Heat shock protein 70 suppresses neuroinflammation induced by α-synuclein in astrocytes.

Neuroinflammation triggered by activation of glial cells plays an important role in the pathophysiology of several neurodegenerative diseases including Parkinson's disease (PD). Besides microglia, astrocytes are also critical in initiating and perpetuating inflammatory process associated with PD. Heat shock protein 70 (Hsp70) is originally described as intracellular chaperone, however, recent study revealed that it had anti-inflammatory effects as well. The present study is designed to investigate whether Hsp70 mediates neuroinflammation in astrocytes. By employing α-synuclein (α-Syn) (A53T) aggregates on primary cultured astrocytes of rats, we found that astrocytes were activated and neuroinflammatory response was triggered, as indicated by over-expression of glial fibrillary acidic protein (GFAP), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), increased production of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). The data also showed that the neuroinflammatory response accompanied up-regulated Hsp70 expression. Moreover, over-expression of Hsp70 through transfection of Hsp70 cDNA plasmids could significantly reduce the production of TNF-α, IL-1ß, and the expression of GFAP, COX-2 as well as iNOS. While inhibition of Hsp70 by VER155008 exacerbated neuroinflammatory response in astrocytes challenged by α-Syn aggregates. Further mechanistic study indicated that c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) signalings were responsible for the neuroinflammation, which was also regulated by Hsp70. These findings demonstrated that Hsp70 was an important modulator in astrocytes induced inflammation, and up-regulation of Hsp70 might be a potential regulating approach for neuroinflammation-related neurodegenerative diseases, such as PD.

The pre-rRNA processing factor Nop53 regulates fungal development and pathogenesis via mediating production of reactive oxygen species.

Botrytis cinerea is a necrotrophic plant fungal pathogen that annually causes enormous economic losses worldwide. The ribosome is an organelle for cellular protein biosynthesis. However, little is known about how the ribosome operates as a machine to mediate microbial pathogenesis. Here, we demonstrate that Nop53, a late-acting factor for 60S ribosomal subunit maturation, is crucial for the pathogen's development and virulence. BcNop53 is functionally equivalent to yeast nop53p. Complementation of BcNOP53 completely restored the growth defect of the yeast Δnop53 mutant. BcNop53 is located in nuclei and disruption of BcNOP53 also dramatically impaired pathogen growth. Deletion of BcNOP53 blocked infection structure formation and abolished virulence of the pathogen, possibly due to reduced production of reactive oxygen species. Moreover, loss of BcNOP53 impaired pathogen conidiation and stress adaptation, altered conidial and sclerotial morphology, retarded conidium and sclerotium germination as well as reduced the activities of cell-wall degradation-associated enzymes. Sclerotium production was, however, increased. Complementation with the wild-type BcNOP53 allele rescued defects found in the ΔBcnop53 mutant. Our work establishes a systematic elucidation of Nop53 in regulating microbial development and pathogenesis, provides novel insights into ribosomal processes that regulate fungal pathogenesis, and may open up new targets for addressing fungal diseases.

The key gluconeogenic gene PCK1 is crucial for virulence of Botrytis cinerea via initiating its conidial germination and host penetration.

The process of initiation of host invasion and survival of some foliar phytopathogenic fungi in the absence of external nutrients on host leaf surfaces remains obscure. Here, we demonstrate that gluconeogenesis plays an important role in the process and nutrient-starvation adaptation before the pathogen host invasion. Deletion of phosphoenolpyruvate carboxykinase gene BcPCK1 in gluconeogenesis in Botrytis cinerea, the causative agent of grey mould, resulted in the failure of the ΔBcpck1 mutant conidia to germinate on hard and hydrophobic surface and penetrate host cells in the absence of glucose, reduction in conidiation and slow conidium germination in a nutrient-rich medium. The wild-type and ΔBcpck1 conidia germinate similarly in the presence of glucose (higher concentration) as the sole carbon source. Conidial glucose-content should reach a threshold level to initiate germination and host penetration. Infection structure formation by the mutants displayed a glucose-dependent fashion, which corresponded to the mutant virulence reduction. Exogenous glucose or complementation of BcPCK1 completely rescued all the developmental and virulence defects of the mutants. Our findings demonstrate that BcPCK1 plays a crucial role in B. cinerea pathogenic growth and virulence, and provide new insights into gluconeogenesis mediating pathogenesis of plant fungal pathogens via initiation of conidial germination and host penetration.

Mechanism of Non-receptor Tyrosine Kinase Src Regulating Neuroinflammation Through Phosphatase and Tensin Homology Protein in Microglia.

