Involucrasin B suppresses airway inflammation in obese asthma by inhibiting the TLR4-NF-κB-NLRP3 pathway.

BACKGROUND: Obese asthma is an asthma phenotype that causes more severe lung inflammation and airway hyperresponsiveness than allergic asthma and it is resistant to conventional therapy. Involucrasin B (IB) is a dihydroflavonoid isolated from Shuteria involucrata (Wall.) Wight & Arn., a traditional "Dai" and "Wa" medicine was used in southern China to treat the "phlegm and wetness of sputum" (obesity disease) as well as lung inflammation. However, whether IB can ameliorate obese asthma remains unclear, and the underlying mechanisms and molecular expression in obese asthma specifically targeted by IB are still not fully understood. METHODS: An in vivo C57BL/6 J mouse model of obese asthma was established using house dust mites (HDMs) and high-fat diet (HFD) as inducers to evaluate the therapeutic effect of IB. An in vitro cell culture of human THP-1 monocytic cell culture was used to investigate the effect of IB after the treatment with lipopolysaccharide (LPS) and palmitic acid (PA). RESULTS: In vivo, we found that intervention with IB improved airway hyperresponsiveness and lung histopathology and significantly inhibited the secretion of relevant inflammatory factors, such as interleukin (IL)-1ß, IL-17A, and IL-22 in bronchoalveolar lavage fluid, and total-IgE and HDM-IgE in serum compared with the model group (HFD+HDM). The findings indicate that IB could decrease the expression of granulocyte receptor 1 (Gr-1) and neutrophil extracellular traps (NETs) in lung tissue, as well as the expression of NLR family pyrin domain containing 3 (NLRP3) and inducible nitric oxide synthase in M1 macrophages (M1). IB also reduced the population of ILC3/Th17 cells, which are responsible for producing IL-17A, a crucial mediator of neutrophil-mediated inflammation, confirming that the therapeutic effect of IB in obesity-related asthma was related to neutrophils and M1 cells. In addition, IB regulated lipid metabolism and inhibited the production of macrophages in adipose tissue. The in vitro results revealed that IB inhibited the secretion of IL-1ß, IL-18, and tumor necrosis factor-α (TNF-α) from THP-1 cells, and the expression of NLRP3-related protein in THP-1 cells compared with the model groups (LPS, PA, and LPS+PA), confirming that the action of IB involved the TLR4-NF-κB-NLRP3 pathway. CONCLUSION: This study demonstrated the therapeutic effect of IB in obese asthma for the first time and further clarified its mechanistic pathway as the TLR4-NF-κB-NLRP3 pathway.

Acupuncture Therapy Modulating "Du" Channel Attenuates Ischemic Stroke-induced Disorders by Modulating REST-mediated miR-21/PDCD4 Signaling Transduction.

Acupuncture is a traditional Chinese therapy with treating potential against cognitive dysfunction. MicroRNA-21-3p (miR-21-3p) is well characterized for its benefits on neural tissues. The current study hypothesizes that the acupuncture aiming "Du" channel could attenuate IS-induced neural disorders by modulating the function of REST/miR-21-3p axis. Complications associated with IS are induced by a middle cerebral artery occlusion (MCAO) model in vivo. The disorders are then handled with the acupuncture with nimodipine as the positive control. It is found that the acupuncture improved cognitive function, reduced brain apoptosis, and increased the viable neuron number of model rats. Additionally, the production of cytokines is also suppressed by the acupuncture. At the molecular level, the level of miR-21-3p was up-regulated, while the level of REST was down-regulated by the acupuncture. The changes in miR-REST/21-3p contributed to the inhibition of PDCD4. Collectively, the findings in the current study highlight that miR-21-3p is associated with the anti-IS function of the acupuncture, which is mediated by the inhibition of REST.

Identifying the origin of Laggera crispata (Vahl) Hepper & J. R. I. Wood based on UPLC fingerprinting and chemometrics.

