This differs from previously described alterations in intrinsic characteristics which coincided with enhanced purinergic currents following SE. These ECS-induced effects point to a “seizure signature” in hippocampal microglia characterized by altered purinergic signaling. These data demonstrate that ictal activity per se can drive alterations in microglial physiology without neuronal injury. These physiological changes, which up until now have been associated with prolonged and damaging seizures, are of added interest as they may be relevant to electroconvulsive therapy (ECT), which remains a gold-standard treatment for depression.Microglia are in a privileged position to both affect and be affected by neuroinflammation, neuronal activity and injury, which are all hallmarks of seizures and the epilepsies. Hippocampal microglia become activated after prolonged, damaging seizures known as status epilepticus (SE). However, since SE causes both hyperactivity and injury of neurons, the mechanisms triggering this activation remain unclear, as does the relevance of the microglial activation to the ensuing epileptogenic processes. In this study, we use electroconvulsive shock (ECS) to study the effect of neuronal hyperactivity without neuronal degeneration on mouse hippocampal microglia. Unlike SE, ECS did not alter hippocampal CA1 microglial density, morphology, or baseline motility. In contrast, both ECS and SE produced a similar increase in ATP-directed microglial process motility in acute slices, and similarly upregulated expression of the chemokine C-C motif chemokine ligand 2 (CCL2). Whole-cell patch-clamp recordings of hippocampal CA1 Epilepsy is the fourth most prevalent neurologic disease, affecting one in 26 people over their lifetime. There is a critical unmet need in understanding basic mechanisms underlying the development of epilepsy (epileptogenesis), given that no disease-modifying treatments are currently available. How specific features of microglial activation contribute to subsequent epileptogenesis, and how seizure activity, per se, triggers changes in microglial responses is understudied. In this study, we demonstrate that hippocampal microglia react acutely to single non-epileptogenic seizures, in ways reminiscent of SE-induced activation. Thus, key features of the microglial activation pattern observed after SE may not be related to the epileptogenic process, and further work is needed to fully characterize the interplay between microglia, seizures, and epilepsy.Neuroinflammation and microglial activation are hallmarks of many neurologic and neurodegenerative diseases, including the epilepsies . As the It is now clear that microglia respond to epileptogenic insults in acquired models of epilepsy . FurtherMuch like other epileptogenic insults to the brain , status Moreover, because experimental SE in rodents also acutely causes extensive hippocampal degeneration  and blooTo isolate the role of acute seizures from that of SE-induced damage, we used electroconvulsive shock (ECS), an acute seizure model that is not associated with epileptogenesis nor neuronal cell death . Repeate3CR1GFP/+ mice, which possess green fluorescent protein in place of one allele of the fractalkine receptor gene, resulting in fluorescently labeled microglia  were obtained from The Jackson Laboratory and backcrossed onto a C57BL/6J  congenic background before this study. A total of 143 mice were used for the experiments in this study. All animals used in this study were housed on a standard 12/12 h light/dark cycle with water and standard chow available ad libitum.We used postnatal day (P)30–P45 male and female CXicroglia . CX3CR1GExperimental and control mice were heterozygous littermates resulting from homozygote × wild-type mating. Littermates were randomized to treatment and control groups. Matched control animals were handled and treated in the same manner except that they received sham/0 mA shocks or saline/vehicle instead of pilocarpine injections (see below). Vehicle and sham control animals were pooled in a single control group for all analyses. The procedures described in this manuscript were performed with the approval of the Georgetown University Animal Care and Use Committee.https://phenome.jax.org/projects/Frankel1/protocol.Maximal (tonic-clonic) seizures were elicited by transcorneal stimulation using an Ugo Basile Electro-Convulsive Device  and custom-built transcorneal electrodes. Animals received 0.5% tetracaine HCl eyedrops (Alcon) 15–30 min before stimulation. Shocks consisting of 0.3 s long trains of 0.9 ms wide square pulses  at 200 Hz reliably evoked a tonic hindlimb extension response lasting between 12 and 20 s with negligible mortality. Stimulation protocols and intensities were adapted from the literature  and the For the SE model : 30 min after pretreating with 1 mg/kg each of scopolamine methylbromide and terbutaline , we inje2, 6.6 mmol/l MgSO4 anhydrate, 1.4 mmol/l NaH2PO4, 26 mmol/l NaHCO3, 25 mmol/l dextrose, and 75 mmol/l sucrose ; 300 μm horizontal hippocampal slices were cut on a Vibratome 3000 Plus Sectioning System (Vibratome), in cold sucrose aCSF as above. Sections were recovered for 30 min at 32°C in normal/recording aCSF solution, containing: 124 mmol/l NaCl, 4.5 mmol/l KCl, 1.2 mmol/l Na2HPO4, 26 mmol/l NaHCO3, 2 mmol/l CaCl2, 1 mmol/l MgCl2, and 10 mmol/l dextrose. Slices were then transferred to room temperature  and equilibrated for >10 min before use. In some experiments, slices were incubated with 1.8 μM sulforhodamine 101 in aCSF for 10 min to mark astrocytes . All experiments were conducted at RT.Twenty-four hours following seizure induction, mice were euthanized by decapitation and brains were rapidly dissected into an ice-cold sucrose aCSF slicing solution as previously described , and in trocytes  for unre2; pH 7.35 adjusted with KOH.Whole-cell patch-clamp recordings were performed under DIC illumination on fluorescently identified ramified cells, with 4.5 to 6.5 MΩ pipettes pulled from Wiretrol II borosilicate glass capillaries (Drummond) filled with an internal solution as described in SCR_011323, Molecular Devices). The LJP correction was only applied to our reported resting membrane potential (RMP) values. Patch-clamp was performed with a MultiClamp700B amplifier (Molecular Devices). Recordings were digitized at 20 kHz and low-pass Bessel-filtered at 2 kHz with a personal computer running Clampex11 and a DigiData1440 (Molecular Devices).Liquid junction potential  was calculated in Clampex11  mode, immediately after break-in to minimize effect of dialysis. All other data were recorded in voltage clamp configuration. Input resistance (IR), access resistance and cell capacitance were calculated from the current response to brief −5 mV hyperpolarizing voltage steps. I/V curves were calculated using 500-ms voltage steps from −60 mV to Vm = −120 mV to +50 mV, every 10 mV.2 and MgCl2) was locally perfused via a custom-made Y-tube apparatus  was dissolved in aCSF to create a 1 mM stock solution, and then some slices were preincubated in 5 μM BBG in aCSF for 30 min . Electroxyz) volumes of horizontal hippocampal slices containing CA1sr were imaged with a long working distance 60× water-dipping objective . Differential interference contrast (DIC) images (on acute and fixed slices) or fluorescent images of NeuN  staining  were used to identify and confirm our region of interest as CA1sr.Confocal Z- or ZT-stacks were taken using a laser scanning microscope system (Thor Imaging Systems Division) equipped with 488/561/642 nm lasers and Green/Red/Far-red filters and dichroics and mounted on an upright Eclipse FN1 microscope (Nikon Instruments); 284 × 284 × 20 μm (z-axis) time lapses were registered using the StackReg plugin  before the motility analyses.For the baseline and responsive motility experiments, we took 1024 × 1024 pixel (px) ZT-stacks of acute slices by imaging 11 planes 2 μm apart every 30 s. If necessary, maximal intensity projection (MIP over the g plugin  in FIJI sr of either naïve slices or in the presence of a 0 mM [ATP] containing (aCSF-only) pipette .Spontaneous motility of microglial processes directly mediates the physical microglia-neuron contact that is a prerequisite for many microglial functions like phagocytosis of synaptic terminals. We imaged this baseline behavior over 20 min in CA1t = 20 min) was then measured and normalized to the area above threshold of the first frame of the movie . The EI through time of each time-lapse movie was then determined.Motility analysis was performed in FIJI by adapting the method described in sr, 10–20 μm deep, and imaged the volume surrounding it for 20 min. We quantified process velocity with the Manual Tracking plugin in FIJI  or pentobarbital (>100 mg/kg) and intracardially perfused with cold PBS. Brains were quickly excised and drop-fixed overnight in 4% paraformaldehyde  + 4% sucrose in PBS; 50 to 100 μm hippocampal slices were cut horizontally using a Vibratome Series 1000 (Vibratome) for immunostaining and morphometry. For FluoroJade C (FJC) studies, fixed brains were cryoprotected overnight in 30% sucrose in PBS before freezing, and sectioned at 25 μm on a cryostat  and immediately mounted on 10–12 gelatin-subbed slides per brain.sr per hemi section.For microglial density quantification we took 2048 × 2048 px Z-stacks of fixed slices from perfused brains by imaging 21 planes 1 μm apart. We analyzed the MIPs across the Z axis, referring to the 3D Z-stack for the manual cell counting analysis if necessary. Cells were manually counted using FIJI in a single 284 × 284 × 20 μm field containing CA1AB_94936, MilliporeSigma) and rabbit anti-NeuN . Slices were washed and then incubated at RT for 1 h with secondary antibodies . Sections were mounted and coverslipped using VectaShield fluorescent mounting media .Following perfusion and sectioning, slices were processed free-floating for immunofluorescence against GFP to better visualize microglia and their fine processes, and against NeuN to mark stratum pyramidale. Sections were blocked and permeabilized for 2 h in 0.5% Triton X-100 and 10% normal goat serum in PBS. Next, slices were incubated overnight at 4°C with mouse anti-GFP  from Z-stacks of fixed anti-GFP stained slices with 41 planes of 4096 × 4096 px taken at 0.5 μm apart. We compared microglia morphometrically by extracting patterns of 3D Sholl crossings, numbers of branching points and primary branches, and total filament tree lengths for each traced cell.Individual microglia were traced using the “FilamentTracer” tool in Imaris 7.4.2  for 2 min. After incubating in a 0.06% potassium permanganate solution for 5 min, followed by a 2 min rinse in ddH2O, samples were stained in an acidified 0.001% FJC working solution for 10 min in the dark. After staining, slides were washed three times for 1 min in ddH2O, then placed on a slide warmer at 40°C until dry before being cleared in xylene for 2 min and coverslipped with D.P.X. mounting medium . Fluorescence photomicrographs from three to five sections per slide were captured on an upright microscope  with a QIClick camera (QImaging), using a standard FITC filter set and a 0.65NA 40× objective (Nikon Instruments). Images were captured by a blinded investigator