Objective To investigate the mechanism of non-receptor tyrosine kinase Src regulating neuroinflammation through phosphatase and tensin homology protein(PTEN)in microglia. Methods BV2 cells were incubated with PTEN inhibitor bpv(HOpic)for 2 hours,and then added with lipopolysaccharide(LPS)to induce neuroinflammation,Western blot was performed to determine the expression of phosphorylated protein kinase B(Akt)to investigate the activity of PTEN. Enzyme-linked immunosorben assay(ELISA)was used to determine the release of tumor necrosis factor α(TNF-α)to assess neuroinflammation.After PTEN inhibitor or Src specific small interfering RNA was added,the change of neuroinflammation was evaluated to study the mechanism of Src regulating neuroinflammation. Results LPS induced significant neuroinflammation in BV2 cells,as indicated by significantly increased expression of p-Akt and release of TNF-α(P<0.001).The PTEN inhibitor signficantly increased Akt phosphorylation(P<0.05)and TNF-α release(P<0.001)in LPS-induced BV2 cells compared to simply LPS-induced cells.The Src small interfering RNA significantly decreased the release of TNF-α(P<0.001)and inhibited PTEN(P<0.001)and Akt(P<0.001)phosphorylation. Conclusion Src kinase may regulate neuroinflammtion response in BV2 cells by regulating the phosphorylation of PTEN.

[Microglial Phagocytosis in the Neurodegenerative Diseases].

Microglia are the resident innate immune cells in the brain. Under endogenous or exogenous stimulates, they become activated and play an important role in the neurodegenerative diseases. Microglial phagocytosis is a process of receptor-mediated engulfment and degradation of apoptotic cells. In addition, microglia can phagocyte brain-specific cargo, such as myelin debris and abnormal protein aggregation. However, recent studies have shown that microglia can also phagocyte stressed-but-viable neurons, causing loss of neurons in the brain. Thus, whether microglial phagocytosis is beneficial or not in neurodegenerative disease remains controversial. This article reviews microglial phagocytosis related mechanisms and its potential roles in neurodegenerative diseases, with an attempt to provide new insights in the treatment of neurodegenerative diseases.

Reabsorption of intervertebral disc prolapse after conservative treatment with traditional Chinese medicine: A case report.

BACKGROUND: Conservative treatments have been reported to diminish or resolve clinical symptoms of lumbar intervertebral disc herniation (LIDH) within a few weeks. CASE SUMMARY: Computed tomography and magnetic resonance imaging (MRI) of the lumbar region of a 25-year-old male diagnosed with LIDH showed prolapse of the L5/S2 disc. The disc extended 1.0 cm beyond the vertebral edge and hung along the posterior vertebral edge. The patient elected a conservative treatment regimen that included traditional Chinese medicine (TCM), acupuncture, and massage. During a follow-up period of more than 12 mo, good improvement in pain was reported without complications. MRI of the lumbar region after 12 mo showed obvious reabsorption of the herniation. CONCLUSION: A conservative treatment regimen of TCM, acupuncture, and massage promoted reabsorption of a prolapsed disc.

Effects of Fluid Shear Stress on Human Intervertebral Disc Nucleus Pulposus Cells Based on Label-Free Quantitative Proteomics.

Objective: To explore the possible mechanism of fluid shear stress on human nucleus pulposus cells based on label-free proteomics technology. Methods: The human nucleus pulposus cell line was purchased and subcultured in vitro. The Flexcell STR-4000 multiflow field cell fluid shear stress loading culture system was used to apply continuous laminar fluid shear stress (12 dyne/cm2, 45 mins) to the monolayer adherent cells. Those without mechanical loading were used as the control group, and those subjected to fluid shear loading were used as the experimental group. Differential protein expression was identified using mass spectrometry identification technology, and bioinformatics analysis was performed using Gene Ontology GO (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes KEGG (Kyoto Encyclopedia of Genes and Genomes). Results: The proteomics results of the experimental group and the control group showed that the total number of mass spectra was 638653, the number of matched mass spectra was 170110, the total number of identified peptides was 32050, the specific peptide was 30564, and the total number of identified proteins was 4745. Comparing the two groups, 47 proteins were significantly differentially expressed, namely, 25 upregulated proteins and 22 downregulated proteins. Bioinformatics analysis showed that significantly different proteins were mainly manifested in cellular process, biological regulation, metabolic process, binding, catalytic activity, cellular components (cell part), organelle part (organelle part), and other molecular biological functions. Conclusion: Using proteomics technology to screen human nucleus pulposus cells after fluid shear stress loading, the differential protein expression provides a basis for further exploration of the mechanism of mechanical factors on nucleus pulposus.

