V-set and immunoglobulin domain containing 4 inhibits oxidative stress, mitochondrial dysfunction, and inflammation to attenuate Parkinson's disease progression by activating the JAK2/STAT3 pathway.

OBJECTIVE: V-set and immunoglobulin domain containing 4 (VSIG4) inhibits neurological dysfunction, microglial M1 polarization, and inflammation to participate in the progression of neurological disorders, but evidence regarding Parkinson's disease (PD) is scarce. The present study intended to investigate the engagement of VSIG4 in PD progression, and the potential mechanism. METHODS: BV-2 cells were treated with 1-Methyl-4-phenylpyridinium (MPP+) to establish PD model. MPP+ treated BV-2 cells were infected with VSIG4 overexpression adenovirus-associated virus (AAV) (oeVSIG4) and negative control AAV (oeNC), and AZD1480 (JAK2 inhibitor) was added to these cells. RESULTS: MPP+ reduced VSIG4 mRNA (P < 0.05) and protein (P < 0.05) in BV-2 cells. Interestingly, VSIG4 reduced malondialdehyde (P < 0.01), reactive oxygen species (P < 0.01), NOD-like receptor family pyrin domain containing 3 (P < 0.05), cleaved-caspase1 (P < 0.05), tumor necrosis factor-α (P < 0.05), and interleukin-1ß (P < 0.05), but increased glutathione (P < 0.05), mitochondrial membrane potential (P < 0.05), phosphorylation (p)-JAK2 (P < 0.05), and p-STAT3 (P < 0.01) in MPP+ treated BV-2 cells, which indicated that VSIG4 inhibited oxidative stress, mitochondrial dysfunction, and inflammation, as well as activated the JAK2/STAT3 pathway in PD model. Moreover, AZD1480 inhibited the JAK2/STAT3 pathway and aggravated oxidative stress, mitochondrial dysfunction, and inflammation in PD model (all P < 0.05). Importantly, AZD1480 attenuated the influence of VSIG4 on oxidative stress, mitochondrial dysfunction, inflammation, and the JAK2/STAT3 pathway in PD model (all P < 0.05). CONCLUSION: VSIG4 suppresses oxidative stress, mitochondrial dysfunction, and inflammation by activating the JAK2/STAT3 pathway, which may be helpful in attenuating PD progression.

LncRNA-ENST00000421645 Upregulates Kank1 to Inhibit IFN-γ Expression and Promote T Cell Apoptosis in Neurosyphilis.

Long non-coding RNAs are involved in many infectious diseases. Our previous studies showed that lncRNA-ENST00000421645 expression is increased in T lymphocytes of neurosyphilis patients compared to healthy controls. However, whether lncRNA-ENST00000421645 has biological functions remains unclear. The current study was undertaken to understand the mechanism of lncRNA-ENST00000421645 in T lymphocyte function in neurosyphilis patients. The lncRNA-ENST00000421645 pull-down assay showed that lncRNA-ENST00000421645 acted on the acetylase NAT10. The chromatin immunoprecipitation (ChIP)-PCR results showed that lncRNA-ENST00000421645 promoted the acetylation of histone H3K27 adjacent to the Kank1 promoter, thereby promoting Kank1 protein expression. Kank1 promotes 14-3-3 protein expression, inhibits NF-kB activation, inhibits IFN-γ secretion by T lymphocytes, and promotes T lymphocyte apoptosis. Taken together, our findings suggest a novel mechanism that LncRNA-ENST00000421645 upregulates Kank1 to inhibit IFN-γ expression and promote T cell apoptosis in neurosyphilis.

JKAP relates to disease risk, severity, and Th1 and Th17 differentiation in Parkinson's disease.

