[Effect of LAG3 deficiency on natural killer cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis].

OBJECTIVE: To investigate the effect of LAG-3 deficiency (LAG3-/-) on natural killer (NK) cell function and hepatic fibrosis in mice infected with Echinococcus multilocularis. METHODS: C57BL/6 mice, each weighing (20 ± 2) g, were divided into the LAG3-/- and wild type (WT) groups, and each mouse in both groups was inoculated with 3 000 E. multilocularis protoscoleces via the hepatic portal vein. Mouse liver and spleen specimens were collected 12 weeks post-infection, sectioned and stained with sirius red, and the hepatic lesions and fibrosis were observed. Mouse hepatic and splenic lymphocytes were isolated, and flow cytometry was performed to detect the proportions of hepatic and splenic NK cells, the expression of CD44, CD25 and CD69 molecules on NK cell surface, and the secretion of interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A. RESULTS: Sirius red staining showed widening of inflammatory cell bands and hyperplasia of fibrotic connective tissues around mouse hepatic lesions, as well as increased deposition of collagen fibers in the LAG3-/-group relative to the WT group. Flow cytometry revealed lower proportions of mouse hepatic (6.29% ± 1.06% vs. 11.91% ± 1.85%, P < 0.000 1) and splenic NK cells (4.44% ± 1.22% vs. 5.85% ± 1.10%, P > 0.05) in the LAG3-/- group than in the WT group, and the mean fluorescence intensity of CD44 was higher on the surface of mouse hepatic NK cells in the LAG3-/- group than in the WT group (t = -3.234, P < 0.01), while no significant differences were found in the mean fluorescence intensity of CD25 or CD69 on the surface of mouse hepaticNK cells between the LAG3-/- and WT groups (both P values > 0.05). There were significant differences between the LAG3-/- and WT groups in terms of the percentages of IFN-γ (t = -0.723, P > 0.05), TNF-α (t = -0.659, P > 0.05), IL-4 (t = -0.263, P > 0.05), IL-10 (t = -0.455, P > 0.05) or IL-17A secreted by mouse hepatic NK cells (t = 0.091, P > 0.05), and the percentage of IFN-γ secreted by mouse splenic NK cells was higher in the LAG3-/- group than in the WT group (58.40% ± 1.64% vs. 50.40% ± 4.13%; t = -4.042, P < 0.01); however, there were no significant differences between the two groups in terms of the proportions of TNF-α (t = -1.902, P > 0.05), IL-4 (t = -1.333, P > 0.05), IL-10 (t = -1.356, P > 0.05) or IL-17A secreted by mouse splenic NK cells (t = 0.529, P > 0.05). CONCLUSIONS: During the course of E. multilocularis infections, LAG3-/- promotes high-level secretion of IFN-γ by splenic NK cells, which may participate in the reversal the immune function of NK cells, resulting in aggravation of hepatic fibrosis.

Effect of IL-17 on pulmonary artery smooth muscle cells and connective tissue disease-associated pulmonary arterial hypertension.

OBJECTIVE: To explore the role of interleukin (IL)-17 in connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH) and to investigate its possible mechanism on pulmonary artery smooth muscle cells (PASMCs). METHODS: Enzyme-linked immunosorbent assay (ELISA) were used to compare levels of serum IL-17 in patients with CTD-PAH and healthy controls (HCs). After treatment for 3 months, the serum IL-17 levels were tested in CTD-PAH. ELISA and immunohistochemistry were used to compare levels of serum IL-17 and numbers of pulmonary artery IL-17+ cells, respectively, in a rat model of monocrotaline-induced PAH and untreated rats. Proliferation, migration, and inflammatory factors expression of PASMCs were assessed after stimulation with different concentrations of IL-17 for various time periods. Proteins in the mitogen-activated protein kinase (MAPK) pathway were examined by western blot. RESULTS: Levels of IL-17 were upregulated in patients with CTD-PAH compared to HCs. After 3 months of treatment, serum IL-17 levels were downregulated with pulmonary artery pressure amelioration. Moreover, serum IL-17 levels and numbers of IL-17+ cells infiltrating lung arterioles were increased in PAH model rats. IL-17 could dose- and time-dependently promote proliferation and migration of PASMCs as well as time-dependently induce IL-6 and intercellular cell adhesion molecule-1 (ICAM-1) expression. The levels of MKK6 increased after IL-17 treatment. Inhibition of MAPK decreased proliferation of PASMCs. CONCLUSION: Levels of IL-17 may increase in CTD-PAH, and IL-17 promotes proliferation, migration, and secretion of IL-6 and ICAM in PASMCs, respectively, which likely involves the p-38 MAPK pathway.

Network pharmacology analysis and experimental validation of Xiao-Qing-Long-Tang's therapeutic effects against neutrophilic asthma.