Expression of ANGPTL4 in Nucleus Pulposus Tissues Is Associated with Intervertebral Disc Degeneration.

OBJECTIVE: Angiopoietin-like protein 4 (ANGPTL4), encoding a glycosylated secreted protein, has been reported to be closely related to many kinds of diseases, including diabetes, tumor, and some musculoskeletal pathologies, such as rheumatoid arthritis, osteoarthritis, and osteoporosis. The aim of the current study is to investigate the role of ANGPTL4 in intervertebral disc degeneration and analyze the association of ANGPTL4 expression with Pfirrmann grades. METHODS: A total of 162 nucleus pulposus tissues were collected from lumbar intervertebral disc herniation patients undergoing interforaminal endoscopic surgery. Real-time quantitative PCR and western blot were performed to determine the mRNA and protein expression of ANGPTL4 in nucleus pulposus samples. Statistical analysis was performed to analyze the association of ANGPTL4 expression with Pfirrmann grades. RESULTS: Based on the clinical data of 162 patients, results showed that Pfirrmann grades were significantly associated with patients' age (r = 0.162, P = 0.047) and were not significantly associated with patients' gender (P > 0.05). RT-qPCR and western blot results showed that the mRNA (r = 0.287, P < 0.05) and protein (r = 0.356, P < 0.05) expressions of ANGPTL4 were both closely associated with Pfirrmann grades. The expression of ANGPTL4 was remarkably increased in the groups of high IVDD Pfirrmann grades. CONCLUSION: The results demonstrated that ANGPTL4 expression was positively associated with the Pfirrmann grades and the severity of intervertebral disc degeneration. ANGPTL4 may be served as a candidate biomarker for intervertebral disc degeneration.

[Finite element analysis of the treatment of cervical spondylotic radiculopathy with three dimensional balanced manipulation].

OBJECTIVE: To explore the biomechanical characteristics of "three-dimensional balanced manipulation" for the treatment of cervical spondylotic radiculopathy(CSR). METHODS: A CSR patient was treated with "three-dimensional balanced manipulation", and the mechanical changes during the manipulation were monitored by mechanical testing system. Using spiral CT to scan the neck of the patient to obtain DICOM data. The three-dimensional finite element model of cervical spondylotic radiculopathy was established by using Mimics software, Geographic Studio software. The "three-dimensional balance manipulation" was simulated and loaded, and the mechanical parameters of each part were replaced into the finite element model, and the finite element analysis was carried out by using ANSYS software to study the internal stress changes and displacement deformation of vertebral body and intervertebral disc under the action of "three-dimensional balance manipulation". RESULTS: The established C3-C7 finite element model of the CSR patient consisted of 5 vertebrae, 4 intervertebral discs and 3 ligaments, involving 153 471 nodes and 64 978 units. The stress of C3-C7 vertebral body was mainly located in anterior and root of C5 spinous processes, arch, vertebral arch and the combination of the two after full loading of manipulation, and the maximum stress was 17.781 MPa. The deformation sites were mainly concentrated in articular processes and anterior transverse processes of C3, superior articular processes and transverse processes of C4, articular processes of C5. The stress of C3-C7 intervertebral disc mainly distributed in the anterior part of C3, 4 intervertebral disc and the nucleus pulposus of C4, 5 and C5, 6. The displace mentextended to the middle and posterior part of C3, 4 nucleus pulposus, around the nucleus of C4, 5 and C5, 6 and anterior part of cervical intervertebral disc. CONCLUSION: The establishment of three-dimensional finite element model of C3-C7 cervical spondylotic radiculopathy can simulate the geometry and material properties of cervical spine, and also accurately reflects the biomechanical characteristics of cervical spine, verifys the internal mechanism of "three-dimensional balanced manipulation" on CSR, proves the safety and effectiveness of treatment, guides more standardized manipulation, and avoids medical accidents.

Transcriptome analysis and functional validation reveal a novel gene, BcCGF1, that enhances fungal virulence by promoting infection-related development and host penetration.

