Flavonoid extracted from Epimedium attenuate cGAS-STING-mediated diseases by targeting the formation of functional STING signalosome.

Hyperactivation of the cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway has been shown to be associated with the development of a variety of inflammatory diseases, and the discovery of an inhibitor of the cGAS-STING signalling pathway holds great promise in the therapeutic interventions. Epimedium flavonoid (EF), a major active ingredient isolated from the medicinal plant Epimedium, has been reported to have good anti-inflammatory activity, but its exact mechanism of action remains unclear. In the present study, we found that EF in mouse bone marrow-derived macrophages (BMDMs), THP-1 (Tohoku Hospital Pediatrics-1) as well as in human peripheral blood mononuclear cells (hPBMC) inhibited the activation of the cGAS-STING signalling pathway, which subsequently led to a decrease in the expression of type I interferon (IFN-ß, CXCL10 and ISG15) and pro-inflammatory cytokines (IL-6 and TNF-α). Mechanistically, EF does not affect STING oligomerization, but inhibits the formation of functional STING signalosome by attenuating the interaction of interferon regulatory factor 3 (IRF3) with STING and TANK-binding kinase 1 (TBK1). Importantly, in vivo experiments, EF has shown promising therapeutic effects on inflammatory diseases mediated by the cGAS-STING pathway, which include the agonist model induced by DMXAA stimulation, the autoimmune inflammatory disease model induced by three prime repair exonuclease 1 (Trex1) deficiency, and the non-alcoholic steatohepatitis (NASH) model induced by a pathogenic amino acid and choline deficiency diet (MCD). To summarize, our study suggests that EF is a potent potential inhibitor component of the cGAS-STING signalling pathway for the treatment of inflammatory diseases mediated by the cGAS-STING signalling pathway.

MXene-based composites as an electrochemical sensor for ultrasensitive determination of ofloxacin.

Sensitive determination of ofloxacin (OFL) is very essential for human health and environmental protection. Here, a novel composite of gold nanoparticles(nAu)@MXene(Ti3C2Tx)/poly-p-aminobenzene sulfonic acid (PABSA) was fabricated on the surface of glassy carbon electrode (GCE) and used to sensitively determine OFL. The results of experiments showed that the obtained nAu@Ti3C2Tx/PABSA/GCE electrode could be used as an electrochemical sensor to directly detect ofloxacin (OFL) by differential pulse voltammetry (DPV). Under the optimal conditions, the proposed electrode displayed a broader linear range and a lower detection limit (LOD) for OFL determination when it was compared to those similar sensors. The linear range was from 5.0 × 10-8 to 5.0 × 10-4 mol/L and the LOD was 3.7 × 10-8 mol/L (S/N = 3). The nAu@Ti3C2Tx/PABSA/GCE electrode also showed good selectivity, repeatability, and reproducibility. Finally, the proposed electrode was used to detect OFL in commercial samples by the standard addition method. The obtained recovery was from 97.3% and 105.7% showing its potential applications in actual sample analysis.

Increased interleukin 1α and interleukin 1ß expression is involved in the progression of periapical lesions in primary teeth.

BACKGROUND: Interleukin 1 (IL-1) is involved in bone resorption. However, the role of IL-1 in periapical lesions characterized by periapical bone destruction in primary teeth has not yet been fully elucidated. This study aimed to detect the distribution and expression of IL-1 in periapical lesions in primary teeth and assess the relationship between the cytokines and the degree of inflammatory cell infiltration. METHODS: A total of 106 chronic periapical lesions in primary teeth were harvested. Haematoxylin and eosin (H&E) staining was used to determine the histological type and the inflammatory cell infiltration grade (mild, moderate, and severe), and immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) were used to detect the distribution and expression of IL-1α and IL-1ß. RESULTS: Of the 106 chronic periapical lesion samples, there were 85 cases of periapical granuloma, accounting for 80.19% of the total samples, and 21 cases of radicular cysts, accounting for 19.81%; no cases of abscess were detected. Immunohistochemistry results showed that both IL-1α and IL-1ß were expressed in periapical granulomas and cysts. ELISA results showed that IL-1α and IL-1ß levels were higher in the periapical granuloma group than in the radicular cyst and normal control groups (P < 0.05). In the periapical granuloma group, IL-1α and IL-1ß were detected at higher levels in the severe inflammatory cell infiltration subgroup than in the mild-inflammatory cell infiltration subgroup (P < 0.05), and IL-1ß expression was also higher in the moderate inflammatory cell infiltration subgroup than in the mild inflammatory cell infiltration subgroup (P < 0.01). A significant positive correlation was observed between the protein expression levels of IL-1α and IL-1ß and the inflammation grade in periapical granulomas from primary teeth (P < 0.05). CONCLUSION: Expression levels of the cytokines IL-1α and IL-1ß in periapical granulomas from primary teeth increased with increasing inflammatory severity and appeared to be a contributing factor to the progression of periapical lesions.

