysis using CD11b/CD18 antibody [ 20 ]. As shown in Fig. 8 , OVA triggered a large accumulation of leukocytes (depicted as brown cells) around the bronchi and in the alveoli. Both the reference drug Dexa and JN-KI3 resulted in a noteworthy decrease in leukocyte recruitment, indicating that JN-KI3 effectively attenuated the infiltration of leukocytes into the lungs. Discussion 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. Conclusions Our study demonstrated that JN-KI3 selectively inhibits PI3Kγ, resulting in targeted disruption of the PI3K/Akt signaling pathway and significant anti-inflammatory effects. Furthermore, the in vivo experiments demonstrated the therapeutic potential of JN-KI3 in mitigating airway inflammation in a murine model of asthma. Thus, JN-KI3 could serve as a promising lead compound for the treatment of asthma. However, JN-KI3 needs further optimization and development to become a candidate drug for use against pulmonary inflammatory diseases. Our study also suggests that the inhibitory effects of PI3Kγ on inflammation may present an additional therapeutic strategy for asthma. Declarations Acknowledgements The study was supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX23_2469), the National Natural Science Foundation of China (No. 21807049, 82001711), the Fundamental Research Funds for the Central Universities (JUSRP51703A), the University-Industry Cooperation Research Project in Jiangsu (No. BY2020432), the Natural Science Foundation of Jiangsu Province (BK20201137), and the Foundation of Wuxi Municipal Health Commission (HB2023023). Author Contributions LJ: Methodology, Data curation, Writing - original draft. MM: Methodology. WX: Methodology, Validation. JZ: Investigation, Conceptualization, Project administration. YCai: Methodology. YChen: Writing - review & editing. JJ: Resources, Supervision. MG: Resources, Writing - review & editing. All authors reviewed the manuscript. Conflict of interest The authors declare no conflict of interest. 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Knight ZA, Chiang GG, Alaimo PJ, Kenski DM, Ho CB, Coan K, et al. Isoform-specific phosphoinositide 3-kinase inhibitors from an arylmorpholine scaffold. Bioorg Med Chem 2004; 12:4749–59. Marwick JA, Chung KF, Adcock IM. Phosphatidylinositol 3-kinase isoforms as targets in respiratory disease. Ther Adv Respir Dis 2010; 4:19–34. Perry MWD, Abdulai R, Mogemark M, Petersen J, Thomas MJ, Valastro B, et al. Evolution of PI3Kgamma and delta Inhibitors for Inflammatory and Autoimmune Diseases. J Med Chem 2019; 62:4783–4814. Cushing TD, Metz DP, Whittington DA, McGee LR. PI3Kdelta and PI3Kgamma as targets for autoimmune and inflammatory diseases. J Med Chem 2012; 55:8559–81. Thomas MJ, Smith A, Head DH, Milne L, Nicholls A, Pearce W, et al. Airway inflammation: chemokine-induced neutrophilia and the class I phosphoinositide 3-kinases. Eur J Immunol 2005; 35:1283–91. Costa C, Martin-Conte EL, Hirsch E. Phosphoinositide 3-kinase p110gamma in immunity. IUBMB Life 2011; 63:707–13. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002; 420:860–867. Miller RL, Grayson MH, Strothman K. Advances in asthma: New understandings of asthma's natural history, risk factors, underlying mechanisms, and clinical management. J Allergy Clin Immunol 2021; 148:1430–1441. Castillo JR, Peters SP, Busse WW. Asthma Exacerbations: Pathogenesis, Prevention, and Treatment. J Allergy Clin Immunol Pract 2017; 5:918–927. Sala V, Della Sala A, Ghigo A, Hirsch E. Roles of phosphatidyl inositol 3 kinase gamma (PI3Kgamma) in respiratory diseases. Cell Stress 2021; 5:40–51. Schneble N, Schmidt C, Bauer R, Muller JP, Monajembashi S, Wetzker R. Phosphoinositide 3-kinase gamma ties chemoattractant- and adrenergic control of microglial motility. Mol Cell Neurosci 2017; 78:1–8. Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L, et al. Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science 2000; 287:1049–1053. Plociennikowska A, Hromada-Judycka A, Borzecka K, Kwiatkowska K. Co-operation of TLR4 and raft proteins in LPS-induced pro-inflammatory signaling. Cell Mol Life Sci 2015; 72:557–581. Li X, Shen J, Jiang Y, Shen T, You L, Sun X, et al. Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells. Int J Mol Sci 2016; 17:1938. Byun J, Su KK, Ju YB. Anti-Inflammatory and Anti-Oxidant Effects of Korean Ginseng Berry Extract in LPS-Activated RAW264.7 Macrophages. Am J Chinese Med 2021;49:719–735. Leon B, Ballesteros-Tato A. Modulating Th2 Cell Immunity for the Treatment of Asthma. Front Immunol 2021; 12:637948. Fahy JV. Type 2 inflammation in asthma–present in most, absent in many. Nat Rev Immunol 2015; 15:57–65. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3856128","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267407833,"identity":"80d981d6-1c18-4989-8f5d-0d4aef80efaf","order_by":0,"name":"Lei Jia","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Jia","suffix":""},{"id":267407834,"identity":"7d0d45fc-447b-4c71-aebf-b085b55ac929","order_by":1,"name":"Mengyun Ma","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Mengyun","middleName":"","lastName":"Ma","suffix":""},{"id":267407835,"identity":"6d60fc34-2b26-4a5a-80a4-70ec865077b0","order_by":2,"name":"Wendian Xiong","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Wendian","middleName":"","lastName":"Xiong","suffix":""},{"id":267407836,"identity":"fcc49ede-9908-4f31-865a-1b797f235dfb","order_by":3,"name":"Jingyu Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYBACxmYIncDA3pBwAC7MQ5QWngNEaoGBBAaJBCQuPi3M7byHX/O21eUZ3Hzw8MDPHbV5/LMbGB+8bWOQN8fpML40a942tmKD2wkJB3vPHC+WuHOA2XBuG4PhzgZcWnjMjHnbeBI3ALUc4G07lthwI4FNmreNIcHgAF4tEokbbh5IOPgXqGX+jQT23wS0GD/mbTNI3HCDIeEwb1sNkJHAxkzIFsY55xISZ55JSDgs23YgceONxGbJOeckDDfg0GLYf8b4w5uyusS+42eSP75tq0ucdyP5IFDERh6XLYYNDGxSkFjgSQASh0E2NwAJCezqgUAeGDUff4CZ7CBT63CqHAWjYBSMgpELACDWZht2YZHGAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangnan