In recent years, the role of the monocyte macrophage system and Toll like receptor 9 (TLR9) in the pathogenesis of allergic diseases has attracted attention, but little is known about the relationship between changes in TLR9 expression on monocytes and macrophages and allergic rhinitis (AR) and allergic asthma (AA). This article uses flow cytometry to analyze the expression changes of TLR9+ monocytes in peripheral blood of patients with AR, AA, and allergic rhinitis combined with asthma (ARA). And the AR and AA models of wild-type (WT) and Fc epsilon RI gene knockout (Fc epsilon RI KO) mice were used to investigate the expression changes of TLR9 in monocytes and macrophages in lung tissue of mice after allergen stimulation. It was found that the proportion of TLR9 + cells in the peripheral blood mononuclear cell population of AR, AA, and ARA patients was significantly increased, indicating an increase in MFI expression representing the number of TLR9 molecules expressed in each mononuclear cell. Artemisia selengensis allergen extract (ASWE), house dust mite allergen extract (HDME), wutong pollen allergen extract (PPE) stimulate to increase the proportion of TLR9+ monocytes in the blood of AR and AA model mice, upregulate the expression level of TLR9 MFI in monocytes, and make the expression level of TLR9 in macrophages in lung tissue higher than that of control mice. In AR and AA mouse models with high-affinity IgE receptor (Fc epsilon RI) KO, ASWE, especially HDME, can also induce upregulation of TLR9 + cell percentage and TLR9 MFI in monocytes. Based on the significant reduction in the proportion of TLR9+ cells and TLR9 MFI expression levels in AR and AA mice compared to wild-type (WT) mice, it can be concluded that the increase in the proportion of blood monocytes induced by allergen extract and the upregulation of TLR9 expression levels in monocytes are achieved through both Fc epsilon RI dependent and non-dependent pathways. Similarly, compared with WT mice, the AA model mice of Fc epsilon RI KO showed a significant increase in the proportion of lung tissue macrophages induced by allergen extract and a significant decrease in the upregulation of TLR9 expression level. In summary, monocytes may participate in the pathophysiological processes of AR, AA, and ARA through upregulation of TLR9 expression. The upregulation of TLR9 expression in lung tissue macrophages further suggests that monocytes and macrophages may form a new inflammatory signal amplification loop in local tissues through high expression of TLR9 under allergen stimulation, providing new ideas for understanding the pathogenesis of allergic airway diseases.

ETHNOPHARMACOLOGICAL RELEVANCE:Andrographolide (Andro), the major bioactive constituent of the traditional Chinese medicinal herb Andrographis paniculata (Burm.f.) Wall. ex Nees, possesses prominent pharmacological activities including anti-inflammatory and immunomodulatory effects. Clinically, it has been applied as a supplementary therapy for inflammatory respiratory conditions., yet its intervention efficacy in allergic rhinitis (AR) and its multidimensional molecular mechanisms involving "anti-inflammatory-barrier protection" remain poorly elucidated. AIM OF THE STUDY:To explore the mechanism by which Andro alleviates AR, evaluate its safety, and predict its potential therapeutic targets. MATERIALS AND METHODS:Public bioinformatic databases were applied to forecast the potential targets and signaling pathways. An OVA-induced mouse model of AR was established, and Andro was administered by intraperitoneal injection. In vitro, an inflammatory injury model was established by stimulating human nasal epithelial RPMI-2650 cells with IL-4/IL-13, and functional blockade experiments were performed using the IL-17-specific inhibitor ixekizumab. Nasal mucosa was collected for transcriptome sequencing, and differentially expressed genes (DEGs) were screened and further analyzed with predicted targets. Pathological changes, inflammatory factors, tight junction proteins, immune cell subsets and key pathway-related proteins were evaluated by immunofluorescence, ELISA, RT-qPCR, IHC, flow cytometry and WB. RESULTS:Network pharmacology analysis indicated that the IL-17 signaling pathway served as the core pathway of Andro against AR. Molecular docking and dynamics simulations show the stable binding between Andro and IL-17A. As demonstrated by a series of in vivo experiments, the administration of Andro resulted in a significant alleviation of nasal symptoms in AR mice. Furthermore, the serum levels of IgE, IL-17A, IL-6, and TNF-α were reduced, and the proportion of splenic CD4+IL-17A+ Th17 cells was dose-dependently decreased as determined by flow cytometry, and inflammatory cells infiltration in the nasal mucosa was suppressed. Meanwhile, Andro significantly inhibited goblet cell hyperplasia and MUC5AC overexpression, while dose-dependently restoring the expression of tight junction proteins ZO-1 and Occludin. In vitro experiments confirmed that Andro dose-dependently reversed IL-4/IL-13-induced inflammatory cytokine secretion, mucus hypersecretion and epithelial barrier damage in RPMI-2650 cells. Combined treatment with Andro and ixekizumab produced no additional additive effect, indicating that the protective effect of Andro may be dependent on the inhibition of the IL-17 signaling pathway. Mechanistically, Andro downregulated the transcription and protein expression of IL-17A, IL-17RA, and Act1, inhibited the phosphorylation of NF-κB p65, and blocked IL-17/NF-κB-mediated mucus hypersecretion and epithelial barrier damage. CONCLUSIONS:The therapeutic mechanism of Andro against AR involves suppressing nasal mucosal inflammation and repairing epithelial barrier function by inhibiting key protein expression in the IL-17/NF-κB pathway and downregulating MUC5AC expression. This targeted regulation effectively ameliorates pathological injury in AR.