Claudin-7 indirectly regulates the integrin/FAK signaling pathway in human colon cancer tissue.

The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in regulating the integrin/focal adhesion kinase (FAK)/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, between colon cancer and adjacent normal tissues. Quantitative real-time reverse transcription-PCR and immunohistochemistry were utilized to verify the results of mRNA and protein expression, respectively. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway components, and the STRING database was used to analyze protein-protein interaction pairs among these proteins. Meta-analysis of expression microarrays in The Cancer Genome Atlas (TCGA) database was used to identify significant correlations between Cldn-7 and components of predicted genes in the integrin/FAK/ERK signaling pathway. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples, and the observed changes for all proteins except Cldn-7 were in agreement with changes in the corresponding mRNA levels. Cldn-7 might indirectly regulate MAPK3 via KRT8 due to KRT8 co-expression with MAPK3 or CLDN7. Our bioinformatics methods supported the hypothesis that Cldn-7 does not directly regulate any genes in the integrin/FAK/ERK signaling pathway. These factors may participate in a common network that regulates cancer progression in which the MAPK pathway serves as the central node.

Biodistribution of PAMAM dendrimer conjugated magnetic nanoparticles in mice.

Fluorescein-loaded magnetic nanoparticles (FMNPs) have been increasingly utilized in nanomedicine due to their unique properties. In this study, polyamidoamine (PAMAM) dendrimer was used to modify the FMNPs through bifunctional polyethylene glycol linker. The obtained PAMAM modified magnetic nanoparticles (PFMNPs) were characterized by transmission electron microscope, thermogravimetric analysis, zeta potential titration, and fourier transform infrared spectroscopy. The effect of PAMAM conjugation on the biodistribution of FMNPs and PFMNPs were investigated by confocal laser scanning microscopy and inductively coupled plasma atomic emission spectrometry, respectively. It was revealed that PAMAM conjugation resulted in a lower uptake of FMNPs in the lung and less aggregation in the liver, whereas a higher uptake in brain and testis. Furthermore, the serum biochemical and the hematological analysis indicated the PFMNPs caused no significant changes in enzymes reflective of inflammatory response or organ toxicity.

A bismuth-based double-network hydrogel-mediated synergistic photothermal-chemodynamic therapy for accelerated wound healing.

Multidrug-resistant bacterial infections present a significant challenge to wound healing. Non-antibiotic approaches such as photothermal therapy (PTT) and chemodynamic therapy (CDT) are promising but have suboptimal anti-bacterial efficacy. Herein, we developed a green bismuth-based double-network hydrogel (Bi@P-Cu) as a PTT/CDT synergistic platform for accelerated drug-resistant bacteria-infected wound healing. Bismuth (Bi) nanoparticles fabricated using a microwave method were used as a highly efficient and biocompatible PTT agent while the integration of a small amount of CDT agent Cu2+ endowed the hydrogel with excellent mechanical and self-healing properties, markedly increased photothermal efficiency, promoted cell migration ability, and negligible toxicity. Importantly, PTT enhanced the production of hydroxyl radicals in CDT and the destruction of bacterial cell membranes, which in turn enhanced the thermal sensitivity of bacteria. This synergistic anti-bacterial effect, together with the demonstrated capability to promote angiogenesis and anti-inflammation as well as enhanced fibroblast proliferation, led to accelerated wound healing in a full-thickness mouse model of resistant bacterial infection. This study provides an effective and safe strategy to eliminate drug-resistant bacteria and accelerate wound healing through green, non-antibiotic, double-network hydrogel-mediated synergistic PTT and CDT.