OBJECTIVE: JNK pathway-associated phosphatase (JKAP) is previously reported to regulate immune/inflammatory process via T-cell signaling, and closely involves in neurological diseases, while its implication in Parkinson's disease (PD) is unknown. Therefore, this study aimed to investigate the correlation of JKAP with Th1/Th2/Th17 cells and their clinical roles in PD patients, and then further explore the effect of JKAP on regulating CD4+ T-cell differentiation in PD. METHODS: Totally 50 PD patients and 50 age-/gender-matched controls were enrolled. Their blood samples were collected and proposed to ELISA and flow cytometry assays for JKAP, Th1, Th2, and Th17 measurements. In vitro, CD4+ T cells were isolated from PD patients then transfected with JKAP overexpression and knockdown Lentivirus, followed by detection of markers (CD25+ cell proportion, CD69+ cell proportion, IFN-γ, IL10, and IL17). RESULTS: JKAP was downregulated in PD patients compared to controls, which also showed good potency to discriminate them. Besides, JKAP negatively correlated with Th1 and Th17 cell proportions, but did not associate with Th2 cell proportion in PD patients; Interestingly, JKAP did not correlated with Th1, Th2, or Th17 cell proportions in controls. Furthermore, JKAP correlated with some parts of unified Parkinson's Disease Rating Scale (UPDRS) and Mini-Mental State Examination (MMSE) score. In vitro, JKAP overexpression repressed CD4+ T-cell activation and its differentiation into Th1 and Th17 cells in PD, while JKAP knockdown appeared opposite effect. INTERPRETATION: JKAP associates with disease risk and severity, correlates with Th1 and Th17 cells, and regulates CD4+ T-cell activation/differentiation in PD.

Eomesodermin in CD4+T cells is essential for Ginkgolide K ameliorating disease progression in experimental autoimmune encephalomyelitis.

Eomesodermin (Eomes), a transcription factor, could suppress the Th17 cell differentiation and proliferation through directly binding to the promoter zone of the Rorc and Il17a gene, meanwhile the expression of Eomes is suppressed when c-Jun directly binds to its promoter zone. Ginkgolide K (1,10-dihydroxy-3,14-didehydroginkgolide, GK) is a diterpene lactone isolated from the leaves of Ginkgo biloba. A previous study indicated that GK could decrease the level of phospho JNK (c-Jun N-terminal kinase). Here, we reported the therapeutic potential of Ginkgolide K (GK) treatment to ameliorate experimental autoimmune encephalomyelitis (EAE) disease progression. Methods: EAE was induced in both wildtype and CD4-Eomes conditional knockout mice. GK was injected intraperitoneally. Disease severity, inflammation, and tissue damage were assessed by clinical evaluation, flow cytometry of mononuclear cells (MNCs), and histopathological evaluation. Dual-luciferase reporter assays were performed to measure Eomes transcription activity in vitro. The potency of GK (IC50) was determined using JNK1 Kinase Enzyme System. Results: We revealed that GK could ameliorate EAE disease progression by the inhibition of the Th17 cells. Further mechanism studies demonstrated that the level of phospho JNK was decreased and the level of Eomes in CD4+T cells was dramatically increased. This therapeutic effect of GK was almost completely interrupted in CD4-Eomes conditional knockout mice. Conclusions: These results provided the therapeutic potential of GK treatment in EAE, and further suggested that Eomes expression in CD4+T cells might be essential in this process.

Long noncoding RNA MALAT1 and its target microRNA-125b are potential biomarkers for Alzheimer's disease management via interactions with FOXQ1, PTGS2 and CDK5.

This study aimed to investigate the intercorrelation among long noncoding RNA MALAT1 (lnc-MALAT1), microRNA-125b (miR-125b), FOXQ1, PTGS2 and CDK5, as well as their correlations with disease risk, severity and progression of Alzheimer's disease (AD). In total, 120 AD patients, 120 Parkinson's disease (PD) patients and 120 controls were enrolled. Cerebrospinal fluid (CSF) samples were collected from 50 AD patients, 50 PD patients and 50 controls; plasma samples were obtained from all participants. Lnc-MALAT1, miR-125b, FOXQ1, PTGS2 and CDK5 were detected by RT-qPCR. CSF lnc-MALAT1/FOXQ1 and plasma lnc-MALAT1 were downregulated, while CSF miR-125b/PTGS2/CDK5 and plasma miR-125b/PTGS2 were upregulated in AD patients compared to PD patients and controls, which differentiated AD patients from PD patients and controls, as demonstrated by ROC curve analyses. In AD patients, CSF/plasma lnc-MALAT1 negatively correlated with miR-125b and PTGS2 but positively correlated with FOXQ1; CSF/plasma miR-125b negatively correlated with FOXQ1 but positively correlated with PTGS2/CDK5. In addition, CSF/plasma lnc-MALAT1 and FOXQ1 correlated with alleviated disease severity, while miR-125b, PTGS2 and CDK5 correlated with exacerbated disease severity, which were manifested by their correlations with MMSE score, Aß42, t-tau and p-tau in AD patients. However, their correlations with MMSE score, Aß42, t-tau and p-tau were weak in PD patients and controls. Notably, CSF but not plasma lnc-MALAT1 and miR-125b could predict the MMSE score decline at 1 year, 2 years and 3 years in AD patients. In conclusion, lnc-MALAT1 and its target miR-125b are potential biomarkers for AD management via their intercorrelation with FOXQ1, PTGS2 and CDK5.

