A time window for rescuing dying retinal ganglion cells.

BACKGROUND: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs). METHODS: prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca2+ were revealed by annexin A5-FITC, Mito-tracker, TMRM, and Fluo 8-AM staining, respectively. The distribution of cytochrome c was investigated by immunofluorescence. The ultrastructure of mitochondria was studied by electron microscopy. RESULTS: Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca2+ was elevated during this stage of reversible mitochondrial injury, but there was no sign of mitochondrial cytochrome c release. CONCLUSIONS: Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.

Clinical Characteristics of Multiple Sclerosis Patients Complicated by Disabilities and Analysis of Risk Factors Related to Disease Progression.

Objective: To analyze the clinical characteristics of multiple sclerosis (MS) patients complicated by disabilities in China, and to discuss the related factors of disease progression. Methods: Ninety-three MS patients presented to our hospital between March 2017 and December 2019 were selected as the research participants to conduct a retrospective analysis. Demographic information, onset time, onset age, clinical symptoms, MS types, and Expanded Disability Status Scale (EDSS) score were collected from all patients, and preliminary observation was made on MS cases in China. Subsequently, patients were grouped according to their sex, onset age and MS types to observe the differences in clinical characteristics of MS under different conditions. Finally, Logistic analysis was conducted to analyze the related factors affecting disease progression in MS patients. Results: MS was likely to occur in all age groups, among which the 30-40 age group had a slightly higher predilection. Women were more predisposed to MS, with motor symptoms as the major clinical presentations. The number of patients with sensory symptoms and the frequency of episodes in the past year were higher in female patients than in male patients (P < .05). Clinical isolated syndrome (CIS) patients had lower baseline ESDD than relapsing remitting MS (RRMS) patients (P < .05). According to Logistic regression analysis, baseline ESDD score and the frequency of episodes in the past year were independent risk factors affecting MS progression (P < .05). Conclusions: The clinical characteristics of MS in the Chinese population are basically similar to those in foreign countries, but RRMS accounts for a relatively low proportion. The ESDD score and the frequency of episodes in the past year are independent risk factors for MS progression.

Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells.

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Author Correction: Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/ EETs and suppressing ER stress.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/EETs and suppressing ER stress.

AIM: CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis. METHODS: Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca(2+) concentrations ([Ca(2+)]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA. RESULTS: Angiotensin II (10(-6) mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca(2+)]i in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules (GRP78, CHOP, p-JNK and cleaved caspase-12) and ER stress-mediated apoptosis. OPD (100, 250 and 500 nmol/L) dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca(2+) homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca(2+) homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca(2+) homeostasis. Furthermore, the intracellular Ca(2+) chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells. CONCLUSION: OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca(2+) homeostasis and ER stress.

Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation.

AIM: Pregnane X receptor (PXR) is a nuclear receptor that regulates a number of genes encoding drug metabolism enzymes and transporters and plays a key role in xeno- and endobiotic detoxification. Ginkgolide B has shown to increase the activity of PXR. Here we examined whether ginkgolide B activated PXR and attenuated xenobiotic-induced injuries in endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with ginkgolide B. The expression of PXR, CYP3A4, MDR1, VCAM-1, E-selectin and caspase-3 were quantified with qRT-PCR and Western blot analysis. Cell apoptosis was analyzed with flow cytometry. Fluorescently labeled human acute monocytic leukemia cells (THP-1 cells) were used to examine cell adhesion. RESULTS: Ginkgolide B (30-300 µmol/L) did not change the mRNA and protein levels of PXR in the cells, but dose-dependently increased nuclear translocation of PXR protein. Ginkgolide B increased the expression of CYP3A4 and MDR1 in the cells, which was partially reversed by pretreatment with the selective PXR signaling antagonist sulforaphane, or transfection with PXR siRNA. Functionally, ginkgolide B dose-dependently attenuated doxorubicin- or staurosporine-induced apoptosis, which was reversed by transfection with PXR siRNA. Moreover, ginkgolide B suppressed TNF-α-induced THP-1 cell adhesion and TNF-α-induced expression of vascular adhesion molecule 1 (VCAM-1) and E-selectin in the cells, which was also reversed by transfection with PXR siRNA. CONCLUSION: Ginkgolide B exerts anti-apoptotic and anti-inflammatory effects on endothelial cells via PXR activation, suggesting that a PXR-mediated endothelial detoxification program may be important for protecting endothelial cells from xeno- and endobiotic-induced injuries.

PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration.