Subthalamic hGAD65 gene therapy and striatum TH gene transfer in a Parkinson's disease rat model.

The aim of the present study is to detect a combination method to utilize gene therapy for the treatment of Parkinson's disease (PD). Here, a PD rat model is used for the in vivo gene therapy of a recombinant adeno-associated virus (AAV2) containing a human glutamic acid decarboxylase 65 (rAAV2-hGAD65) gene delivered to the subthalamic nucleus (STN). This is combined with the ex vivo gene delivery of tyrosine hydroxylase (TH) by fibroblasts injected into the striatum. After the treatment, the rotation behavior was improved with the greatest efficacy in the combination group. The results of immunohistochemistry showed that hGAD65 gene delivery by AAV2 successfully led to phenotypic changes of neurons in STN. And the levels of glutamic acid and GABA in the internal segment of the globus pallidus (GPi) and substantia nigra pars reticulata (SNr) were obviously lower than the control groups. However, hGAD65 gene transfer did not effectively protect surviving dopaminergic neurons in the SNc and VTA. This study suggests that subthalamic hGAD65 gene therapy and combined with TH gene therapy can alleviate symptoms of the PD model rats, independent of the protection the DA neurons from death.

The fecal microbiota of gravidas with fetal growth restriction newborns characterized by metagenomic sequencing.

BACKGROUND: Fetal growth restriction (FGR) is a complex obstetric complication with various causes and of great harm. However, the specific pathogenesis of FGR is unclear, which limits its effective treatment. Gut microbiota dysbiosis was found to be important in pathogenesis of various diseases. However, its role in FGR development remains unclear and needs to be clarified. METHODS: In our case-control study, we recruited eight FGR and eight control female participants and collected their fecal samples in third trimester before delivery. We performed metagenomic sequencing and bioinformatic analysis to compare the gut microbiota composition and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between the two groups. RESULTS: Our results showed that totally 20 gut microbes were significantly different between two groups (p<0•05), and the correlation analysis found that g__Roseomonas and g__unclassified_f__Propionibacteriaceae were significantly positive correlated with both maternal body mass index (BMI) before delivery, placental weight, and neonatal birth weight (BW) percentile (all p<0•05), while g__Marinisporobacter and g__Sphingomonas were significantly negative correlated with both neonatal BMI and neonatal BW percentile (all p<0•05). Through KEGG pathway analysis, we found that the abundance of the Nitrogen metabolism pathway decreased significantly (p<0•05) whereas the abundance of the Amoebiasis pathway increased significantly in the FGR group (p<0•05). CONCLUSION: In this study, we demonstrated that the occurrence of FGR is associated with the change of gut microbiota of pregnant women.

Tunable mechanical behavior of collagen-based films: A comparison of celluloses in different geometries.

Collagen (Col) films were reinforced by celluloses in different geometries: microcrystalline cellulose (MCC), cellulosic fines (CF), cellulose nanofiber (CNF) and cellulose nanocrystals (CNC). The reinforcement mechanisms were investigated by the elastoplasticity and fracture appearance. Compared with the fracture stress of collagen film (67.5 MPa), the Col-CNF films effectively borne the stress (95.8 MPa) by intercrystalline fracture, ascribing the abundant hydrogen bonding and mechanical locking between cellulose and collagen. The toughness of Col-CF films was increased by the interfibrillar slippage of CF and pull-off of CF within the matrix, improving the strain-to-break from 8.37% to 12.13%. The films added with MCC and CNC weaken the mechanical behavior, due to the defects and lack of mechanical locking. Besides, the effects of celluloses' geometries on the thickness, density, water-tightness, thermal stability, crystallinity and FTIR of films were also investigated. These provide the evidence that the geometries of fillers diversely improve the behaviors of collagen film offering strategies for the film with adjustable mechanical properties.

Four methods to analyze H3K27M mutation in diffuse midline gliomas.