In this study, thirty-four samples of Laggera crispata (Vahl) Hepper & J. R. I. Wood from five main production areas in Yunnan Province, were collected for experimentation. UPLC- PDA was used to generate fingerprints and the common peaks were analysed through R and SIMCA-P. L. crispata from different origins can be distinguished by OPLS-DA and PCA. The VIP values were compared, and 8 characteristic components with great differences were obtained. It was confirmed that the two characteristic components were chrysosplenetin and artemisetin, and the quantitative analysis was performed with these two compounds from L. crispata samples with different origins. Based on the variance analysis results, the most significant difference in the content of chrysosplendin and artemisin was in Lancang and Honghe and Lancang and Simao, respectively. The chrysosplenetin can be used as an important indicator for quality control and to trace the origin of L. crispata.

Baiheqingjin formula reduces inflammation in mice with asthma by inhibiting the PI3K/AKT/NF-κb signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Baiheqingjin Decoction (BHQJ), which consists of 7 traditional Chinese herbs including Baibu (Stemona tuberosa Lour.), Hezi (Terminalia chebula Retz.), Mahuang (Ephedra sinica Stapf.), Ziwan (Aster tataricus L. f.), Dilong (Pheretima), Sangbaipi (Morus alba L.), and Xianhecao (Agrimonia pilosa Ledeb.). BHQJ is commonly used for treating cough asthma, and variant cough-variant asthma as it, is effective in improving asthma symptoms and reducing airway inflammation. AIM OF THE STUDY: To investigate the mechanisms of BHQJ in treating allergic asthma. MATERIALS AND METHODS: We collected information about the components and targets of 6 Chinese medicines (excluding Pheretima) from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Additionally, we obtained genes associated with asthma from six disease databases. To create a protein-protein interaction network, we conducted an intersection analysis using differentially expressed genes derived from RNA transcriptome data. Subsequently, we carried out Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. To validate the findings from network pharmacology and transcriptomics, we established an allergic asthma mouse model induced by ovalbumin and conducted in vivo experiments. RESULTS: Using network pharmacology and transcriptomics analyses, we identified the pathways including the PI3K/AKT signaling pathway, and NF-κB signaling pathway. Among these, the involvement of the PI3K/AKT/NF-κB signaling pathway in various pathological processes of asthma, such as airway inflammation, smooth muscle contraction, and excessive mucus production, are well-documented. Histopathological examinations indicated that BHQJ had the potential to mitigate inflammatory cell infiltration and the excessive growth of goblet cells in the airways of asthmatic mice, consequently reducing mucus secretion. Results from Western blot demonstrated that BHQJ could inhibit the activation of the PI3K/AKT/NF-κB pathway at the protein levels. Enzyme-linked immunosorbent assay findings revealed that BHQJ could reduce the production of typical "type 2 asthma" cytokines and immunoglobulin (Ig) E in the blood. These discoveries imply that BHQJ has the potential to reduce the release of inflammatory cytokines and suppress the overactivation of the PI3K/AKT/NF-κB signaling pathway, thus offering a therapeutic approach for asthma. CONCLUSION: Our research offers initial insights into the fundamental mechanisms through which BHQJ treats asthma. This study reveals the potential mechanism of BHQJ in treating asthma, particularly its role in reducing inflammatory cytokines, mucus production, and cell infiltration, as well as inhibiting the expression of PI3K/AKT/P65 phosphorylated protein. These findings indicate the potential of BHQJ in treating asthma. In summary, our study provides preliminary insights into the asthma treatment mechanism of BHQJ and provides guidance for future research.

Inhibitory effects of catalpol on DNCB-induced atopic dermatitis and IgE-mediated mast cells reaction.

