[Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway].

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1ß, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(ß-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and ß-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in rat serum, increased VEGF and ß-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and ß-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1ß. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing ß-EP levels.

Function and therapeutic potential of transient receptor potential ankyrin 1 in fibrosis.

The transient receptor potential (TRP) protein superfamily is a special group of cation channels expressed in different cell types and signaling pathways. In this review, we focus on TRPA1 (transient receptor potential ankyrin 1), an ion channel in this family that exists in the cell membrane and shows a different function from other TRP channels. TRPA1 usually has a special activation effect that can induce cation ions, especially calcium ions, to flow into activated cells. In this paper, we review the role of TRPA1 in fibroblasts. To clarify the relationship between fibroblasts and TRPA1, we have also paid special attention to the interactions between TRPA1 and inflammatory factors leading to fibroblast activation. TRPA1 has different functions in the fibrosis process in different organs, and there have also been interesting discussions of the mechanism of TRPA1 in fibroblasts. Therefore, this review aims to describe the function of TRP channels in controlling fibrosis through fibroblasts in different organ inflammatory and immune-mediated diseases. We attempt to prove that TRPA1 is a target for fibrosis. In fact, some clinical trials have already proven that TRPA1 is a potential adjuvant therapy for treating fibrosis.

Polyphyllin D induces apoptosis and protective autophagy in breast cancer cells through JNK1-Bcl-2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Polyphyllin D (PD), an active component from rhizome of Paris polyphylla Sm, root and rhizome, shows a strong anti-cancer activity in several cancers. However, whether autophagy is involved in PD-induced cell death in breast cancer cells and its molecular mechanism has not yet been elucidated. AIM OF THE STUDY: To explore the anti-tumor effects of PD in breast cancer and the underlying mechanisms. MATERIALS AND METHODS: PD was isolated from P. polyphylla Sm and confirmed by HPLC and NMR. The role of PD in cell viability, apoptosis, autophagy in breast cancer cells were determined. RESULTS: PD shows significant anti-tumor activity by inhibit cell proliferation and induce caspase-dependent apoptosis in breast cancer cells. Moreover, PD treatment could induce autophagy by activation of JNK1/Bcl-2 pathway. Importantly, blocking of autophagy by using autophagy inhibitor 3-methyladenine (3-MA) dramatically increase PD-induced apoptosis as evidence by the increased percentage of apoptotic cell death. The anti-tumor effects of PD also investigated in vivo. The results showed that the combinatory treatment of PD with autophagy inhibitor significantly promote PD-induced apoptosis. CONCLUSION: PD could induce caspase-dependent apoptosis and cyto-protectvie autophagy by activation of JNK1/Bcl-2 pathway in breast cancer cells. Combination with an autophagy inhibitor significantly enhance cytotoxic effect of PD and this combination may be a promising candidate for breast cancer therapy.

Intrinsic bioactivity of black phosphorus nanomaterials on mitotic centrosome destabilization through suppression of PLK1 kinase.

Although nanomaterials have shown promising biomedical application potential, incomplete understanding of their molecular interactions with biological systems prevents their inclusion into mainstream clinical applications. Here we show that black phosphorus (BP) nanomaterials directly affect the cell cycle's centrosome machinery. BP destabilizes mitotic centrosomes by attenuating the cohesion of pericentriolar material and consequently leads to centrosome fragmentation within mitosis. As a result, BP-treated cells exhibit multipolar spindles and mitotic delay, and ultimately undergo apoptosis. Mechanistically, BP compromises centrosome integrity by deactivating the centrosome kinase polo-like kinase 1 (PLK1). BP directly binds to PLK1, inducing its aggregation, decreasing its cytosolic mobility and eventually restricting its recruitment to centrosomes for activation. With this mechanism, BP nanomaterials show great anticancer potential in tumour xenografted mice. Together, our study reveals a molecular mechanism for the tumoricidal properties of BP and proposes a direction for biomedical application of nanomaterials by exploring their intrinsic bioactivities.

Reinforcing the Combinational Immuno-Oncotherapy of Switching "Cold" Tumor to "Hot" by Responsive Penetrating Nanogels.