Monascin, a natural and safe drug, exhibits potent anti-inflammatory, hypolipidemic, and antioxidative effects. However, their therapeutic potential and underlying mechanisms in allergic rhinitis (AR) remain unexplored. The intranasal delivery of monascin for treating AR faces the challenge of rapid clearance. Herein, to achieve sustained and prolonged release, we developed monascin-encapsulated poly(lactic co glycolic acid) nanoparticles (PLGA-MS) by the mechanical double emulsion technique, enabling effective local delivery of monascin for the treatment of AR. Histopathological staining, flow cytometry, ELISA, and network pharmacology were used to assess therapeutic effects and potential mechanisms of monascin in treating AR. In vitro and in vivo experiments revealed that PLGA-MS exhibited enhanced stability, excellent drug encapsulation capacity, and sustained drug release. PLGA-MS treatment showed a significant therapeutic effect in AR mice by decreasing inflammatory cells in nasal tissue and regulating IgE, histamine, and a variety of cytokines. PLGA-MS treatment decreased T helper 2 cells (Th2) and T helper 17 cells (Th17) while promoting and increasing Tregs (regulatory T cells). Further analysis showed elevated levels of IL-10 and TGF-β expression in Tregs post-treatment. Results of the CCK-8 assay, pathological analysis of vital organs, and serum analysis of liver and kidney functions demonstrated the biosafety of PLGA-MS. Additionally, network pharmacology identified immune response pathways that may support PLGA-MS's immunomodulatory effects in AR. Our study presented a biomaterial-facilitated intranasal delivery system for monascin, offering an effective therapeutic strategy for AR.

Background It is reported that CD123 + HLA-DR- cells in PBMC are basophils, and CD203c, CD63, and FcεRI molecules are activation markers of basophils. However, little is known of CD123 + HLA-DR-cells in blood granulocytes. Objective To investigate the presence of CD123 + HLA-DR- cells in the blood granulocytes and peripheral PBMC of patients with allergic rhinitis (AR), as well as the impact of allergens on the cell membrane markers of basophils. Methods Flow cytometry was used to detect the expression of the membrane molecules. Results While CD123 + HLA-DR- PBMCs are representative of basophils, their presence did not significantly change in patients with AR. In contrast, both the percentage and number of CD123 + HLA-DR- granulocytes, which make up only up to 50% of basophils, were significantly increased in patients with seasonal (sAR) and perennial AR (pAR). CD63+, CD203c+, and FcεRIα+ cells within CD123 + HLA-DR- granulocytes also showed enhanced activity in patients with AR. Allergen extracts from house dust mite allergen extract (HDME) and Artemisia sieversiana wild extract further increased the number of CD123 + HLA-DR- cells in granulocytes of sAR and pAR patients, as well as in PBMCs of pAR patients. Conclusions The use of CD123 + HLA-DR- granulocytes and PBMC may not be sufficient for diagnosing AR. Allergens could potentially contribute to the development of AR by influencing the number of CD123 + HLA-DR- cells, as well as the expression of CD63, CD203c, and FcεRIαin these cells.