Atopic dermatitis (AD) is a chronic, inflammatory cutaneous disease driven by immune dysregulation. Catalpol is an iridoids, possessing anti-inflammatory, antioxidant, and neuroprotective activities. It can be added to food as a dietary supplement. To evaluate the effects and mechanisms of catalpol on AD, both in vitro and in vivo studies were conducted. It was found that catalpol downregulated the phosphorylation of Lyn and Syk to inhibit various downstream pathways, including intracellular Ca2+ elevation, cytokines generation, and histamine release, which ultimately controlled mast cell (MCs) degranulation. The results showed that catalpol alleviated AD-like skin lesions and MC infiltration via regulation of pro-Th2 and Th2 cytokines in vivo. Furthermore, this compound reduced the levels of IgE in AD mice and improved allergic reactions in PCA mice. The results provided that catalpol was potentially developed as a dietary supplement to improve AD and other atopic diseases.

Daphnetin contributes to allergen-induced Th2 cytokine expression and type 2-immune responses in atopic dermatitis and asthma.

Daphne koreana Nakai is a cherished medicinal plant in the Changbai Mountain region of China. It can be incorporated into medicinal meals and used for various skin diseases by infiltrating liquor. Daphnetin (7,8-dihydroxycoumarin, Dap.) is a main constituent of D. koreana Nakai, which has been used to treat inflammatory conditions and immune disorders due to its numerous pharmacological activities, including anti-oxidant, anti-inflammatory, analgesic, etc. Atopic dermatitis (AD) and allergic asthma are typical diseases of type 2-immune responses. In the present study, the therapeutic potential of Dap. against AD and allergic asthma was investigated using animal and cell experiments. AD-like lesions were induced by repeated application of 1-chloro-2,4-dinitrobenzene (DNCB) to the shaved dorsal skin of BALB/c mice. Ovalbumin (OVA) induction was utilized to establish a mouse asthma model. A passive cutaneous anaphylaxis (PCA) mouse ear model and immunoglobulin E (IgE)/bovine serum albumin (BSA)-stimulated RBL-2H3 cells were used for in vitro assays. The skin lesions and serum and tissue homogenates of the mice were analyzed using histological analysis, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), respectively, in order to investigate the anti-AD effects of Dap. Histological analysis was performed on the allergic asthma model to observe inflammatory cell infiltration in the lung tissues. Total IgE and OVA-specific IgE in the serum were measured by ELISA. The levels of inflammatory cytokines in BALF were detected by ELISA. In addition, ELISA and western blotting were performed for the in vitro analysis of RBL-2H3 cells. The results showed that Dap. inhibited the development of DNCB-induced AD-like lesions in the BALB/c mice by reducing the severity of the lesions, epidermal thickness and mast cell infiltration; this was accompanied by reduced levels of IgE and inflammatory cytokines [interleukin (IL)-4, IL-5, IL-9, IL-13, IL-33 and thymic stromal lymphopoietin (TSLP)]. In the allergic asthma model, Dap. reduced the number of infiltrated inflammatory cells in the lung tissues. Moreover, the levels of total serum IgE and OVA-specific IgE were reduced in the high daphnetin dose groups (Dap., -100 mg kg-1). Dap. administered at a dose of -100 mg kg-1 decreased the levels of inflammatory cytokines (IL-4, IL-5, IL-9, IL-13, IL-33 and TSLP in BALF). Furthermore, Dap. administered to IgE-sensitized mice effectively attenuated the IgE-triggered PCA reaction. In vitro, Dap. decreased the expression levels of histamine, IL-4, IL-6, IL-13, MIP-1α and INF-α, and reduced the protein expression levels of phosphorylated MAPKs, P-Lyn and P-syk in the RBL-2H3 cells. Therefore, Dap. can be represented as a potential therapeutic strategy for the treatment of allergic inflammatory conditions via immunoregulation.

Baicalin Mitigates the Neuroinflammation through the TLR4/MyD88/NF-κB and MAPK Pathways in LPS-Stimulated BV-2 Microglia.