The histone H3 K27M mutation has been frequently reported in the majority of diffuse midline gliomas, which is considered as a prognostic and predictive biomarker. A number of different methods and platforms including pyrosequencing (PSQ), sanger sequencing, immunohistochemistry (IHC), Mass array and NGS (Next Generation Sequencing) have been used to detect H3K27M mutation in diffuse midline gliomas. However, controversy remains about the most appropriate method to use for analyzing H3K27M status. The H3K27 M mutation status of a total of 50 diffuse midline gliomas was examined using PSQ, sanger sequencing, IHC and Mass array in parallel. Using PSQ as a recommended standard method, the sensitivity, specificity and correlation with the other assays were calculated. Among 50 diffuse midline glioma cases, the H3K27M mutation was positive in 64 %, 66 %, 62 % and 62 % of the cases by PSQ, IHC, sanger sequencing and mass array, respectively. The sensitivity and specificity of IHC were 100 % and 94.4 %, respectively. The sensitivity and specificity of sanger sequencing and mass array were both 96.9 % and 100 %, respectively. This study demonstrated that IHC is an effective and rapid detection method for routine use in pathology laboratories for the identification of H3K27M mutation. A combination of IHC and sanger sequencing assays can provide 100 % sensitivity and specificity for the prediction of H3K27M status.

A top-down approach to improve collagen film's performance: The comparisons of macro, micro and nano sized fibers.

The severe reduction of mechanical strength of collagen once it is extracted or dissociated from animal tissues and no additional crosslinking approaches are conducted, impede its application in biodegradable and edible food packaging. Here, for the first time, high pressure homogenization (HPH) was used to prepare diverse sized fibers and the related fibers-composed films' performance were investigated. These fibers have a diversity of effects on film performance. The films prepared with smaller sized fibers had a more uniform and denser structure. The mechanical and the water barrier properties of the films improved significantly as the fiber size decreased. No obvious change in FTIR and thermal properties suggests that the improved film performance is mainly attributed to the physical entanglement and non-covalent bonds. Given the forementioned benefits of the films, control of fiber size can be a potential industrial approach for producing collagenous materials in edible food packaging.

LncRNA SNHG4 promotes the increased growth of endometrial tissue outside the uterine cavity via regulating c-Met mediated by miR-148a-3p.

Long noncoding RNAs (lncRNAs) modulate endometriosis. The current study investigated the mechanisms and effects of SNHG4 on endometriosis. The qRT-PCR was conducted to examine the miR-148a-3p and SNHG4 expressions in endometriosis tissues. The 5-ethynyl-2'-deoxyuridine incorporation assay and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay were used to measure the rate of cell proliferation. The association between miR-148a-3p, SNHG4 and c-Met was confirmed via bioinformatical approach and luciferase reporter gene assay. Also, the function of SNHG4 on the growth of endometriotic lesions was investigated in vivo. The SNHG4 expression was considerably upregulated in endometriosis tissues, whereas the level of miR-148a-3p expression was reduced. In addition, SNHG4 can be considered as ceRNAs that bind miR-148a-3p and rise the proliferation activity of HESCs by downregulating miR-148a-3p. Furthermore, silencing SNHG4 could downregulate the c-Met level by enhancing miR-148a-3p expression, and finally inhibiting endometriosis development in vivo. LncRNA SNHG4 promotes the increased growth of endometrial tissue outside the uterine cavity via regulating c-Met mediated by miR-148a-3p, which may be used as diagnostic biomarker as well as molecular target in the treatment of endometriosis.

Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress.

Parkinson's disease (PD) is a progressive neurodegenerative disorder. Although the precise mechanism remains unclear, mounting evidence suggests that oxidative stress plays an important role in the pathogenesis of PD. DJ-1 gene is associated with oxidative stress and mutations in DJ-1 are involved in an autosomal recessive, early onset familial form of PD. The ERK1/2 signaling pathway contributes to neuroprotection during oxidative stress. However, the correlation between DJ-1 and the ERK1/2 signaling pathway remains unknown. To test for an association of DJ-1 with the ERK1/2 signaling pathway, we transfected wild-type and L166P mutated DJ-1 into COS-7 and MN9D cells. The results showed that over-expression of WT-DJ-1 dramatically enhanced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2. Meanwhile, WT-DJ-1, but not L166P-DJ-1 inhibited the expression of protein phosphatase 2A (PP2A), an inhibitor of the ERK1/2 signaling pathway. Moreover, over-expression of WT-DJ-1 increased cell viability and decreased cell sensitivity to H2O2-induced neurotoxicity. Inhibition of the ERK1/2 signaling pathway with a MEK1/2 inhibitor reversed these changes. We conclude that DJ-1 does affect the ERK1/2 signaling pathway and change the susceptibility of cells to oxidative stress.