Background: The present work illustrates the role of multi-walled carbon nanotubes in photothermal therapy. Nasopharyngeal carcinoma (NPC) is a malignant tumor of the head and neck with significant ethnic and geographic differences, and conventional treatment options are no longer suitable to improve the prognosis and survival of patients. Photothermal therapy (PTT) has emerged as a new strategy for oncology treatment in recent years and is now used in the treatment of many common cancers. Multi-walled carbon nanotubes (MWCNT) have been used to advantage in several fields due to their excellent thermal conductivity. The aim of this paper is to investigate the promotion of apoptosis of nasopharyngeal cancer cells by multi-walled carbon nanotubes as an adjuvant nanomaterial for nasopharyngeal cancer photothermal therapy. Methods: Carboxylated multi-walled carbon nanotubes and prepared multi-walled carbon nanotube-hyaluronic acid (MWCNT-HA) composites were used for cell proliferation-related experiments such as CCK-8 assay, live-dead staining and flow cytometric analysis and inverted fluorescence microscopy to determine the expression level of apoptotic factors and confocal microscopy cell morphology analysis on nasopharyngeal carcinoma CNE-1 cells under near-infrared laser irradiation. The effects of multi-walled carbon nanotubes on the proliferation and apoptosis of tumor cells under NIR response were elucidated, and the mechanism of apoptosis was explored. Results: TEM and SEM demonstrated that MWCNT had good appearance morphology and the temperature rise curve indicated excellent photothermal stability. And MWCNT and MWCNT-HA could significantly inhibit the proliferation of tumor cells and change the normal morphology of cells under NIR laser irradiation. Cellular immunofluorescence analysis confirmed that MWCNT-HA significantly upregulated the expression level of apoptosis factor Caspase-3 and significantly downregulated the expression level of anti-apoptosis factor Bcl-2. Conclusion: In this study, MWCNT inhibited the proliferation of tumor cells and promoted apoptosis through the use of multi-walled carbon nanotubes as an adjuvant nanomaterial for photothermal therapy. In addition, multi-walled carbon nanotubes could inhibit the mitochondrial pathway of CNE-1 cells to cause cell death. These studies suggest that multi-walled carbon nanotubes can function as efficient photothermal conversion materials for tumor photothermal therapy.

背景 近年来,研究表明趋化因子受体 6(chemokine receptor 6,CCR6)可能与过敏性疾病中免疫细胞的募集有关,但CCR6在过敏性鼻炎(allergic rhinitis,AR)中的作用尚不明确.目的 检测AR患者外周血中Treg表面CCR6表达情况和AR小鼠鼻黏膜中CCR6的分布和表达,探究CCR6在AR中趋化募集免疫细胞的作用机制.方法 留取AR患者和健康人群(HC组)的外周静脉血样本,流式细胞仪检测并比较Treg所占比例及其表面CCR6的表达情况.制备AR小鼠模型,苏木精和伊红染色观察AR小鼠和对照组小鼠鼻黏膜嗜酸性粒细胞浸润情况,免疫组织化学染色检测鼻黏膜中CCR6蛋白的表达.结果 AR患者外周血CD4+CD25+FoxP3+Treg细胞占比(2.92％±1.83％vs5.23％±2.58％)和CCR6阳性细胞占比(30.02％±11.50％vs50.20％±11.67％)较HC组明显降低(P均＜0.01),AR患者CCR6与Treg细胞呈正相关(r=0.889,P＜0.01).小鼠AR模型组鼻黏膜中嗜酸性粒细胞数[24.0(14.5,31.5)vs 0(0,1.8)]和CCR6阳性细胞数量(136.25±26.04 vs 81.00±23.08)较对照组明显增多(P均＜0.01),CCR6表达与嗜酸性粒细胞数呈正相关(r=0.766,P＜0.01).结论 CCR6在AR外周血和鼻黏膜中的变化提示CCR6参与AR发病过程,可能与诱导免疫细胞趋化聚集有关.