Baicalin (BA) is a major flavone from Scutellaria baicalensis Georgi and has showed significant curative effects in Parkinson's and Alzheimer's diseases. In the present study, we investigated the effects of BA on antineuroinflammation and related signaling cascade in lipopolysaccharide- (LPS-) induced BV-2 microglial model. The results showed that BA significantly attenuated inflammatory mediators (NO, iNOS, IL-1ß, COX-2, and PGE2) and suppressed the expression of miR-155. More crucially, BA could regulate the expression of related proteins in Toll-like receptor 4 (TLR4)/myeloid differentiation protein 88 (MyD88)/nuclear factor κB (NF-κB) pathway and suppress the phosphorylation of mitogen-activated protein kinase (MAPK) family. In addition, molecular docking analysis indicated that BA binds to the amino acids Lie 63 and Tyr 65 of TLR4 by π-σ and π-π T-shaped interaction. Thus, BA suppressed the LPS-stimulated neuroinflammation in BV-2 microglia by blocking the TLR4-mediated signal transduction through TLR4/MyD88/NF-κB and MAPK pathways and inhibiting the miR-155 expression. Our findings demonstrated that BA could be a valuable therapeutic for the treatment of neuroinflammation and neurodegenerative diseases.

Vasicine alleviates 2,4-dinitrochlorobenzene-induced atopic dermatitis and passive cutaneous anaphylaxis in BALB/c mice.

Atopic dermatitis (AD), a type of skin inflammation, is associated with immune response mediated by T-helper 2 (Th2) cells, and mast cells. Vasicine is an alkaloid isolated from Adhatoda vasica, a popular Ayurvedic herbal medicine used for treating inflammatory conditions. In the present study, the anti-AD effects of vasicine were evaluated on 2,4-dinitrochlorobenzene-induced AD-like skin lesions in BALB/c mice. The potential anti-allergic effects of vasicine were also assessed using the passive cutaneous anaphylaxis (PCA) test. The results showed that the oral administration of vasicine improved the severity of AD-like lesional skin by decreasing histopathological changes and restoring epidermal thickness. Vasicine also inhibited the infiltration of mast cells in the skin and reduced the levels of pro-Th2 and Th2 cytokines as well as immunoglobulin E in the serum. Finally, vasicine inhibited the expression of pro-Th2 and Th2 cytokines in skin tissues, indicating the therapeutic potential of vasicine for AD.

Therapeutic Effect of Renifolin F on Airway Allergy in an Ovalbumin-Induced Asthma Mouse Model In Vivo.

Renifolin F is a prenylated chalcone isolated from Shuteria involucrata, a traditional minority ethnic medicine used to treat the respiratory diseases and asthma. Based on the effects of the original medicine plant, we established an in vivo mouse model of allergic asthma using ovalbumin (OVA) as an inducer to evaluate the therapeutic effects of Renifolin F. In the research, mice were sensitized and challenged with OVA to establish an allergic asthma model to evaluate the effects of Renifolin F on allergic asthma. The airway hyper-reactivity (AHR) to methacholine, cytokine levels, ILC2s quantity and mircoRNA-155 expression were assessed. We discovered that Renifolin F attenuated AHR and airway inflammation in the OVA-induced asthmatic mouse model by inhibiting the regulation of ILC2s in the lung, thereby, reducing the upstream inflammatory cytokines IL-25, IL-33 and TSLP; the downstream inflammatory cytokines IL-4, IL-5, IL-9 and IL-13 of ILC2s; and the co-stimulatory factors IL-2 and IL-7; as well as the expression of microRNA-155 in the lung. The findings suggest a therapeutic potential of Renifolin F on OVA-induced airway inflammation.

Molecular Mechanism of Crataegi Folium and Alisma Rhizoma in the Treatment of Dyslipidemia Based on Network Pharmacology and Molecular Docking.