BACKGROUND: Xiao-Qing-Long-Tang (XQLT), a classical Chinese herbal medicine formula, has been extensively used for allergic asthma treatment. However, there is limited research on its anti-inflammatory effects and mechanisms specifically in neutrophilic asthma (NA). PURPOSE: This study aims to investigate the potential therapeutic effects of XQLT against NA using a combination of network pharmacology and experimental validation. STUDY DESIGN: By utilizing traditional Chinese medicine and disease databases, we constructed an XQLT-asthma network to identify potential targets of XQLT for NA. In the experimental phase, we utilized an ovalbumin (OVA)/lipopolysaccharide (LPS)-induced model for neutrophilic asthma and examined the therapeutic effects of XQLT. RESULTS: Our research identified 174 bioactive components within XQLT and obtained 140 target genes of XQLT against asthma. Functional enrichment analysis revealed that these target genes were primarily associated with inflammation and cytokines. In the experimental validation, mice induced with OVA-LPS showcased eosinophilic and neutrophilic cell infiltration in peri-bronchial areas, elevated levels of IL-4 and IL-17 in both serum and lung, increased percentages of Th2 and Th17 cells in the spleen, as well as elevated levels of CD11b+ and CD103+ dendritic cells (DCs) within the lung. Treatment with XQLT effectively reduced IL-4 and IL-17 levels, decreased the percentages of Th2, Th17, CD11b+, and CD103+ DCs, and improved inflammatory cell infiltrations in lung tissues. These findings serve as a foundation for the potential clinical application of XQLT in neutrophilic asthma.

KRT6A Inhibits IL-1ß-Mediated Pyroptosis of Keratinocytes via Blocking IL-17 Signaling.

Keratin 6A (KRT6A) is involved in the pathogenesis of various skin diseases. However, the reports on the roles of KRT6A in atopic dermatitis (AD) are limited. This study aimed to investigate the potentials of KRT6A in AD. mRNA levels were detected by RT-PCR. Cytokine release was determined by ELISA. Protein expression was determined using Western blot. Cell viability was determined by CCK-8. Cytotoxicity was detected by LDH assay. Cell death was determined by TUNEL. The pyroptosis of keratinocytes was detected using flow cytometry. We found that KRT6A was overexpressed in AD patients. Moreover, KRT6A was stimulated after exposed to proinflammatory cytokines. Overexpressed KRT6A suppressed inflammatory response, while KRT6A knockdown exerted the opposite effects. Overexpressed KRT6A suppressed inflammation-induced pyroptosis of keratinocytes. Additionally, KRT6A negatively regulated interleukin-17a (IL-17a) expression, blocking IL-17 signaling. IL-17a overexpression antagonized the effects of KRT6A and promoted pyroptosis of keratinocytes. In conclusion, KRT6A exerted protective functions in AD via regulating IL-17 signaling. This KRT6A/IL-17 may be a novel target for AD.

Oleuropein Has Modulatory Effects on Systemic Lipopolysaccharide-Induced Neuroinflammation in Male Rats.

BACKGROUND: Neuroinflammation induced by systemic inflammation is a risk factor for developing chronic neurologic disorders. Oleuropein (OLE) has antioxidant and anti-inflammatory properties; however, its effect on systemic inflammation-related neuroinflammation is unknown. OBJECTIVES: This study aimed to determine whether OLE protects against systemic lipopolysaccharide (LPS)-induced neuroinflammation in rats. METHODS: Six-wk-old Wistar rats were randomly assigned to 1 of the following 5 groups: 1) control, 2) OLE-only, 3) LPS + vehicle, 4) OLE+LPS (O-LPS), and 5) a single-dose OLE + LPS (SO-LPS group). OLE 200 mg/kg or saline as a vehicle was administered via gavage for 7 d. On the seventh day, 2.5 mg/kg LPS was intraperitoneally administered. The rats were decapitated after 24 h of LPS treatment, and serum collection and tissue dissection were performed. The study assessed astrocyte and microglial activation using glial fibrillary acidic protein (GFAP) and CD11b immunohistochemistry, nod-like receptor protein-3, interleukin (IL)-1ß, IL-17A, and IL-4 concentrations in prefrontal and hippocampal tissues via enzyme-linked immunosorbent assay, and total antioxidant/oxidant status (TAS/TOS) in serum and tissues via spectrophotometry. RESULTS: In both the O-LPS and SO-LPS groups, LPS-related activation of microglia and astrocytes was suppressed in the cortex and hippocampus (P < 0.001), excluding cortical astrocyte activation, which was suppressed only in the SO-LPS group (P < 0.001). Hippocampal GFAP immunoreactivity and IL-17A concentrations in the dentate gyrus were higher in the OLE group than those in the control group, but LPS-related increases in these concentrations were suppressed in the O-LPS group. The O-LPS group had higher cortical TAS and IL-4 concentrations. CONCLUSIONS: OLE suppressed LPS-related astrocyte and microglial activation in the hippocampus and cortex. The OLE-induced increase in cortical IL-4 concentrations indicates the induction of an anti-inflammatory phenotype of microglia. OLE may also modulate astrocyte and IL-17A functions, which could explain its opposing effects on hippocampal GFAP immunoreactivity and IL-17A concentrations when administered with or without LPS.

Vascular dysfunction and arterial hypertension in experimental celiac disease are mediated by gut-derived inflammation and oxidative stress.