Although immuno-oncotherapy in clinic has gained great success, the immunosuppressive tumor microenvironment (TME) existing in the "cold" tumor with insufficient and exhausted lymphocytes may result in a lower-than-expected therapeutic efficiency. Therefore, a properly designed synergistic strategy that can effectively turn the "cold" tumor to "hot" should be considered to improve the therapeutic effects of immuno-oncotherapy. Herein, TME-responsive penetrating nanogels (NGs) were developed, which can improve the delivery and penetration of the co-loaded resiquimod (R848) and green tea catechin (EGCG) in tumors by a nano-sized controlled releasing system of the soluble cyclodextrin-drug inclusion complex. Consequently, the NGs effectively promoted the maturation of dendritic cells, stimulated the cytotoxic T lymphocytes (CTLs), and decreased the PD-L1 expression in tumors. The combination of NGs with the OX40 agonist (αOX40) further synergistically enhanced the activation and infiltration of CTLs into the deep tumor and inhibited the suppression effects from the regulatory T cells (Tregs). As a result, an increased ratio of active CTLs to Tregs in tumors (20.66-fold) was achieved with a 91.56% tumor suppression effect, indicating a successful switch of "cold" tumors to "hot" for an immunologically beneficial TME with significantly improved anti-tumor immune therapeutics. This strategy could be tailored to other immuno-oncotherapeutic approaches to solve the urgent efficiency concerns of the checkpoint-based treatment in clinic.

Material basis and molecular mechanisms of Dachengqi decoction in the treatment of acute pancreatitis based on network pharmacology.

BACKGROUND: Dachengqi decoction (DCQD) is a classical prescription in traditional Chinese medicine (TCM). It has been used to treat abdominal pain and acute pancreatitis (AP) for thousands of years in China. OBJECTIVE: To predict the active components and signaling pathway of DCQD and to further explore the potential molecular mechanism of DCQD as a treatment of AP using network pharmacology. METHODS: Network pharmacology and bioinformatics were used to determine the active components of DCQD and its potential target in the treatment of AP. The AP model was induced by Cerulein (Cer) combined with lipopolysaccharide (LPS). The pharmacodynamic basis of DCQD in the treatment of AP was evaluated in vitro and in vivo and Western blot analysis and immunofluorescence were used to determine the molecular mechanism of DCQD. RESULTS: Screening using relevant databases and topological analysis revealed 71 active components and 535 potential target proteins in DCQD. In addition, 445 differential genes for AP were also screened. Pathway enrichment analysis, PPI network analysis and transcription factor prediction showed that DCQD played an important role in the PI3K-Akt signal pathway, and 17 DCQD monomers were found in this signal pathway. In the AP model, DCQD promoted pancreatic acinar cell apoptosis, reduction in inflammation, and regulation of the PI3K-AKT signaling pathway. DCQD inhibited the expression of p-AKT and p- NF-kB proteins in pancreatic tissue of the AP model both in vitro and in vivo. CONCLUSION: This study reveals that 17 active components of DCQD improve AP by regulating the PI3K/AKT signaling pathway and promoting apoptosis and suppressing pathological injury and inflammation.

Antifungal Activities of Volatile Secondary Metabolites of Four Diaporthe Strains Isolated from Catharanthus roseus.

Four endophytic fungi were isolated from the medicinal plant, Catharanthus roseus, and were identified as Diaporthe spp. with partial translation elongation factor 1-alpha (TEF1), beta-tubulin (TUB), histone H3 (HIS), calmodulin (CAL) genes, and rDNA internal transcribed spacer (ITS) region (TEF1-TUB-HIS--CAL-ITS) multigene phylogeny suggested for species delimitation in the Diaporthe genus. Each fungus produces a unique mixture of volatile organic compounds (VOCs) with an abundant mixture of terpenoids analyzed by headspace solid-phase microextraction (SPME) fiber-GC/MS. These tentatively-detected terpenes included α-muurolene, ß-phellandrene, γ-terpinene, and α-thujene, as well as other minor terpenoids, including caryophyllene, patchoulene, cedrene, 2-carene, and thujone. The volatile metabolites of each isolate showed antifungal properties against a wide range of plant pathogenic test fungi and oomycetes, including Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, and Phytophthora cinnamomi. The growth inhibition of the pathogens varied between 10% and 60% within 72 h of exposure. To our knowledge, the endophytic Diaporthe-like strains are first reported from Catharanthus roseus. VOCs produced by each strain of the endophytic Diaporthe fungi were unique components with dominant monoterpenes comparing to known Diaporthe fungal VOCs. A discussion is presented on the inhibitive bioactivities of secondary metabolites among endophytic Diaporthe fungi and this medicinal plant.