Background: Dyslipidemia has become a critical global issue for public health, with elevating prevalence and morbidity closely related to many cardiovascular diseases (CVD) with high incidence rates. Crataegi Folium (known as Shanzhaye in China, SZ, the leaves of Crataegus pinnatifida Bge. var. major N.E. Br. or Crataegus pinnatifida Bge) and Alisma rhizoma (known as Zexie in China, ZX, the dried tuber of Alisma orientale (Sam.) Juzep or Alisma plantago-aquatica Linn), a classic combination of herbs, have been widely used to treat dyslipidemia. However, the therapeutic mechanism of this pair still remains unclear. Hence, this study aimed to elucidate the molecular mechanism of the Shanzhaye-Zexie herb pair (SZHP) in the treatment of dyslipidemia with the use of a network pharmacology analysis approach. Methods: Active compounds, targets of the SZHP, and targets for dyslipidemia were screened based on the public database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed on the database for annotation, visualization, and integrated discovery (DAVID 6.8). The compound-target-disease-pathway network was visualized using the Cytoscape software, and SYBYL was used for molecular docking. Results: Twelve active compounds in the SZHP were screened out, which were closely connected to 186 dyslipidemia-related targets. The network analysis revealed that sitosterol, stigmasterol, isorhamnetin, kaempferol, and quercetin might be candidate agents and CCND1, CASP3, HIF1A, and ESR1 genes were potential drug targets. GO analysis revealed 856 biological processes (BP), 139 molecular functions (MF), and 89 cellular components (CC). The KEGG pathway enrichment analysis indicated that the lipid level and atherosclerosis might influence the treatment of dyslipidemia. Molecular docking showed that quercetin bound well to CCND1, HIF1A, MYC, AKT1, and EGFR genes. These findings were in accord with the prediction obtained through the network pharmacology approach. Conclusions: This study revealed the primary pharmacological effects and relevant mechanisms of the SZHP in treating dyslipidemia. Our findings may facilitate the development of the SZHP or its active compounds as an alternative therapy for dyslipidemia. Still, more pharmacological experiments are needed for verification.

[Consensus on collaborative ethical review of multi-center clinical trials of new drugs of traditional Chinese medicine (version 1.0)].

At present, the issues regarding multi-center clinical trials of new drugs of traditional Chinese medicine(TCM) remain: the lack of agreement on the content and scope of the ethical review among the ethics committee members of the center and the participating units results in repeated review, which leads to a time-consuming ethical review process. Moreover, the review capabilities of the ethics committees of various research centers are uneven, which is not necessarily beneficial to the protection of subjects' rights and safety. In view of the existing problems, to improve the efficiency of ethical review of multi-center clinical trials of new drugs of TCM and avoid repeated reviews, the TCM Clinical Evaluation Professional Committee of Chinese Pharmaceutical Association organized experts to formulate the "Consensus on collaborative ethical review of multi-center clinical trials of new drugs of TCM(version 1.0)"(hereinafter referred to as "Consensus"). The "Consensus" is formulated in accordance with the requirements of relevant documents such as but not limited to "the opinions on deepening the reform of the evaluation and approval system to encourage the innovation of pharmaceutical medical devices", "the regulations of ethical review of biomedical research involving human subjects". The "Consensus" covers the scope of application, formulation principles, conditions for the ethics committee of the center, sharing of ethical review resources, scope and procedure of collaborative review, rights and obligations, etc. The aims of the "Consensus" is to preliminarily explore and establish a scientific and operable ethical review procedure. Additionally, on the basis of fully protecting the rights and interests of the subjects, a collaborative ethical review agreement needs to be signed to clarify the ethical review responsibilities of all parties, to avoid repeated review, and to improve the efficiency and quality of ethical review in multi-center clinical trials of new drugs of TCM.

A novel TSC1 frameshift mutation c.1550_1551del causes tuberous sclerosis complex by aberrant splicing and nonsense-mediated mRNA degradation (NMD) simultaneously in a Chinese family.

