Bisphenol A (BPA) causes multiorgan toxicity via oxidative stress, inflammation, and apoptosis, and the effect of nutritionally relevant elevation of casein intake is unknown. To evaluate whether a nutritionally relevant elevation of dietary casein (18% vs. 14%) attenuates BPA-induced multiorgan toxicity in rats, male Wistar rats were divided into four groups (n = 8/group) and treated for 35 days: control (14% casein), casein 18% alone, BPA (50 mg/kg/day +14% casein), and BPA + 18% casein. We measured oxidative and inflammatory markers, renal function, metabolic parameters, hematological parameters, histopathology and immunohistochemistry in kidney, spleen, and pancreas. BPA significantly increased malondialdehyde, interleukin-6, tumor necrosis factor-alpha, creatinine, kidney injury molecule-1, insulin resistance, white blood cells, neutrophils, and monocytes, and decreased reduced glutathione, glutathione S-transferase, superoxide dismutase, catalase, interleukin-4, serum soluble CD4 and CD8 protein levels, hemoglobin, red blood cells, platelets, and lymphocytes. BPA also caused renal tubular necrosis, splenic degeneration, pancreatic fibrosis, and upregulated caspase-3 and inducible nitric oxide synthase. Co-administration of 18% casein partially attenuated these effects, reducing malondialdehyde by 35-40%, creatinine by 40%, and insulin resistance by 53%, and restoring antioxidant enzymes and glucose tolerance. However, several parameters remained significantly different from controls. Nutritionally relevant elevation of casein (18%) provides significant partial protection against BPA-induced toxicity, supporting dietary protein modulation as a supportive strategy.

This study investigated the effects of Qingzao Jiufei Decoction on M2 macrophage polarization in bleomycin(BLM)-induced pulmonary fibrosis(PF) mice via the janus kinase 1(JAK1)/signal transducer and activator of transcription 6(STAT6) signaling pathway. Sixty C57BL/BJ mice were randomly divided into normal group, model group, pirfenidone group(200 mg·kg~(-1)), and low-, medium-, and high-dose Qingzao Jiufei Decoction groups(4.9, 9.8, and 19.6 g·kg~(-1)). The PF model was established in the model and treatment groups by intratracheal instillation of BLM(5 mg·kg~(-1)). Mice in each group were administered the same volume of normal saline or the corresponding dose of Qingzao Jiufei Decoction by gavage for 21 consecutive days. After 21 days, blood gas analysis was performed. ELISA was used to detect the levels of interleukin-4(IL-4), interleukin-13(IL-13), arginase-1(Arg-1), transforming growth factor-β(TGF-β), and interleukin-10(IL-10) in bronchoalveolar lavage fluid(BALF). HE and Masson staining were performed to observe pathological changes in lung tissue and for histopathological scoring. Immunofluorescence staining was used to detect the expression of α-smooth muscle actin(α-SMA), type Ⅰ collagen(COL-1), the macrophage marker CD11b, and the M2 macrophage marker CD206. Western blot and RT-qPCR were used to detect the expression of JAK1/STAT6 signaling pathway-related proteins and mRNA, as well as M2 macrophage-related proteins and mRNA, in lung tissue. The results showed that, compared with the normal group, the levels of IL-4, IL-13, Arg-1, TGF-β, and IL-10 in BALF were significantly increased in the model group(P<0.01). Histopathological staining revealed extensive inflammatory cell infiltration and increased collagen fiber deposition in the alveolar septa, accompanied by significantly increased Szapiel alveolitis scores and Ashcroft fibrosis scores(P<0.01). Immunofluorescence staining showed significantly increased fluorescence intensity of α-SMA, COL-1, CD11b, and CD206. The mRNA expression levels of JAK1, STAT6, CD206, and Arg-1, as well as the protein expression levels of phosphorylated(p)-JAK1, p-STAT6, CD206, and Arg-1 in lung tissue, were significantly increased(P<0.01). Compared with the model group, treatment with Qingzao Jiufei Decoction significantly reduced the levels of IL-4, IL-13, Arg-1, TGF-β, and IL-10 in BALF(P<0.05, P<0.01), markedly improved pathological changes in lung tissue, and downregulated the expression of α-SMA, COL-1, CD11b, and CD206 detected by immunofluorescence staining. In addition, Qingzao Jiufei Decoction significantly downregulated the mRNA expression of JAK1, STAT6, CD206, and Arg-1, as well as the protein expression of p-JAK1, p-STAT6, CD206, and Arg-1 in lung tissue(P<0.05, P<0.01). In conclusion, Qingzao Jiufei Decoction exerts a protective effect against BLM-induced PF in mice, and its mechanism may be related to inhibition of M2 macrophage polarization through regulation of the JAK1/STAT6 signaling pathway.

