Two zinc finger proteins, VdZFP1 and VdZFP2, interact with VdCmr1 to promote melanized microsclerotia development and stress tolerance in Verticillium dahliae.

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Small-molecule arone protects from neuroinflammation in LPS-activated microglia BV-2 cells by targeting histone-remodeling chaperone ASF1a.

Histone post-translational modifications (PTMs) have been shown to be highly associated with inflammation response, suggesting a therapeutic significance of pharmacologically editing histone PTMs. Currently reported anti-inflammation small-molecules mainly target histone PTMs writers or erasers for methylation, phosphorylation, and acetylation. Although histone chaperones also appear to be involved in inflammation signaling cascades, whether small-molecules could target histone chaperones to show anti-inflammation effects has still been rarely discovered. In this study, natural product artone was found to show obvious inhibitory effects on microglia-mediated neuroinflammation by directly targeting ASF1a, which is a histone-remodeling chaperone. Mechanism study revealed that artone modulated histone H3 PTMs profile by down-regulating acetylation and trimethylation modification levels at sites K4, K9, K18 and K27. Artone-dependent regulations on PTMs further caused an effective inhibition on transcription factor NF-κB assembling to promoters of pro-inflammatory cytokine genes including Tnf-α, Il-6 and Rgs3, indicating a distinctive anti-neuroinflammation mechanism. Collectively, we reported artone as the first small-molecule targeting histone-remodeling chaperone ASF1a for anti-neuroinflammation. Moreover, these findings broaden our knowledge of histone chaperone as a druggable target protein for neuroinflammation inhibition, and open a new avenue to novel therapy strategy for inflammation-associated neurological disorders.

Aglaia odorata Lour. extract inhibit ischemic neuronal injury potentially via suppressing p53/Puma-mediated mitochondrial apoptosis pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Aglaia odorata Lour. is a traditional Chinese medicinal plant possessing properties of improving blood circulation, and it is widely used in the treatment of dizziness, traumatic injuries and bruises. AIM OF STUDY: In this study, we are aimed to investigate the cerebral protection effect of the extracts from leaves of Aglaia odorata Lour. (ELA) and the potential mechanism in vivo and in vitro. MATERIALS AND METHODS: The therapeutic effect of ELA on ischemic cerebral stroke was measured on a middle cerebral artery occlusion (MCAO) rat model. Protective effect of ELA on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cells was measured by MTT assay. The apoptotic cells were observed by Hoechst 33258 staining and acridine orange/ethidium bromide double staining assay. Mitochondria were observed by Mitotracker staining assay. The mitochondrial membrane potential was determined by JC-1 staining assay. Western blot was used to investigate the effects of ELA on apoptosis-related proteins. RESULTS: We showed that ELA was an effective neuroprotective agent. In vivo experiments, ELA exerted significant protective effect on MCAO model. TTC staining showed that ELA could reduce cerebral infarction area against MCAO insult. HE and Nissl's staining indicated that ELA could reverse the damage of cortex and hippocampus caused by MCAO. In vitro experiments, ELA showed significant protective effect on OGD/R-induced PC12 cells by reducing the number of apoptotic cells, increasing mitochondrial membrane potential, and reducing superoxide aggregation, further suppressing mitochondrial caspase-9/3 apoptosis pathway. Moreover, protective effect of ELA on mitochondrial function may be exerted by inhibiting p53/Puma signal pathway. CONCLUSION: Our results suggest that ELA exerts a marked neuroprotective effect against cerebral ischemia potentially via suppressing p53/Puma-mediated mitochondrial caspase-9/3 apoptosis pathway.

[Neuroprotective effects and mechanism of ethanol extract of Cistanche tubulosa against oxygen-glucose deprivation/reperfusion].

To investigate the inhibitory effects and mechanism of Cistanche tubulosa ethanol extract( CTEE) against oxygen-glucose deprivation/reperfusion( OGD/R)-induced PC12 cells neuronal injury. In this study,OGD/R-induced PC12 cells were used to explore the neuroprotective effects of CTEE( 12. 5,25,50 mg·L-1) by detecting cell viability with MTT assay,apoptosis with AO/EB and Hoechst 33258,mitochondrial membrane potential changes with JC-1 staining,mitochondrial oxidative stress with MitoSOX staining,as well as the apoptosis-related protein expression( PARP,cleaved PARP,caspase-3,cleaved caspase-3,Bax,Bcl-2) with Western blot. RESULTS:: showed that CTEE effectively protected OGD/R-induced neuronal injury and increased the survival rate of PC12 cells.AO/EB and Hoechst 33258 staining showed that CTEE could effectively inhibit apoptosis. Moreover,JC-1 and MitoSOX staining results showed that CTEE decreased mitochondrial stress and mitochondrial membrane potential imbalance in PC12 cells in a concentration-dependent manner. Meanwhile,CTEE could obviously suppress the activation of key proteins in mitochondrial apoptosis pathway such as caspase-3 and PARP,and significantly inhibit the rise of Bax and down-regulation of Bcl-2. In conclusion,CTEE has obvious protective effects on OGD/R-induced PC12 cells neuronal injury,potentially via inhibiting mitochondrial oxidative stress and apoptosis-related signaling pathway.