Targeting MicroRNA-125b Promotes Neurite Outgrowth but Represses Cell Apoptosis and Inflammation via Blocking PTGS2 and CDK5 in a FOXQ1-Dependent Way in Alzheimer Disease.

This study aimed to explore the molecular regulatory network among microRNA-125b (miR-125b), forkhead box Q1 (FOXQ1), prostaglandin-endoperoxide synthase 2 (PTGS2), and cyclin-dependent kinase 5 (CDK5), as well as their effects on cell apoptosis, neurite outgrowth, and inflammation in Alzheimer disease (AD). Rat embryo cerebral cortex neurons and nerve growth factor-stimulated PC12 cells were insulted by Aß1-42 to construct two AD cellular models. Negative control (NC) inhibitor, miR-125b inhibitor, NC siRNA, FOXQ1 siRNA, PTGS2 siRNA, and CDK5 siRNA were transferred into the two AD cellular models alone or combined. Then, cell apoptosis, neurite outgrowth, proinflammatory cytokines, miR-125b, FOXQ1, PTGS2, and CDK5 expressions were detected. MiR-125b inhibition facilitated neurite outgrowth but suppressed cell apoptosis and proinflammatory cytokines (tumor necrosis factor-α, interleukin 1ß, and interleukin 6); meanwhile, it upregulated FOXQ1 but downregulated PTGS2 and CDK5. Furthermore, FOXQ1 inhibition promoted cell apoptosis and proinflammatory cytokines but repressed neurite outgrowth; PTGS2 inhibition achieved the opposite effects; CDK5 inhibition attenuated cell apoptosis, whereas it less affected neurite outgrowth and inflammation. Notably, FOXQ1 inhibition attenuated, whereas PTGS2 inhibition elevated the effect of miR-125b inhibition on regulating neurite outgrowth, cell apoptosis, and proinflammatory cytokines. As for CDK5 inhibition, it enhanced the effect of miR-125b inhibition on regulating cell apoptosis, but less impacted the neurite outgrowth and proinflammatory cytokines. Additionally, PTGS2 inhibition and CDK5 inhibition both reversed the effect of FOXQ1 inhibition on regulating cell apoptosis, neurite outgrowth, and proinflammatory cytokines. In conclusion, targeting miR-125b alleviates AD progression via blocking PTGS2 and CDK5 in a FOXQ1-dependent way.

Tp47 induces cell death involving autophagy and mTOR in human microglial HMO6 cells.

BACKGROUND: Tp47 can induce immune cells to produce numerous inflammatory factors, some of which can trigger autophagy. Increased autophagy has a dual effect on cell survival. However, whether Tp47 induces autophagy in microglia is unknown. OBJECTIVE: To evaluate the potential role of Tp47 in microglia. METHODS: After treatment with Tp47, autophagy-related proteins were assessed in HMO6 human microglial cells by flow cytometry, Western blotting and immunofluorescence. Cell death was assessed by flow cytometry and trypan blue staining. Changes in mTOR pathway proteins were explored by using Western blotting. RESULTS: After treatment with Tp47, a gradual increase in total LC3 expression was observed as a dose- and time-dependent accumulation of its active form, LC3-II (P < 0.05), but P62 expression was downregulated (P < 0.05). Moreover, microglial mortality gradually increased in a dose- and time-dependent manner. 3-Methyladenine (3-MA), a specific inhibitor of PI3KC3, reversed autophagy and cell death. The mortality rate of HMO6 microglial cells treated with Tp47 was approximately 13.7 ±â€¯2%, and the basal expression of p-mTOR, p-p70s6k and p-S6 in these cells was significantly downregulated by Tp47. Moreover, the mortality rate of microglia was significantly reduced after mTOR agonist intervention. CONCLUSION: In human microglial HMO6 cells, Tp47 induces autophagy- and mTOR pathway-dependent cell death.