The aim of this paper was to explore the effect and mechanism of Regan Saibisitan Granules(RGS) on improving airway inflammatory responses in asthmatic mice by regulating the mitogen-activated protein kinase(MAPK)/nuclear factor-κB(NF-κB) signaling pathway. Female Balb/c mice were randomly divided into a blank group, a model group, low-, medium-and high-dose RGS groups(0.75, 1.5, and 3 g·kg~(-1)), dexamethasone group(3 mg·kg~(-1)), and a Feining Tablets group(0.72 g·kg~(-1)) according to body weight. The asthma model was established by ovalbumin(OVA) sensitization combined with aerosol challenge. The numbers of white blood cells(WBC), eosinophils(EOS), neutrophils(NEU), and lymphocytes(LYM) in bronchoalveolar lavage fluid(BALF) were counted. The levels of interleukin-4(IL-4), interleukin-5(IL-5), interleukin-13(IL-13), interferon-γ(INF-γ) in BALF, as well as the secretion of immunoglobulin E(IgE), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) in serum, were detected using enzyme-linked immunosorbent assay(ELISA) kits. The pathological changes of lung tissue were observed by hematoxylin-eosin and periodic acid-Schiff staining to comprehensively evaluate the ameliorative effect of RGS on inflammation in asthmatic mice. The protein expression levels of phosphorylated extracellular signal-regulated kinase 1/2(p-ERK1/2), phosphorylated c-Jun N-terminal kinase(p-JNK), phosphorylated mitogen-activated protein kinase(p-p38), phosphorylated NF-κB(p-NF-κB), phosphorylated inhibitor of NF-κB α(p-IκBα), cleaved caspase-3 and B-cell lymphoma/leukemia-2-associated X protein(Bax) in lung tissue were detected by Western blot. The results showed that compared with the model group, the numbers of WBC, EOS, NEU and LYM in BALF of the RGS groups, the Feining Tablets group and the dexamethasone group decreased, and the content of IL-4, IL-5, IL-13 in BALF as well as the levels of IgE, TNF-α and IL-1β in serum significantly decreased. In addition, inflammatory cell infiltration, airway wall thickening, and mucus secretion were significantly alleviated, and the levels of p-ERK1/2, p-JNK, p-p38, p-NF-κB, p-IκBα, as well as the protein levels of cleaved caspase-3 and Bax in lung tissue of the RGS groups decreased. In conclusion, RGS may inhibit inflammatory response and cell apoptosis by regulating the MAPK/NF-κB signaling pathway, thereby alleviating airway inflammation in asthmatic mice.

Host bone microenvironment is a critical factor for successful bone regeneration during implantation. In this work, we identified a spatiotemporal mismatch at drilling-induced injury sites where initial apoptosis outpaces macrophage recruitment, leading to apoptotic cell accumulation and oxidative stress that impairs osteogenic differentiation. To mitigate this phenomenon, we developed a synergistic strategy co-delivering interleukin 4 (IL-4) and arginine (Arg) to reprogram the microenvironment. Mechanistically, IL-4 recruits and polarizes macrophages toward a pro-reparative M2 phenotype, while Arg fuels the arginase-1 metabolic pathway to sustain continual efferocytosis and secretion of pro-regenerative cytokines. In a mouse bone injury model, transcriptomic and histological analyses confirmed that this strategy accelerates apoptotic clearance and establishes an M2-dominant environment, significantly enhancing early bone regeneration. Altogether, this work proposes a novel therapeutic paradigm targeting macrophage efferocytosis to resolve early microenvironmental imbalances, providing a promising therapeutic approach to promote bone defect healing and offering novel insights into optimizing host microenvironments for osseointegration following implantation.