using the same imaging conditions throughout. FJC-positive cells in each image were manually counted by two blinded investigators. Cell counts were averaged from at least three sections per animal.Briefly, after drying, slides were treated with basic ethanol solution for 5 min before transfer into 70% ethanol for 2 min, then rinsed in distilled/deionized water  approach with anti-Cd11b MicroBeads , tightly adhering to the manufacturer’s standard protocol for single cell dissociation and microglial isolation from adult brains, except for omission of the erythrocyte lysis step.g for 10 min at 4°C , and the supernatant aspirated. The cell pellet was resuspended in 3100 µl of PBS and mixed with 900 µl of debris removal solution; 4 ml of cold PBS was then overlaid on top, and the tubes were centrifuged for 10 min at 3000 × g. Three phases formed, the top two were discarded and cold PBS was added to the tube to bring the final volume to 15 ml, before being centrifuged at 1000 × g for 10 min and aspirated completely. The pellet was resuspended in 10 ml cold 0.5% BSA in PBS, and centrifuged at 300 × g for 10 min. The supernatant was again aspirated completely, and the cell pellet was carefully resuspended in 90 µl cold 0.5% BSA, to which 10 µl of MicroBeads were added before incubation in the dark for 15 min. The cells were washed in cold 0.5% BSA and centrifuged at 300 × g for 5 min, supernatant was aspirated completely, and the pellet was resuspended in 500 µl 0.5% BSA. Microglia were magnetically isolated through MACS MS columns (130-042-201) placed into MiniMACS Separator magnets (130-042-102). Unlabeled cells were collected in the original flow-through and after 3 × 500 µl washes. Labeled microglial cells were collected by flushing the column after removal from the magnetic field.Mice were anesthetized and perfused transcardially with cold PBS. Brains were rapidly dissected on ice and stored in MACS tissue storage solution (130-100-008) until dissociation could proceed (<15 min). Brain tissues were sliced six to eight times with a sterile scalpel, then placed together with the enzyme mix from the adult brain dissociation kit (130-107-677) in a C tube (130-093-237) for processing in the gentleMACS Octo Dissociator with Heaters (130-096-427) using the recommended program (37C_ABDK_01). The dissociated tissue was resuspended in PBS and applied to a 30 µm SmartStrainer (130-098-458). All subsequent steps were performed at 4°C except for the magnetic column separation. The resulting single-cell suspension was centrifuged at 300 × g and 4°C. The aqueous phase was transferred to a new tube with 0.5 ml isopropanol  and RNA was precipitated by incubating for 10 min at RT. After being centrifuged for 10 min at 12,000 × g and 4°C, the supernatant was discarded, and the RNA washed twice by resuspending the pellet in 75% ethanol  in UltraPure Nuclease-free ddH2O , then vortexing briefly and then spinning down for 5 min at 7,500 × g and 4°C. After the second wash was removed, RNA was air-dried for 10 min before resuspension in 25 μl RNase-free water. Samples were then incubated at 60°C for 10–15 min before RNA quantity and purity were determined by standard spectrophotometry methods . If necessary, RNA samples were stored at –80°C before cDNA synthesis and qPCR analysis downstream.After MACS isolation, positive fractions were immediately lysed with 0.8 ml cold TRIzol LS  and homogenized by thorough pipette and vortex mixing. After a 5 min initial incubation, 0.2 ml chloroform  was added and samples were incubated for 2–3 min before being centrifuged for 15 min at 12,000 × cDNA was synthesized from RNA samples using random hexamer primers in the SuperScript IV First Strand Synthesis system  according to the manufacturer’s directions. cDNA samples were stored at –20°C before qPCR analysis; 10% of the 20 μl RT reaction output was used in a 20 μl qPCR reaction for each technical replicate.Gene: Unique AssayID): Actb: qMmuCEP0039589; Tmem119: qMmuCEP0042925; Tnf: qMmuCEP0028054; Ccl2: qMmuCEP0056726; P2rx1: qMmuCIP0031612; P2rx4: qMmuCIP0028782; P2rx7: qMmuCIP0042331; P2ry6: qMmuCIP0029813; P2ry12: qMmuCEP0057087.Hydrolysis probe-based qPCR reactions were run in multiplex on a 4-channel  Mic cycler instrument (Bio Molecular Systems) using mouse assays from the PrimePCR line (Bio-Rad) as follows  following the manufacturer’s directions for multiplex assays . Multiplex assay combinations were validated by comparing their results to those from parallel singleplex reactions (data not shown). Threshold cycle was automatically determined and averaged across replicates by the cycler manager software (Bio Molecular Sciences). Fold changes were determined using the 2After decapitation, hippocampi were dissected in ice-cold PBS, flash frozen in isopentane cooled in dry ice (−78°C), and lysed . Lysates were probed for total protein using a Pierce BCA assay  and for CCL2/monocyte chemoattractant protein 1 (MCP-1) using a Quantikine CCL2 ELISA  assay according to manufacturers’ directions.SCR_016137, Microsoft) and Prism . Results are presented as mean ± SEM. N is number of animals and n is number of slices/fields or number of cells for the patch-clamp electrophysiology studies. We did not detect any sex differences in microglial density, morphology and motility, or in microglial gene expression, and thus combined data across sexes for further statistical analysis. Similarly, wherever applicable, control groups for both seizure models  were combined.Our primary question of interest was the degree to which ECS would alter microglial responses. The impact of SE on the same endpoints we measure for ECS have been previously reported and replicated by others . For compost hoc multiple comparisons with Tukey’s test , by Kruskal–Wallis non-parametric test followed by multiple comparisons with Dunn’s correction , or by unpaired t tests for all other comparisons. We established statistical significance at p < 0.05, applied recommended multiple comparison corrections where appropriate and computed all p values from two-tailed distributions. Exact p values are provided whenever made available by the statistics software (Prism).Data were statistically analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test , by one-way ANOVA followed by 3CR1GFP/+ mice and studied green fluorescent protein-labeled microglia in hippocampal area CA1sr.To characterize the microglial response to neuronal hyperactivity/seizures we employed ECS to induce maximal seizures on CXN = 13 mice, n = 56 fields) had a mean cell density of 27 ± 2.9 microglia/106 μm3 of volume imaged, compared to 28 ± 2.7 in the ECS group  = 0.949, p = 0.34, t test; by animal: t(23) = 0.367, p = 0.717, t test; data not shown).A single ECS-induced tonic-clonic seizure did not result in observable differences in microglial density or baseline motility 24 h after the seizures in the CA1sr hippocampal region. As shown in the examples in N = 9) and ECS-exposed animals , but significantly elevated in animals that underwent SE  = 53.1, p = 0.00000003), that was driven by the SE group, which differed from both control (q(18) = 13.13, p = 0.00000008, Tukey’s test) and ECS groups (q(18) = 11.89, p = 0.0000003, Tukey’s test). Control and ECS groups did not differ from each other (q(18) = 0.612, p = 0.902, Tukey’s test). Thus, as expected, single ECS did not cause acute neurodegeneration, a profile different from that following SE, which is associated with high levels of degeneration.To verify that our ECS treatment was not associated with neuronal damage in the hippocampus, we counted FJC-positive cells. FJC is a polyanionic dye that selectively marks degenerating neurons . The numN = 7 and 6 animals; n = 17 and 12 slices; t(27) = 0.269, p = 0.79, t test; To evaluate baseline motility, we calculated an EI by dividing the mean area of process extensions by the mean area of retracted processes  over an sr after perfusion, fixation, and immunofluorescent amplification of GFP. Representative tracings are shown in To investigate the effect of our experimental treatment on microglial morphology we reconstructed confocal z-stacks of individual cells in CA1F = 1761, p < 10−15), with significantly fewer crossings from 2 to 33 μm from the cell body  = 13.4, p = 0.0000000001, Tukey’s test; q(35) = 12.4, p = 0.0000000007, Tukey’s test; q(35) = 12.73, p = 0.0000000004, Tukey’s test; p > 0.9 at all radii, Tukey’s multiple comparisons tests; q(35) = 2.422, p = 0.22, Tukey’s test; q(35) = 1.79, p = 0.43, Tukey’s test; q(35) = 2.747, p = 0.142, Tukey’s test; Microglia from control animals were highly ramified and had long and complex processes with regular branching, as is expected under physiologic conditions. Compared to controls, and unlike ECS animals, SE animals displayed clear morphologic activation of hippocampal microglia, as has been previously described by others . The 3D Thus, ECS did not induce any observable microglial activation as measured by microglial density, spontaneous motility or morphology. Given that “activated” microglia are at once a result, target, and source of proinflammatory molecules, we wanted to verify that our model was associated with low relative expression levels of CCL2 , a chemokine whose expression was significantly upregulated after SE . CCL2 haN = 16 hippocampi from 16 animals). One-way ANOVA revealed a statistically significant treatment group effect  = 6.13, p = 0.0056). This effect was driven by a significant increase in CCL2 in the ECS  = 4.48, p = 0.0091) and SE  = 3.61, p = 0.041) groups, as compared to the control group by Tukey’s test. There was no significant difference in CCL2 protein expression between the ECS and the SE groups (q(32) = 0.416, p = 0.95). When microglial RNA samples were analyzed by qPCR, two-way repeated measures mixed model ANOVA analysis showed significant effects of treatment group  = 5.311, p = 0.03), interaction between treatment group and gene  = 5.815, p = 0.008) as well as of gene assayed  = 5.156, p = 0.01). As shown in Ccl2 to be significantly changed: in accordance with the increased CCL2 protein expression in ECS hippocampi  = 3.959, p = 0.001, N = 5/group). No differences were found in relative expression of the microglial marker Tmem119  = 0.175, p > 0.99, N = 6/group) or the pro-inflammatory cytokine Tnf  = 0.039, p > 0.99, N = 6/group).Surprisingly, as shown in ppocampi A, Ccl2 mand consequence of both neuronal hyperexcitability and injury  = 17.9, p < 10−15). Control slices  displayed a mean process velocity of 2.7 ± 0.04 μm/min, compared to 4.3 ± 0.12 μm/min for 38 slices from 10 ECS animals, and 4.4 ± 0.23μm/min for 17 slices from five SE animals  = 15.7, p = 0.0000000004; C vs SE: q(91) = 13.1, p = 0.0000000004; ECS vs SE: q(91) = 0.793, p = 0.84). ECS enhancement of ATP-directed microglial process motility was relatively much smaller but still statistically significant when the concentration of ATP in the patch pipette solution was 1 mM  = 3.79, p = 0.0013, t test, n = 10 slices per group, from N = 3 animals per group). A difference between groups was not detectable when the pipette contained a saturating concentration of ATP  = 0.233, p = 0.82, t test, n = 10 and n = 9 slices per group, from N = 3 animals per group).In