The Ethanolic Extract of Caesalpinia sappan Heartwood Inhibits Cerebral Ischemia/Reperfusion Injury in a Rat Model Through a Multi-Targeted Pharmacological Mechanism.

Background: Caesalpinia sappan L. (C. sappan) is a traditional Chinese medicinal plant. The dried heartwood of C. sappan (also known as Sappan wood) has been widely used for the folkloric medical treatment of ischemic cerebral stroke in China. However, the detailed underlying pharmacological mechanism still remains largely unexplored. Methods: In this study, a middle cerebral artery occlusion (MCAO) rat model was employed to elucidate the mechanism of the anti-cerebral ischemic effects of C. sappan ethanolic extract (CEE). Moreover, systemic multi-target identification coupled with gene ontology biological process (GO BP) and reactome pathway analysis was used to investigate the potential neuroprotective mechanism. Furthermore, the presumed mechanism was confirmed through biological analysis by determining the effects of CEE on the identified signaling pathways in PC12 cells model-induced by oxygen-glucose deprivation/reperfusion (OGD/R). Results: Our study demonstrates that CEE (both through in vivo administration at a dosage of 300 mg/kg and through in vitro incubation at a dosage of 2.4 µg/mL) is a neuroprotective agent that can effectively inhibit neuronal damage, promote synaptic generation, and suppress the activation of neutrophils, microglia, and astrocytes. Moreover, the neuroprotective mechanism of CEE is mediated via regulating 150 potential target proteins, which are associated with 6 biological processes and 10 pathways, including JAK-STAT, HSP90 and DNA damage/telomere stress. Conclusion: CEE can exert neuroprotective effect through multi-target pharmacological mechanisms to prevent ischemia/reperfusion-induced cerebral injury.

Total saponins of Albiziae Cortex show anti-hepatoma carcinoma effects by inducing S phase arrest and mitochondrial apoptosis pathway activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Albiziae Cortex (AC) is a widely used traditional medicine in China. It is possess various properties to treat insomnia, traumatic injuries, diuresis, sthenia, and confusion. Total saponins of Albiziae Cortex (TSAC) are the most abundant bioactive components of AC, which were reported to show significant anti-tumor effects in vivo and in vitro. But the underlying mechanism of TSAC remained to be revealed. AIM OF STUDY: In this study, we investigated the anti-hepatoma carcinoma effects and the potential mechanism of TSAC in vivo and in vitro. MATERIALS AND METHODS: We first purified TSAC from crude extracts and characterized the major bioactive compounds by high performance liquid chromatography (HPLC). Effects of TSAC on viability of various hepatoma carcinoma cell lines were measured by MTT. Inhibition on cell proliferation was analysed using colony formation assay. Cell cycle distribution was revealed by flow cytometry. The apoptotic cells were observed by Hoechst 33258 staining and acridine orange (AO)/ethidium bromide (EB) double staining. Microstructures of apoptotic cells were examined by Transmission electron microscopy (TEM). The mitochondrial membrane potential were determined by JC-1 staining. Western blot was used to investigate the effects of TSAC on apoptosis-related proteins, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax), and S-phase related protein cyclin A, cyclin E and cyclin-dependent kinases 2 (CDK2). Effects on tumor growth was assessed by H22-bearing ICR mice. RESULTS: TSAC significantly decreased the hepatoma carcinoma cell viability and inhibited HepG2 cell colony formation in a concentration-dependent manner. We also found that TSAC inhibited HepG2 cell growth via induction of S phase arrest. Further study showed that TSAC significantly down-regulated the expressions of cyclin A, cyclin E and CDK2 in HepG2 cells. Meanwhile, TSAC could effectively induce mitochondria-dependent caspase apoptosis pathway activation. Furthermore, TSAC increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2. In vivo assay showed that the anti-tumor effects of TSAC were significantly augmented without increasing toxicity in H22-bearing ICR mice. CONCLUSION: TSAC could inhibit cell proliferation through inducing S phase arrest and activate cell apoptosis via mitochondria-dependent apoptosis pathway. Therefore, TSAC could be a promising agent in clinical trials for anti-hepatoma carcinoma treatment.