The pleiotropic anti-inflammatory cytokine interleukin 4 (IL-4) not only modulates immune responses but also plays important roles in the nervous system by mediating itch, antinociception and neuroregeneration. Here, we assessed the expression and function of IL-4, its receptors, and downstream signaling pathways in human induced pluripotent stem cell derived nociceptors (iNocs), organotypic mouse spinal cord cultures, and in in vivo models. In silico analyses of human and mouse dorsal root ganglion (DRG) single cell transcriptomes revealed that a specific subpopulation of murine and human sensory neurons expressed IL4R and IL13RA1 (but very little IL2RG) receptor subunits highlighting conserved neuron responsiveness to IL-4. In iNocs, exogenous IL-4 induced de novo formation of presynaptic vGLUT1-positive puncta. Pharmacological inhibition of Janus kinases 1/2, STAT6, protein kinase C isoforms, or the translation initiation factor eIF4E significantly attenuated the IL-4-associated increase in vGLUT1-positive puncta. Transcriptomic profiling of iNocs revealed IL-4-regulated biological processes associated with neuronal development and synaptic organization. Consistently, IL-4 increased mean-firing-rate in iNocs and increased vGLUT1 expression in mouse organotypic spinal cord cultures treated with IL-4, in vitro. Intrathecal or intranasal IL-4 treatment enhanced both, inhibitory and excitatory synaptic activity in acute spinal cord slices, and this was associated with a decrease in mechanical and thermal sensitivity but normal sensorimotor coordination in vivo. These findings position IL-4 as key regulator of presynaptic glutamatergic organization for primary afferent neuron connectivity in the spinal dorsal horn and support a critical role for IL-4 in the excitation - inhibition balance of pain processing spinal neuronal networks.

Despite advances in understanding colorectal cancer (CRC) biology, how tumor-cell signaling coordinates epithelial-mesenchymal transition (EMT) with macrophage polarization remains incompletely defined. We investigated whether NOTCH2 expression is associated with EMT and with a GATA3/IL-4-dependent macrophage-polarizing phenotype in CRC. NOTCH2 expression and prognostic associations were analyzed using TCGA data and validated in 20 paired CRC and adjacent normal tissue (ANT) specimens. Gain- and loss-of-function experiments in HCT116 cells assessed proliferation, migration, invasion, EMT markers, GATA3, and IL-4. A Transwell co-culture system containing transfected HCT116 cells and peripheral blood mononuclear cells (PBMCs) was used to evaluate CD206-positive M2-like macrophage polarization, and IL-4 neutralization was used to test the contribution of IL-4. NOTCH2 was increased in CRC tissues, and higher NOTCH2 expression was associated with poorer overall survival. NOTCH2 overexpression increased CRC-cell proliferation, migration, invasion, Vimentin, GATA3, and IL-4, while reducing E-cadherin; NOTCH2 silencing produced the opposite changes. Clinical tissues showed increased NOTCH2, GATA3, IL-4, CD163, and Vimentin, together with reduced E-cadherin. In co-culture, NOTCH2 overexpression increased the proportion of CD206-positive M2-like cells, whereas NOTCH2 knockdown reduced it. IL-4 neutralization significantly attenuated the increase in CD206-positive cells induced by NOTCH2 overexpression. These findings support associations among NOTCH2-associated malignant phenotypes, EMT, altered GATA3/IL-4 expression, and IL-4-dependent M2-like macrophage polarization in CRC, while direct transcriptional regulation of the GATA3/IL-4 axis remains to be established.

Chronic wound healing remains a persistent challenge clinically, particularly in diabetic wounds, where hyperglycemia drives sustained activation of the AGE-RAGE-associated signaling within the wound bed, precipitating excessive ROS generation and persistent inflammation that together stall tissue repair and erode quality of life. To address this, we developed a core-shell microneedle (GS + IL-4@MN) patch loading Gymnema sylvestre (GS) extract and interleukin-4 (IL-4) as a promising therapeutic strategy. The patch comprised a 10 × 10 array of conical needles and the optimized shell-loading concentration of GS was determined to be 1.0 mg/mL. The GS-loaded outer shell can suppress the release of pro-inflammatory mediators, scavenge ROS, and strengthen antioxidant defenses, thereby attenuating local inflammation; the IL-4-encapsulated core induces macrophage polarization toward the M2 phenotype, mitigates the pro-inflammatory microenvironment, and modulates T-cell immunity, while enhancing cellular proliferation and migration to support tissue regeneration. In diabetic wound models, GS + IL-4@MN achieved wound-closure rates of 64.72 ± 6.3% at day 3 and 85.80 ± 3.6% at day 7, and re-epithelialization reached 91.52 ± 2.1% by day 14. Moreover, collagen deposition increased to 79.38 ± 4.3%, CD31-positive neovascularization reached 70.50 ± 3.7%, and the M1/M2 ratio decreased to 0.3, indicating marked inflammatory resolution and regenerative activation. In summary, this core-shell MN system achieves multidimensional regulation of the pathological microenvironment of diabetic wounds through a dual mechanism: outer-layer inflammatory suppression and inner-layer microenvironment reset, and offers a new avenue for promoting diabetic wound healing.

