Arbidol attenuates liver fibrosis and activation of hepatic stellate cells by blocking TGF-ß1 signaling.

Chronic liver diseases (CLD) impact over 800 million people globally, causing about 2 million deaths annually. Arbidol (ARB), an indole-derivative used to treat influenza virus infection, was extensively used during COVID-19 pandemic in China. In recent years, studies have shown that ARB, compared to other antiviral drugs, exhibits greater liver-protective efficacy, indicating a potential hepatoprotective effect beyond its antiviral activity. However, the mechanism remains unclear. In this study, we investigated the impact of ARB on liver injury/fibrosis in bile duct ligated (BDL) mice and its effect on spontaneous and transforming growth factor ß1 (TGF-ß1)-induced activation of primary cultured hepatic stellate cells (HSCs). Oral administration of ARB significantly ameliorated BDL-induced liver injury/fibrosis as reflected by decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced collagen deposition, and diminished mRNA expression of fibrosis markers. ARB notably inhibited spontaneous and TGF-ß1-induced activation of primary cultured HSCs. Moreover, ARB also drastically attenuated mRNA expression levels of platelet-derived growth factor receptor (Pdgfr), transforming growth factor-beta receptor (Tgfbr) 1, Tgfbr2, matrix metalloproteinase (Mmp)-2, and Mmp-9 in activated HSCs. We further demonstrate that ARB mitigated Smad2/3 phosphorylation in both TGF-ß1 treated HSCs and BDL mice. These data together demonstrate that the therapeutic efficacy of ARB on liver fibrosis is independent of its antiviral activity and likely is achieved by blocking TGF-ß1 signaling-mediated HSC activation.

Influence of bifenthrin exposure at different gestational stages on the neural development.

Perinatal exposure to bifenthrin (BF) alters neurodevelopment. However, the most susceptible time period to BF exposure and the possible mechanisms are not clear. In the current study, pregnant female mice were treated with BF (0.5 mg/kg/d) at three different stages [gestational day (GD) 0-5, 6-15 and 16-birth (B)] and neurologic deficits were evaluated in offspring mice. BF exposure at GD 16-B significantly altered the locomotor activity and caused learning and memory impairments in 6-week-old offspring. Gestational BF exposure also caused neuronal loss in the region of cornu ammonis of hippocampi of 6-week-old offspring. Interestingly, neurobehavioral impairments and neuronal loss were not observed in offspring at 10-week-old. BF exposure at GD 16-B also decreased protein levels of VGluT1, NR1 and NR2A while increased the protein levels of NR2B and VGAT1, as well as the gene levels of Il-1ß, Il-6 and Tnf-α in hippocampi of 6-week-old offspring. Collectively, these data demonstrate that gestational exposure to a low dose BF causes neurodevelopmental deficits that remit with the age and the late-stage of pregnancy is the most susceptible time window to BF exposure. Imbalance in excitatory/inhibitory neuronal transmission, altered expression levels of NMDA receptors and increased neural inflammation may be associated with BF prenatal exposure-triggered neurobehavioral impairments.

Corydecumine G inhibits microglia activation via MAPK pathway in a rat model of neuropathic pain.

BACKGROUND AND PURPOSE: Microglial activation plays an important role in the onset and progression of neuropathic pain by producing a variety of pro-inflammatory cytokines that interact with neurons to enhance neuronal hyperexcitability. Corydalis decumbens (Thunb.) pers., a traditional Chinese medicine has been used to treat mild cancer pain, dementia and to remit cerebral ischemia in clinics. Phenylphthalide isoquinolines are the major type of metabolites of C. decumbens and one of the derivatives, Corydecumine G (Cor G) has been shown to inhibit neuronal excitability. The present study aims to investigate the analgesic efficacy of Cor G in neuropathic pain rat model, the effects of Cor G on microglia activation and the possible mechanisms. EXPERIMENTAL APPROACH: Neuropathic pain was modeled using chronic constriction sciatic nerve injury (CCI) in rats. Western blot, immunofluorescence, and qRT-PCR were used to evaluate the levels of protein and mRNA. KEY RESULTS: Intraperitoneal administration of Cor G concentration-dependently ameliorates mechanical and thermo allodynia, suppresses CCI-induced p38/ERK phosphorylation and spinal cord microglia activation, and attenuates the expression levels of NO, inos, Tnf-α, Pge2 in dorsal horn of L4-L6 spinal cord on the ligation side in CCI rats. Pretreatment with 30 µM Cor G decreased LPS-induced BV2 microglia activation, which occurred via the inos, Tnf-α, Il-1ß, Il-6 and phospho-p38/ERK pathways. CONCLUSIONS AND IMPLICATIONS: Taken together, we suggest that Cor G, the specific phthalide isoquinoline from traditional Chinese medicine Corydalis Decumbentis Rhizoma, may be promising for treatment of neuropathic pain.