Interleukin (IL)‐18 is a potentially important molecule in allergic rhinitis (AR). However, expressions of IL‐18, IL‐18 binding protein isoform a (IL‐18BPa) and IL‐18 receptor alpha (IL‐18Rα) in AR blood monocytes remain obscure. We, therefore, investigated IL‐18, IL‐18BPa and IL‐18Rα expressions in monocytes using flow cytometry, murine AR model and quantitative real‐time polymerase chain reaction (PCR). The results showed that the numbers of IL‐18 + monocytes increased, whereas IL‐18BPa + monocytes decreased in the peripheral blood of AR patients. It was also observed that Platanus pollen extract provoked elevated expressions of IL‐18 and IL‐18Rα in the monocytes of AR patients. House dust mite extract, Artemisia sieversiana wild extract and Platanus pollen extract enhanced IL‐18Rα protein and mRNA expression in the isolated primary monocytes from AR patients. Using ELISA kits, we observed that the levels of total IL‐18 and free IL‐18 in the plasma of perennial AR (pAR) and seasonal AR (sAR) patients were elevated, and the molar concentration ratio of free IL‐18BPa/free IL‐18 was 16.5 for healthy control subjects and 9.7 for patients with sAR, indicating that IL‐18 likely plays a role in sAR. In the murine AR model, the number of IL‐18Rα + monocytes increased in the blood, and the number of IL‐18Rα + macrophages increased in the nasal lavage fluid of WT mice. In conclusion, IL‐18 may serve as a causative factor for AR.

Increased expression of substance P (SP) and neurokinin-1 receptor (NK1R) has been noticed in patients with allergic rhinitis (AR) and allergic asthma (AA). However, little is known of the expression of SP and NK1R in monocytes and B cells of AR and AA. In the present study, the expression levels of SP and NK1R were determined by flow cytometry and mouse AR and AA models. The results showed that both percentages of SP+ monocytes and SP+ B cells, and mean fluorescence intensity (MFI) of SP in monocytes were elevated in the blood of AA and AR combined with AA (ARA) patients. Similarly, the percentages of NK1R+ monocytes were elevated in the blood of AR, AA, and ARA patients. Allergens Artemisia sieversiana wild allergen extract (ASWE), house dust mite extract (HDME), and Platanus pollen allergen extract (PPE) increased the expression density of SP molecules (determined by MFI) in an individual monocyte of AR patients. HDME and PPE appeared to enhance SP and NK1R expression in the B cells of ARA and AR patients. In the mouse AR and AA models, the percentages of NK1R+ monocytes and B cells were elevated in blood following OVA (ovalbumin) sensitization and challenge. Knocking out the FcεRI molecule completely abolished the OVA-induced upregulation of expression of NK1R in monocytes and B cells of AA mice. In conclusion, upregulated expressions of SP and NK1R may contribute to the pathogenesis of airway allergy.

Background: Recently, it has been reported that Toll-like receptor 7 (TLR7) agonists can improve allergic rhinitis (AR) symptoms by up-regulation of Th1 cytokine release and suppression of Th2 cell functions. However, little is known of the expression of TLR7 in basophils of AR.Objective: To explore the expression of TLR7 in basophils of AR, and influence of allergens on TLR7 expression.Methods: The expression levels of TLR7 in basophils of patients with AR were determined by flow cytometry, and the influence of allergens on TLR7 expression was examined by real time (q) PCR.Results: The percentages of TLR7+CCR3+ cells, TLR7+CD123+HLA-DR- cells and TLR7+CCR3+CD123+HLA-DR- cells in blood granulocyte and mononucleated cell populations of the patients with AR were increased, respectively compared with HC subjects. TLR7 MFI on CCR3+ cells, CD123+HLA-DR- cells and CCR3+CD123+HLA-DR- cells were enhanced. Allergens Der p1 and OVA provoked upregulation of TLR7 expression at both protein and mRNA levels and IL-13 production in KU812 cells. House Dust Mite extract (HDME), Artemisia sieversiana wild allergen extract (ASWE), IL-31, IL-33, IL-37, and TSLP provoked elevation of IL-6 release from KU812 cells following 2 h incubation period.Conclusions: The percentage of TLR7+ basophils and TLR7 expression intensity in a single basophil are both increased in the blood of patients with AR, indicating that basophils likely contribute to the pathogenesis of AR via TLR7. Trial Registration: Trial registry: Chinese clinical trial; registration number: ChiCTR-BOC-16010279.