AIMS: We examined the cardiovascular effects of celiac disease (CeD) in a humanized mouse model, with a focus on vascular inflammation, endothelial dysfunction, and oxidative stress. METHODS AND RESULTS: NOD.DQ8 mice genetically predisposed to CeD were subjected to a diet regime and oral gavage to induce the disease (gluten group vs. control). We tested vascular function, confirmed disease indicators, and evaluated inflammation and oxidative stress in various tissues. Plasma proteome profiling was also performed. CeD markers were confirmed in the gluten group, indicating increased blood pressure and impaired vascular relaxation. Pro-inflammatory genes were upregulated in this group, with increased CD11b+ myeloid cell infiltration and oxidative stress parameters observed in aortic and heart tissue. However, heart function remained unaffected. Plasma proteomics suggested the cytokine interleukin-17A (IL-17A) as a link between gut and vascular inflammation. Cardiovascular complications were reversed by adopting a gluten-free diet. CONCLUSION: Our study sheds light in the heightened cardiovascular risk associated with active CeD, revealing a gut-to-cardiovascular inflammatory axis potentially mediated by immune cell infiltration and IL-17A. These findings augment our understanding of the link between CeD and cardiovascular disease providing clinically relevant insight into the underlying mechanism. Furthermore, our discovery that cardiovascular complications can be reversed by a gluten-free diet underscores a critical role for dietary interventions in mitigating cardiovascular risks associated with CeD.

Prostaglandin E2 promotes Th17 differentiation induces corneal epithelial cell apoptosis and participates in the progression of dry eye.

This study is mainly based on T helper type 17 (Th17) cells analysis of the mechanism of prostaglandin E2 (PGE2) promoting the progression of dry eye (DE). Scopolamine and dry environment were used to induce mice DE model. Celecoxib was used to inhibit PGE2. Corneal epithelial cells and CD4+ T cells were used to construct a co-culture system. The osmotic pressure was increased by adding NaCl to simulate DE in vitro. AH6809 and E7046 were used to pre-culture to inhibit EP2/4 in T cells to verify the effect of exogenous PGE2 on Th17 cell differentiation and corneal epithelial cell apoptosis. The function of Th17 cells was analyzed by detecting RORγt and interleukin-17 (IL-17). PGE2 was instilled on the ocular surface to induce DE symptoms of mice. AH6809 and E7046 were used to inhibit EP2/4. The corneal epithelial cell apoptosis was observed by TUNEL. The proportion of Th17 cells in corneal tissue and draining lymph nodes (DLNs) was detected by flow cytometry. In DE mice, the concentration of PGE2 and IL-17 increased in tears, and the proportion of Th17 increased, while inhibition of PGE2 alleviated the symptoms of DE and inhibited Th17 differentiation. Hypertonic environment induces corneal epithelial cells to secrete PGE2. PGE2 promoted the expression of EP2/4 and the differentiation of Th17 cells in vitro. The hypertonic environment promoted PGE2 level and the apoptosis of corneal epithelial cells in the co-culture system. PGE2 alone did not cause corneal epithelial cell apoptosis, while PGE2 promoted apoptosis by promoting Th17. Blocking EP2/4 reduced the induction of Th17 differentiation by PGE2 and the promoted corneal epithelial cell apoptosis. Animal experiments showed that exogenous PGE2 induced DE symptoms. Blocking EP2/4 not only inhibited the proportion of Th17, but also alleviated the apoptosis of corneal epithelial cells caused by PGE2. PGE2 induces aggravation of inflammation by promoting the level of Th17 in the ocular surface, and causes corneal epithelial cell apoptosis, thereby participating in the progression of DE.

Fecal microbiota transplantation regulates the microbiota-gut-spinal cord axis to promote recovery after spinal cord injury.

Spinal cord injury (SCI) is devastating for patients, and currently lacks effective treatments. Dysbiosis commonly occurs after SCI and has significant immunomodulatory effects, but its impact on recovery remains unclear. The current study investigated the effects and mechanisms of fecal microbiota transplantation (FMT) in SCI. FMT was administered in a rat model of SCI and spinal pathology, inflammatory cytokines, and gut microbiome composition were assessed. Flow cytometry identified a source of interleukin (IL)-17 in spinal cord tissues, and carboxyfluorescein succimidyl ester labeling tracked γδ T cell migration. In vitro coculture was used to analyze the regulatory mechanisms of γδ T cells. Seahorse analysis was used to profile dendritic cell (DC) metabolism. Here we show that FMT improved spinal pathology and dampened post-injury inflammation. It also corrected post-SCI dysbiosis, increasing levels of the beneficial bacterium Akkermansia. The therapeutic effects of FMT were mediated by IL-17 produced by γδ T cells. FMT regulated γδ T cells via DC-T regulatory cell interaction, and induced metabolic reprogramming in DCs. These findings suggest that FMT represents a promising therapeutic approach for SCI, with potential to target IL-17+ γδ T cells. Elucidating the interconnected pathways between microbiota, immunity, and the spinal cord may facilitate novel treatment strategies.

Gut microbiota-derived 12-ketolithocholic acid suppresses the IL-17A secretion from colonic group 3 innate lymphoid cells to prevent the acute exacerbation of ulcerative colitis.