BACKGROUND: Tuberous sclerosis complex (TSC), belongs to autosomal dominant genetic disorder, which affects multiple organ systems in the body, including the skin, brain, lungs, kidneys, liver, and eyes. Mutations in TSC1 or TSC2 was proved to be associated with these conditions. METHODS: Gene-panel Sequence of NGS was used to detect the mutation in a Chinese family. The research further investigates whether aberrant splicing and nonsense-mediated mRNA degradation (NMD) could serve as a mechanism cause by TSC1 mutation. MINI-Gene assay apply by pcMINI-TSC1wt/mut plasmids delivered in HeLa and 293T cell lines. Recombinant plasmids expressing wild-type and mutant-type EGFP-TSC1 were constructed and transiently transfected into human embryonic kidney cells 293T by lipofectamine. Real-time PCR and Western Blot were performed to analyze the expression of mRNAs and proteins of EGFP-TSC1 and NMD factor UPF1. RESULTS: The gene test verified a novel heterozygous TSC1 frameshift mutation (TSC1 c.1550_1551del) in the proband and her mother. From MINI-Gene assay, the agarose gel showed that both the mutant and wild-type mRNA possess two main bands, indicating two splicing modes, named band A and B, respectively. The mutation c.1550_1551del has not produced new splicing site, but there is a selective splicing in varying degree significantly after mutation. On the contrary, function validation assay showed that cells transfected with the mutant TSC1 plasmids expressed significantly lower TSC1 in mRNAs and proteins levels, compared with the wild-type TSC1 plasmid transfection. A translation inhibitor cycloheximide and small interfering RNA of UPF1 (siRNA-UPF1) increased mRNA or protein expression of TSC1 significantly in cells transfected with the mutant plasmids. CONCLUSION: Our study demonstrated that the novel TSC1 frameshift mutation (TSC1 c.1550_1551del) trigger aberrant splicing and NMD simultaneously, causing decrease of hamartin, then, leading to tuberous sclerosis complex formation.

Tranexamic Acid Inhibits Angiogenesis and Melanogenesis in Vitro by Targeting VEGF Receptors.

Melasma is a common but complex skin condition concerning cosmetic problems. Tranexamic acid (TA) has been proved to be effective in treatment of melasma with still unclear mechanisms. Here, we show that VEGF165 enhanced the expression of VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2 and NRP-1) in human umbilical vein endothelial cells (HUVECs), which was attenuated by TA. VEGF165 also promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in HUVECs, which was again abolished by TA. TA further showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting cell proliferation, migration, invasion and tube formation of HUVECs induced by VEGF165, suggesting that TA could inhibit angiogenesis by targeting VEGFRs in HUVECs. In addition, VEGF165 enhanced the expression of VEGFRs and promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2 in normal human melanocytes, which were also attenuated by TA. Furthermore, TA showed similar effects to neutralization of VEGFR-1 and VEGFR-2 in inhibiting tyrosinase activity, melanin production and even melanogenic proteins induced by VEGF165, suggesting that TA could reduce melanogenesis via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that TA can inhibit angiogenesis and melanogenesis in vitro at least in part by targeting VEGFRs, which may offer a new understanding of the pathogenesis of melasma as well as the molecular mechanism for TA in treatment of the disease.

Activation of VEGF receptors in response to UVB promotes cell proliferation and melanogenesis of normal human melanocytes.

VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and signaling via VEGFRs extends beyond the classical roles in blood vessel formation. We previously showed VEGFRs were also expressed in epidermal keratinocytes and activation of VEGFR-2 by ultraviolet B (UVB) was involved in the pro-survival mechanism. Here, we show that both VEGF165 and UVB enhanced the expression of VEGFRs (including VEGFR-1, VEGFR-2 and NRP-1) in normal human melanocytes, and increased expression of VEGFRs by UVB was mediated through hypoxia and oxidative stress. Also, VEGF165 and UVB promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2, and UVB-induced phosphorylation of VEGFR-1 and VEGFR-2 required PKA but not P38 MAPK. In addition, UVB and VEGF165 contributed to the over-expression of melanogenic proteins in melanocytes, which could be reduced by neutralization of VEGFR-1 and/or VEGFR-2. UVB, but not VEGF165 promoted cell proliferation, while neutralization of VEGFR-1 and/or VEGFR-2 abolished this effect. UVB showed stronger than VEGF165 in promoting tyrosinase activity and melanin production, while neutralization of VEGFR-2 was stronger in reducing these effects than that of VEGFR-1. Furthermore, tranexamic acid (TA) decreased tyrosinase activity and melanin production via inhibiting activation of VEGFRs and subsequent expression of melanogenic proteins in melanocytes. Taken together, we demonstrate that VEGFRs are functionally involved in UVB-induced melanogenesis, and TA can inhibit melanogenesis at least in part by targeting VEGFRs in melanocytes.

Correction: Ruxolitinib sensitizes ovarian cancer to reduced dose Taxol, limits tumor growth and improves survival in immune competent mice.

[This corrects the article DOI: 10.18632/oncotarget.21541.].

Ruxolitinib sensitizes ovarian cancer to reduced dose Taxol, limits tumor growth and improves survival in immune competent mice.

Background: Chemotherapy initially reduces the tumor burden in patients with ovarian cancer. However, tumors recur in over 70% of patients, creating the need for novel therapeutic approaches. Methods: We evaluated Ruxolitinib, an FDA-approved JAK 1/2 kinase inhibitor, as a potential adjunctive therapy for use with low-dose Taxol (Paclitaxel) by assessing the impact on in vitro proliferation and colony formation of ID8 cells or human TOV-112D ovarian cancer cells, as well as flow cytometric measurement of surface markers associated with cellular stress and stemness by ID8 cells. The syngeneic ID8 murine model of ovarian cancer was used to assess the impact of Ruxolitinib and Taxol, individually and in combination, on tumor initiation and growth, as well as capacity to extend survival. Results: Ruxolitinib (≤10 µM) sensitized both ID8 and TOV-112D cells to low concentrations of Taxol (≤5 nM), limiting cell proliferation and colony formation in vitro. Mechanistically, we demonstrated that Taxol induced expression of stress and stemness markers including GRP78 and CD133 was significantly reduced by addition of Ruxolitinib. Finally, we demonstrated that a single administration of a low-dose of Taxol (10 mg/Kg) together with daily Ruxolitinib (30 mg/Kg; which is equivalent to plasma concentrations of ∼ 0.01 µM steady-state) limited ID8 tumor growth in vivo and significantly extended median survival up to 53.5% (median 70 v 107.5 days) as compared to control mice. Conclusion: Together, these data support the use of Ruxolitinib in combination with low-dose Taxol as a therapeutic approach with the potential for improved efficacy and reduced side effects for patients with recurrent ovarian cancer.

[Generalized interaction LASSO based on alternating direction method of multipliers for liver disease classification].

Features and interaction between features of liver disease is of great significance for the classification of liver disease. Based on least absolute shrinkage and selection operator (LASSO) and interaction LASSO, the generalized interaction LASSO model is proposed in this paper for liver disease classification and compared with other methods. Firstly, the generalized interaction logistic classification model was constructed and the LASSO penalty constraints were added to the interactive model parameters. Then the model parameters were solved by an efficient alternating directions method of multipliers (ADMM) algorithm. The solutions of model parameters were sparse. Finally, the test samples were fed to the model and the classification results were obtained by the largest statistical probability. The experimental results of liver disorder dataset and India liver dataset obtained by the proposed methods showed that the coefficients of interaction features of the model were not zero, indicating that interaction features were contributive to classification. The accuracy of the generalized interaction LASSO method is better than that of the interaction LASSO method, and it is also better than that of traditional pattern recognition methods. The generalized interaction LASSO method can also be popularized to other disease classification areas.