Dachengqi Decoction Attenuates Intestinal Vascular Endothelial Injury in Severe Acute Pancreatitis in Vitro and in Vivo.

BACKGROUND/AIMS: Dachengqi decoction (DCQD) is a well-known traditional Chinese herbal drug with strong anti-inflammatory effects. Angiopoietin-1 (Ang-1) plays a vital role in maintaining the stability and integrity of the vascular wall and prevents vascular leakage due to inflammatory mediators. Our previous work found that DCQD protects against pancreatic injury in rats with severe acute pancreatitis (SAP). This study aims to investigate the effects of DCQD on intestinal endothelial damage in both damaged human umbilical vein endothelial cells (HUVECs) and SAP rats. METHODS: HUVECs were randomly divided into four groups: control group, TNF-α group, TNF-α plus Ang-1 group (Ang-1 group), and TNF-α plus DCQD group (DCQD group). Cells were incubated for 6 h, 12 h, and 24 h, before collection. The treatment concentration of DCQD was decided based on a Cell Counting Kit-8 (CCK-8) assay. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TEER). Apoptosis was analyzed by flow cytometry. mRNA and protein expression of aquaporin 1 (AQP-1), matrix metalloproteinase 9 (MMP9), and junctional adhesion molecule-C (JAM-C) was evaluated by RT-PCR, immunocytofluorescence, and western blot. Forty male Sprague-Dawley rats were randomized into a control group, SAP group, SAP plus Ang-1 group (Ang-1 group), and SAP plus DCQD group (DCQD group). SAP was induced by intraperitoneal injection of cerulein and lipopolysaccharide (LPS), while the control group received 0.9% saline solution. Evans blue was injected through the penile vein and the rats were then sacrificed 12 h after modeling. Levels of serum amylase, TNF-α, IL-1ß, IL-2, and IL-6 were determined by using ELISA. Intestinal tissue was analysed by histology, and capillary permeability in the tissues was evaluated by Evans blue extravasation assay. Protein and mRNA expression of AQP-1, MMP9, and JAM-C were assessed by immunohistofluorescence, western blot, and RT-PCR. RESULTS: DCQD reduced the permeability of HUVEC induced by TNF-α in vitro. Furthermore, DCQD altered the mRNA and protein levels of JAM-C, MMP9, and AQP-1 in HUVECs after TNF-α induction. SAP intestinal injury induced by cerulein combined with lipopolysaccharides was concomitant with increased expression of JAM-C and MMP9, and reduced expression of AQP-1 in intestinal tissue. Pretreatment with DCQD attenuated SAP intestinal injury and lowered the levels of serum amylase, TNF-α, IL-1ß, IL-2, and IL-6 effectively. Our study demonstrated that DCQD decreased the expression of JAM-C and MMP9 and increased the expression of AQP-1 both in vitro and in vivo. CONCLUSION: DCQD can reduce capillary endothelial damage in acute pancreatitis-associated intestinal injury and the mechanism may be associated with the regulation of endothelial barrier function-associated proteins AQP-1, MMP9, and JAM-C.

Gold nanoclusters as switch-off fluorescent probe for detection of uric acid based on the inner filter effect of hydrogen peroxide-mediated enlargement of gold nanoparticles.