Intestinal microbiota dysbiosis and metabolic disruption are well-known as the primary triggers of ulcerative colitis (UC). However, their role in regulating the group 3 innate lymphoid cells (ILC3s), which are essential for intestinal health, remains unexplored during the development of disease severity. Here, our results showed that the microbiota structure of patients with severe UC (SUCs) differed from those with mild UC (MiUCs), moderate UC (MoUCs), and healthy controls (HCs). Microbes producing secondary bile acids (SBAs) and SBAs decreased with the aggravation of UC, and a strong positive correlation existed between them. Next, fecal microbiota transfer was used to reproduce the human-derived microbiota in mice and decipher the microbiota-mediated inflammatory modulation during an increase in disease severity. Mice receiving SUC-derived microbiota exhibited enhancive inflammation, a lowered percentage of ILC3s, and the down-regulated expressions of bile acid receptors, including vitamin D receptor (VDR) and pregnane X receptor (PXR), in the colon. Similar to clinical results, SBA-producing microbes, deoxycholic acids (DCA), and 12-ketolithocholic acids (12-KLCA) were diminished in the intestine of these recipients. Finally, we compared the therapeutic potential of DCA and 12-KLCA in preventing colitis and the regulatory mechanisms mediated by ILC3s. 12-KLCA but not DCA represented a strong anti-inflammatory effect associated with the higher expression of VDR and the lower secretion of IL-17A from colonic ILC3s. Collectively, these findings provide new signatures for monitoring the acute deterioration of UC by targeting gut microbiota and bile acid metabolism and demonstrate the therapeutic and preventive potential of a novel microbiota-derived metabolite, 12-KLCA.

Enhanced Innate and Acquired Immune Responses in Systemic Sclerosis Primary Peripheral Blood Mononuclear Cells (PBMCs).

Chronic immune activation in systemic sclerosis is supported by the production of a plethora of cytokines with proven regulatory activities of the immune responses. This study aimed to explore PBMCs' cytokine profiles in SSc patients versus controls, as well as to investigate the balance between pro- and anti-inflammatory cytokines in association with disease duration. PBMCs were isolated from 18 SSc patients and 17 controls and further subjected to in vitro stimulation with lipopolysaccharide and heat-killed Candida albicans. Cytokine production was measured after 24 h and 7 days, respectively, using ELISA kits for interleukin (IL)-1ß, IL-1 receptor antagonist (IL-1Ra), IL-6, tumor necrosis factor (TNF), IL-10, IL-17, and interferon-gamma (IFN-gamma). IL-1 ß, IL-6, and TNF levels were increased in SSc patients compared with healthy volunteers irrespective of the stimulus used. IL-1Ra and Il-17 concentrations were not statistically different between groups, even though a trend toward higher levels in patients compared with their matched controls was also observed. Most cytokines demonstrated a stable course with disease progression, except for IL-10 levels, which declined over time. In conclusion, the results of this pilot study reveal that in patients with SSc a persistently enhanced immune response is established and maintained regardless of stimulus or disease duration.

Notopterol Suppresses IL-17-Induced Proliferation and Invasion of A549 Lung Adenocarcinoma Cells via Modulation of STAT3, NF-κB, and AP-1 Activation.

Interleukine-17 is a proinflammatory cytokine that promotes lung cancer growth and progression though the activation of the STAT3, NF-κB, and AP-1 signaling pathways. Therefore, blocking the IL-17-induced oncogenic pathway is a new strategy for the treatment of lung cancer. Notopterol, a furanocoumarin, has demonstrated anti-tumor effects in several types of tumors. However, its molecular function in relation to the IL-17-induced proliferation and invasion of A549 lung adenocarcinoma cells remains unknown. Here, notopterol exhibited an inhibitory effect on IL-17-promoted A549 cell proliferation and induced G0/G1 cell cycle arrest. Western blot analysis revealed that notopterol inhibited the expression of cell-cycle-regulatory proteins, including cyclin D1, cyclin E, CDK4, and E2F. Moreover, notopterol blocked IL-17-induced A549 cell migration and invasion by regulating the epithelial-mesenchymal transition (EMT) and reducing the expression of extracellular degradation enzymes. At the molecular level, notopterol treatment significantly down-regulated the IL-17-activated phosphorylation of Akt, JNK, ERK1/2, and STAT3, leading to a reduced level of transcriptional activity of NF-κB and AP-1. Collectively, our results suggest that notopterol blocks IL-17-induced A549 cell proliferation and invasion through the suppression of the MAPK, Akt, STAT3, AP-1, and NF-κB signaling pathways, as well as modulating EMT.

Avian safety guardian: Luteolin restores Mycoplasma gallisepticum-induced immunocompromise to improve production performance via inhibiting the IL-17/NF-kB pathway.