Fabric-phase sorptive extraction coupled with ion mobility spectrometry for on-site rapid detection of PAHs in aquatic environment.

The contamination of water is a high risk to human health, so there is an urgent need to rapidly detect water pollution in the field. Ion mobility spectrometry (IMS) is suitable for on-site analysis with the merit of rapid analysis and compact size. In this study, we developed a new method which coupled fabric phase sorptive extraction (FPSE) with IMS for rapid detection of polycyclic aromatic hydrocarbons (PAHs) in water present in the field. Polydimethylsiloxane (PDMS) was coated on the glass fiber cloth through a sol-gel reaction. After extracting the PAHs in water, the fabric coated PDMS could be directly put into the inlet of IMS instrument for thermal desorption. The PAHs were analyzed by the IMS instrument operated in the positive ion mode with a corona discharge (CD) ionization source. The primary parameters affecting extraction efficiency such as extraction time, extraction temperature, and ionic strength were investigated and optimized by using phenanthrene (Phe), benzo[a]anthracene (BaA) and benzo[a]pyrene (BaP) as model compounds. Under the optimal conditions, the FPSE-IMS detection limits were 5 ng ml-1,8 ng ml-1 and 10 ng ml-1 respectively. Satisfactory recoveries were obtained in the range from 80.5% to 100.5% by testing the spiked real water samples and validated by the standard method（HJ487-2009）. Based on the results, the method of FPSE-IMS could be feasibly applied for monitoring the water quality on-site and providing early warning in the field.

Variant of EOMES Associated with Increasing Risk in Chinese Patients with Relapsing-remitting Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a common central nervous system autoimmune disorder. Increasing number of genome-wide association study (GWAS) analyses hint that MS is strongly associated with genetics. Unfortunately, almost all the GWAS analyses were Caucasian population based. Numbers of risk loci might not be replicated in Chinese MS patients. Hence, we performed a MassArray Assay to genotype the previously reported variants located in the transcription regulation genes in order to elucidate their role in the Chinese MS patients. METHODS: One hundred and forty-two relapsing-remitting MS (RRMS) patients and 301 healthy controls were consecutively collected from September 2, 2008, to June 7, 2013, as stage 1 subjects. Eight reported transcription regulation-related single-nucleotide polymorphisms (SNPs) were genotyped using the Sequenom MassArray system. In stage 2, another 44 RRMS patients and 200 healthy controls were consecutively collected and Sanger sequenced from April 7, 2015, to June 29, 2017, for the validation of positive results in stage 1. Differences in allele and genotype frequencies between patients and healthy controls, odds ratios, and 95% confidence intervals were calculated with the Chi-square test or Fisher's exact test. Hardy-Weinberg equilibrium was tested also using the Chi-square test. RESULTS: In stage 1 analysis, we confirmed only one previously reported risk variant, rs11129295 in EOMES gene. We found that the frequency of T/T genotype was much higher in MS group (χ2 = 10.251, P = 0.005) and the T allele of rs11129295 increased the risk of MS (χ2 = 10.022, P = 0.002). In stage 2 and combined analyses, the T allele of rs11129295 still increased the risk of MS (χ2 = 4.586, P = 0.030 and χ2 = 16.378, P = 5.19 × 10-5, respectively). CONCLUSIONS: This study enhances the knowledge that the variant of EOMES is associated with increasing risk in Chinese RRMS patients and provides a potential therapeutic target in RRMS.

Toxicity assessment of the extractables from multi-layer coextrusion poly ethylene bags exposed to pH=5 solution containing 4% benzyl alcohol and 0.1 M sodium acetate.