Simultaneous transcriptome analyses of both host plants and pathogens, and functional validation of the identified differentially expressed genes (DEGs) allow us to better understand the mechanisms underlying their interactions. Here, we analyse the mixed transcriptome derived from Botrytis cinerea (the causal agent of grey mould) infected tomato leaves at 24 hr after inoculation, a critical time point at which the pathogen has penetrated and developed in the leaf epidermis, whereas necrotic symptoms have not yet appeared. Our analyses identified a complex network of genes involved in the tomato-B. cinerea interaction. The expression of fungal transcripts encoding candidate effectors, enzymes for secondary metabolite biosynthesis, hormone and reactive oxygen species (ROS) production, and autophagy-related proteins was up-regulated, suggesting that these genes may be involved in the initial infection processes. Specifically, tomato genes involved in phytoalexin production, stress responses, ATP-binding cassette transporters, pathogenesis-related proteins, and WRKY DNA-binding transcription factors were up-regulated. We functionally investigated several B. cinerea DEGs via gene replacement and pathogenicity assays, and demonstrated that BcCGF1 was a novel virulence-associated factor that mediates fungal development and virulence via regulation of conidial germination, conidiation, infection structure formation, host penetration, and stress adaptation. The fungal infection-related development was controlled by BcCGF-mediated ROS production and exogenous cAMP restored the mutant infection-related development. Our findings provide new insights into the elucidation of the simultaneous tactics of pathogen attack and host defence. Our systematic elucidation of BcCGF1 in mediating fungal pathogenesis may open up new targets for fungal disease control.

The Separation of Antler Polypeptide and Its Effects on the Proliferation and Osteogenetic Differentiation of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND: Colla Cornus Cervi (CCC) has been used as a traditional Chinese medicine in the treatment of osteoporosis and osteonecrosis of the femoral head. However, the bioavailability of CCC is seriously limited owing to its large molecular weight and complex ingredients. In the present study, antler polypeptide was separated from CCC, and the effects of antler polypeptide on rat bone marrow mesenchymal stem cells (BMSCs) were investigated. METHODS: Antler polypeptide was separated from Colla Cornus Cervi by ultrafiltration into different samples according to the molecular weight. The total peptide content of these samples was determined by the biuret method. The content of antler polypeptide in different samples was quantified by high-performance liquid chromatography (HPLC). The effects of antler polypeptide at different concentrations on the proliferation, cell cycle, alkaline phosphatase activity, and BMP7 expression of BMSCs were investigated. RESULTS: Antler polypeptide was separated by ultrafiltration into different samples: A (molecular weight <800 Da), B (molecular weight 800-1500 Da), and C (molecular weight >1500 Da). The total peptide contents of A, B, and C were 0.602 mg/mL, 8.976 mg/mL, and 38.88 mg/mL. Antler polypeptide B eluted at 14.279∼15.351 min showed that the content of antler polypeptide was significantly higher than that of A and C with a peak area of 933.80927. The BMSCs proliferation rate (84.66%) of polypeptide B was the highest at the concentration of 1.578 × 10-2 g/mL. Antler polypeptide B significantly promoted the proliferation of BMSCs with a proliferation index of 38.68%, which was significantly higher than that of the other groups. Antler polypeptide B significantly enhanced the activity of alkaline phosphatase in BMSCs compared to that of the blank group (P < 0.001). Antler polypeptide B increased the BMP7 protein expression in BMSCs. CONCLUSIONS: Results suggested that antler polypeptide may promote the proliferation and osteogenic differentiation of BMSCs. Our study lays an experimental foundation for the further development and application of antler polypeptide in medicine.

A Novel Parkinson's Disease Drug Candidate with Potent Anti-neuroinflammatory Effects through the Src Signaling Pathway.

Numerous drug treatments are available for Parkinson's disease (PD), an age-related neurodegenerative disease, but most cause serious side effects. Therefore, novel therapeutic strategies that halt disease progression and allow for long-term administration are urgently needed. Neuroinflammation critically contributes to the pathogenesis of PD. Here, we report the discovery and optimization of phloroglucinol derivatives, a novel class of anti-neuroinflammatory compounds. Structural modifications of the hit compound 3-methyl-1-(2,4,6-trihydroxyphenyl)butan-1-one produced 43 derivatives, including a preclinical candidate (compound 21), that exhibited potent in vitro anti-neuroinflammatory effects, good blood-brain barrier penetration, and desirable safety margins in mice at a median lethal dose (LD50) >5000 mg/kg. Its in vivo efficacy was demonstrated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and MPTP/probenecid (prob)-induced subacute and chronic PD models, respectively, and α-synuclein transgenic mice. Mechanistic studies revealed neuroinflammation inhibition by targeting Src/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt signaling might be promising. We highlighted the potential usefulness of phloroglucinol derivatives in PD treatment.