Human umbilical cord blood-derived non-hematopoietic stem cells suppress lymphocyte proliferation and CD4, CD8 expression.

One concern in the use of transplantation of non-hematopoietic stem cells from human umbilical cord blood (CB-nHSCs) is the possibility of rejection by the host's immune system. This study shows that both CB-nHSCs and their progenies after passaging, neuronal differentiation or IFN-gamma treatment have no significant effects on proliferation of xenogenic T lymphocytes. CB-nHSCs transplanted into the striatum of SD rat are shown to induce a lower level of CD4 and CD8 expression in the brain and in the peripheral blood and to survive better in the brain than SH-SY5Y cells. The results indicate that both undifferentiated and differentiated CB-nHSCs all have weak immunogenicity.

Comparison of the labeling efficiency of BrdU, DiI and FISH labeling techniques in bone marrow stromal cells.

Cells are generally labeled during in vivo implantation studies enabling the cells to be traced. The relationship between the labeling efficiency and cellular proliferation after transplantation is critical for the interpretation of data obtained by detection of the signals on tissue sections. Here, we compare cellular labeling methods of rat marrow stromal cells that were labeled with 5-bromo-2-deoxyuridine (BrdU), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and fluorescence in situ hybridization (FISH). Our data show that (i) BrdU uniformly labeled the nuclei, (ii) DiI-labeled cells had many dots or stained clear and uniform when a longer exposure time was used during detection and (iii) FISH labeled the cells with dots along the edges of the nuclei. The labeling efficiency was 94.1+/-8.6%, 97.6+/-3.4% and 90.5+/-3.0%, in BrdU, DiI- and FISH-labeled cells, respectively. After sub-culturing of labeled cells, the percentage of BrdU-positive cells was found to be 71.9+/-18.0% and 18.4+/-6.9%, after the first and second passages, respectively. The percentage of DiI-labeled cells detected depended on the exposure time: a long exposure time (>10 s) resulted in identification of 95.1+/-4.0% and 94.5+/-3.9% DiI-positive cells after the first and second sub-cultures, respectively. The percentage of FISH-positive cells was found to be 87.0+/-3.0% and 89.1+/-9.7%. The BrdU labeling signal quickly decreased over time. Thus, BrdU should only be used to temporarily label dividing cells. In contrast, our data indicate that DiI and FISH labeling may be used to steadily trace cells during in vivo experiments. To our knowledge, this is the first time that the effects of different labeling methods over time have been examined during a cell transplantation study.

Establishment of an immortalized GABAergic neuronal progenitor cell line from embryonic ventral mesencephalon in the rat.

Effective cell replacement therapies for neurological disease require neuron-restricted precursors as grafted cells. The problem of obtaining sufficient grafts for transplantation can be resolved by creating an appropriate immortalized cell line. In the present study, a thermally controlled immortalized GABAergic neuronal progenitor cell line (RMNE6) was established from E13 rat ventral mesencephalon cells immortalized using the temperature-sensitive mutant of SV40 large T antigen (ts-TAg). RMNE6 cells proliferated rapidly and expressed a neuron-like phenotype at the permissive temperature (33 degrees C), but eventually stopped growing at the non-permissive temperature (39 degrees C). Expression of the neuronal markers PSA-NCAM, beta-tubulin III and MAP2 by RMNE6 cells was confirmed by RT-PCR or immunocytochemistry. Furthermore, these cells exhibited functional GABAergic neuron properties, as evidenced by the expression of glutamate decarboxylase (GAD) as well as the synthesis and release of the neurotransmitter GABA in a calcium-dependent manner. Moreover, RMNE6 cells spontaneously expressed and secreted several neurotrophic factors, such as NGF, BDNF, NT-3, NT-4/5, and GDNF. The cells survived well and kept expression of SV40 Tag, GAD65/67 and GABA in the striatum, at least 28 days after being transplanted in the rat brain. Tumorigenesis assays confirmed the safety of the immortalized cell line in vivo. Taken together, the results support the use of RMNE6 cells as an ideal cell model for transplantation research aimed at the treatment and prevention of neurodegenerative disease.