It was reported that the expression of Toll‐like receptor (TLR) 9 may be related to Th2‐type allergic inflammation including allergic rhinitis (AR). However, little is known about the expression of TLR9 in the basophils in AR. In the present study, the expression of TLR9 was examined by flow cytometry analysis, and the expression of TLR9 mRNA in KU812 was determined by quantitative real‐time PCR. The results showed that the percentage of TLR9 + CCR3 + cells in blood granulocytes increased by 46% in patients with AR, but not in peripheral blood mononuclear cells (PBMCs). Allergens namely Dermatophagoide allergen extract (DAE) and Platanus pollen allergen extract (PPAE) upregulated the expression of TLR9 in CCR3 + granulocytes by 76% and 84%, respectively. DAE and PPAE also enhanced the proportions of TLR9 + CD123 + HLA‐DR − cells and TLR9 + CCR3 + CD123 + HLA‐DR − cells in granulocytes and PBMCs of patients with AR. In order to investigate the actions of allergens on basophils, KU812 cells were used. It was observed that all KU812 cells expressed TLR9, and the expression intensity of TLR9 in a single KU812 cell was elevated by CpG. IL‐37, IL‐31, IL‐33, Artemisia sieversiana wild allergen extract (ASWAE), DAE, OVA and Der p 1 induced an increase in the expression of TLR9 mRNA and IL‐6 production in KU812 cells. It was shown that the percentage of TLR9‐expressing basophils increased in the blood of ovalbumin (OVA)‐sensitized mice. In conclusion, an increased expression of TLR9 and the production of IL‐6 in basophils implicate that the contribution of basophils to AR is likely via TLR9.

目的 探讨鼻内镜高选择性鼻部神经阻断术对中重度变应性鼻炎(AR)患者的疗效及血清血管活性肠肽(VIP)表达的影响.方法 选取行鼻内镜高选择性鼻部神经阻断术治疗的中、重度AR患者30例,分别于术前、术后1年用症状VAS评分评估手术疗效,同时检测患者外周血血清中VIP浓度的变化,并分析两者的相关性.结果 与术前比较,鼻内镜高选择性鼻部神经阻断术后1年各症状VAS评分均下降,显效率为53.3％(16/30),有效率为46.7％(14/30);VIP水平明显降低[(18.42±0.64)pg/mL vs(39.89±1.30)pg/mL,P<0.05],且与VAS评分呈直线正相关(r2=0.77).结论 高选择性鼻部神经阻断术可有效改善中重度变应性鼻炎患者的临床症状,其机制可能与降低患者体内VIP的水平有关.

目的 探讨鼻内镜下高选择性鼻部神经(翼管神经鼻后神经丛+咽支+筛前神经分支)阻断术治疗中、重度变应性鼻炎的临床疗效.方法 回顾性分析于北部战区空军医院耳鼻喉科自2017年1月至2019年12月行高选择性鼻部神经阻断术的32例中、重度变应性鼻炎患者的临床资料.记录并比较患者术前、术后主观症状视觉模拟量表(VAS)评分、过敏性鼻炎鼻结膜炎生存质量(RQLQ)评分.结果 所有患者均顺利完成治疗和随访,随访率100.0％(32/32),均无严重并发症发生.与术前比较,术后3个月与术后2年患者的主观症状VAS评分及生活质量RQLQ评分均显著降低,差异均有统计学意义(P<0.05).术后2年的整体有效率为87.5％(28/32),低于术后3个月的100.0％(32/32),但差异无统计学意义(P>0.05).结论 鼻内镜下高选择性鼻部神经阻断术治疗中、重度变应性鼻炎患者临床效果良好,是一种安全、有效的治疗方式.