Neurodegenerative disorders are characterized by the progressive loss of structure and function of neurons, often including the death of the neuron. Previously, we reported that, by removing the cell death stimulus, dying/injured neurons could survive and recover from the process of regulated cell death, even if the cells already displayed various signs of cellular damage. Now we investigated the role of mitochondrial dynamics (fission/fusion, biogenesis, mitophagy) in both degeneration and in recovery of neuronal cells. In neuronal PC12 cells, exposure to ethanol (EtOH) induced massive neurite loss along with widespread mitochondrial fragmentation, mitochondrial membrane potential loss, reduced ATP production, and decreased total mitochondrial volume. By removing EtOH timely all these mitochondrial parameters recovered to normal levels. Meanwhile, cells regrew neurites and survived. Study of the mitochondrial dynamics showed that autophagy was activated only during the cellular degeneration phase (EtOH treatment) but not in the recovery phase (EtOH removed), and it was not dependent on the Parkin/PINK1 mediated mitophagy pathway. Protein expression of key regulators of mitochondrial fission, phospho-Drp1Ser616 and S-OPA1, increased during EtOH treatment and recovered to normal levels after removing EtOH. In addition, the critical role of PGC-1α mediated mitochondrial biogenesis in cellular recovery was revealed: inhibition of PGC-1α using SR-18292 after EtOH removal significantly impeded recovery of mitochondrial damage, regeneration of neurites, and cell survival in a concentration-dependent manner. Taken together, our study showed reversibility of mitochondrial morphological and functional damage in stressed neuronal cells and revealed that PGC-1α mediated mitochondrial biogenesis played a critical role in the cellular recovery. This molecular mechanism could be a target for neuroprotection and neurorescue in neurodegenerative diseases.

Sinomenine ameliorates fibroblast-like synoviocytes dysfunction by promoting phosphorylation and nuclear translocation of CRMP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and arthritic pain. Sinomenine (SIN), derived from the rhizome of Chinese medical herb Qing Teng (scientific name: Sinomenium acutum (Thunb.) Rehd. Et Wils), has a longstanding use in Chinese traditional medicine for treating rheumatoid arthritis. It has been shown to possess anti-inflammatory, analgesic, and immunosuppressive effects with minimal side-effects clinically. However, the mechanisms governing its effects in treatment of joint pathology, especially on fibroblast-like synoviocytes (FLSs) dysfunction, and arthritic pain remains unclear. AIM: This study aimed to investigate the effect and underlying mechanism of SIN on arthritic joint inflammation and joint FLSs dysfunctions. MATERIALS AND METHODS: Collagen-induced arthritis (CIA) was induced in rats and the therapeutic effects of SIN on joint pathology were evaluated histopathologically. Next, we conducted a series of experiments using LPS-induced FLSs, which were divided into five groups (Naïve, LPS, SIN 10, 20, 50 µg/ml). The expression of inflammatory factors was measured by qPCR and ELISA. The invasive ability of cells was detected by modified Transwell assay and qPCR. Transwell migration and cell scratch assays were used to assess the migration ability of cells. The distribution and content of relevant proteins were observed by immunofluorescence and laser confocal microscopy, as well as Western Blot and qPCR. FLSs were transfected with plasmids (CRMP2 T514A/D) to directly modulate the post-translational modification of CRMP2 protein and downstream effects on FLSs function was monitored. RESULTS: SIN alleviated joint inflammation in rats with CIA, as evidenced by improvement of synovial hyperplasia, inflammatory cell infiltration and cartilage damage, as well as inhibition of pro-inflammatory cytokines release from FLSs induced by LPS. In vitro studies revealed a concentration-dependent suppression of SIN on the invasion and migration of FLSs induced by LPS. In addition, SIN downregulated the expression of cellular CRMP2 that was induced by LPS in FLSs, but increased its phosphorylation at residue T514. Moreover, regulation of pCRMP2 T514 by plasmids transfection (CRMP2 T514A/D) significantly influenced the migration and invasion of FLSs. Finally, SIN promoted nuclear translocation of pCRMP2 T514 in FLSs. CONCLUSIONS: SIN may exert its anti-inflammatory and analgesic effects by modulating CRMP2 T514 phosphorylation and its nuclear translocation of FLSs, inhibiting pro-inflammatory cytokine release, and suppressing abnormal invasion and migration. Phosphorylation of CRMP2 at the T514 site in FLSs may present a new therapeutic target for treating inflammatory joint's destruction and arthritic pain in RA.

Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release.

Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing and phosphatidylserine (PS) exposure at the plasma membrane. It is still poorly understood which events mark the point of no return for dying neurons. Here we analyzed the neuronal cell line SH-SY5Y expressing cytochrome C (Cyto.C)-GFP. Cells were exposed temporarily to ethanol (EtOH) and tracked longitudinally in time by light and fluorescent microscopy. Exposure to EtOH induced elevation of intracellular Ca2+ and reactive oxygen species, cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing, PS exposure and Cyto.C release into the cytosol. Removing EtOH at predetermined time points revealed that all phenomena except Cyto.C release occurred in a phase of neuronal cell death in which full recovery to a neurite-bearing cell was still possible. Our findings underscore a strategy of treating chronic neurodegenerative diseases by removing stressors from neurons and harnessing intracellular targets that delay or prevent trespassing the point of no return.