Mycoplasma gallisepticum (MG) is a major pathogen causing chronic respiratory disease (CRD) in chickens. Exposure to MG poses a constant threat to chicken health and causes substantial economic losses. Antibiotics are the main treatment for MG infections, but have to struggle with antibiotic residues and MG resistance. To date, no safe and more effective prevention or treatment for MG infections has been identified. Luteolin (Lut) is a natural flavonoid compound known for its excellent anti-viral, anti-bacterial, immunoregulatory, and anti-inflammatory pharmacological activities. Herein, we established an MG-infected model using partridge shank chickens and chicken-like macrophages (HD11 cells) to investigate the effect and potential mechanism of Lut against MG-induced immune damage. According to our findings, Lut significantly inhibited the expression of MG adhesion protein (pMGA1.2) in vivo and in vitro. Lut effectively mitigated the MG-induced decrease in body weight gain, feed conversion ratio, survival rate, and serum IgG and IgA levels. Lut directly repaired MG-induced spleen and thymus damage by histopathological analysis. Furthermore, network pharmacology analysis revealed that Lut most probably resisted MG infection through the IL-17/NF-kB pathway. In vivo and in vitro experiments, Lut significantly suppressed the increase in key protein IL-17A, TRAF6, p-p65, and p-IkBα in the IL-17/NF-kB pathway. Meanwhile, Lut markedly alleviated MG-induced the increase of pro-inflammatory cytokines TNF-α, IL-6, IL-1ß, pro-apoptotic genes caspase3 and caspase9, while promoting the expression of anti-apoptotic genes Bcl-2 and Bcl-XL. In summary, Lut effectively suppressed MG colonization, alleviated MG-induced the production performance degradation, inflammatory responses, and immune damage by inhibiting the IL-17/ NF-kB pathway. This study indicates Lut can serve as a safe and effective antibiotic alternative drug for preventing and treating MG-induced CRD. It also provides new evidence to explore the molecular mechanisms of MG infection.

Gentiopicroside-Loaded Chitosan Nanoparticles Inhibit TNF-α-Induced Proliferation and Inflammatory Response in HaCaT Keratinocytes and Ameliorate Imiquimod-Induced Dermatitis Lesions in Mice.

Purpose: In this study, we aimed to report the biological characteristics of the first successful synthesis of gentiopicroside-loaded chitosan nanoparticles and to evaluate the therapeutic effects and preliminary mechanisms of gentiopicrin-loaded chitosan on psoriasis-like cell and mouse models. Methods: Gentiopicroside-loaded chitosan nanoparticles (CHI-GEN) were prepared, and their biological characteristics were evaluated. HaCaT keratinocytes were stimulated with TNF-α to establish a psoriatic keratinocyte model. MTT assay and flow cytometry were used to measure cell viability and apoptosis, respectively. mRNA levels of K17, VEGF A, and IL-6 and IL-23A were detected using qRT-PCR. These tests were used to preliminarily assess the effects of CHI-GEN on keratinocyte proliferation and inflammation. Imiquimod was used to construct a psoriasis-like mice model. The severity of psoriasis was scored based on the psoriasis area severity index (PASI), H&E staining was used to observe the histological changes and the level of inflammation and cell proliferation of skin lesions was evaluated by measuring the mRNA levels of K17, IL-23A, and IL-17A using qRT-PCR. Results: The average particle size of CHI-GEN nanoparticles was approximately 100 nm, and the zeta potential was 2.69 ± 0.87 mV. The cumulative release was 67.2% in solutions of pH 5.5 at 24 h. GEN reduced TNF-α-induced excessive proliferation of HaCaT keratinocytes and downregulated mRNA levels of K17, VEGF A, and inflammatory cytokines IL-6 and IL-23A, which was more obvious in the CHI-GEN treatment group. Additionally, CHI-GEN significantly improved the severity of skin lesions in psoriasis-like mice and downregulated the mRNA expressions of IL-6, IL-23A, and IL-17A in mice skin lesions. Conclusion: In conclusion, we successfully prepared gentiopicrin-chitosan nanoparticles. Our results show that these nanoparticles have anti-psoriasis activity, inhibits keratinocyte proliferation and improves symptoms in psoriasis model mice and can be used to develop an effective strategy for the treatment of psoriasis.

Melittin inhibits tumor cell migration and enhances cisplatin sensitivity by suppressing IL-17 signaling pathway gene LCN2 in castration-resistant prostate cancer.

BACKGROUND: Melittin is a small molecule polypeptide extracted from the abdominal cavity of bees, which is used to treat inflammatory diseases and relieve pain. However, the antitumor effect of melittin and its mechanisms remain unclear, especially in castration-resistant prostate cancer (CRPC). METHODS: Through CCK-8 assay, colony formation assay, wound healing assay and Transwell migration assay, we explored the effect of melittin on CRPC cell lines. In addition, with microarray analysis, gene ontology analysis and kyoto encyclopedia of genes and genomes analysis, this study identified key genes and signaling pathways that influence the growth of PC-3 cells. Meanwhile, the effect of melittin on CRPC was also verified through subcutaneous tumor formation experiments. Finally, we also tested the relevant indicators of human prostate cancer (PCa) specimens through immunohistochemistry and H＆E stating. RESULTS: Here, melittin was verified to inhibit the cell proliferation and migration of CPRC. Moreover, RNA-sequence analysis demonstrated that Interleukin-17 (IL-17) signaling pathway gene Lipocalin-2 (LCN2) was downregulated by melittin treatment in CRPC. Further investigation revealed that overexpression of LCN2 was able to rescue tumor suppression and cisplatin sensitivity which melittin mediated. Interestingly, the expression of LCN2 is highly related to metastasis in PCa. CONCLUSIONS: In brief, our study indicates that LCN2 plays an oncogenic role in CRPC and melittin may be selected as an attractive candidate for CRPC therapy.