University","correspondingAuthor":true,"prefix":"","firstName":"Jingyu","middleName":"","lastName":"Zhu","suffix":""},{"id":267407837,"identity":"058156e2-a12b-4de7-a705-063fc02ceb4c","order_by":4,"name":"Yanfei Cai","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Yanfei","middleName":"","lastName":"Cai","suffix":""},{"id":267407838,"identity":"d2d1b442-2111-4a86-9aa7-a5a586ddfd82","order_by":5,"name":"Yun Chen","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Chen","suffix":""},{"id":267407839,"identity":"eadaee15-bd9b-4002-959b-822e834bb0ea","order_by":6,"name":"Jian Jin","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Jin","suffix":""},{"id":267407840,"identity":"4fd1776a-996d-42de-96f9-43e7a0f07ce3","order_by":7,"name":"Mingzhu Gao","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"prefix":"","firstName":"Mingzhu","middleName":"","lastName":"Gao","suffix":""}],"badges":[],"createdAt":"2024-01-12 07:48:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3856128/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3856128/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49834809,"identity":"ec0326b8-3f55-4865-93b3-158dd5366c2d","added_by":"auto","created_at":"2024-01-18 18:13:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":264099,"visible":true,"origin":"","legend":"\u003cp\u003eThe workflow of this study.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/81671f3270ed9d4082fee010.png"},{"id":49834810,"identity":"77ca87d0-6b59-4b81-a486-4fe873ed9155","added_by":"auto","created_at":"2024-01-18 18:13:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":382240,"visible":true,"origin":"","legend":"\u003cp\u003eRAW264.7 cells were treated with JN-KI3 at different concentration from 0 to 10 μM for \u003cstrong\u003e(A)\u003c/strong\u003e 24 h, \u003cstrong\u003e(B) \u003c/strong\u003e48 h and \u003cstrong\u003e(C)\u003c/strong\u003e 72 h, followed by assessment of cell viability using the MTT method. (\u003cstrong\u003eD\u003c/strong\u003e) RAW264.7 cells were treated with JN-KI3 at different concentrations from 0 to 10 μM for 30 min, followed by C5a stimulation (12.5 nM, 5 min). The protein levels of C5a-induced phosphorylated Akt Ser473 (pAkt473) were determined by western blot, with IPI-549 (1 μM) utilized as the positive (P).\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/561bfc1dc59a72f52221c776.png"},{"id":49834816,"identity":"066aaf6f-3187-4264-82fa-3542dcbe40db","added_by":"auto","created_at":"2024-01-18 18:13:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":311795,"visible":true,"origin":"","legend":"\u003cp\u003eRAW264.7 cells were exposed to JN-KI3, \u003cstrong\u003e(A)\u003c/strong\u003e at various concentrations ranging from 0 to 10 μM for 30 min, and \u003cstrong\u003e(B)\u003c/strong\u003e for varying durations from 0 to 6 h at a concentration of 5 μM, followed by LPS stimulation (500 ng/mL, 30 min). The protein levels of C5a-induced phosphorylated Akt Ser473 (pAkt473) were determined by western blot, with IPI-549 (1 μM) utilized as the positive (P).\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/7eaf299d995f8b243695c127.png"},{"id":49834814,"identity":"8f1ab56f-aeb5-4c74-ba57-10cba317f67a","added_by":"auto","created_at":"2024-01-18 18:13:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":571318,"visible":true,"origin":"","legend":"\u003cp\u003eRAW264.7 cells were exposed to JN-KI3 at different concentrations ranging from 0 to 10 μM for 30 min, followed by stimulation with 500 ng/mL LPS for 24 h. The mRNA expression levels of \u003cstrong\u003e(A) \u003c/strong\u003eTNF-α, \u003cstrong\u003e(B)\u003c/strong\u003e IL-6, and \u003cstrong\u003e(C)\u003c/strong\u003eIL-1β were analyzed by quantitative PCR. The levels of protein expression for \u003cstrong\u003e(D) \u003c/strong\u003eTNF-α,\u003cstrong\u003e (E)\u003c/strong\u003e IL-6, and \u003cstrong\u003e(F)\u003c/strong\u003e IL-1β were analyzed using ELISA kits. Data represent the means ± SEMs based on three independent experiments, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/51770bf1ceb5293c56fd694f.png"},{"id":49834812,"identity":"9ea872cd-2e4a-479e-a744-022401e47c00","added_by":"auto","created_at":"2024-01-18 18:13:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1526671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Establishment of different experimental asthma models in mice. \u003cstrong\u003e(B)\u003c/strong\u003e Enumeration of inflammatory cells in BALF following exposure to dexamethasone (Dexa) at 5 mg/kg and JN-KI3 at 20, 40, and 80 mg/kg, including total white blood cells (WBCs), neutrophils (Neu), monocytes (Mon), lymphocytes (Lym), eosinophils (Eos), and basophils (Bas). \u003cstrong\u003e(C)\u003c/strong\u003e Western blot analysis was conducted to assess OVA-induced Akt Ser473 phosphorylation in lung fragments isolated from mice treated with JN-KI3 at 40 and 80 mg/kg. Data represent the means ± SEMs based on three independent experiments, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/34388cdbd0fef814f4c1548e.png"},{"id":49835734,"identity":"721c1a5b-5b70-4fa4-906f-1ea2eb5a5f61","added_by":"auto","created_at":"2024-01-18 18:29:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":134653,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytokine levels in BALF upon exposure to dexamethasone (Dexa) at 5 