A non-target analysis was developed for the analysis of extractables from multi-layer coextrusion bags exposed to 4% benzyl alcohol solution and 0.1 M sodium acetate at pH = 5 for defined periods (15 day, 45 day and 90 day) according to manufacturer instructions based on the ultra-performance liquid chromatography (UPLC) quadrupole-time of flight mass spectrometry (Q-TOF MS). In order to confirm the extractables, principal component analysis (PCA) was used to indicate the differences among samples of different periods. Then, the extractables were identified based on searching the self-built library or online searching. The total content of extractables of 90 day samples was 589.78 µg/L, and the content was in the range of acceptable levels for pharmaceutical manufacturers. The risk assessment of the extractables were evaluated by Toxtree and T.E.S.T. software to avoid the animals bioexperiment.

Lipidomics to investigate the pharmacologic mechanisms of ginkgo folium in the hyperuricemic rat model.

Hyperuricemia caused by purine metabolic abnormalities is reported to have close correlation with lipid metabolic disorders. Ginkgo folium, a frequently-used lipid-lowering medicine, has significant anti-hyperuricemia effects. However, it is poorly known about the interaction between lowering uric acid and regulation of lipid metabolic disorders. In this study, hyperuricemic rat model was induced by orally administration with fructose. Ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) combined with pattern recognition approaches were used to determine different lipid metabolites in serum of control group, model group, and different doses of ginkgo folium groups. Principal component analysis (PCA) was applied to analyze the MS data to assess the establishment of model, partial least squares-discriminate analysis (PLS-DA) and independent samples T-test were performed to indicate the differences between different groups of rats and to find biomarkers. Metabolomics pathway analysis (MetPA) was introduced to reveal the pharmacologic mechanisms of ginkgo folium. 19 potential biomarkers associated with hyperuricemia were found. After intervened by ginkgo folium, these biomarkers were returning to normal level. Among these biomarkers, 13 lipid biomarkers were significantly reversed. Ginkgo filum can lower uric acid via adjusting back the level of PCs and LPCs, which suggested that its treatment mechanisms may be related to reducing the activity of PLA2. In sum, the lipidomics analysis in the system level have enhanced our understanding to pathogenesis of hyperuricemia and the results suggested that ginkgo folium could alleviate the abnormal metabolic status of hyperuricemia. These results demonstrated a new mechanism for lowering uric acid, which was helpful to the early treatment for hyperuricemia.

Prediction of biomarkers of therapeutic effects of patients with lung adenocarcinoma treated with gefitinib based on progression-free-survival by metabolomic fingerprinting.

Lung carcinoma is one of the most frequently diagnosed malignancy and threats human life and health. In clinical practice, gefitinib, one of the most well-known epidermal growth factor receptor tyrosine kinase inhibitors, was frequently used in the treatment of non-small cell lung carcinoma. However, this drug is not useful for all non-small cell patients. In this study, the biomarkers were found out to predict the therapeutic effects of gefitinib for lung carcinoma patients. Serum samples were collected from patients with advanced lung adenocarcinoma. The ultra-high performance liquid chromatography (UHPLC)-quadrupole-time of flight mass spectrometry (Q-TOF MS) was conducted to obtain the metabolic data for each patient. Partial least squares-discriminate analysis (PLS-DA) was performed to indicate the differences between metabolites of patients, and Cox proportional hazards regression analysis was used to eliminate the interference of the patient's gender, age, smoking history and disease stage. Thus, differential biomarkers were found. The combination of these biomarkers was statistically significant predictors based on progression-free survival. If these biomarkers can be further confirmed by the clinic, it could suggest the proper therapeutic schedule, and help to reduce patients' economic burden and medication side effects.

Variants of Interleukin-7/Interleukin-7 Receptor Alpha are Associated with Both Neuromyelitis Optica and Multiple Sclerosis Among Chinese Han Population in Southeastern China.