Molecular modeling and rational design of disulfide-stapled self-inhibitory peptides to target IL-17A/IL-17RA interaction.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine implicated in diverse autoimmune and inflammatory disorders such as psoriasis and Kawasaki disease. Mature IL-17A is a homodimer that binds to the extracellular type-III fibronectin D1:D2-dual domain of its cognate IL-17 receptor A (IL-17RA). In this study, we systematically examined the structural basis, thermodynamics property, and dynamics behavior of IL-17RA/IL-17A interaction and computationally identified two continuous hotspot regions separately from different monomers of IL-17A homodimer that contribute significantly to the interaction, namely I-shaped and U-shaped segments, thus rendered as a peptide-mediated protein-protein interaction (PmPPI). Self-inhibitory peptides (SIPs) are derived from the two segments to disrupt IL-17RA/IL-17A interaction by competitively rebinding to the IL-17A-binding pocket on IL-17RA surface, which, however, only have a weak affinity and low specificity for IL-17RA due to lack of the context support of intact IL-17A protein, thus exhibiting a large flexibility and intrinsic disorder when splitting from the protein context and incurring a considerable entropy penalty when rebinding to IL-17RA. The U-shaped segment is further extended, mutated and stapled by a disulfide bridge across its two strands to obtain a number of double-stranded cyclic SIPs, which are partially ordered and conformationally similar to their native status at IL-17RA/IL-17A complex interface. Experimental fluorescence polarization assays substantiate that the stapling can moderately or considerably improve the binding affinity of U-shaped segment-derived peptides by 2-5-fold. In addition, computational structural modeling also reveals that the stapled peptides can bind in a similar mode with the native crystal conformation of U-shaped segment in IL-17RA pocket, where the disulfide bridge is out of the pocket for avoiding intervene of the peptide binding.

Punicalagin alleviates the hyperproliferation of keratinocytes in psoriasis through inhibiting SKP2 expression.

Psoriasis is a chronic inflammatory skin disorder characterized by abnormal keratinocytes proliferation and multiple immune cells infiltration in the dermis and epidermis. Although most psoriasis-related researches have been concentrated on the interleukin-23 (IL-23)/interleukin-17 (IL-17) axis, new data suggest that keratinocytes also play a pivotal role in psoriasis. Previously, we found that punicalagin (PUN), a bioactive ellagitannin extracted from Pericarpium Granati (the pericarpium of Punica granatum L.), exerts a therapeutic effect on psoriasis. However, the underlying mechanism, especially its potential modulatory effect on keratinocytes, remains obscure. Our study aims to reveal the potential regulatory effect and its underlying cellular mechanism of PUN on the hyperproliferation of keratinocytes. We used tumor necrosis factor α (TNF-α), IL-17A and interleukin-6 (IL-6) to induce abnormal proliferation of HaCaT cells (Human Keratinocytes Cells) in vitro. Then, we evaluated the effects of PUN through MTT assay, EdU staining and cell cycle detection. Finally, we explored the underlying cellular mechanisms of PUN via RNA-sequencing, WB in vitro and in vivo. Here, we found that PUN can directly and dose-dependently decrease TNF-α, IL-17A and IL-6-induced abnormal proliferation of HaCaT cells in vitro. Mechanically, PUN suppresses the hyperproliferation of keratinocytes through repressing S-phase kinase-associated protein 2 (SKP2) expression in vitro and in vivo. Moreover, overexpression of SKP2 can partly abolish PUN-mediated inhibition of aberrantly proliferative keratinocytes. These results illustrate that PUN can reduce the severity of psoriasis through directly repressing SKP2-mediated abnormal proliferation of keratinocytes, which gives new insight into the therapeutic mechanism of PUN on psoriasis. Moreover, these findings imply that PUN might be a promising drug candidate for the treatment of psoriasis.

[Effect and mechanism of Bovis Calculus on ulcerative colitis by inhibiting IL-17/IL-17RA/Act1 signaling pathway].