MicroRNAs(miRNAs) are maladjusted in multifarious malignant tumor and can be considered as both carcinogens and tumor-inhibiting factor. In the present study, we analyzed the miRNAs expression profiles and clinical information of 481 patients with head and neck squamous cell carcinoma (HNSCC) through the TCGA dataset to identify the prognostic miRNAs signature. A total of 114 significantly differentially expressed miRNAs (SDEMs) were identified, consisting of 60 up-adjusted and 54 down-adjusted miRNAs. The Kaplan-Meier survival method identified the prognostic function of 2 miRNAs (miR-4652-5p and miR-99a-3P). Univariate and multivariate Cox regression analyses indicated that the 2 miRNAs were significant prognostic elements of HNSCC. Furthermore, bioinformatic analysis was conducted by means of 4 online gene predicted toolkits to recognize the target genes, and enrichment analysis was performed on the target genes by DAVID. The outcomes depicted that target genes were correlated with calcium, as well as cell proliferation, circadian entrainment, EGFR, PI3K-Akt-mTOR, and P53 signaling pathways. Finally, the PPI network was conducted in view of STRING database and Cytoscape. Eight hub genes were identified by CytoHubba and MCODE app, respectively, CBL, SKP1, H2AFX, HGF, POLR2F, UBE2I, VAMP2, and GNAI2 genes. As a result, we identified 2 miRNAs signatures, 8 hub genes, and significant signaling pathways for estimating the prognosis of HNSCC. In order to further explore the molecular mechanism of HNSCC occurrence and development, more comprehensive basic and clinical studies are needed.

Background: Interleukin (IL)-18 is emerging as an attractive participant in allergic rhinitis (AR). However, correlation of IL-18 with IL-18 binding protein (BP) in plasma, and expression of IL-18, IL-18BP and IL-18 receptor (R) in AR blood monocytes remain obscure. We therefore investigated IL-18, IL-18BP and IL-18R expression in monocytes using flow cytometric analysis, murine AR model, and quantitative real-time PCR in the present study. Results: Plasma IL-18 and IL-1β in AR patients was higher than those in healthy control subjects. Free (f)IL-18 had a high correlation with IL-18BP, IL-1β and TNF-α in AR plasma. Proportion of IL-18 + monocytes was increased, whereas IL-18BP + monocytes were decreased in blood of patients with AR. It was found that Platanus pollen allergen extract provoked the elevated expression of IL-18 and IL-18R in AR blood monocytes. Dermatophagoides pteronyssinus , Artemisia sieversiana wild and Platanus pollen allergen extracts enhanced IL-18R protein and mRNA expression in isolated primary monocytes from AR patients. Moreover, numbers of macrophages and IL-18R + macrophages in nasal lavage fluid (NLF) were increased, and levels of IL-18 in both plasma and NLF were elevated in AR mice. Conclusions: These suggest that IL-18 is likely to participate in the development of AR as a causative factor; therefore, it could be a therapeutic target for AR.

目的 研究过敏性鼻炎(allergic rhinitis,AR)患者外周血滤泡性辅助T细胞(follicular helperT cells,Tfhs)中可诱导共刺激分子(inducible co-stimulator molecule,ICOS)的表达情况及ICOS阻断剂对AR患者外周血Tfhs IL-21表达的影响.方法 收集AR患者和健康人(healthy controls,HCs)的外周血,流式细胞仪检测HCs和AR患者外周血中Tfhs ICOS表达水平及ICOS阻断剂对AR患者外周血Tfhs IL-21表达的影响.结果 AR患者外周血中Tfhs的数量和ICOS+ Tfhs的比例显著高于HCs(P＜0.05).此外,ICOS阻断剂可显著抑制AR患者外周血Tfhs分泌IL-21(P＜0.05).结论 AR患者外周血Tfhs的数量及ICOS表达升高,ICOS阻断剂可抑制Tfhs分泌IL-21,提示Tfhs可能通过ICOS相关机制参与AR发病.