BACKGROUND: Neuromyelitis optica (NMO) and multiple sclerosis (MS) are autoimmune demyelinating diseases of the central nerve system. Interleukin-7 (IL-7) and interleukin-7 receptor alpha (IL-7Rα) were proved to be important in the pathogenesis of both diseases because of the roles they played in the differentiations of autoimmune lymphocytes. The variants of both genes had been identified to be associated with MS susceptibility in Caucasian, Japanese and Korean populations. However, the association of these variants with NMO and MS has not been well studied in Chinese Southeastern Han population. Here, we aimed to evaluate the association of six IL-7 variants (rs1520333, rs1545298, rs4739140, rs6993386, rs7816065, and rs2887502) and one variant of IL-7RA (rs6897932) with NMO and MS among Chinese Han population in southeastern China. METHODS: Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MassARRAY system) and Sanger sequencing were used to determine the variants of IL-7 and IL-7RA in 167 NMO patients, 159 MS patients and 479 healthy controls among Chinese Han population in southeastern China. Samples were excluded if the genotyping success rate <90%. RESULTS: Statistical differences were observed in the genotypes of IL-7 rs1520333 in MS patients and IL-7RA rs6897932 in NMO patients, compared with healthy controls (P = 0.035 and 0.034, respectively). There was a statistically significant difference in the genotypes of IL-7 rs2887502 between MS and NMO patients (P = 0.014). And there were statistically significant differences in the rs6897932 genotypes (P = 0.004) and alleles (P = 0.042) between NMO-IgG positive patients and healthy controls. CONCLUSIONS: The study suggested that among Chinese Han population in southeastern China, the variant of IL-7RA (rs6897932) was associated with NMO especially NMO-IgG positive patients while the variant of IL-7 (rs1520333) with MS patients. And the genotypic differences of IL-7 rs2887502 between MS and NMO indicated the different genetic backgrounds of these two diseases.

Variants of CYP27B1 are associated with both multiple sclerosis and neuromyelitis optica patients in Han Chinese population.

Multiple sclerosis (MS) and neuromyelitis optica (NMO) are chronic demyelinating diseases of the central nervous system (CNS). Recently, variants of vitamin D metabolizing genes, including rs12368653, rs10876994, rs118204009 and rs703842 in CYP27B1, and rs2248359 in CYP24A1 have been identified to be associated with the pathogenicity of MS in Caucasian populations. However, these results have not been replicated in Han Chinese population. Here we investigated the association of these variants with MS and NMO susceptibility in 149 MS patients, 110 NMO patients and 294 healthy controls using MassARRAY system and Sanger sequencing. We found that the frequencies of the A allele of rs703842 were higher in MS patients than controls (p=0.032), and statistical differences were observed in the genotypes of both rs703842 (p=0.013) and rs10876994 (p=0.001) between NMO patients and controls. In addition, we found difference in the genotype of rs12368653 between MS patients and controls (p=0.008). However, no difference was found in rs2248359 among these three groups. The reported rare mutation p.R389H (rs118204009) was not found in our study. In conclusion, our study suggested that variants of CYP27B1 were associated with both MS and NMO patients in Han Chinese population.

Variants of autophagy-related gene 5 are associated with neuromyelitis optica in the Southern Han Chinese population.

Neuromyelitis optica (NMO) and multiple sclerosis (MS) are autoimmune demyelinating diseases of the central nervous system. The discovery of NMO immunoglobulin G (NMO-IgG) antibody has improved the clinical definition of NMO. Recently, the autophagy-related genes (ATGs) have been proved to be associated with several autoimmune and inflammation diseases. Increased T cell expression of ATG5 may be correlated with the pathogenesis of inflammatory demyelination in MS. However, the association of ATG5 variants with MS and NMO patients has not been well studied. In this study, five ATG5 variants were genotyped in 144 MS patients, 109 NMO patients and 288 controls in the Han Chinese population. In the cohort of NMO patients, we observed that the CC genotype of rs548234 increased susceptibility to NMO (p = 0.016), while the allele T of rs548234 (p = 0.003) and the allele A of rs6937876 (p = 0.009) acted as protective factors for NMO-IgG positive NMO patients. However, no association was found between ATG5 variants and MS patients. These results indicated that ATG5 variants are associated with NMO but not MS patients, which may provide a clue for further clarifying the autoimmune mechanisms of autophagy-related pathogenesis in NMO.