stark contrast to the responses in control slices, slices from ECS and SE animals displayed similarly enhanced microglial process motility toward 3 mM ATP in a patch pipette, 24 h after the seizures. One-way ANOVA revealed a significant effect of treatment group  = 0.165, p = 0.69, two-way ANOVA, n = 16 and n = 12 cells; t(29) = 0.635, p = 0.53, t test, n = 17 and n = 14; t(32) = 0.348, p = 0.73, t test, n = 20 and n = 14; t(33) = 1.02, p = 0.316, t test, n =21 and n = 14; N = 3 and N = 4 mice for the control and ECS groups, respectively) and in accordance with published ranges  = 5.07, p = 0.000079, t test, n = 12 and n = 8; t(21) = 3.23, p = 0.0041, t test, n = 12 and n = 11; Local perfusion of 1 mM Na-ATP through a Y-tube application device rapidly and reproducibly evoked inward currents in cells from control slices which reversed near 0 mV and were potentiated by divalent cation-free aCSF, putatively identifying them as cationic purinergic currents mediated by P2X receptors . ECS sign = 6 and 11 cells from N = 2 animals/group, Kruskal–Wallis test, H(3) = 27.8, p = 0.000004; p = 0.000002). Given that the currents evoked in control slices incubated with aCSF were small , this group did not differ following incubation in BBG .Next, we sought to identify the receptors underlying these enhanced currents by preincubation of slices with the purinergic inhibitor BBG (5 μM) which preferentially blocks P2X7 over P2X4 and P2X1 . MicroglH(3) = 29.9, p = 0.000001). Multiple comparisons tests revealed significant differences in ATP-evoked current densities from: cells from control slices exposed to BBG versus those that were not BBG-treated  and cells from ECS slices exposed to BBG versus those that incubated in control aCSF . These findings are consistent with higher affinity, larger conductance, or increased number of P2X7-containing receptors in hippocampal microglia post-ECS.Similarly, as summarized in F = 2.213, p = 0.149), of interaction between treatment group and gene  = 1.377, p = 0.270) or of gene assayed  = 0.8903, p = 0.484). There were no statistical differences in relative expression between control and ECS microglial samples  for: P2rx1  = 0.2205, p > 0.99, N = 6/group), P2rx4  = 0.4724, p > 0.99, N = 6/group), P2rx7  = 2.476, p = 0.102, N = 6/group), P2ry6  = 0.5179, p > 0.99, N = 6/group), or P2yr12  = 1.026, p > 0.99, N = 6/group). The lack of changes in transcript levels suggest that translational or posttranslational alterations account for the enhanced P2X7-dependent currents we observed after ECS.We next sought to quantify the relative expression of various purinergic receptor genes in microglia after ECS. As illustrated in Ccl2/CCL2, a chemokine with an established role in neuron-glia-inflammation crosstalk in healthy states and in seizures/epilepsy. Moreover, ECS enhanced ATP-responsive motility and ATP-evoked currents in microglia, in the absence of measurable changes in receptor expression. Thus, brief, non-injurious seizures (ECS) partially recapitulate the activation state seen after prolonged, injurious seizures (SE). This overlap suggests that for a subset of features, seizure activity per se, and not neuronal damage, drives microglial responses.Here, we report a spectrum of changes in hippocampal microglia in response to maximal ECS seizures. This ECS-induced “activation” state features changes that partially overlap with those seen after SE. ECS (unlike SE) did not cause observable differences in hippocampal microglial proliferation/density, morphology, spontaneous motility, or intrinsic electrophysiological properties. On the other hand, similarly as after SE, ECS resulted in increased gene and protein expression of + cells deep in the slice and the quality of those recordings. Moreover, our findings with SE mirror those previously reported by others using multiple slice preparation approaches. It does remain possible that either ECS or SE makes neurons more vulnerable to the slicing protocol, meaning that some of our observations could arise from a seizure/slicing interaction.A potential concern raised regarding evaluation of microglia in acute brain slices is that the slicing process itself may alter microglial state or response. Following others , we emplhttp://www.microgliasinglecell.com/). This potential confound is mostly mitigated by the finding that different models of SE induction are associated with similar microglial activation ] or in voltage-activated currents following ECS. These data are consistent with the lack of an effect on morphology and baseline motility, since IR and Cm directly reflect cell membrane properties, and changes in voltage-activated potassium currents underlie pathogenic microglial-neuron contacts resulting in cell death after SE . Thus, oCcl2 mRNA in microglia after ECS. This change was not accompanied by increased levels of Tnf mRNA, another pro-inflammatory cytokine whose expression is increased post-SE  is a canonically pro-inflammatory signaling molecule that has been strongly implicated in the post-SE neuronal injury  as well  post-SE . Interes post-SE . AlthougSE, inflammation, and subsequent injury/epileptogenesis are intricately linked through at least two related signaling pathways: fractalkine and interleukin (IL)-1β. Neuronal-microglial fractalkine signaling, and subsequent astrocytic and neuronal IL-1R activation have been extensively implicated in the pathogenesis mechanism observed after SE , as wellCcl2 after ECS is of particular interest given the fact that this chemotactic cytokine has been found to directly enhance purinergic signaling in microglia by promoting trafficking of receptors like P2X4 to the microglial plasmalemma  or its equally well-known deleterious effects (confusion or amnesia).In conclusion, we describe here a state of microglial activation in the mouse hippocampal area CA1


Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date.pro or 3Dpol were tested. The interaction between EV71 3Cpro and quercetin was predicted and calculated by molecular docking.The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cpro, blocking viral replication, but not the activity of the protease, 2Apro, or the RNA polymerase, 3Dpol. Modeling of the molecular binding of the 3Cpro-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity.Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cpro activity, thereby blocking EV71 replication.Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3C Enterovirus 71 (EV71) is a non-enveloped, positive-sense, single stranded RNA virus belonging to the family Picornaviridae, and a major pathogen causing hand, foot, and mouth disease in children under 5 years of age. EV71 was first isolated in California, USA –7. CliniNo approved direct-acting antiviral drug is available for EV71 infection to date . Thus, tThe RNA genome of EV71 encodes a large precursor polypeptide that is processed by viral protease to generate viral structural  and nonstructural  proteins . ProteasQuercetin, one of the most widely distributed flavonoids in plants, has been studied for many years because of its anti-inflammatory and anti-cancer properties , and haspro activity, blocking EV71 replication. These findings are of potential relevance to the development of clinical therapeutics against EV71 infections.Here, we studied the inhibitory activity of quercetin against EV71 and explored its anti-EV71 modes of action. We observed that quercetin showed powerful therapeutic effects, exhibited a degree of preventive activity against EV71, and also inhibited viral adsorption. Quercetin potently inhibited EV71 3C2 atmosphere. EV71 (strain SK-EV006) with GFP (EV71-GFP) was kindly supplied by Prof. Bo Zhang from Wuhan Institute of Virology. Live EV71 strain wuhan/3018/2010virus was kindly provided by Prof. YingZhu  and propagated in the RD cell line. Virus titer was determined by the standard TCID50 method  and a reporter plasmid, pCMV-FLuc, harboring firefly luciferase under the control of the CMV enhancer/promoter region, were co-transfected into RD cells seeded in 24-well plates using Lipofectamine 2000 transfection reagent (Life Technologies). Serial concentrations of quercetin were added 5 h post-transfection. After 24 h, cell lysates were collected and analyzed to determine FLuc activity using a Firefly luciferase assay system, following the manufacturer’s instructions .Escherichia coli BL21 (DE3) after induction using isopropyl β-D-1-thiogalactopyranoside, and purified by affinity chromatography using a Ni-NTA column . The purified proteins were concentrated to 1 mg/mL in 20 mM Tris-HCl (pH 7.0), 500 mM NaCl, 2 mM DTT buffer for storage.The purification of EV71 3C protease was based on a previously reported protocol . Brieflypro is a specific protease that recognizes peptide substrates containing a Q-G junction [A(I) = A(0) × {1-([I])/([I] + IC50)}, where A(I) is the difference between the enzyme activity with quercetin concentration [I] and A(0), which is the enzyme activity without inhibitor.EV71 3Cjunction . Thus, tjunction . Reactio2, 60 μM ZnCl2, 5 μM UTP, 0.4 μCi/μL [α-32P]UTP, 1.8 μM dT15-primer, 0.15 μM poly (rA) 300 and various concentrations of quercetin. Reactions were incubated for 5–10 min at 30 °C, and then quenched by adding 2 × gel loading buffer . After separating the reactions on 6 M urea polyacrylamide denaturing gels, RNA products were measured using a Cyclone Plus storage phosphor system (PerkinElmer).The detailed protocol for the expression of EV71 3D polymerase and the polymerase elongation assay has been previously reported . RNA eloThe X-ray structure of EV71 3C protease (PDB no. 3SJO) was prepared using a Protein Preparation Wizard and Maestro 9.3 . The model of EV71 3C protease structure (EV71/wuhan/3018/2010) was used as a template to predicate whether quercetin affects 3C protease. The study of docking of EV71 3C protease with quercetin was performed using AUTODOCK 4.2.6, with atomic affinity potential energy calculated using AUTOGRID supporting software.P values were < 0.05.The data shown are the means ± standard deviations (SD) of three independent experiments. The levels of significance for the differences between the test and control groups were analyzed using Student t-test, and the differences were considered significant if The molecular structure of quercetin is presented in Fig. To further evaluate the anti-EV71 activity of quercetin, the inhibitory effects on the cytopathogenicity effect (CPE) induced by viral infection were examined in both RD and Vero cells by assessment of cell viability. The cytotoxic effects of quercetin were also evaluated. The results showed that quercetin exhibited low cytotoxicity in RD and Vero cells, with 50% cytotoxic concentration (CC50) values > 200 μM Fig. , and potPrevious studies have shown that EV71 induces apoptosis in infected cells . We examTo identify the stage in the viral life cycle that was affected by quercetin, assays were performed using three different treatment protocols Fig. . As show50) method. RD cells were infected with EV71 (100 and 1000 TCID50) and treated with 40 and 80 μM quercetin. After 2 h adsorption at 37 °C, the inoculum was discarded, the cells were washed and harvested following freeze-thaw cycles, and subjected to virus titration. Cells infected with EV71 in the absence of quercetin were used as the virus control. As illustrated in Fig. Similar results were obtained when quercetin was added to RD cells concurrently with EV71 Fig. , suggestQuercetin exhibited powerful therapeutic effects, with treatment at a concentration of 80 μM leading to cell viability of almost 100% Fig. , indicatTo further understand the mechanisms of quercetin against EV71 propagation in cells, a time-of-addition experiment was performed. For this experiment, 50 μM quercetin was added to infected cultures at different time periods  and the reductions in virus yield, relative to those of untreated cultures, were determined at 12 h p.i. As shown in Fig. To further investigate the effect of quercetin on EV71 replication, the efficacy of quercetin in inhibiting viral progeny yield, viral RNA synthesis, and translation of viral protein were analyzed. Infected cells treated with or without 50 μM quercetin were harvested 4, 8, 16 and 32 h p.i. and progeny viral yields were determined using the Reed and Muench method. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and indirect immunofluorescence analysis of harvested cells were also carried out to determine the relative amounts of viral RNA and viral protein, respectively.As shown in Fig. The influence of quercetin on EV71 replication at the level of translation was also determined. Viral protein immunofluorescence foci were absent in mock-infected control cells Fig. , demonstpro cleaves viral polyproteins and cellular factors, and has multiple roles in regulation of various viral and cellular processes, including viral replication and cytopathogenesis  no. 3OSY) using this method. The binding free energy was calculated  140–147 , in addition to the two assays described above. GFP reporter infection assays, using a full-length infectious clone, allowed screening for inhibitors of any step(s) of the replication cycle of EV71. CPE detection, MTT and virus yield assays reflected changes in cellular phenotypes, metabolism, and viral multiplication, respectively. The similarity of the EC50s obtained using these different approaches suggested that quercetin alone did not inhibit cell growth and could effectively inhibit viral infection.Furthermore, we determined the CC50 values at a maximum quercetin concentration of 200 μM, because when the concentration was higher than 200 μM, a large amount of particles would adsorb on the cell surface, resulting in an inability to visualize the state of the cells, or to accurately determine cell viability using the MTT method. The value of CC50 for quercetin was > 200 μM, which was the same as the positive control, ribavirin. Quercetin exhibited dose-dependent antiviral effects against EV71, which meant quercetin could inhibit EV71 infection only when it reached a certain concentration. Actually, quercetin had an EC50 of 12.1 μM for RD cells and 8.8 μM for Vero cells against EV71 infection, with selective indexes of > 16.6 and > 22.8, respectively. An approximately ten-fold inhibitory activity against EV71 relative to ribavirin was observed. Selectivity index (SI) values ≥4 are considered suitable for antiviral agents , which spro, and the results verified that flavonoids with similar structures exert different mechanisms against EV71. Further study of the affinity of quercetin for 3Cpro is warranted to ascertain whether the compound affects other viral or cellular functions crucial for EV71 replication.An internal ribosomal entry site (IRES) required for viral protein translation is a potential EV71 drug target. As previously reported, several flavonoids, including kaempferol and hesperetin, exhibit antiviral activity against EV71 through inhibition of the IRES; however, quercetin does not inhibit IRES-driven translation . Anotherpro and fluorogenic substrate were used to evaluate the effect of quercetin on protease activity. The amounts of quercetin required to inhibit purified EV71 3Cpro activity in vitro were higher than those required to block EV71 replication, this is because the higher concentrations of 3Cpro present in vitro required more quercetin for inhibition.In this study, recombinant 3Cpro is a multifunctional protein involved in binding viral RNA and RNA replication, in addition to a number of other biological processes. It has also been reported that 3Cpro can impair host RNA processing and polyadenylation by cleavage of the cellular CstF-64 protein, leading to enhanced viral RNA replication [pro activity.Viral 3Clication . The 3C lication . In thispro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is vital for SARS-CoV replication, and quercetin displays strong inhibition of 3CLpro [pro and quercetin and calculated the binding energy using molecular docking. Docking calculations between 3Cpro and other flavonoids have been reported previously, and the accurate prediction of biomolecular conformation and binding energies of protein-ligand complexes exhibited similarities, including common binding sites Leu4, Leu8, Ser111, Met112, Phe113, and Pro115 [pro and other flavonoids (or its phosphate ester), they have not been identified as involved in biological catalysis to date. Analysis of the 3Cpro-quercetin interaction revealed that quercetin interacted with the 3Cpro catalytic residues, His40, Glu71 and Cys147 (rank 7 and rank 8 in Table 2), which is different to other flavonoids, this binding may decrease EV71 3Cpro activity. Moreover, for the initial manner of binding, predicted by the docking results (rank 0 in Table 2), quercetin was predicted to interact with numerous residues in 3Cpro, including Tyr138, Phe140, Ala144, His161, Ile162, Gly163, Gly164, Gln168, Gly169, and Phe170. The molecular binding model of the 3Cpro-quercetin complex revealed that quercetin may stably insert into the substrate-binding pocket of EV71 3Cpro via numerous hydrogen bond interactions. Similar binding mechanisms have been reported for the interactions between flavonoids and 3CLpro in SARS-CoV [pro inhibitors.The 3C-like protease (3CLf 3CLpro . Quercetf 3CLpro . In thisd Pro115 , 46. TheSARS-CoV , but notEV71 infection is often accompanied by the apoptosis of host cells, and it can be a mechanism for viral spread to neighboring cells, which may promote culminating a lytic infection and cause viremia and severe central nervous system complications. Here, quercetin was found to protect host cells from apoptosis induced by viral infections, suggesting that quercetin may play an important role in EV71-induced critically severe and fatal cases.pro activity, thereby blocking EV71 replication.Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Based on our findings, quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, this study demonstrates that quercetin potently inhibits EV71 3C


Meiotic recombination hotspots control the frequency and distribution of Spo11 (Rec12)-initiated recombination in the genome. Recombination occurs within and is regulated in part by chromatin structure, but relatively few of the many chromatin remodeling factors and histone posttranslational modifications (PTMs) have been interrogated for a role in the process.ade6-M26 hotspot or a basal recombination control were purified approximately 100,000-fold under native conditions from meiosis; then, associated proteins and histone PTMs were identified by mass spectrometry. Proteins and PTMs enriched at the hotspot included known regulators , validating the approach. The abundance of individual histones varied dynamically during meiotic progression in hotspot versus basal control MiniCs, as did a subset of 34 different histone PTMs, implicating these as potential regulators. Measurements of basal and hotspot recombination in null mutants confirmed that additional, hotspot-enriched proteins are bona fide regulators of hotspot activation within the genome. These chromatin-mediated regulators include histone H2A-H2B and H3-H4 chaperones , subunits of the Ino80 complex , a DNA helicase/E3 ubiquitin ligase (Rrp2), components of a Swi2/Snf2 family remodeling complex , and a nucleosome evictor (Fft3/Fun30).We developed a chromatin affinity purification and mass spectrometry-based approach to identify proteins and histone PTMs that regulate recombination hotspots. Small (4.2 kbp) minichromosomes (MiniCs) bearing the fission yeast Overall, our findings indicate that a remarkably diverse collection of chromatin remodeling factors and histone PTMs participate in designating where meiotic recombination occurs in the genome, and they provide new insight into molecular mechanisms of the process.The online version of this article (10.1186/s13072-018-0233-x) contains supplementary material, which is available to authorized users. In meiosis, cells express the broadly conserved Rec12/Spo11 (topoisomerase II-like) protein which, along with other components of the basal meiotic recombination machinery, catalyzes the formation of dsDNA breaks (DSBs) that initiate meiotic recombination . As withSequence-specific DNA binding proteins, such as the Atf-Pcr1 heterodimer, Bas1, and Prdm9, regulate hotspots , 5–7. ThM26 DNA sites in the genome [Interestingly, all factors known to help position meiotic recombination at hotspots display context variable penetrance, indicating that they must function in concert  with other, yet unidentified factors to promote recombination locally , 23, 24.e genome , only abe genome . This pre genome , 28, 29,e genome , 30, ande genome , 32. Adde genome , 20. To ade6-M26 of fission yeast  [M26 DNA site (M375 or BC) lacks hotspot activity but supports basal levels of recombination, permitting one to determine whether a given factor  is specific to hotspot activation or affects more generally the basal recombination machinery [In this study, we sought to define as comprehensively as possible the local epiproteome (proteins and histone PTMs) of a well-defined, DNA sequence-dependent meiotic recombination hotspot, ast Fig. . BindingACGT-3′)  activateACGT-3′) , 26, 34.ACGT-3′) , 20, 35.achinery –38. Sincachinery , we reasM26 hotspot, and confirmation that newly identified, broadly conserved, hotspot-enriched factors are bona fide regulators of hotspots.We report the development of a way to purify discrete, unit-length segments of chromatin to near homogeneity, the discovery of numerous, dynamic changes in protein occupancy and histone modifications at the ade6 locus in the genome. Following optimization, we were able to enrich ade6 chromatin fragments up to 100-fold, relative to those from loci elsewhere in the genome, but we deemed this level of purification inadequate to meet our goals (even a 1000-fold enrichment would be inadequate). The reason is straightforward and is germane to all such studies. If a chemically cross-linked, 12,500-kbp genome is sheared into chromatin fragments approximately 1 kbp in length and if the target fragment is enriched 1000-fold, then greater than 90% of proteins in the purifications would come from genomic regions other than the target locus of interest. To increase the likelihood of discovering factors associated specifically with hotspot chromatin, relative to basal control chromatin, we needed to increase substantially the degree of purification.We