mg/kg and JN-KI3 at 20, 40, and 80 mg/kg: \u003cstrong\u003e(A)\u003c/strong\u003e IL-4, \u003cstrong\u003e(B)\u003c/strong\u003e IL-5, and \u003cstrong\u003e(C)\u003c/strong\u003e IL-13. Data represent the means ± SEMs based on three independent experiments, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/e48d6b397439145b91f2a347.png"},{"id":49835422,"identity":"a35be8cf-972a-4851-9726-179ad541477f","added_by":"auto","created_at":"2024-01-18 18:21:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2145917,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of lung tissue was performed 24 h after the final OVA challenge. \u003cstrong\u003e(A)\u003c/strong\u003e Hematoxylin and Eosin staining (H\u0026amp;E), Bar = 200 μM, \u003cstrong\u003e(B)\u003c/strong\u003e Representative Masson’s trichrome staining, Bar = 200 μM, and \u003cstrong\u003e(C)\u003c/strong\u003e Periodic acid-schiff (PAS) staining, Bar = 100 μM. \u003cstrong\u003e(D) \u003c/strong\u003eQuantitative scoring of H\u0026amp;E, Masson’s trichrome, and PAS staining.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/7d25afd47daaaf5ec776a49f.png"},{"id":49834815,"identity":"0066cbfa-7233-44f1-9346-be98f2a6f3fc","added_by":"auto","created_at":"2024-01-18 18:13:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1652368,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry analysis of lung sections using CD11b/CD18 antibody: \u003cstrong\u003e(A) \u003c/strong\u003econtrol mice, \u003cstrong\u003e(B) \u003c/strong\u003eOVA-treated, \u003cstrong\u003e(C) \u003c/strong\u003edexamethasone-treated (Dexa, 5 mg/kg, intraperitoneal injection), \u003cstrong\u003e(D)\u003c/strong\u003e JN-KI3-treated (80 mg/kg, oral administration). The presence of brown cells indicates CD11b/CD18-positive leukocyte infiltration. Bar = 100 μM.\u003c/p\u003e","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3856128/v1/1512081667e16863f373290b.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating the anti-inflammatory potential of JN-KI3: the therapeutic role of PI3Kγ- selective inhibitors in asthma treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhosphoinositide 3-kinase (PI3K) is a lipid kinase involved in signal transduction. It controls various biological processes, including cellular growth, proliferation, and differentiation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. PI3Ks can be categorized into class I, II, and III according to their substrates [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Class I PI3Ks, which have attracted significant attention, are further divided into class IA (α, β, and δ) and IB (γ) based on their regulatory proteins and tissue specificity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Class IA PI3Ks are triggered by a cascade of receptor tyrosine kinases (RTK), while class IB PI3K is activated by G protein-coupled receptors (GPCR) such as C5a, initiating downstream signaling pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In contrast to the wide distribution of PI3Kα and β, PI3Kγ and PI3Kδ are predominantly expressed in the immune system, specifically the leukocytes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Their selective expression of γ and δ closely ties them to human immune function, with the two subtypes fulfilling non-redundant roles [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Accumulated research has found that mutations in the genes encoding both PI3Kγ and δ result in primary immunodeficiency diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As the sole representative of class IB, PI3Kγ plays a key role in regulating leukocyte migration, including neutrophils and eosinophils, and is involved in mediating the recruitment of certain immune cells to inflammation sites [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As a result, PI3Kγ has been implicated in various inflammatory conditions (such as asthma, allergies, rheumatoid arthritis, pulmonary fibrosis, etc.), immunodeficiency diseases, cardiovascular disease, and cancer-related inflammation. Consequently, there is a growing interest in the identification of PI3Kγ-selective inhibitors for the treatment of inflammatory diseases [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAsthma, one of the most common chronic airway inflammatory diseases in the world, has caused significant harm to the health of patients and it can even threaten the lives of patients if certain treatment measures are not taken [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Allergic asthma, being the predominant form of asthma, is distinguished by airway hyperresponsiveness and remodeling. Research indicates a critical involvement of T-helper type 2 (Th2) cells in the pathogenesis of asthma [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Upon encountering allergens or antigens presented by antigen-presenting cells (e.g., dendritic cells), Th2 cells initiate allergic reactions and produce cytokines, including interleukin-4 (IL-4), IL-5, and IL-13 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This process leads to the infiltration of eosinophils into the airways, contributing to the development of allergic asthma. PI3Kγ, as a key factor influencing leukocyte migration, may also be involved in modulating the behavior of eosinophils, which are a pivotal element in the pathogenesis of allergic asthma [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recently, some PI3Kγ inhibitors have been reported to be useful for the treatment of asthma [\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, PI3Kγ is anticipated to serve as a potential therapeutic