Lamin A/C might be involved in the EMT signalling pathway.

We have previously reported a heterogeneous expression pattern of the nuclear membrane protein lamin A/C in low- and high-Gleason score (GS) prostate cancer (PC) tissues, and we have now found that this change is not associated with LMNA mutations. This expression pattern appears to be similar to the process of epithelial to mesenchymal transition (EMT) or to that of mesenchymal to epithelial transition (MET). The role of lamin A/C in EMT or MET in PC remains unclear. Therefore, we first investigated the expression levels of and the associations between lamin A/C and several common EMT markers, such as E-cadherin, N-cadherin, ß-catenin, snail, slug and vimentin in PC tissues with different GS values and in different cell lines with varying invasion abilities. Our results suggest that lamin A/C might constitute a type of epithelial marker that better signifies EMT and MET in PC tissue, since a decrease in lamin A/C expression in GS 4 + 5 cases is likely associated with the EMT process, while the re-expression of lamin A/C in GS 5 + 4 cases is likely linked with MET. The detailed GS better exhibited the changes in lamin A/C and the EMT markers examined. Lamin A/C overexpression or knockdown had an impact on EMT biomarkers in a cell model by direct regulation of ß-catenin. Hence, we suggest that lamin A/C might serve as a reliable epithelial biomarker for the distinction of PC cell differentiation and might also be a fundamental factor in the occurrence of EMT or MET in PC.

Effect of Dandelion Extracts on the Proliferation of Ovarian Granulosa Cells and Expression of Hormone Receptors.

BACKGROUND: In the current society, infertility related to age has become a social problem. The in vitro fertilization (IVF) success rate in women with poor ovarian response (POR) is very low. Dandelion extract T-1 (DE-T1) is an effective component of the extract from the leaves and stems of Taraxacum officinale, which is one of the medicines used in some patients with POR, but its molecular mechanism remains unclear. METHODS: Following IVF, ovarian granulosa cells (GCs) of sixty patients were extracted and divided into normal ovarian response (NOR) and POR groups. GCs were cultured in a dose-dependent and time-dependent manner with DE-T1, proliferation of GCs was determined by Cell Counting Kit-8 assay, and mRNA levels of insulin-like growth factor 1 receptor (IGF-1R), luteotropic hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), LHR, and CYP19A1 (aromatase) were determined by quantitative polymerase chain reaction. Progesterone and estradiol (E2) concentrations were determined by enzyme-linked immunosorbent assay. RESULTS: The cell viability gradually increased with the progressive increase in the DE-T1 concentration. Compared with the control group (without DE-T1), the mRNA expressions of FSHR, LHR, IGF-1R, and CYP19A1 were upregulated after the addition of DE-T1, especially in the 2.5% DE-T1 group (P < 0.01). The expression of IGF-1R was upregulated approximately 25 times (24.97 ± 4.02 times) in the POR group with 2.5% DE-T1. E2 and progesterone levels increased with the increasing DE-T1 concentration. There were highly significant differences in the E2 and progesterone secretion between the NOR and POR groups (P < 0.01). CONCLUSION: DE-T1 may promote steroid hormone synthesis by promoting GC proliferation and upregulating GC receptor expression, thereby improving ovarian endocrine function.

Astrocytes protect MN9D neuronal cells against rotenone-induced oxidative stress by a glutathione-dependent mechanism.