first applied published methods for chromatin affinity purification with mass spectrometry (ChAP-MS)  and CRISade6 in a plasmid is like that in the chromosome [E. coli plasmid backbone, would provide several advantages. First, since they are extrachromosomal elements, there would be no need to shear chromosomes to liberate the target locus. Second, the omission of shearing would obviate the need for prior chemical cross-linking of proteins to DNA and would streamline the process, allowing us to purify chromatin rapidly under native conditions. Third, the large differences in size  between MiniCs and chromosomes might improve partitioning, and hence degree of enrichment, during purifications. Fourth, since the MiniCs are of unit length, they would lack the heterogeneity intrinsic to sheared chromatin and would, correspondingly, provide a more discrete readout of associated factors.The chromatin structure of romosome , and epiromosome . We reasARS), the ade6 gene, and eight copies of the LacO DNA site  and basal control (M375 or BC) alleles of ade6 differ at only two base pairs and by the presence or absence of the M26 DNA site to which the hotspot-activating Atf1-Pcr1 heterodimer binds  and basal control (M375) alleles of ade6 each contain a stop codon near the 5′ end of the ade6 ORF  ag. sup9e , 46, we sup9e strain [sup9e, as initially defined [sup35-F592S [sup9e and sup35-F592S were effective suppressors of adenine auxotrophy caused by the M26 allele, neither one effectively suppressed M375 [M375. We therefore created a new basal control allele, ade6-BC, that harbors the same (suppressible) stop codon as ade6-M26, but that lacks the DNA binding site for the hotspot-activating Atf1-Pcr1 heterodimer  supports the induction of synchronous meiosis in S. pombe  of standard induction protocols and monitored the timing of the two meiotic divisions. Strains harboring the hotspot and basal control MiniCs were highly proficient for meiosis, and importantly, they each displayed equivalent timing .–52.ts prLacO DNA sites. To generate the affinity capture reagent, we expressed and purified a fusion protein that contains LacI, a hexahistidine (6xHis) tag, and protein A (prA)  relative to DNA from the genome (act1).We purified MiniCs from whole-cell extracts under native, stringent (300 mM KCl) conditions by affinity capture, taking advantage of the high-affinity, multivalent interactions between Lac repressor (LacI) and The qPCR analyses of unfractionated material revealed that MiniCs are maintained at a copy number of about 20 per cell Fig. : Fig. S2This does not mean that all of the recovered proteins were components of, or were interacting with, MiniCs in vivo. As is the case for every affinity purification and immuno-purification experiment that is conducted, many proteins associate artifactually following homogenization of cell contents. The magnetic bead-adsorbed MiniCs are, for example, essentially mixed bed ion exchange matrices to which other positively and negatively charged proteins can bind. However, our well-matched hotspot and basal control alleles Fig. c provideM26) to basal control (BC). We used three independent biological replicates except for the 1-h time point, for which there were duplicates. Proteins in each sample were identified and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses of tryptic peptides using a Thermo Orbitrap Fusion Tribrid mass spectrometer. For broader depth of coverage, the peptides were first fractionated by LC under basic conditions (bLC), and then, those fractions were concatenated . In total, 437 concatenated fraction pools were each analyzed by LC–MS/MS, which identified 21,362 unique peptides corresponding to 2721 proteins. The abundance of each protein was determined using intensity-based absolute quantification (iBAQ), and the abundance of a given peptide harboring one or more histone PTMs was determined relative to all occurrences of that peptide [To discover factors enriched at the hotspot, we conducted affinity purifications of MiniC chromatin from four time points of synchronous meiosis spanning from induction (0 h) to the time (3 h) just before the first meiotic division  between all pairwise comparisons of samples, taking into account the abundance of each protein in each sample  for each protein detected at each time point of meiosis. While majority of the proteins were of similar abundance in hotspot and basal control, subsets of proteins were enriched in either the hotspot or the basal control Fig. b. This iM26-bearing MiniC serves as a good surrogate for its genomic counterpart and that the M26 allele remains an active hotspot in this context. We conclude that our MiniC-AP-MS approach can identify bona fide regulators of recombination hotspots.Given that hotspot activation likely involves an ordered sequence of potentially transient events within chromatin that culminates in the formation of Rec12 (Spo11)-catalyzed DSBs, we constructed a list of proteins that were enriched at least twofold at the hotspot, for one or more of the meiotic time points, and that were annotated as being either nuclear or associated with chromatin see Fig. d. The idbona fide regulators of meiotic recombination hotspots within chromosomes.Our screen for hotspot-enriched factors also uncovered a large collection of additional, candidate regulatory proteins  [M26 DNA sites elsewhere [As expected for purified chromatin, histones were among the most abundant proteins detected. There were dynamic changes in the relative occupancy of histones H2A, H2A.Z, H2B, H3, and H4 between hotspot and basal control MiniCs  , 57 and lsewhere . The newbona fide regulators of recombination hotspots in chromosomes.We searched our histone peptide datasets for well-characterized histone PTMs: acetylation (ac), ubiquitination (ub), phosphorylation (ph), and mono-, di-, and tri-methylation . This revealed changes in the relative abundance and temporal dynamics of histone PTMs in hotspot versus basal control MiniCs Fig. b. For exWe also uncovered a constellation of additional histone PTMs that were enriched or depleted in the hotspot MiniC, relative to basal control, at one or more of the time points of meiosis Fig. b but thaino80) is essential, we chose to analyze two genes encoding two non-essential subunits of the complex (arp5 and arp8). Our selection of candidates was also guided by the proteomics results on histone occupancy and PTMs, as follows.To test whether factors that were enriched within the hotspot MiniC actually regulate meiotic recombination, we selected nine different proteins with known or putative roles in chromatin remodeling. Each of these proteins was enriched at the hotspot, relative to basal control, at one or more time points of meiosis in at least one experiment, or was a component of a well-defined protein complex with multiple subunits enriched at the hotspot, or both  and basal control (M375) alleles of ade6 were crossed to a tester strain (M210) and spore colonies were genotyped to determine the frequency of Ade + recombinants . However, the ablation of Hip1 reduced substantially the frequency of recombination at M26  recombination at M375. We obtained the same result using additional hotspot and control alleles (our unpublished observations). Therefore, Arp5 exhibits dual specificity as a repressor of basal recombination and as an activator of hotspot recombination. These phenotypes are, in each case, attributable presumptively to the local binding of Arp5 because it was enriched at the hotspot early in meiosis (1 h) and at the basal control later in meiosis (3 h)  or a basal recombination control (BC) that differ by only two base pairs , discrete, small segments of chromatin 4.2 kbp) bearing a meiotic recombination hotspot , it is not without limitations. There are likely false negatives due to the dissociation of labile proteins during purification under the native, stringent conditions employed. In addition, some proteins and (in particular) histone PTMs that are present can escape detection due to paucity of tryptic peptides that fall within the mass-to-charge gates of MS instrumentation. Other histone PTMs were missed simply because we focused on well-characterized PTMs . Components of the basal recombination machinery, such as Rec12, were also not detected at meaningful levels in our samples, which is not surprising because DSBs occur in only about 1% of DNA molecules harboring ade6-M26 . In addiThe first criterion, on the screen’s ability to identify known regulators, was satisfied at the level of sequence-specific DNA binding proteins, histone- and chromatin-modifying enzymes, histone PTMs, and changes in histone/nucleosome occupancy.M26 DNA site is essential for hotspot activity at ade6-M26 and other M26 DNA sites in the genome [Binding of the Atf1-Pcr1 (Mts1–Mts2) heterodimer  to the Me genome , 25, 80,M26 protein–DNA complex-dependent, meiotically induced, transient accumulation (as determined by ChIP) of histone H3K14 acetylation at the hotspot [The previously reported, Atf1-Pcr1- hotspot , 20 was cis, changes in chromatin structure during activation of the hotspot.A histone acetyltransferase that writes the H3K14ac mark, Mst2 , and an M26 hotspot [Recently, ChIP analyses of a single meiotic time point revealed that the occupancy of the histone H2A variant H2A.Z is low at the  hotspot . This fiAt each of the levels described above, findings made using MiniC-AP-MS Figs. ; 4 are cThe second criterion, on whether newly discovered factors are required for biological activity in vivo, was satisfied by comparing rates of meiotic recombination in the genomes of wild-type cells and null mutants Fig. .ade6-M26  to both the hotspot and basal control  that mediate the exchange of individual histones within nucleosomes –70, subuM26 Fig. . For sev, Hip1 thOur findings  increase substantially the number of chromatin remodeling factors known to help position meiotic recombination at hotspots, and they provide new mechanistic insight. The requirement for histone chaperones  Fig. a, providbona fide regulators of hotspot activity.Our analyses also uncovered a constellation of differentially abundant histone PTMs Fig. b, includS. pombe (our unpublished observations). Fundamental aspects of hotspot control in fission yeast—including its regulation in trans by signal transduction pathways [cis [i.e., they are insufficient to promote recombination at many of their locations in the genome). Multiple epigenetic and chromatin factors function in concert  to position meiotic recombination at hotspots.Overall, our findings indicate that a remarkably diverse collection of chromatin remodeling factors and  histone modifications participate in designating where meiotic recombination occurs in the fission yeast genome. To the extent tested, these factors also regulate other DNA sequence-dependent hotspots of pathways , 82, 83 ays [cis  by DNA says [cis –14, 25, ays [cis , 37, 57—E. coli plasmid pBluescript II KS- and, after DNA sequencing to confirm the correct structure and to eliminate any clones with spurious mutations, that plasmid was digested with NotI to liberate the S. pombe sequences. The linear MiniC DNA fragment was isolated by gel electrophoresis, was ligated at very low