target for asthma. However, there are limited preclinical studies using PI3Kγ inhibitors for asthma, highlighting the urgent need to develop newer and more efficient PI3Kγ inhibitors for the treatment of asthma [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Recently, we identified a novel PI3Kγ inhibitors, JN-KI3, utilizing a machine learning-based multi-conformational structure virtual screening approach. The kinase test demonstrated that JN-KI3 remarkably inhibited PI3Kγ outside the cellular matrix. Additionally, JN-KI3 specifically inhibited the PI3K/Akt signaling pathway in a time- and concentration-dependent manner [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, the article comprehensively investigates the anti-inflammatory and anti-asthma effects of this lead compound in \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models. The study flow is outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eThe positive compounds, IPI-549 and dexamethasone (Dexa), were purchased from Top-science (TargetMol, USA) and Chenxin (Shandong, China). JN-KI3 was obtained from ChemDiv Inc. (San Diego, USA). Thiazolyl Blue Tetrazolium Bromide (MTT) was purchased from Solarbio (Beijing, China). Complement component 5a (C5a) was obtained from R\u0026amp;D Systems (California, USA). Lipopolysaccharide (LPS) and ovalbumin (OVA) were purchased from Sigma (St Louis, USA). A PrimeScrip\u0026trade; RT reagent kit and QuantiNova\u0026trade; SYBR\u0026reg; Green PCR Master Mix were obtained from TaKaRa Bio (Tokyo, Japan) and QIAGEN (Germany). Tumor necrosis factor-α (TNF-α), IL-6, and IL-1β ELISA kits were purchased from Cusabio biotech (Wuhan, China). The primary antibodies against phosphorylated Akt serine 473 (pAkt473) and total Akt were purchased from Cell Signaling Technology (Beverly, USA). The HRP-conjugated secondary antibody was purchased from Proteintech Group, Inc. (Wuhan, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were sourced from the Cell Bank of the Shanghai Institute of Cell Biology and Biochemistry, Chinese Academy of Sciences (Shanghai, China). RAW264.7 cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Sigma, USA), supplemented with 10% fetal bovine serum (Gibco, USA), and 100 \u0026micro;g/mL penicillin-streptomycin in 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability assay\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were plated at a density of 8 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells per well in a 96-well plate and incubated overnight. The cytotoxicity experiments were divided into different groups: the control group was treated with an equal volume of PBS, while the test group was exposed to JN-KI3 at various concentrations (0.16, 0.32, 0.64, 1.25, 2.5, 5, and 10 \u0026micro;M) for 72 h at 37\u0026deg;C. Then, 10 \u0026micro;L of 5 mg/mL MTT was added to each well, followed by a 4 h incubation. Subsequently, 100 \u0026micro;L MTT buffer was added to each well. The absorbance was measured at 550 nm with spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot assay\u003c/h2\u003e \u003cp\u003eRAW264.7 cells were seeded in 6-well plates at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well and treated with different concentrations of JN-KI3 (0, 1, 2.5, 5, and 10 \u0026micro;M), in addition to the positive compound IPI-549 (1 \u0026micro;M). After incubation for 1 h, 12.5 \u0026micro;g/ml of C5a was added for 10 min. The cells were washed three times with PBS and then added 100 \u0026micro;L RIPA lysis buffer (Thermo Scientific, USA), which contained protease and phosphatase inhibitors. After full lysis on ice, the supernatant was collected by 12,000 rpm centrifugation for 20 min, and the protein concentration was determined by BCA protein assay kit (Sangon Biotech, China). Loading buffer was added to all protein samples prepared for western blot assays and boiled at 100\u0026deg;C for 5 min. The protein samples were resolved using 10% SDS-PAGE and subsequently transferred onto nitrocellulose filter (NC) membranes. The membranes were blocked with 5% skim milk diluted in 1 x TBST with 0.3% Tween 20 for 2 h. After that, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies (phosphorylated Akt serine 473, 1:2000; total Akt, 1:1000). After washing the membrane three times with TBST, all the membranes were incubated with the corresponding secondary antibody at room temperature for 2 h. The blots were visualized using ECL solution after the membranes had been washed three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eRAW264.7 cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were treated with JN-KI3 at concentrations of 0, 1, 2.5, 5, and10 \u0026micro;M for 1h, followed by incubation with 500 ng/mL LPS for 16 h. The total RNA was then extracted using Trizol Reagent (Thermo Scientific, USA), followed by reverse transcription into cDNA and amplification by qRT-PCR. The thermocycling program consisted of holding at 95\u0026deg;C for 2 min, followed by 40 cycles of 5 s at 95\u0026deg;C and 10 s at 60\u0026deg;C. The primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer sequences used for RT-PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer sequence (5\u0026prime; to 3\u0026prime;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTNF-α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026prime;-GCCCACGTCGTAGCAAACCA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR: 5\u0026prime;-GCAGGGGCTCTTGACGGCAG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIL-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026prime;-TAGTCCTTCCTACCCCAATTTCC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR: 