Entamoeba histolytica infection exhibits a range of clinical consequences, spanning from asymptomatic carriage to severe symptomatic disease. Despite comprehensive research on symptomatic E. histolytica infection, limited attention has been given to asymptomatic cases, notably in interpreting the immune activation and regulatory pathways that allow parasite sustainability without observable disease. This study aimed to evaluate the participation of pro-inflammatory cytokines [tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ)], anti-inflammatory cytokines (interleukin-10 [IL-10], interleukin-4 [IL-4]) and mucosal antibodies (secretory IgA [SIgA]) in distinguishing sexes, symptomatic and asymptomatic infections and elucidating their role in amoebiasis. Only serum SIgA exhibited significant sex-based differences among all immune parameters, suggesting the impact of sex hormones on the systemic immune regulation rather than the localized immunity. Serum cytokine levels were assessed and statistically evaluated across symptomatic, asymptomatic and control groups. TNF-α and IFN-γ levels were significantly increased in the symptomatic group (p < 0.01), reflecting a robust inflammation associated with tissue destruction and clinical symptoms. Conversely, IL-10 was significantly greater in the asymptomatic group, indicating a protective role in dampening intense inflammation and sustaining parasite persistence without obvious clinical symptoms. IL-4 levels did not differ significantly among groups, suggesting limited involvement of this cytokine in the immunopathology of amoebiasis. Similarly, SIgA concentrations in saliva, urine and serum specimens were consistently heightened in the symptomatic group, indicating mucosal immune promotion. Receiver operating characteristic (ROC) curve analysis demonstrated that cytokine ratios (TNF-α/IL-10, IFN-γ/IL-10) with serum, urinary and salivary SIgA levels showed high discrimination between symptomatic and asymptomatic groups, thereby demonstrating strong prognostic accuracy. These findings emphasize the crucial role of cytokine balance and mucosal antibody reactions in determining the clinical trajectory of E. histolytica infection, highlighting the clinical utility of quantifying these biomarkers for the diagnosis and differentiation of amoebiasis.

Effective regeneration of critical-sized bone defects requires the coordinated reconstruction of immune homeostasis, vascular networks, and osteogenic matrix formation. However, conventional bone-regenerative materials frequently provide structural support or single-pathway osteoinduction, while failing to actively orchestrate the multicellular repair microenvironment. Here, we develop a hydrogel-assisted immunoregenerative strategy based on hypoxia-programmed apoptotic-mimetic small extracellular vesicles derived from M2-like macrophages. Interleukin-4-polarized macrophages were subjected to hypoxic preconditioning to generate M2-HP-Apo-sEVs with increased phosphatidylserine exposure, enhanced cellular internalization, and amplified immunomodulatory potency. Compared with normoxic M2-sEVs, M2-HP-Apo-sEVs more effectively reprogrammed inflammatory macrophages toward a pro-resolving phenotype, suppressed inflammatory cytokine production, and enhanced reparative cytokine secretion. The vesicles further promoted osteogenic differentiation of bone marrow mesenchymal stem cells and stimulated endothelial migration and capillary-like network formation, demonstrating coordinated osteo-immuno-angiogenic activity. To enable sustained local delivery, M2-HP-Apo-sEVs were incorporated into photocrosslinkable GelMA hydrogels and implanted into critical-sized calvarial defects. The GelMA-M2-HP-Apo-sEV system substantially enhanced bone regeneration, vascularization, and osteogenic matrix deposition while attenuating local inflammation. This work establishes a cell-free vesicle-hydrogel platform that integrates donor-cell hypoxic programming, apoptotic-mimetic membrane signaling, and local biomaterial delivery for immunoregenerative bone repair.