MicroRNA-335 (miR-335) is implicated in several pathophysiological processes, including tumorigenesis, lipid metabolism and ischemic stroke; however, whether miR-335 plays a role in modulating myocardial ischemia reperfusion injury (MIRI) is still unknown. This study is aimed to explore the role and mechanism of miR-335 in the pathophysiological process of MIRI. Specifically, miR-335 mimics or a chemically modified agomiR-335 were transfected or injected into H9c2 cells and Wistar rats to upregulate miR-335 expression in vitro and in vivo, respectively. The effects of miR-335 overexpression on hypoxia/reoxygenation (H/R)-treated cardiomyocytes and ischemia/reperfusion (I/R)-exposed heart samples were investigated by a Cell Counting Kit-8 assay, flow cytometry, TTC staining and a TUNEL assay. The target of miR-335 was identified using a luciferase reporter assay. The expression of heme oxygenase 1 (HO-1) and inducible nitric oxide synthase (iNOS) was detected by reverse transcription-quantitative polymerase chain reaction and western blotting. The results showed that miR-335 expression in cardiomyocytes and the myocardium was downregulated during MIRI but was induced by hypoxic/ischemic postconditioning. MiR-335 overexpression led to an increase in cell viability and a reduction in the apoptosis of H/R-treated cardiomyocytes. Meanwhile, myocardial infarct size and the apoptosis of I/R-exposed heart tissues were decreased in response to miR-335 upregulation. Furthermore, we identified that hypoxia inducible factor 1-alpha subunit inhibitor (HIF1AN), a suppressor of hypoxia inducible factor 1-alpha (HIF-1α) stabilization and transcriptional activity, is a novel target of miR-335. MiR-335 overexpression enhanced the transcriptional activity of HIF-1α, increased the expression of HO-1 and iNOS, and inhibited mitochondrial permeability transition pore (MPTP) opening. In conclusion, we are the first to demonstrate that upregulation of miR-335 ameliorates MIRI by targeting HIF1AN. Thus, miR-335 may be a new therapeutic target for the treatment of MIRI.

The aim of the present study was to observe the effects of cytokine signaling suppressor 1 (SOCS1)-silenced dendritic cells (DCs) pulsed with epidermal growth factor receptor (EGFR) fusion protein on the activation of T lymphocyte and cytotoxic T-lymphocyte (CTL) activity against Hep-2 cells. DCs were derived from the medullary cells of mice and authenticated by flow cytometry (FCM). Recombinant glutathione-S-transferase (GST)-EGFR fusion protein was produced and purified. After being pulsed with it, DCs were modified by recombinant SOCS1-siRNA adenoviral to silence SOCS1 gene expression. The maturation of DCs was evaluated by FCM. The effects of modified DCs on T-cell proliferation were assessed by MTT assay. The killing effects against Hep-2 cells of CTL were assessed by lactate dehydrogenase (LDH) release assay. High-purity DCs from the medullary cells of mice were obtained. Compared with the control, EGFR-pulsed DCs displayed higher expression of cell surface molecules, including CD83, CD860 and HLA-DR. The MTT assay revealed that all of the EGFR-pulsed, SOCS1‑silenced and EGFR-pulsed plus SOCS1-silenced DCs had an enhanced capacity to stimulate T-lymphocyte proliferation. As expected, EGFR-pulsed plus SOCS1-silenced DCs had the strongest effects on T-cell proliferation. The splenic T cells isolated from both EGFR-pulsed DC-immunized mice and EGFR-pulsed plus SOCS1-silenced DC-immunized mice enhanced the cytotoxicity against Hep-2 cells, while T cells from EGFR‑pulsed plus SOCS1-silenced DC-immunized mice exhibited significantly higher cytotoxicity than those from EGFR-DC-immunized mice. The EGFR-pulsed SOCS1‑siRNA-silenced DCs had the strongest effects on activation of T-cell proliferation and the CTL activity against Hep-2 cells.

A rapid, specific and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determination of isofraxidin, rosmarinic acid and kaempferol-3-O-glucuronide in rat plasma using warfarin as an internal standard (IS). Separation was conducted on a Thermo Hypersil GOLD C18 column with linear gradient elution using methanol and water. Mass spectrometric detection was conducted using selected reaction monitoring (SRM) via an electrospray ionization (ESI) source. All analytes exhibited good linearity within their concentration ranges (r > 0.9990). The lower limits of quantitations of isofraxidin, rosmarinic acid, and kaempferol-3-O-glucuronide were 1.31, 0.67 and 0.92 ng/mL, respectively. Intra- and inter-day precisions of these investigated components exhibited an RSD within 11.7%, and the accuracy ranged from -12.5 to 15.0% at all QC levels. The developed method was successfully applied to a pharmacokinetic study of isofraxidin, rosmarinic acid, and kaempferol-3-O-glucuronide in rats after oral administration of Herba Sarcandrae Extract.