This study aimed to elucidate the effect and underlying mechanism of Bovis Calculus in the treatment of ulcerative colitis(UC) through network pharmacological prediction and animal experimental verification. Databases such as BATMAN-TCM were used to mine the potential targets of Bovis Calculus against UC, and the pathway enrichment analysis was conducted. Seventy healthy C57BL/6J mice were randomly divided into a blank group, a model group, a solvent model(2% polysorbate 80) group, a salazosulfapyridine(SASP, 0.40 g·kg~(-1)) group, and high-, medium-, and low-dose Bovis Calculus Sativus(BCS, 0.20, 0.10, and 0.05 g·kg~(-1)) groups according to the body weight. The UC model was established in mice by drinking 3% dextran sulfate sodium(DSS) solution for 7 days. The mice in the groups with drug intervention received corresponding drugs for 3 days before modeling by gavage, and continued to take drugs for 7 days while modeling(continuous administration for 10 days). During the experiment, the body weight of mice was observed, and the disease activity index(DAI) score was recorded. After 7 days of modeling, the colon length was mea-sured, and the pathological changes in colon tissues were observed by hematoxylin-eosin(HE) staining. The levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), interleukin-6(IL-6), and interleukin-17(IL-17) in colon tissues of mice were detected by enzyme-linked immunosorbent assay(ELISA). The mRNA expression of IL-17, IL-17RA, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1ß, CXCL1, CXCL2, and CXCL10 was evaluated by real-time polymerase chain reaction(RT-PCR). The protein expression of IL-17, IL-17RA, Act1, p-p38 MAPK, and p-ERK1/2 was investigated by Western blot. The results of network pharmacological prediction showed that Bovis Calculus might play a therapeutic role through the IL-17 signaling pathway and the TNF signaling pathway. As revealed by the results of animal experiments, on the 10th day of drug administration, compared with the solvent model group, all the BCS groups showed significantly increased body weight, decreased DAI score, increased colon length, improved pathological damage of colon mucosa, and significantly inhibited expression of TNF-α,IL-6,IL-1ß, and IL-17 in colon tissues. The high-dose BCS(0.20 g·kg~(-1)) could significantly reduce the mRNA expression levels of IL-17, Act1, TRAF2, TRAF5, TNF-α, IL-6, IL-1ß, CXCL1, and CXCL2 in colon tissues of UC model mice, tend to down-regulate mRNA expression levels of IL-17RA and CXCL10, significantly inhibit the protein expression of IL-17RA,Act1,and p-ERK1/2, and tend to decrease the protein expression of IL-17 and p-p38 MAPK. This study, for the first time from the whole-organ-tissue-molecular level, reveals that BCS may reduce the expression of pro-inflammatory cytokines and chemokines by inhibiting the IL-17/IL-17RA/Act1 signaling pathway, thereby improving the inflammatory injury of colon tissues in DSS-induced UC mice and exerting the effect of clearing heat and removing toxins.

[Effects of interleukin-17A on liver and kidney injury and prognosis in septic mice].

OBJECTIVE: To explore the effect of interleukin-17A (IL-17A) on liver and kidney injury and prognosis in septic mice. METHODS: A total of 84 SPF male C57BL/6 mice were randomly divided into sham operation group (Sham group), cecal ligation and puncture (CLP) induced sepsis model group (CLP group), and IL-17A intervention group. IL-17A intervention group were then divided into five subgroups according to the dose of IL-17A (0.25, 0.5, 1, 2, 4 µg). Mice in the IL-17A intervention group were intraperitoneally injected with the corresponding dose of IL-17A 100 µL immediately after surgery. The other groups were intraperitoneally injected with 100 µL phosphate buffer solution (PBS). The survival rate of mice was observed at 7 days, and peripheral blood and liver, kidney and spleen tissues were collected. According to the 7-day survival, another 18 mice were randomly divided into Sham group, CLP group, and 1 µg IL-17A intervention group. Peripheral blood samples were collected at 12 hours and 24 hours after CLP, and the mice were sacrificed to obtain liver, kidney, and spleen tissues. The behavior and abdominal cavity of each group were observed. The levels of peripheral blood liver and kidney function indexes and inflammatory factors were detected. The histopathological changes of liver and kidney were observed under light microscope. The peripheral blood and spleen tissues were inoculated in the medium, the number of bacterial colonies was calculated, and the bacterial migration of each group was evaluated in vitro. RESULTS: Except for the Sham group, the 7-day survival rate of mice in the 1 µg IL-17A intervention group was the highest (75.0%), so this condition was selected as the intervention condition for the subsequent study. Compared with Sham group, the liver and kidney functions of CLP group were significantly damaged at each time point after operation. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (SCr) reached the peak at 24 hours after operation, and the liver and kidney pathological scores reached the peak at 7 days after operation, the levels of inflammatory cytokines interleukin (IL-17A, IL-6, IL-10) reached the peak at 12 hours after operation, and tumor necrosis factor-α (TNF-α) reached the peak at 24 hours after operation. In addition, a large number of bacteria proliferated in the peripheral blood and spleen, which reached the peak on day 7. Compared with the CLP group, exogenous administration of 1 µg IL-17A significantly delayed the rising trend of each index in the early stage of sepsis [24-hour ALT (U/L): 166.95±5.20 vs. 271.30±6.11, 24-hour AST (U/L): 599.42±7.25 vs. 1 013.27±3.37, 24-hour BUN (mg/L): 815.4±26.3 vs. 1 191.2±39.4, 24-hour SCr (µmol/L): 29.34±0.87 vs. 60.75±3.83, 7-day liver pathological score: 2.50 (2.00, 3.00) vs. 9.00 (8.50, 9.00), 7-day kidney pathological score: 1.00 (1.00, 2.00) vs. 5.00 (4.50, 5.00), 12-hour IL-17A (ng/L): 105.21±0.31 vs. 111.28±1.37, 12-hour IL-6 (ng/L): 83.22±1.01 vs. 108.88±0.99, 12-hour IL-10 (ng/L): 731.54±3.04 vs. 790.25±2.54, 24-hour TNF-α (µg/L): 454.67±0.66 vs. 576.18±0.76, 7-day peripheral blood colony count (CFU/mL): 600 (400, 600) vs. 4 200 (4 200, 4 300), 7-day spleen tissue colony count (CFU/g): 4 600 (4 400, 4 600) vs. 23 400 (23 200, 23 500), all P < 0.05]. CONCLUSIONS: Appropriate dose (1 µg) of exogenous IL-17A can reduce the lethal inflammatory response induced by CLP and improve the ability of bacterial clearance, thereby alleviating liver and kidney injury and improving the 7-day survival rate of septic mice.