DNA concentration (1 ng/ml) to favor intramolecular ligation (circularization) over intermolecular ligation (concatemerization), and was transformed into S. pombe [Standard recombinant DNA methods were used to construct minichromosomes (MiniCs) with components shown in Fig. S. pombe . The uniade6 DNA in the chromosome, the MiniCs, and in MiniCs from steps of affinity purification was normalized to a single-copy genomic control, act1, and relative abundance values were calculated using the double-delta Ct method [ade6 were: 5′-CAATTGGGCCGAATGATGGT-3′ and 5′-TTTCGTAACGGCTGCCAAGG-3′; those for act1 were 5′-GAAATCGCAGCGTTGGTTAT-3′ and 5′-ACGCTTGCTTTGAGCTTCAT-3′.Quantitative, real-time PCR  was used to measure MiniC copy number and degree of enrichment during purifications. The relative abundance of t method . The seqS. pombe strains used in this study are provided in Additional file ade6-D1 sup35-F592S strain background, which suppresses the ade6 mutations in the MiniCs [Genotypes of e MiniCs . Recombie MiniCs , 26.ts repressor, as described [pombe minimal media [g for 5 min at 4 °C), washed once with ice-cold ddH2O, collected by centrifugation, resuspended in the residual liquid, and snap-frozen by drizzling into liquid N2. The frozen cell “popcorn” was stored at − 80 °C until processed further.Meiosis was induced by thermal inactivation of the Pat1-114escribed , but wital media  containiTo monitor meiotic progression, cells (1 ml of culture) were taken at hourly time points, fixed with 70% ethanol, and stored at − 20 °C. Following rehydration with sodium citrate, cells were spread onto microscope slides, dried with heat, and then stained with 1 μg/ml of 4,6-diamidino-2-phenylin-dole (DAPI) and antifade solution (0.25% DABCO in 75% glycerol/25% PBS). Cells were visualized and analyzed with the EVOS FL Auto Imaging System using phase contrast and fluorescence microscopy. A minimum of 100 cells were counted for each sample at each time point.2). The resulting frozen cell lysate powder was stored at − 80 °C until processed further. For each affinity purification, 6 g of frozen cell lysate powder was thawed on ice; all subsequent steps were carried out on ice or at 4 °C. To the 6 g (approximately 6 ml) of thawed lysate, we added 25 ml of buffer A150 . The A150 buffer was supplemented with protease inhibitors , phosphatase inhibitors , deacetylase inhibitors (500 µM butyric acid), 0.5% (v/v) Triton X-100, 125 µM spermidine, and 50 µM spermine. The solution was centrifuged at 2600×g for 5 min, and the supernatant was collected; then, that material was centrifuged at 12,000×g for 30 min. The supernatant was collected as clarified whole-cell extract (WCE), 20 µL of aliquots was saved (to measure amounts of protein and DNA), and the remaining WCE was used for affinity purification. The WCE was pre-cleared with 500 µL of IgG-Sepharose beads (GE Lifesciences), equilibrated in A150 buffer, for 15 min on a rotator at 4 °C. The Sepharose beads were removed by two rounds of centrifugation, each for 5 min at 2600×g. Six hundred ng of LacI-6xhis-prA fusion protein was added to the WCE, which was then incubated on a rotator at 4 °C for 15 min. Twelve milligrams of Rabbit IgG-conjugated magnetic Dynabeads (Thermo Fisher) was added, and the sample was incubated for 2 h on a rotator at 4 °C. The Dynabeads  were first collected by centrifugation for 5 min at 2600×g. All but 1 ml of supernatant was removed, and the Dynabeads were resuspended in the remaining liquid. That material was then split into 3 separate 1.7-ml Eppendorf tubes, the magnetic beads were collected using a magnetic rack, and the supernatant was removed. The same process was used to wash the beads sequentially 3 times, for 5 min each, with 1 ml of A300 buffer  per tube. For the final wash, the beads were transferred to new 1.7-ml Eppendorf tubes. A small aliquot of beads (5% v/v in wash buffer) was collected to test for enrichment of target DNA. The remaining beads (95% v/v) were pooled into one tube, recovered magnetically, resuspended in 30 µL of 1 × SDS-PAGE loading buffer, brought to 100 µL volume with ddH2O, and incubated at 95 °C for 10 min. The eluant was separated from the Dynabeads by centrifugation, collected, and transferred to a new Eppendorf tube. This process was repeated one more time to ensure that no beads were collected. The samples were dried overnight in a DNA 120 SpeedVac concentrator (Thermo Fisher) at room temperature.Frozen cells were lysed under cryogenic conditions using a Retsch MM301 ball mill, at power setting of 30 Hz, with 5 cycles of 3 min each . The individual fractions were then consolidated into 12, 18, or 24 super-fractions, each of which was then further fractionated by reverse phase chromatography on a Jupiter Proteo resin (Phenomenex) on an in-line 150 × 0.075 mm column using a nanoAcquity UPLC system (Waters). Peptides were eluted using a 60-min gradient from 97:3 to 65:35 buffer A:B ratio . Eluted peptides were ionized by electrospray (2.15 kV) followed by MS/MS analysis using higher-energy collisional dissociation (HCD) on an Orbitrap Fusion Tribrid mass spectrometer (Thermo) in top-speed data-dependent mode. MS data were acquired using the FTMS analyzer in profile mode at a resolution of 240,000 over a range of 375–1500 m/z. Following HCD activation, MS/MS data were acquired using the ion trap analyzer in centroid mode and normal mass range with precursor mass-dependent normalized collision energy between 28.0 and 31.0. Proteins were identified by database search using MaxQuant (Max Planck Institute) with a parent ion tolerance of 3 ppm and a fragment ion tolerance of 0.5 Da. Carbamidomethylation of cysteine residues was used as a fixed modification. Acetylation of protein N-termini and oxidation of methionine were selected as variable modifications. Protein abundance was calculated using the intensity-based absolute quantification (iBAQ) algorithm [Purified proteins were reduced, alkylated, and digested using filter-aided sample preparation . Trypticlgorithm –55. HistAdditional file 1. Contains supplementary information germane to but not essential for inclusion in the main text: Fig. S1, purification and DNA binding of LacI-6xHis-PrA fusion protein; Fig. S2, optimization of conditions for purification of MiniCs; Fig. S3, normalization of MS data for differences in protein yield; Fig. S4, Pearson correlation coefficients for all pairwise combinations; Table S1, genotypes of S. pombe strains used; Table S2, nuclear and chromatin-associated proteins enriched at the hotspot.


Terminalia bellirica (Gaertn.) Roxb. fruit (TBF) is a widely planted traditional medicinal herb in Tibet. We aimed to determine the most active substance-enriched extract by comparing the in vitro antioxidant activities of different extract fractions of TBF that were subsequently extracted by petroleum ether, chloroform, ethyl acetate, and n-butanol after initial extraction by 95% ethanol. The main compounds of the ethyl acetate extract fraction (EF) were analyzed via HPLC-MS. Gallic acid (GA) was obtained from EF to determine in vitro antifibrotic activity based on the traditional usage of TBF. After HSC-T6 cells were incubated with GA, extracellular secreted levels of fibrosis-associated cytokines, such as collagen I, collagen III, TGF-β1, and hydroxyproline, were estimated by ELISA. Gene and protein expressions of PDGFR, CTGF, NF-κB, MMP-2, TIMP-1, TIMP-2, α-SMA, and the Bcl-2/Bax family were determined by quantitative PCR and western blot. The proapoptotic effect of GA was further investigated by annexin V-PI and TUNEL staining. These results indicate that EF has prominent in vitro antioxidant activity among four extract fractions, and its main component, GA, manifests antifibrosis activity and its potential mechanism of action includes inhibition of cytokine secretion and collagen synthesis, as well as proapoptosis of HSCs. TBF is an important medicinal herb most frequently used in the Tibetan medicine system and also used as a folk medicine by Uighurs and Mongolians. Modern pharmacological research on Pilile mainly focuses on antioxidant, antidiabetic, antihyperlipidemic, anti-HIV-1, antimalarial, antifungal, antispasmodic, and bronchodilatory properties [People living in the Qinghai-Tibet Plateau have heavy diets consisting mostly of meat and rarely eat vegetables due to the limitations brought forth by high altitude and the special geographical environment that supports the agriculture of green buckwheat and animal husbandry. These environmental features and diet as well as genetic variation are reasons why Tibetans have high risk of cardiovascular disease, hepatobiliary diseases, and oxidative stress . Tibetanoperties –7. Howevoperties . To quicoperties , is a naoperties , anti-inoperties , antifiboperties , and antoperties  and induoperties . Neverthα-smooth muscle actin (α-SMA) and ECM proteins such as collagen I and platelet-derived growth factor (PDGF) [Liver fibrosis results from an imbalance regulation between synthesis and degradation of extracellular matrix (ECM) proteins during a scarring process . HSC actr (PDGF) . Thus, ir (PDGF) . Therefoin vitro antioxidant activities of different extract fractions of TBF by a series of chemical reactions: to identify the main components of the ethyl acetate extract fraction of TBF by the HPLC-MS approach, to investigate the antifibrotic effect of GA in vitro by determining the expressions of ECM-associated proteins, and to study the role of GA played in inducing HSC-T6 apoptosis by fluorescent labeling.In this study, we aim to compare the α,α-diphenyl-β-picrylhydrazyl (DPPH) were purchased from Sigma . 