5\u0026prime;-TTGGTCCTTAGCCACTCCTTC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIL-1β\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026prime;-TGTGCAAGTGTCTGAAGCAGC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR: 5\u0026prime;-TGGAAGCAGCCCTTCATCTT-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF: 5\u0026prime;-AGCCTCGTCCCGTAGACAA-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR: 5\u0026prime;-AATCTCCACTTTGCCACTGC-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eELISA assay\u003c/h2\u003e \u003cp\u003eRAW264.7 cells (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) were treated with various concentrations of JN-KI3 (0, 1, 2.5, 5, and 10 \u0026micro;M) for 1 h. Following a 1 h incubation, cells were stimulated with LPS at a concentration of 500ng/mL for 16 h. The supernatant was taken out to measure the levels of TNF-α, IL-6, and IL-1β by using an ELISA kit according to the manufacturer\u0026rsquo;s instructions. A450 nm were determined on a Tecan microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimal\u003c/h2\u003e \u003cp\u003eFemale BALB/c mice (6\u0026ndash;8 weeks, 19\u0026ndash;21 g) were acquired from Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China) and housed under normal conditions as follows: 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity, and 12 h day/night cycle. All mice were given plenty of water and food. The protocol of animal experiments was approved by the Animal Ethics Committee of Jiangnan University (JN.No20220315b0360530[087]).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eInduction of an asthma mice model\u003c/h2\u003e \u003cp\u003eThe mice were randomly divided into 6 groups (6 mice/group): (1) control; (2) OVA-treated; (3) Dexa-treated (5 mg/kg); (4) JN-KI3-treated (20 mg/kg); (5) JN-KI3 (40 mg/kg), and (6) JN-KI3 (80 mg/kg) groups. To induce asthma, each mice was intraperitoneally injected with 100 \u0026micro;l OVA mixture (20 \u0026micro;g OVA and 2 mg aluminum hydroxide adjuvant in 0.1 mL PBS) in both the model and administration groups. The mice in the control group received the same dose of aluminum hydroxide adjuvant on days 1 and 14 for sensitization. Subsequently, the OVA-challenged mice were exposed to ultrasonic atomization of 5% OVA for 20 min from day 22 to day 28 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. The mice were sacrificed 24 h after the final challenge.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBronchoalveolar lavage fluid (BALF) collection and leukocyte counts\u003c/h2\u003e \u003cp\u003eThe bronchial tubes of the mice were ligated on the right side, and the left lung was washed with 0.5 mL of PBS and repeated three times to collect BALF. The collected BALF was separated at 800 g for 10 min at 4\u0026deg;C. The supernatant was stored at -80\u0026deg;C for subsequent ELISA analysis, and the cell precipitation was resuspended with 100 \u0026micro;l PBS for cell counting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eLung tissue samples were collected for western blot analysis to measure Akt phosphorylation levels. After protein extraction using RIPA, equal amounts were loaded into the wells of an SDS-PAGE gel and subsequently transferred to NC membranes. Immunoblotting analyses were then performed to assess the levels of pAkt473 and total Akt protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of IL-5, IL-13 and OVA-IgE levels\u003c/h2\u003e \u003cp\u003eThe concentration of IL-5 and IL-13 in the BALF, as well as OVA-specific IgE in the serum, were measured by ELISA according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis of lung tissue\u003c/h2\u003e \u003cp\u003eThe right lung was excised and fixed with 4% paraformaldehyde. After dehydration, the lung tissue was embedded in paraffin, sliced into 3 \u0026micro;m sections, and subjected to staining with hematoxylin-eosin (HE), Masson\u0026rsquo;s Trichrome (Masson), and periodic acid Schiff (PAS). HE staining was used to evaluate lung inflammation, and then a blind method was utilized to assess the level of inflammation using the following criteria: 0, normal; 1, occasional presence of inflammatory cells; 2, presence of inflammatory cells surrounding the bronchi or blood vessels; 3, 2\u0026ndash;4 layers of inflammatory cells surrounding the bronchi or blood vessels; 4, presence of more than 4 layers of inflammatory cells around most bronchi or blood vessels [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Masson trichrome was employed to assess collagen deposition and airway remodeling. The area occupied by collagen was quantified using ImageJ software to calculate the percentage of collagen fibers [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, PAS staining was used to observe airway mucus production. Goblet cell proliferation was quantified using a five-point scoring system as follows: 0, \u0026lt; 5% goblet cells; 1, 5\u0026ndash;25%; 2, 25\u0026ndash;50%; 3, 50\u0026ndash;75%; 4, \u0026gt; 75%. A minimum of 8 bronchi and parenchyma regions were counted per lung tissue sample [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) and analyzed using Graphpad prism 9.0 software (GraphPad Software Inc., San Diego, CA, United States). One-way ANOVA analysis was conducted to assess the differences between groups. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 versus the control group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eImpact of JN-KI3 on PI3Kγ-dependent cellular functions\u003c/h2\u003e \u003cp\u003eThe structure of JN-KI3 is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The enzyme inhibition assay revealed that JN-KI3 exhibits greater selectivity towards PI3Kγ in comparison to the other three isoforms. In order to eliminate chemical toxic interference, the MTT assay was performed to determine the cytotoxic effect of JN-KI3 on RAW264.7 cells following 24, 48, and 72 h of treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C, at a high concentration of 10 \u0026micro;M, JN-KI3 did not induce toxicity in RAW264.7 cells.\u003c/p\u003e \u003cp\u003eTo further validate the selectivity of JN-KI3 among class I PI3Ks within a cellular context, we examined the PI3Kγ-dependent cellular potencies in C5a-stimulated RAW264.7 cells. C5a can stimulate GPCR signals via PI3Kγ. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, compared to the unstimulated condition, C5a induced significant phosphorylation of Akt at Ser473 (pAkt473). IPI-549, a well-established selective PI3Kγ inhibitor, served as the positive control for inhibiting Akt phosphorylation. Notably, IPI-549 considerably suppressed Akt phosphorylation, and JN-KI3 also showed concentration-dependent inhibition of Akt phosphorylation while not affecting total Akt expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffect of JN-KI3 on PI3K/Akt signaling pathways in LPS-induced RAW264.7 cells\u003c/h2\u003e \u003cp\u003eThe LPS-induced RAW264.7 macrophage inflammatory model is a widely utilized model for inflammation. Studies have shown that LPS binds to Toll-like receptors (TLR) secreted by macrophages, leading to the activation of the PI3K/Akt signaling pathway, subsequently resulting in the release of a variety of inflammatory factors that mediate signaling [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the level of pAkt473 was significantly increased by LPS compared to the control, suggesting activation of the PI3K signaling pathways by LPS, whereas Akt phosphorylation was significantly suppressed by JN-KI3 in a concentration- and time- dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of JN-KI3 on expression of TNF-α, IL-6 and IL-1β in LPS-induced RAW264.7 macrophages.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs an important link in the immune system, macrophages play a crucial role in the host's response to pathogen infection and inflammation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. LPS induces classical activation of macrophages (M1), leading to the destruction of pathogens and the production of pro-inflammatory mediators such as TNF-α, IL-6, and IL-1β [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. As pro-inflammatory cytokines play a key role in the inflammatory process, we investigated the inhibition of classical pro-inflammatory cytokines by JN-KI3. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the mRNA expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β significantly increased in RAW264.7 cells following LPS treatment. After treatment of varied concentrations of JN-KI3, the mRNA expression of these three pro-inflammatory factors decreased by varying degrees compared to the control group, exhibiting concentration-dependence behavior.\u003c/p\u003e \u003cp\u003eThen, an ELISA assay was performed to measure the protein levels of these three pro-inflammatory factors in LPS-induced RAW264.7 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, LPS significantly enhanced the protein expression of these pro-inflammatory factors, whereas the levels of IL-1β and IL-6 in the JN-KI3-treated group exhibited concentration-dependent reduction compared to those in the control group. Surprisingly, while JN-KI3 exhibited concentration-dependent inhibition of TNF-α expression at low concentrations, it seemed to lose its inhibitory effect at high concentrations. By establishing an extracellular inflammatory cell model with RAW264.7 cells, we preliminarily confirmed that JN-KI3 can attenuate inflammatory responses by inhibiting the LPS-induced activation of PI3K/Akt pathways in RAW264.7 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of JN-KI3 on the infiltration of inflammatory cells in BALF through the PI3K signaling pathway\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the anti-inflammatory and anti-asthmatic effects, as well as the potential mechanism of action of JN-KI3, an \u003cem\u003ein vivo\u003c/em\u003e study was conducted using an OVA-induced asthma mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). After 24 h of the last-day OVA stimulation, BALF was collected from the mice to evaluate the impact of Dexa and JN-KI3 on the recruitment of inflammatory cells in the lungs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, compared with the control group, the inflammatory cells in the lungs of the OVA-stimulated group substantially increased, while the cell count exhibited a noticeable reduction in response to JN-KI3 in a dose-dependent manner. In addition, there was a significant increase in the counts of neutrophils, eosinophils, and macrophages in the OVA group of mice. Conversely, these inflammatory cells were significantly reduced in the JN-KI3-treated group, particularly under JN-KI3 administration at 80 mg/kg, displaying an effect similar to the positive control, Dexa. Therefore, JN-KI3 was able to exert an inhibitory effect on the infiltration of inflammatory cell in the lungs of asthmatic mice.\u003c/p\u003e \u003cp\u003eThen, the inhibition of Akt phosphorylation in lung tissue homogenates of mice after treatment with JN-KI3 was analyzed using western blot analysis. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC demonstrates a significant increase in Akt protein phosphorylation, while the total Akt level remained constant. The administration of JN-KI3 resulted in a concentration-dependent reduction in Akt phosphorylation, indicating that orally administered JN-KI3 accessed the lung tissues and interacted with the target protein, subsequently inhibiting PI3K/Akt signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of JN-KI3 on cytokine levels in BALF\u003c/h2\u003e \u003cp\u003eOVA-induced asthma is primarily characterized by the release of pro-inflammatory Th2 cytokines, including IL4, IL-5, and IL-13 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Consequently, the expression levels of these pro-inflammatory factors in BALF were initially assessed using the ELISA assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the expression of all three cytokines was significantly higher in the OVA-treated group compared to the lower expression level in the control group. The positive control group Dexa demonstrated a clear inhibitory effect on pro-inflammatory factors. After oral administration of JN-KI3, the levels of these pro-inflammatory factors decreased to varying degrees in a dose-dependent manner. At a high concentration of JN-KI3 (80 mg/kg), the anti-inflammatory efficacy was equivalent to that of Dexa, the positive control group. These results suggest that JN-KI3 has the potential to reduce OVA-induced Th2 cytokine infiltration in lung tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology examination of the lungs\u003c/h2\u003e \u003cp\u003eTo gain further insight into the pathological changes and the extent of lung inflammation, we performed lung section analysis on all 4 groups of mice. Upon H\u0026amp;E staining, it was observed that the OVA-stimulated group showed a substantial infiltration of inflammatory cells in the bronchial and perivascular connective tissues of the lungs compared to the control group, (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Whereas the accumulation of inflammatory cells was significantly reduced following treatment with Dexa and JN-KI3 treatment. Analysis of bronchial collagen distribution was conducted using Masson\u0026rsquo;s trichrome staining. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, lung tissues exhibited excessive collagen deposition around the bronchi following OVA stimulation, with varying degrees of reduction in collagen accumulation around the trachea observed after treatment with Dexa or JN-KI3. To determine the potential of JN-KI3 in reducing mucus production in the lungs of OVA-treated mice, lung tissues were subjected to PAS staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). After OVA stimulation, there was a substantial increase in goblet cell metaplasia and mucus secretion in the lungs of mice. In contrast, mucus production was significantly reduced after treatment with Dexa and JN-KI3. The analysis of the mice lung sections revealed that JN-KI3 exhibited an inhibitory effect on airway remodeling and demonstrated therapeutic potential for treating asthma in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffects of JN-KI3 on OVA-induced white blood cell infiltration into lung tissue\u003c/h2\u003e \u003cp\u003eAsthma is an inflammatory pulmonary disease characterized by heightened leukocyte infiltration into the airways and diminished respiratory function [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To evaluate the effect of JN-KI3 on OVA-induced peribronchial leukocyte recruitment, mice lung tissue sections were subjected to immunohistochemical analysis using CD11b/CD18 antibody [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, OVA triggered a large accumulation of leukocytes (depicted as brown cells) around the bronchi and in the alveoli. Both the reference drug Dexa and JN-KI3 resulted in a noteworthy decrease in leukocyte recruitment, indicating that JN-KI3 effectively attenuated the infiltration of leukocytes into the lungs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA 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 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, PI3K-selective inhibitors are progressively demonstrating their advantages [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Recent studies have identified PI3Kγ as a promising target for treating inflammatory and autoimmune diseases owing to its predominant expression in leukocytes [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It has been discovered that PI3Kγ\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e neutrophils and macrophages demonstrate a certain level of chemotactic impairment when stimulated by GPCR agonists such as C5a [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInflammation represents a complex series of defense responses initiated by the body to shield against internal and external factors [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Hence, the focus of this study was to assess the anti-inflammatory properties of JN-KI3 and confirm its preliminary therapeutic effects on asthma.\u003c/p\u003e \u003cp\u003eTo assess the potential \u003cem\u003ein vitro\u003c/em\u003e 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 \u0026micro;M), 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 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Studies have found that macrophages, when stimulated in the inflammatory response, secrete TNF-α, IL-1β, and IL-6 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. 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 \u003cem\u003ein vitro\u003c/em\u003e results suggest that JN-KI3 may yield anti-inflammatory effects by specifically inhibiting the PI3Kγ signaling pathway.