Sinomenine regulates immune cell subsets: Potential neuro-immune intervene for precise treatment of chronic pain.

Chronic pain is a disease of long-lasting pain with unpleasant feelings mediated by central and (or) peripheral sensitization, its duration usually lasts more than 3 months or longer than the expected recovery time. The patients with chronic pain are manifested with enhanced sensitivity to noxious and non-noxious stimuli. Due to an incomplete understanding of the mechanisms, patients are commonly insensitive to the treatment of first line analgesic medicine in clinic. Thus, the exploration of non-opioid-dependent analgesia are needed. Recent studies have shown that "sinomenine," the main active ingredient in the natural plant "sinomenium acutum (Thunb.) Rehd. Et Wils," has a powerful inhibitory effect on chronic pain, but its underlying mechanism still needs to be further elucidated. A growing number of studies have shown that various immune cells such as T cells, B cells, macrophages, astrocytes and microglia, accompanied with the relative inflammatory factors and neuropeptides, are involved in the pathogenesis of chronic pain. Notably, the interaction of the immune system and sensory neurons is essential for the development of central and (or) peripheral sensitization, as well as the progression and maintenance of chronic pain. Based on the effects of sinomenine on immune cells and their subsets, this review mainly focused on describing the potential analgesic effects of sinomenine, with rationality of regulating the neuroimmune interaction.

Design of a Spark Big Data Framework for PM2.5 Air Pollution Forecasting.

In recent years, with rapid economic development, air pollution has become extremely serious, causing many negative effects on health, environment and medical costs. PM2.5 is one of the main components of air pollution. Therefore, it is necessary to know the PM2.5 air quality in advance for health. Many studies on air quality are based on the government's official air quality monitoring stations, which cannot be widely deployed due to high cost constraints. Furthermore, the update frequency of government monitoring stations is once an hour, and it is hard to capture short-term PM2.5 concentration peaks with little warning. Nevertheless, dealing with short-term data with many stations, the volume of data is huge and is calculated, analyzed and predicted in a complex way. This alleviates the high computational requirements of the original predictor, thus making Spark suitable for the considered problem. This study proposes a PM2.5 instant prediction architecture based on the Spark big data framework to handle the huge data from the LASS community. The Spark big data framework proposed in this study is divided into three modules. It collects real time PM2.5 data and performs ensemble learning through three machine learning algorithms (Linear Regression, Random Forest, Gradient Boosting Decision Tree) to predict the PM2.5 concentration value in the next 30 to 180 min with accompanying visualization graph. The experimental results show that our proposed Spark big data ensemble prediction model in next 30-min prediction has the best performance (R2 up to 0.96), and the ensemble model has better performance than any single machine learning model. Taiwan has been suffering from a situation of relatively poor air pollution quality for a long time. Air pollutant monitoring data from LASS community can provide a wide broader monitoring, however the data is large and difficult to integrate or analyze. The proposed Spark big data framework system can provide short-term PM2.5 forecasts and help the decision-maker to take proper action immediately.

Advanced treatment of dyeing wastewater towards reuse by the combined Fenton oxidation and membrane bioreactor process.

The performance of combined Fenton oxidation and membrane bioreactor (MBR) process for the advanced treatment of an effluent from an integrated dyeing wastewater treatment plant was evaluated. The experimental results revealed that under the optimum Fenton oxidation conditions (initial pH 5, H2O2 dosage 17 mmol/L, and Fe2+ 1.7 mmol/L) the average total organic carbon (TOC) and color removal ratios were 39.3% and 69.5% after 35 min of reaction, respectively. Results from Zahn-Wallens Test also represented that Fenton process was effective to enhance the biodegradability of the test wastewater. As for the further purification of MBR process, TOC removal capacity was examined at different hydraulic retention times (HRT) of 10, 18 and 25 hr. Under the optimum HRT of 18 hr, the average TOC concentration and color of the final MBR effluent were 16.8 mg/L and 2 dilution time, respectively. The sludge yield coefficient was 0.13 g MLSS/g TOC and TOC degradation rate was 0.078 kg TOC/(m3 x day). The final effluent of MBR can meet the reuse criteria of urban recycling water - water quality standard for miscellaneous water consumption GBT18920-2002.

Ophiopogonin D Increases SERCA2a Interaction with Phospholamban by Promoting CYP2J3 Upregulation.