Chronic treatment with IL-25 increases renal M2 macrophages and reduces renal injury in obese Dahl salt-sensitive rats during the prepubescent stage.

Recently, we have reported that the early progression of proteinuria in the obese Dahl salt-sensitive (SS) leptin receptor mutant (SSLepRmutant) strain was associated with increased renal macrophage infiltration before puberty. Macrophages can be divided into two distinct phenotypes: M1 (proinflammatory) and M2 (anti-inflammatory). Moreover, previous studies have demonstrated that interleukin (IL)-25 converts resting macrophages and M1 into M2. Therefore, the present study examined whether treatment with IL-25 would reduce the early progression of renal injury in SSLepRmutant rats by increasing renal M2. We also investigated the impact of IL-25 on M2 subtypes: M2a (wound healing/anti-inflammatory), M2b (immune mediated/proinflammatory), M2c (regulatory/anti-inflammatory), and M2d (tumor associated/proangiogenic). Four-wk-old SS and SSLepRmutant rats were treated with either control (IgG) or IL-25 (1 µg/day ip every other day) for 4 wk. The kidneys from SSLepRmutant rats displayed progressive proteinuria and renal histopathology versus SS rats. IL-25 treatment had no effect on these parameters in SS rats. However, in the SSLepRmutant strain, proteinuria was markedly reduced after IL-25 treatment. Chronic treatment with IL-25 significantly decreased glomerular and tubular injury and renal fibrosis in the SSLepRmutant strain. Although the administration of IL-25 did not change total renal macrophage infiltration in both SS and SSLepRmutant rats, IL-25 increased M2a by >50% and reduced M1 by 60% in the kidneys of SSLepRmutant rats. Overall, these data indicate that IL-25 reduces the early progression of renal injury in SSLepRmutant rats by inducing M2a and suppressing M1 and suggest that IL-25 may be a therapeutic target for renal disease associated with obesity. NEW & NOTEWORTHY For the past few decades, immune cells and inflammatory cytokines have been demonstrated to play an important role in the development of renal disease. The present study provides strong evidence that interleukin-25 slows the early progression of renal injury in obese Dahl salt-sensitive rats before puberty by increasing systemic anti-inflammatory cytokines and renal M2a macrophages.

MicroRNA-31 regulates TNF-α and IL-17A co-induced-endothelial cell apoptosis by repressing E2F6.

Vascular endothelial cell (VEC) apoptosis is the fundamental cause of pulmonary arterial hypertension. MicroRNA-31 (MiR-31) is a novel target for hypertension treatment. However, the role and mechanism of miR-31 in the apoptosis of VECs remain unclear. The purpose of this study is to determine whether miR-31 plays an important role in VEC apoptosis as well as the detailed mechanisms involved. We found that pro-inflammatory cytokines IL-17A and TNF-α were highly expressed in serum and aorta, and the expression of miR-31 was significantly increased in aortic intimal tissue from Angiotensin II (AngII)- induced hypertensive mice (WT-AngII) compared with control mice (WT-NC). In vitro, co-stimulation of VECs with IL-17A and TNF-α resulted in increased expression of miR-31 and VEC apoptosis. MiR-31 inhibition strikingly decreased TNF-α and IL-17A co-induced VEC apoptosis. Mechanistically, in IL-17A and TNF-α co-stimulated VECs (co-induced VECs), we found that the activation of the NF-κB signal effectively increased the expression of miR-31. Dual-luciferase reporter gene assay revealed that miR-31 directly targeted and inhibited the expression of the E2F transcription factor 6 (E2F6). The expression of E2F6 was decreased in Co-induced VECs. MiR-31 inhibition significantly alleviated the decreased expression of E2F6 in co-induced VECs. Consistent with the co-stimulated effect of IL-17A and TNF-α on VECs, transfection of siRNA E2F6 induced cell apoptosis without the stimulation of the above cytokines. In conclusion, TNF-α and IL-17A generated in the aortic vascular tissue and serum from Ang II-induced hypertensive mice could trigger VECs apoptosis by the miR-31/E2F6 axis. To sum up, our study suggests that the key factor between cytokine co-stimulation effect and VEC apoptosis was miR-31/E2F6 axis, which was mainly regulated by NF-қB signaling pathway. This gives us a new sight to treat hypertension-associated VR.