\u003c/p\u003e \u003cp\u003eFunctional studies have substantiated the significant role of PI3Kγ in regulating inflammation, particularly in lung diseases [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. 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\u0026amp;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.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study demonstrated that JN-KI3 selectively inhibits PI3Kγ, resulting in targeted disruption of the PI3K/Akt signaling pathway and significant anti-inflammatory effects. Furthermore, the \u003cem\u003ein vivo\u003c/em\u003e experiments demonstrated the therapeutic potential of JN-KI3 in mitigating airway inflammation in a murine model of asthma. Thus, JN-KI3 could serve as a promising lead compound for the treatment of asthma. However, JN-KI3 needs further optimization and development to become a candidate drug for use against pulmonary inflammatory diseases. Our study also suggests that the inhibitory effects of PI3Kγ on inflammation may present an additional therapeutic strategy for asthma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (KYCX23_2469), the National Natural Science Foundation of China (No. 21807049, 82001711), the Fundamental Research Funds for the Central Universities (JUSRP51703A), the University-Industry Cooperation Research Project in Jiangsu (No. BY2020432), the Natural Science Foundation of Jiangsu Province (BK20201137), and the Foundation of Wuxi Municipal Health Commission (HB2023023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLJ: Methodology, Data curation, Writing - original draft. MM: Methodology. WX: Methodology, Validation. JZ: Investigation, Conceptualization, Project administration. YCai: Methodology. YChen: Writing - review \u0026amp; editing. JJ: Resources, Supervision. MG: Resources, Writing - review \u0026amp; editing. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhu J, Li K, Yu L, Chen Y, Cai Y, Jin J, et al. Targeting phosphatidylinositol 3-kinase gamma (PI3Kgamma): Discovery and development of its selective inhibitors. 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Nat Rev Immunol 2015; 15:57\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PI3Kγ-selective inhibitor, JN-KI3, anti-inflammation, airway inflammatory disease, asthma","lastPublishedDoi":"10.21203/rs.3.rs-3856128/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3856128/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eIntroduction\u003c/b\u003e Asthma is a chronic airway inflammatory disease of the airways characterized by the involvement of numerous inflammatory cells and factors. Therefore, targeting airway inflammation is one of the crucial strategies for developing novel drugs in the treatment of asthma. Phosphoinositide 3-kinase gamma (PI3Kγ) has been demonstrated to have a significant impact on inflammation and immune responses, thus emerging as a promising therapeutic target for airway inflammatory disease, including asthma.\u003c/p\u003e \u003cp\u003e \u003cb\u003eObjective and method\u003c/b\u003e There are few studies reporting on the therapeutic effects of PI3Kγ-selective inhibitors in asthma disease. In this study, we investigated the anti-inflammatory and therapeutic effects of PI3Kγ-selective inhibitor JN-KI3 for treating asthma by utilizing both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e approaches, thereby proving that PI3Kγ-selective inhibitors could be valuable in the treatment of asthma.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e In RAW264.7 macrophages, JN-KI3 effectively suppressed C5a-induced Akt phosphorylation in a concentration-dependent manner, with no discernible toxicity observed in RAW264.7 cells. Furthermore, JN-KI3 can inhibit the PI3K/Akt signaling pathway in lipopolysaccharide-induced RAW264.7 cells, leading to the suppression of transcription and expression of the classical inflammatory cytokines in a concentration-dependent manner. Finally, an ovalbumin-induced murine asthma model was constructed to evaluate the initial therapeutic effect of JN-KI3 for treating asthma. Oral administration of JN-KI3 inhibited the infiltration of inflammatory cells and the expression of T-helper type 2 cytokines in bronchoalveolar lavage fluid, which was associated with the suppression of the PI3K signaling pathway. Lung tissue and immunohistochemical studies demonstrated that JN-KI3 inhibited the accumulation of inflammatory cells around the bronchus and blood vessels, as well as the secretion of mucus and excessive deposition of collagen around the airway. In addition, it reduced the infiltration of white blood cells into the lungs.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e JN-KI3 shows promise as a candidate for the treatment of asthma. Our study also suggests that the inhibitory effects of PI3Kγ on inflammation could offer an additional therapeutic strategy for pulmonary inflammatory diseases.\u003c/p\u003e","manuscriptTitle":"Evaluating the anti-inflammatory potential of JN-KI3: the therapeutic role of PI3Kγ- selective inhibitors in asthma treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 18:13:21","doi":"10.21203/rs.3.rs-3856128/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"766f98bc-2cf3-4e75-a4bf-5a8e20036ae3","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-24T19:59:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-18 18:13:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3856128","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3856128","identity":"rs-3856128","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}