[Macrophage activation by low molecular weight saccharides from Cistanche deserticola].

To investigate the immune activation effect and mechanism of low molecular weight saccharides from Cistanche deserticola(LMSC) on mouse peritoneal macrophages, RAW264.7 cells. The RAW264.7 cells were divided into the normal control group, LPS positive control group, and LMSC treatment groups. The RAW264.7 cells were treated with various concentrations of LMSC from 3.91 to 62.5 g•L ⁻¹. The neutral red assay was employed to detect the phagocytic activity of macrophages. NO release was detected by using NO kit, and macrophage activation associated protein expression levels (TNF-α, IL-6, IKKß, p-IKKß, IκBα, p-IκBα, NF-κB, and p-NF-κB) were detected by Western blot. Results showed that LMSC had an activation effect on macrophages; it can significantly increase the release of NO in RAW264.7 cells and promote the expression of cytokines TNF-α and IL-6. Moreover, LMSC significantly increased the phosphorylation of IKKß, IκBα, and NF-κB p65. Furthermore, mannitol's one of the main constituents in LMSC significantly enhanced the phagocytic activity of macrophages. These results showed that LMSC could activate macrophages by up-regulating the NF-κB signaling pathway, and mannitol may be one of the main active components in LMSC.

[Targets identification and mechanism analysis for macrophage activation of low molecular weight saccharides from Cistanche deserticola based on molecular affinity chromatography].

This study aims to investigate the targets and targets-involved mechanism for the macrophage activation of low molecular weight saccharides from Cistanche deserticola (LMSC). The phagocytic activity and NO release of RAW264.7 cells were detected, and results showed that LMSC exerts immune activation effect by significantly increasing the phagocytic activity and NO release. LMSC-conjugated epoxy-activated sepharose beads were prepared as affinity reagent to capture the target proteins. Twenty-four proteins such as Eef2 were identified by LC-MS/MS analysis. Pathway enrichment analysis showed LMSC activated RAW264.7 cells by regulating Fcgamma receptor dependent phagocytosis, TNF-alpha NF-κB signaling pathway, glycolysis/gluconeogenesis and the citric acid cycle and respiratory electron transport pathway.

Protosappanin A exerts anti-neuroinflammatory effect by inhibiting JAK2-STAT3 pathway in lipopolysaccharide-induced BV2 microglia.

Microglial activation and resultant neuroinflammatory response are implicated in various brain diseases including Alzheimer's disease and Parkinson's disease. Treatment with anti-neuroinflammatory agents could provide therapeutic benefits for such disorders. Protosappanin A (PTA) is a major bioactive ingredient isolated from Caesalpinia sappan L.. In this work, the anti-neuroinflammatory effects of PTA on LPS-stimulated BV2 cells were investigated and the underlying mechanisms were explored. Results showed that PTA significantly inhibited the production of TNF-α and IL-1ß in LPS-activated BV2 microglia. Moreover, the mRNA expressions of IL-6, IL-1ß, and MCP-1 were reduced by PTA in a dose-dependent manner. Furthermore, PTA suppressed JAK2/STAT3-dependent inflammation pathway through down-regulating the phosphorylation of JAK2 and STAT3, as well as STAT3 nuclear translocation against LPS treatment. These observations suggested a novel role for PTA in regulating LPS-induced neuroinflammatory injuries.

[Effect of skeletal muscle contraction on bone capillary permeability: an experimental study in rabbits].

OBJECTIVE: To assess the effect of muscle contraction on bone capillary permeability. METHODS: Thirty female New Zealand white rabbits were randomly divided into the experimental and control groups (n=15). The bone chambers were implanted medially into the right tibiae of the rabbit, and in the experimental group, the rabbits were stimulated by transcutaneous electrical nerve stimulator (TENS) at 4 Hz (1 h/day, 6 days/week) starting from week 2. From week 3, intravital microscopic observation was carried out weekly till week 10, and the vasculature in the tibiae was observed under fluorescent microscope with FITC and RITC staining. All the images were analyzed by Metamorph program. RESULTS: The bone capillary permeability increased significantly in the experimental group. CONCLUSION: Skeletal muscle contraction can increase the capillary permeability.