Herein we report a novel switch-off fluorescent probe for highly selective determination of uric acid (UA) based on the inner filter effect (IFE), by using poly-(vinylpyrrolidone)-protected gold nanoparticles (PVP-AuNPs) and chondroitin sulfate-stabilized gold nanoclusters (CS-AuNCs) as the IFE absorber/fluorophore pair. In this IFE-based fluorometric assay, the newly designed CS-AuNCs were explored as an original fluorophore and the hydrogen peroxide (H2O2) -driven formed PVP-AuNPs can be a powerful absorber to influence the excitation of the fluorophore, due to the complementary overlap between the absorption band of PVP-AuNPs and the emission band of CS-AuNCs. Under the optimized conditions, the extent of the signal quenching depends linearly on the H2O2 concentration in the range of 1-100µM (R2 =0.995) with a detection limit down to 0.3µM. Based on the H2O2-dependent fluorescence IFE principle, we further developed a new assay strategy to enable selective sensing of UA by using a specific uricase-catalyzed UA oxidation as the in situ H2O2 generator. The proposed uricase-linked IFE-based assay exhibited excellent analytical performance for measuring UA over the concentration ranging from 5 to 100µM (R2=0.991), and can be successfully applied to detection of UA as low as 1.7µM (3σ) in diluted human serum samples.

Self-adjuvanted nanovaccine for cancer immunotherapy: Role of lysosomal rupture-induced ROS in MHC class I antigen presentation.

MHC class I (MHC I) antigen presentation of exogenous antigens (so called "cross presentation") is a central mechanism of CD8(+) cytotoxic T lymphocyte (CTL) responses essential for successful vaccine-based cancer immunotherapy. The present study constructed amphiphilic pH-sensitive galactosyl dextran-retinal (GDR) nanogels for cancer vaccine delivery, in which dextran was conjugated with all-trans retinal (a metabolite of vitamin A) through a pH-sensitive hydrazone bond, followed by galactosylation to acquire dendritic cell (DC)-targeting ability. Our results showed that pH-sensitive GDR nanogel was a self-adjuvanted vaccine carrier that not only promoted DC maturation through activating retinoic acid receptor (RAR) signaling, but also facilitated antigen uptake and cytosolic antigen release in DCs. Furthermore, pH-sensitive GDR nanogel effectively augmented MHC I antigen presentation and evoked potent anti-cancer immune responses in vivo. More importantly, we first reported that nanoparticle-triggered lysosome rupture could directly induce ROS production in DCs, which was found to be essential for augmenting proteasome activity and downstream MHC I antigen presentation. Hence, DC-targeted pH-sensitive GDR nanogels could be a potent delivery system for cancer vaccine development. Triggering lyososomal rupture in DCs with pH-sensitive nanoparticles might be a plausible strategy to elevate intracellular ROS production for promoting antigen cross presentation, thereby improving cancer vaccine efficacy.

Dachengqi Decoction Attenuates Inflammatory Response via Inhibiting HMGB1 Mediated NF-κB and P38 MAPK Signaling Pathways in Severe Acute Pancreatitis.

BACKGROUND/AIMS: Severe acute pancreatitis (SAP) is a sudden inflammation of the pancreas. The traditional Chinese medicine formula Dachengqi decoction (DCQD) is proven to be beneficial in the comprehensive treatment for pancreatitis patients in clinical practice. However, the molecular mechanism of DCQD on SAP remains unclear. High mobility group box 1(HMGB1) that functions as a damage-associated molecular pattern molecule (DAMP) has attracted much interest. METHODS: In this study, we used lipopolysaccharide (LPS) and cerulein to induce severe acute pancreatitis in C57BL/6 mice with subsequent administration with low, medium and high dose (2.3 g/kg, 7 g/kg and 21 g/kg, respectively) of DCQD. RESULTS: DCQD treatment improved the pathological score and decreased serum amylase and lipase in a dose-dependent manner. In addition, it suppressed the immune cell-induced secretion of HMGB1 and its translocation from the nucleus to the cytoplasm, thus repressing the expression of IL-6 and TNF-α. Further, pretreatment with DCQD decreased responses of TLRs, and suppressed the activation of NF-κB and p38 MAPK pathway. CONCLUSION: Decreasing the secretion of HMGB1 could reduce pro-inflammatory cytokines, which may help cutting down the risks of development from localized pathological changes to a systemic inflammatory response syndrome and even lead to multiple organ failure.