Paeonol Suppresses Neuroinflammatory Responses in LPS-Activated Microglia Cells.

In this work, we assessed the anti-inflammatory effects of paeonol (PAE) in LPS-activated N9 microglia cells, as well as its underlying molecular mechanisms. PAE had no adverse effect on the viability of murine microglia N9 cell line within a broad range (0.12∼75 µM). When N9 cell line was activated by LPS, PAE (0.6, 3, 15 µM) significantly suppressed the release of proinflammatory products, such as nitric oxide (NO), interleukin-1ß (IL-1ß), and prostaglandin E2 (PGE2), demonstrated by the ELISA assay. Moreover, the levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were significantly reduced in PAE-treated N9 microglia cells. We also examined some proteins involved in immune signaling pathways and found that PAE treatment significantly decreased the expression of TLR4, MyD88, IRAK4, TNFR-associated factor 6 (TRAF6), p-IkB-α, and NF-kB p65, as well as the mitogen-activated protein kinase (MAPK) pathway molecules p-P38, p-JNK, and p-ERK, indicating that PAE might act on these signaling pathways to inhibit inflammatory responses. Overall, we found that PAE had anti-inflammatory effect on LPS-activated N9 microglia cells, possibly via inhibiting the TLR4 signaling pathway, and it could be a potential drug therapy for inflammation-associated neurodegenerative diseases.

A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma.

BACKGROUND: Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer, their production and administration is costly and labor-intensive. We developed a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting mesothelin (MSLN), which is overexpressed on ovarian cancer and mesothelioma cells, to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is a potent immune activator that stimulates monocytes and DCs, enhances DC aggregation and maturation and improves cross-priming of T cells mediated by DCs. METHODS: Binding of this fusion protein with MSLN on the surface of tumor cells was measured by flow cytometry and fluorescence microscopy. The therapeutic efficacy of this fusion protein was evaluated in syngeneic and orthotopic mouse models of papillary ovarian cancer and malignant mesothelioma. Mice received 4 intraperitoneal (i.p.) treatments with experimental or control proteins post i.p. injection of tumor cells. Ascites-free and overall survival time was measured. For the investigation of anti-tumor T-cell responses, a time-matched study was performed. Splenocytes were stimulated with peptides, and IFNγ- or Granzyme B- generating CD3+CD8+ T cells were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo CD8+ cell depletion. We further determined if the fusion protein increases DC maturation and improves antigen presentation as well as cross-presentation by DCs. RESULTS: We demonstrated in vitro that the scFvMTBHsp70 fusion protein bound to the tumor cells used in this study through the interaction of scFv with MSLN on the surface of these cells, and induced maturation of bone marrow-derived DCs. Use of this bifunctional fusion protein in both mouse models significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T-cell dependent immune responses. We also demonstrated in vitro and in vivo that the fusion protein enhanced antigen presentation and cross-presentation by targeting tumor antigens towards DCs. CONCLUSIONS: This new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin.

[Primary study on mechanism of baicalin on the Th1/Th2 response in murine model of asthma].

OBJECTIVE: To study the mechanism of baicalin on the cytokines of Th1/Th2 in murine model of asthma. METHODS: The murine model of asthma was induced by OVA. Different doses of baicalin were orally administered to the mice respectively. The spleen cells were cultured 3 days for the measurement of IFN-gamma, IL-4, IL-5 and IL-10 by ELISA. After 2 days of culture, the spleen cells were treated with Trizol for extraction of total RNA. The gene expressions of T-bet, GATA-3 and STAT-6 were analyzed by RT-PCR. RESULTS: The treatment with baicalin obviously decreased the production of IL-4 and IL-5 and the gene expression of GATA-3, STAT-6, but increased the production of IL-10. CONCLUSION: Baicalin may modulate the Th1/Th2 balance mainly by altering the gene expressions of GATA-3 and STAT-6 in vivo and increasing the production of IL-10.