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was purchased from Fluka . Linoleic acid was purchased from Alfa Aesar . Butylated hydroxytoluene (BHT) and vitamin C (Vc) were obtained from China Medicine (Group) Shanghai Chemical Reagent Corp. (China). HPLC-grade acetonitrile was purchased from Fisher Scientific (China). Gallic acid (GA), rutin, corilagin, and ellagic acid were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . Chebulinic acid was purchased from International Laboratory . All chemicals used for analysis were of analytical reagent grade. The source of cell culture reagents, ELISA kits, and PCR reagents was previously described in Chen et al. [β-actin primary antibody (60008-1-Ig) was purchased from Proteintech Group Inc. . Primary antibodies specific to connective tissue growth factor (CTGF) (sc-14939), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (sc-8008), matrix metallopeptidase 2 (MMP-2) (sc-10736), tissue inhibitor of metalloproteinase 1 (TIMP-1) (sc-365905), tissue inhibitor of metalloproteinase 2 (TIMP-2) (sc-365671), B cell lymphoma 2 (Bcl-2) (sc-7382), and Bcl-2-associated X protein (Bax) (sc-7480) were obtained from Santa Cruz Biotechnology Inc. . Horseradish peroxidase (HRP) secondary antibodies (goat anti-mouse and goat anti-rabbit) were purchased from Abcam Inc. . Annexin V/PI staining was performed using an apoptosis kit from MultiSciences Biotech Co. Ltd. . TUNEL staining was performed using in situ cell death detection Kit-POD from Roche Diagnostics Corp. .The Folin-Ciocalteu reagent and n et al. . PrimersTBF was commercially provided by the Tibetan Traditional Medicine Pharmaceutical Factory . Dr. Youwei Wang (corresponding author) authenticated the voucher specimen (no. 14649), which was stored in the Traditional Chinese Medicine Specimens Museum founded by the School of Pharmaceutical Sciences, Wuhan University.n-butanol). The petroleum ether fraction (PF), chloroform fraction (CF), ethyl acetate fraction (EF), and n-butanol fraction (BF) were obtained, and the yield rates were 0.69%, 0.12%, 10.91%, and 3.00%, respectively.200 g TBF was crushed by a grinder (powder size ≤ 0.25 mm) and subjected to heat reflux extraction using 95% ethanol. After extracting and filtering three times, the combined extract solvents were rotary evaporated at 55°C for 1 h to become concentrated. Subsequently, the residues were lyophilized by a freeze dryer . The yield rate of ethanol extraction was 23.42%. Further successive extraction of the ethanol extract was performed using different solvents with increasing polarity , and BHT and Vc were used as references at the same concentration. In the ABTS assay, PF, CF, EF, and BF solutions were prepared at various concentrations , and BHT and Vc were used as references at the same concentration.Methods and calculation formulas were done without modification as previously published . Brieflyμg/mL) were incubated in a water bath for 10 min at 25°C, and then 150 μL of 3 mM pyrogallic acid was added. The absorbance was measured at 30 s interval at 325 nm. The autooxidation rate constant (Kb) of pyrogallic acid was calculated by A325nm vs. time. A control group equal to a blank group refers to no test extracts being added; BHT and Vc were used as positive controls. The Kb value was used to evaluate test extracts' ability to scavenge superoxide radicals.The capacity of PF, CF, EF, and BF to scavenge superoxide radicals was examined by a pyrogallol autooxidation system with slight modifications . Brieflyμg/mL) of PF, CF, EF, and BF solutions were prepared following the method described in [Various concentrations . The β-carotene solution (4 mL) was transferred to a 500 mL round-bottomed flask and then added with 80 mg of linoleic acid and 800 mg of Tween 80. 200 mL of ddH2O was added and mixed well after chloroform was evaporated. 3.0 mL of the resulting linoleic acid-β-carotene solution was transferred to a reaction tube containing 0.2 mL of PF, CF, EF, or BF solution (400 μg/mL) and incubated in a water bath at 50°C. The control was not added to PF, CF, EF, and BF sample solutions. Absorbance readings at 470 nm were recorded at 30 min intervals for 120 min. Higher absorbance equates to a higher inhibitory effect of LPO. BHT and Vc were selected as positive controls.6 mg of 2O. The mixtures were incubated at 40 ± 1°C in Eppendorf tubes to avoid light. Afterwards, an aliquot (0.1 mL) from the reaction mixture was mixed with 75% ethanol (9.7 mL), 30% ammonium thiocyanate (0.1 mL), and 20 mM ammonium ferrous sulfate solution . After 3 min, the absorbance was measured at 500 nm. The degree of linoleic acid oxidation was measured by the ferric thiocyanate method at 24 h intervals for 12 days.Antioxidative activity of each fraction was measured in a linoleic acid model system. Briefly, 0.1 mg of PF, CF, EF, and BF powder dissolved in 1 mL ethanol, respectively, was mixed with 1 mL of 2.5% linolenic acid in ethanol, 2 mL of 50 mM phosphate buffer (PBS), and 1 mL ddHw/v) trichloroacetic acid  and 0.67% (w/v) 2-thiobarbituric acid  and heated at 100°C for 10 min. After it was cooled to room temperature, the mixture was centrifuged at 1000 g for 10 min. The absorbance of the resulting supernatant was measured at 532 nm with a spectrophotometer. BHT was selected as references in both FTC and TBA assays.At the last day of the FTC method, the TBA assay was employed to determine the antioxidant activity of PF, CF, EF, and BF. Briefly, 1 mL of fraction solution that was prepared the same as the FTC method above was mixed with 20% . The flow rate was 1.0 mL/min. The column temperature was set at 25°C and the detection wavelength at 275 nm. The binary mobile phase comprised both solvents A (0.1% formic acid-water) and B (acetonitrile). The gradient elution started with 5% solvent B for 6 min, from 5% to 15% solvent B (6 min), constant at 15% solvent B (6 min), from 15% to 20% solvent B (7 min), constant at 20% solvent B (15 min), and finally from 20% to 5% solvent B in 5 min. The aliquots (20 μL) of the standards and sample solutions were injected for HPLC analysis after the chromatographic system was equilibrated with 5% B for 15 min.We employed HPLC to define the optimal chromatographic separation condition before performing HPLC-MS analysis, which was performed on a Shimadzu LC-20AT HPLC instrument with a UV detector using a WAT054275-Waters Symmetry CμL. By solvent splitting, 20% eluent was allowed to flow into the MS instrument with an ESI interface in the negative ion mode. The optimized instrumental parameters were set as follows: desolvation temperature, 230°C; source temperature, 120°C; cone voltage, 20 V; capillary voltage, 1.2 kV; desolvation gas (N2) flow rate, 900 L/h; auxiliary gas (He) flow rate, 40 L/h; and scan range, m/z 100–1000 amu.The main compounds of EF were analyzed via a HPLC system  equipped with the same column above and an ion trap mass spectrometer  tandem system. The binary mobile phase comprised both solvents A (0.1% formic acid-water) and B (acetonitrile). The gradient elution started with 5% solvent B for 6 min, from 5% to 15% solvent B (6 min), constant at 15% solvent B (6 min), from 15% to 20% solvent B (7 min), constant at 20% solvent B (15 min), and finally from 20% to 5% solvent B in 10 min. The flow rate was 1.0 mL/min. The injection volume was 10 Rat HSC-T6 cells were purchased and transferred from Shuguang Hospital, Shanghai University of Traditional Chinese Medicine. Cells were thawed quickly and cultured appropriately by following the previously described method .μg/mL) were prepared with DMSO in DMEM by a serial dilution method. The procedure of the MTT assay is referenced in Chen et al. [μL/well of suspended cells was seeded in a 96-well plate and added with 100 μL/well of different doses GA to meet the final concentrations . Absorbance in each well was measured at 570 nm with a microplate reader. Cell viability was calculated as follows: cell viability (%) = (ODsample − ODblank)/(ODcontrol − ODblank) × 100, where ODsample is the absorbance of the wells containing cells and GA or 0.1% dimethyl sulfoxide (DMSO), ODcontrol is the absorbance of the wells containing cells without GA or 0.1% DMSO, and ODblank is the absorbance of the wells containing DMEM.Different doses of GA  and grown to ~80% confluence. GA was solubilized in DMSO and diluted with DMEM to achieve concentrations of 25, 50, and 100 μg/mL and to make the concentration of DMSO 0.1% (v/v). In order to exclude cytotoxicity of DMSO, DMEM containing 0.1% DMSO was used as a negative control. Then, 1 mL of GA at different concentrations was added to each well to meet the final concentrations of 12.5, 25, and 50 μg/mL, respectively . After a day of GA treatment, cell culture supernatants were collected in new Eppendorf tubes for subsequent ELISA experiments. Extracellular secreted levels of collagen I, collagen III, TGF-β1, and hydroxyproline were determined by following the manufacturer's instructions. Absorbance was read using a plate reader at 450 nm. Adherent cells were kept for further PCR and western blot analysis.5 × 10Total RNA was extracted using TRIzol reagent following the instructions from the manufacturer. The real-time PCR procedure was conducted according to the method described by our previous study .After the cell culture medium was aspirated, the adherent HSC-T6 cells were rinsed gently with PBS three times. Protein extraction and western blot analysis were conducted according to our previous study .5 suspended cells/mL were seeded in six-well plates (2 mL/well) and grown to ~80% confluence. Each well contained a piece of a coverslip to let cells grow on it. Afterwards, 2 mL of GA at different concentrations was added to each well to meet the final concentrations of 12.5, 25, and 50 μg/mL, respectively . After a day of GA treatment, culture medium was sucked by a vacuum, and the coverslips were then taken out and rinsed gently in PBS. The annexin V-PI staining procedure was conducted according to our previous study [5 × 10us study .μL TUNEL reaction mixture was added to each sample on the coverslip and incubated in a dark humidified chamber at 37°C for 60 min. After rinsing with PBS three times and drying the area around the samples, 50 μL of converter-POD was added to each coverslip and the coverslips were incubated in a dark humidified chamber at 37°C for 60 min. Then, the coverslips were rinsed with PBS three times and mounted on glass slices with glycerol. The coverslips were examined using a fluorescence microscope  with an excitation wavelength in the range of 450-500 nm and a detection wavelength in the range of 515-565 nm.As described above, cells on coverslips were untreated or treated with three doses of GA for 24 h. Culture medium was sucked by a vacuum, and the coverslips were then taken out and rinsed gently in PBS and fixed in 4% paraformaldehyde for 1 h at the room temperature. After rinsing twice with PBS, the coverslips were soaked in 0.2% Triton X-100 for 5 min. Then, the coverslips were rinsed with PBS twice and the area around the samples was dried. Then, 50 p < 0.05. Statistical differences were analyzed by one-way ANOVA and the LSD test (IBM SPSS Statistics 20.0).All data are mean ± SD represented by the error bars. Means were considered significantly different when 50 values of 13.88 ± 0.27 μg/mL, 11.04 ± 0.85 μg/mL, 1.10 ± 0.08 μg/mL, and 6.78 ± 0.23 μg/mL, while the IC50 value of Vc and BHT was 6.68 ± 1.14 μg/mL and 19.36 ± 0.24 μg/mL, respectively. This showed that the ability of EF to scavenge DPPH free radical was superior to that of Vc and BHT. BF has a comparable DPPH free radical scavenging ability with Vc while PF and CF are valued between the Vc and BHT. Naik et al. reported that the IC50 value of the aqueous extract of TBF for DPPH free radical scavenging was 10.00 μg/mL [50 value of the 70% methanol extract of TBF for DPPH free radical scavenging was 1.45 ± 0.02 μg/mL [50 value of the ethyl acetate extract for DPPH free radical scavenging was 1.40 μg/mL. All the IC50 values they reported for DPPH free radical scavenging were higher than what we reported (1.10 ± 0.08 μg/mL). This can be explained by the fact that different solvent extractions have different effects on concentrating active compounds; the extraction with ethyl acetate after ethanol extraction is considered a better way to concentrate active compounds in TBF. In short, the TBF purchased from Tibet has excellent antioxidation effects after undergoing extraction by our method. Such exceptional antioxidation effect has never been reported by other researchers.The DPPH radical was used to evaluate the scavenging activity of antioxidants by accepting hydrogen atom or electron donation resulting in a bleaching of a purple-colored methanol solution. As shown in 00 μg/mL . Hazra e02 μg/mL . The IC550 = 0.82 ± 0.09 μg/mL) and BF (IC50 = 1.89 ± 0.04 μg/mL) were found to be very effective free radical scavengers, while PF (IC50 = 5.30 ± 0.53 μg/mL) and CF (IC50 = 2.03 ± 0.10 μg/mL) showed weaker scavenging abilities relative to Vc (IC50 = 1.38 ± 0.15 μg/mL) and BHT (IC50 = 1.63 ± 0.03 μg/mL). EF was valued as having the best ABTS free radical scavenging ability among the four extract fractions, even stronger than Vc and BHT. ABTS radical cation instantaneously formed once potassium persulfate was added to an ABTS solution. Phani Kumar et al. reported that the ethyl acetate fraction of the ethanolic extract of Terminalia arjuna skin had an IC50 value of 25 ± 1.2 μg/mL for ABTS free radical scavenging [50 = 0.82 ± 0.09 μg/mL, proving that EF is significantly better at scavenging ABTS free radicals.ABTS is also a widely accepted free radical compound to estimate the scavenging capacity of antioxidants. In avenging , while o·O2−) is a ubiquitously generated free radical in vivo, which can be produced from pyrogallic acid spontaneous oxidation under alkaline conditions, and also disappears rapidly by reacting with the hydrodioxyl radical under acidic conditions. The fractions can hinder the autooxidation reaction of pyrogallic acid in this in vitro system due to their abilities to scavenge ·O2− radicals  indicates better scavenging of ·O2− radicals. As indicated in ·O2− radical scavenging of CF, EF, and BF was higher than that of BHT, while PF has an equivalent scavenging ability with BHT. However, Vc has the strongest ability to scavenge ·O2− compared to all other fractions. EF showed the best scavenging ability among all the four fractions, and we compared its Kb value with that of other extracts investigated in our lab. Huang et al. reported that the Kb value of the superoxide anion radical scavenging by the 70% methanol extract of Halenia elliptica was 10.87 ± 0.02 (×10−4 A/s) [Kb value of the superoxide anion radical scavenging by the 70% ethanol extract of Meconopsis integrifolia was 3.93 ± 0.30 (×10−4 A/s) [Halenia elliptica but slightly less than that of the 70% ethanol extract of Meconopsis integrifolia.The superoxide anion radical  . Zhou et0−4 A/s) . These s3+/ferricyanide complex reduces to the ferrous form (Fe2+) when antioxidants are present, and Fe2+ concentration can be monitored by measuring the formation of Perl's Prussian blue at 700 nm [The reducing power of a substance is associated with its potential antioxidant activity. The Fet 700 nm . A highet 700 nm , a conclβ-carotene and linoleic acid, β-carotene discolors rapidly in the absence of an antioxidant to scavenge free radicals. The linoleic acid free radical, formed by drawing a hydrogen atom from its diallylic ethylene groups, attacks the highly unsaturated β-carotene molecules [β-carotene becomes oxidized and loses its chromophore and characteristic orange color. The antioxidants present in the system can impede the extent of β-carotene bleaching by neutralizing the linoleic acid free radical. As a result, the absorbance decreases rapidly in samples lacking sufficient antioxidants, while samples with antioxidants have their orange color retained. β-carotene among the four fractions. From the results of the β-carotene bleaching assay, we concluded that the positive control Vc was not suitable for the lipid peroxidation system, so only BHT was used as a positive control in the subsequent anti-LPO experiments.Because of the coupled oxidation of olecules . Consequ2+ to form Fe3+, which has a maximum absorbance at 500 nm with SCN−. In our assay, the formation of peroxides and oxidation of Fe2+ were quenched on day 8 due to shortage of linoleic acid. Therefore, the absorbance at 500 nm begins reducing after day 8. Accordingly, the oxidation of linoleic acid would be slow in the presence of antioxidants. The effect of fractions on preventing the peroxidation of linoleic acid is shown in The FTC method was used to measure the amount of peroxide produced during the linoleic acid oxidation. Peroxides react with Fep < 0.05). This may be due to the fact that PF performed best in the process of inhibiting lipid peroxidation in the early stage (FTC assay), so that no excessive MDA and other substances were produced in this system. Sarin et al. reported that oleic acid glyceride (61.5%) dominates in Terminalia bellirica Roxb. seed oil [The TBA method is a measure of the final products of the lipid peroxidation assay, including small molecules such as aldehydes, ketones, acids, and hydrocarbons, which have maximum absorption peaks between 532 and 535 nm. The lower the absorbance value, the less the end products of LPO. seed oil . Low-denseed oil . The preIn summary, EF has relatively prominent in vitro antioxidant activity in both free radical scavenging and antilipid peroxidation systems among the four fractions. It is worth exploring the molecular basis of this important extract regarding whether EF can achieve extremely excellent antioxidant effects.″,4″-di-O-galloyl-α-L-rhamnopyranosyl) ellagic acid (3), chebulanin (4), corilagin (5), 3,4,6-tri-O-galloyl-β-D-Glc (6), chebulagic acid (7), methyl neochebulagate (8), 1,3,4,6-tetra-O-galloyl-β-D-Glc (9), ellagic acid (11), chebulinic acid (12), methyl neochebulinate (13), 1,2,3,4,6-penta-O-galloyl-β-D-Glc (14), and 3′-O-methyl-4-O-(β-D-xylopyranosyl) ellagic acid (16) were identified based on the molecular weight given by MS and the previous report [The liquid chromatogram of the EF was obtained by Shimadzu HPLC at 275 nm . The tot-β-D-Glc , ellagicThe 14 compounds identified by HPLC-MS can be classified into five categories: (1) gallic acid and simple gallate esters , (2) chebulic acid and chebulic ellagitannins , (3) nonchebulic ellagitannins (5), (4) ellagic acid and derivatives (11), and (5) ellagic glycosides (3 and 16). This shows that EF is enriched in a large amount of phenolic compounds. Many studies have reported that phenolic compounds play an indispensable role in antioxidant activity , 29. TheThe enrichment effect of ethyl acetate on gallic acid and simple gallic acid ester compounds is well known. Gallic acid (GA) and ellagic acid in mango kernel and longan seed have been shown to have good activity in scavenging ABTS free radicals . EF may 50 value was determined as 41.39 μg/mL after coincubating with GA for 24 h  GA treatment, the type I collagen content in the ECM was significantly reduced compared to the control group  by GA in a dose-dependent manner . In Figures β1 appears to be the most important one. In our previous study, we proved that GA in medium and high doses could significantly reduce TGF-β1 and hydroxyproline in rats with liver fibrosis [β1 by siRNA and shRNA might be an efficient approach against liver fibrosis [After medium-dose  in decreasing PDGFR gene expression (μg/mL of GA (κB mRNA (μg/mL), and the effect of GA on the mRNA level of TIMP-2 was more pronounced , showing a significant dose-dependent effect (μg/mL GA being the strongest. p < 0.01) the mRNA expression of Bcl-2 while p < 0.01) the mRNA expression of Bax. Interestingly, in Figure 5(i), 12.5 μg/mL GA treatment resulted in the highest mRNA expression of Bax while 50 μg/mL GA showed the second highest effect. However, there were no significant differences between the three treatment groups. All of the above experiments showed that 0.1% DMSO treatment has no effect on expressions of the target genes. In summary, we evaluated the effect of GA on reducing the accumulated ECM in HSC-T6 cells by monitoring the gene expressions of fibrotic cytokines and proapoptotic proteins. The protein expression of CTGF, NF-κB, MMP-2, TIMP-1, TIMP-2, Bcl-2, and Bax has similar alteration trends to those of their mRNA expressions as shown in β induces the expression of CTGF that independently promotes fibroblast proliferation and matrix deposition [α1(I) collagen and fibronectin synthesis by both dermal and lung fibroblasts in culture and promote significant matrix remodeling of fibroblast-populated three-dimensional collagen lattices [κB was detected as a protein that could complex to a 10 bp site in the κ light chain enhancer, called κB [κB is present in the cell in its inactive form, bound to an inhibitory protein called IκB [κB, such as growth factors, bacteria, viruses, UV radiation, and oxidative stress [κB is present in activated fibrotic cells and is closely related to the progression of fibrosis [β1 was proven to regulate the expressions of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs), which promote ECM degradation and ECM synthesis, respectively [Lithospermum erythrorhizon, has been proven to significantly reduce TIMP-1 expression and improve MMP-2 expression of either mRNA expression or protein level in a CCl4-induced or bile duct ligation-induced liver fibrosis model [α-SMA is an actin isoform and a specific marker for smooth muscle cell differentiation, which has been used to identify aHSCs to show a myofibroblastic phenotype [Phyllanthus emblica L. fruits and its major compound ellagic acid were able to markedly reduce protein levels of α-SMA in HSC-T6 cells [μM of GA treatment caused an imbalance between Bcl-2 and Bax, which induced the apoptosis of hypertrophic scar fibroblasts [After the cells were treated with various doses of GA for 24 h, we evaluated the expression of platelet-derived growth factor receptor (PDGFR) mRNA to analyze the response of cells to GA. The decrease in PDGFR gene expression caused the decrease in the cell response to PDGF, and GA at each concentration played a significant role (pression . There wmL of GA , but alt(κB mRNA . We also(κB mRNA . Compare Figures . The mRNt effect  with theposition . CTGF halattices . NF-κB walled κB . When unlled IκB . Many fae stress . The litfibrosis , 42. TGFectively . Shikoniis model . α-SMA ihenotype . Lu et aT6 cells . DrivingT6 cells . BCL-B, T6 cells . Similarroblasts . Their rμg/mL of GA for 24 h, cells were stained with annexin-V/FITC and PI. The results indicate that the amount of apoptotic HSC-T6 cells that were cocultured with GA significantly increased in a dose-dependent manner compared to the untreated cells (Figures μg/mL of GA. This is because the highest concentration of GA used is greater than the IC50 value we obtained from the MTT assay. Cell viability was only 30% when the cells were undergoing 50 μg/mL GA treatment. Wang et al. observed similar fluorescent images when they tested whether curcumin had the ability to protect against thioacetamide-induced hepatic fibrosis by inducing apoptosis of damaged hepatocytes [Since GA simultaneously upregulated proapoptosis genes and downregulated antiapoptosis genes, we next evaluated the ability of GA to promote apoptosis of hepatocytes using annexin V-PI and TUNEL assay staining on HSC-T6 cells. After treatment with 12.5, 25, and 50 atocytes . Taken tIn summary, our study first provides evidences that EF of TBF possesses excellent antioxidant capacity, especially in its outstanding free radical scavenging ability; one out of sixteen active constituents of EF, GA, effectively reduced the accumulated ECM in cultured HSC-T6 cells. GA mediates gene expression and protein levels of the Bcl-2/Bax family to induce HSC-T6 cell apoptosis. All of these can be considered potential antifibrotic mechanisms of GA.