Ophiopogonin D (OPD), a compound from the Chinese herb Radix Ophiopogonis, reportedly induces increased levels of cytochrome P450 2J3 (CYP2J3)/epoxyeicosatrienoic acids (EETs) and Ca2+ in rat cardiomyocytes. Little is known regarding the specific mechanism between CYP2J3 and Ca2+ homeostasis. Here, we investigated whether CYP2J3 is involved in the protective action of OPD on the myocardium by activating the Ca2+ homeostasis-related protein complex (SERCA2a and PLB) in H9c2 rat cardiomyoblast cells. The interaction between SERCA2a and PLB was measured using fluorescence resonance energy transfer. OPD attenuated heart failure and catalyzed the active transport of Ca2+ into the sarcoplasmic reticulum by inducing the phosphorylation of PLB and promoting the SERCA2a activity. These beneficial effects of OPD on heart failure were abolished after knockdown of CYP2J3 in a model of heart failure. Together, our results identify CYP2J3 as a critical intracellular target for OPD and unravel a mechanism of CYP2J3-dependent regulation of intracellular Ca2+.

A rapid and sensitive UPLC-MS/MS method for the determination of flibanserin in rat plasma: application to a pharmacokinetic study.

BACKGROUND: In this work, we aim to develop and validate a fast, simple, and sensitive method for the quantitative determination of flibanserin and the exploration of its pharmacokinetics. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was the method of choice for this investigation and carbamazepine was selected as an internal standard (IS). The plasma samples were processed by one-step protein precipitation using acetonitrile. The highly selective chromatographic separation of flibanserin and carbamazepine (IS) was realised using an Agilent RRHD Eclipse Plus C18 (2.1 × 50 mm, 1.8 µ) column with a gradient mobile phase consisting of 0.1% formic acid in water and acetonitrile. The analytes were detected using positive-ion electrospray ionization mass spectrometry via multiple reaction monitoring (MRM). The target fragment ions were m/z 391.3 → 161.3 for flibanserin and m/z 237.1 → 194 for carbamazepine (IS). The method was validated by linear calibration plots over the range of 100-120,000 ng/mL for flibanserin (R2 = 0.999) in rat plasma. RESULTS: The extraction recovery of flibanserin was in the range of 91.5-95.8%. The determined inter- and intra-day precision was below 12.0%, and the accuracy was from - 6.6 to 12.0%. No obvious matrix effect and astaticism was observed for flibanserin. The target analytes were long-lasting and stable in rat plasma for 12 h at room temperature, 48 h at 4 °C, 30 days at - 20 °C, as well as after three freeze-thaw cycles (from - 20 °C to room temperature). The proposed method has been fully validated and successfully applied to the pharmacokinetic study of flibanserin.

Antidepressant-like effect of low molecular proanthocyanidin in mice: involvement of monoaminergic system.

Proanthocyanidin is a phenolic product present in plants which has antioxidant, antinociceptive and neuroprotective properties, without inducing significant toxicological effects. The present study tested the hypothesis that low molecular proanthocyanidin from grapes that has optimized bioavailability, would exert antidepressant-like activities in behavioral despair tests. The results suggested that oral administration proanthocyanidin at doses of 25 and 50mg/kg for 7days significantly reduced the duration of immobility in both the tail suspension and forced swimming tests. The doses that affected the immobile response did not affect locomotor activity. In addition, the neurochemical and neuropharmacological assays showed that proanthocyanidin produced a marked increase of 5-HT levels at 25 and 50mg/kg in three brain regions, the frontal cortex, hippocampus and hypothalamus. Noradrenaline and dopamine levels were also increased when higher dose of proanthocyanidin (50mg/kg) administration both in the frontal cortex and hippocampus. These effects were similar to those observed for the classical antidepressant imipramine (10mg/kg, i.p.). Moreover, Our study suggested that proanthocyanidin (12.5, 25 and 50mg/kg) dose dependently inhibited monoamine oxidase-A (MAO-A) activity, while MAO-B inhibitory activity was also found at higher doses (25 and 50mg/kg) after 7days administration. MAO-A selective inhibitor, moclobemide (20mg/kg, i.g.) produced MAO-A inhibition of 70.5% in the mouse brain. These findings suggest that the antidepressant-like effects of proanthocyanidin may involve the central monoaminergic neurotransmitter systems.

Antidepressant-like effect of trans-resveratrol: Involvement of serotonin and noradrenaline system.

The antidepressant-like effect of trans-resveratrol, a phenolic compound present in polygonum cuspidatum, was evaluated through behavioral and neurochemical methods. trans-Resveratrol (20, 40 and 80 mg/kg, via gavage) significantly decreased the immobility time in mouse models of despair tests, but did not influence locomotor activity. Two behavioral models and neurochemical assays suggested that trans-resveratrol produced a significant increase in serotonin and noradrenaline levels at 40 or 80 mg/kg in brain regions. In addition, trans-resveratrol dose dependently inhibited MAO-A activity. These findings indicate that the antidepressant-like effect of trans-resveratrol might be related to serotonergic and noradrenergic activation.