Astrocytes maintain homeostasis of neuronal microenvironment, provide metabolic and trophic support to neurons and modulate neuronal responses to injury. Rotenone specifically inhibits mitochondrial complex I, and long exposure to rotenone may increase the risk for Parkinson's disease (PD) and cause Parkinsonism. However, little is known about the role of astrocytes in the process of rotenone-induced dopaminergic neuron injury. In order to investigate this issue, we used MN9D cells as a cell model of dopaminergic neurons and rotenone as a toxin to initiate mitochondrial deficiency. MN9D cells treated with the normal medium or astrocyte-conditioned medium (ACM) were exposed to different concentrations of rotenone for different time followed by cell viability measurement by MTT assay. Besides, various concentrations of ACM and temporally different treatments were devised to evaluate protective efficiency of ACM. Growth curve of cells in the normal medium or ACM was continuously assessed by cell counting for 8 d. The influence of rotenone and ACM on cellular oxidative stress was determined by DCFH-DA staining followed by flow cytometric analysis. Glutathione (GSH) content after treatment of ACM or rotenone was measured by GSH assay kit. Our results showed that rotenone decreased viability of MN9D cells in a dose-dependent manner and ACM treatment significantly attenuated rotenone toxicity at each concentration. No significant difference in growth rate was observed between the normal medium and ACM treatment. Four concentrations of ACM, namely 1/3ACM, 1/2ACM, 2/3ACM and pure ACM, all displayed protection, increasing cell viability to (124.15+/-0.79)%, (126.59+/-0.82) %, (125.84+/-0.61) % and (117.15+/-1.63) % of the cells exposed directly to rotenone, respectively. Treatment with ACM through the whole experiment except the initial 24 h, 24 h before or at the same time of rotenone addition all exerted protective effects, with cell viability being (110.11+/-2.52)%, (113.30+/-2.36) %, (114.42+/-2.00)% of the cells exposed directly to rotenone, respectively. Conversely, ACM treatment 12 h after rotenone addition had no protective effect, with cell viability being (102.54+/-1.36)% of the cells exposed directly to rotenone. Moreover, ACM treatment up-regulated GSH level in MN9D cells nearly twofold. Incubation with 100 nmol/L rotenone for 24 h depleted GSH level by nearly two thirds of the control, but ACM treatment mitigated the drop of GSH level, maintaining its content at (147.83+/-0.63)% of the control. Consistent with GSH change, rotenone administration resulted in a positive rate of 96.24% of DCF staining, implying a great extent of oxidative stress, whereas treatment with ACM reduced the extent of oxidative stress to a positive rate of 78.31%. Taken together, these findings suggest that astrocytes protect MN9D cells from oxidative stress caused by rotenone, and GSH partially accounts for the protection. Therefore, astrocytes may play a protective role in the process of PD.

[Overexpression of alpha-synuclein in SH-SY5Y cells partially protected against oxidative stress induced by rotenone].

Both genetic and environmental factors are involved in the pathogenesis of Parkinsonos disease (PD). Epidemiological studies showed that environmental factors shared with the common mechanisms of resulting in alpha-synuclein aggregation by inhibiting complex I of mitochondria and leading to oxidative stress. To investigate the relationship between alpha-synuclein and oxidative stress, we used human dopaminergic SH-SY5Y cells transfected with alpha-synuclein-enhanced green fluorescent protein (EGFP). alpha-synuclein gene expression was determined by immunocytochemistry and real-time quantitative PCR. Both SH-SY5Y and alpha-synuclein overexpressed SH-SY5Y (SH-SY5Y/Syn) cells were treated with various concentrations of rotenone for different time. Cell viability and oxidative stress were detected by MTT assay and DCF assay. Superoxide dismutase (SOD) activity was assessed with xanthine peroxidase method. Cell apoptosis was detected with flow cytometry. Results showed that alpha-synuclein gene was constantly overexpressed in SH-SY5Y/Syn cells. After treatment with rotenone, both cell viability and complex I activity in these cells were reduced in a concentration-dependent manner. Oxidative stress was also found in these cells. Compared with SH-SY5Y cells, SOD activity in SH-SY5Y/Syn cells was increased distinctly (P<0.05) and alpha-synuclein significantly attenuated rotenone-induced cell apoptosis. These results suggest that the alpha-synuclein overexpression in SH-SY5Y cells has a tendency to partially resist oxidative stress induced by rotenone and this response may assist cell survival.