Influence of perinatal deltamethrin exposure at distinct developmental stages on motor activity, learning and memory.

BACKGROUND: Perinatal exposure to deltamethrin (DM) causes attention-deficit/ hyperactivity disorder-like behaviors. However, the vulnerable time window to DM exposure and the possible mechanism are obscure. We aimed to identify the critical window(s) at perinatal stages for DM exposure and the possible mechanism. METHOD: Pregnant mice were exposed to DM (0.5 mg/kg) at three different prenatal stages [gestational day (GD) 0-5, 6-15 and 16-birth (16-B)] and early postnatal stage (PD 0-10). Locomotor activity, learning and memory were evaluated using open field and Y-maze test, respectively. Nissl staining and western blots were used to examine the neuronal loss and the protein expression, respectively. RESULTS: Perinatal exposures to DM had no effect on reproductive and growth index of offspring. However, mice receiving DM exposure during GD 16-B displayed significantly higher mortality suggesting GD 16-B is the most vulnerable time window to DM exposure. Prenatal but not early postnatal DM exposure impaired locomotor activity, learning and memory, and caused neuron loss in the dentate gyrus of male offspring. However, neither prenatal nor postnatal DM exposure affected mouse behavior of female offspring. Prenatal DM exposures decreased the protein levels of NR2A and NR2B in both hippocampi and cerebral cortices of male offspring. However, female mice receiving DM exposure at GD 16-B but not other stages displayed increased expression levels of NR2A and NR2B in hippocampi. CONCLUSION: Prenatal but not early postnatal DM exposure impairs the neuron development in male but not female mice. Altered NMDA receptor expression may correlate to DM-induced behavioral deficits.

Polysubstituted Cyclopentene Benzamides and Dianthramide Alkaloids from Delphinium anthriscifolium Hance.

Thirteen new benzamide alkaloids, delphiniumines A-M (1-13), together with one known analogue (14), were isolated from Delphinium anthriscifolium Hance. All of the structures were determined by spectroscopic and spectrometric analyses. Absolute configuration for 1 was established using experimental and calculated ECD data, as well as by X-ray crystallography analysis. Compound 1 possesses a previously undescribed polysubstituted cyclopentene carbon framework. Compound 2 was isolated as an artifact from 1 during the extraction process. Compound 7 is glycosylated with a ß-D-glucose unit. Compound 13 bears a chlorine substituent. At a concentration of 10 µM, compounds 6, 8, and 10-12 suppressed LPS-induced NO production in RAW264.7 cells with inhibition rates ranging from 40.3% to 78.8%.

Targeted Discovery of Amantamide B, an Ion Channel Modulating Nonapeptide from a South China Sea Oscillatoria Cyanobacterium.

Amantamide B (1) is a new linear nonapeptide analogue of the cyanobacterial natural product amantamide A (2), and both have methyl ester and butanamide termini. These compounds were discovered in this study from the organic extract of a tropical marine filamentous cyanobacterium, Oscillatoria sp., collected around the Paracel Islands in the South China Sea. The use of LC-MS/MS molecular networking for sample prioritization and as an analytical dereplication tool facilitated the targeted isolation of 1 and 2. These molecules were characterized by spectroscopy and spectrometry, and configurational assignments were determined using chemical degradation and chiral-phase HPLC analysis. Compounds 1 and 2 modulated spontaneous calcium oscillations without notable cytotoxicity at 10 µM in short duration in vitro testing on primary cultured neocortical neurons, a model system that evaluates neuronal excitability and/or the potential activity on Ca2+ signaling. Both molecules were also found to be moderately cytotoxic in longer duration bioassays, with in vitro IC50 values of 1-10 µM against CCRF-CEM human T lymphoblastoid cells and U937 human histiocytic lymphoma cells. These formerly undiscovered bioactivities of known compound 2 expand upon its previously reported function as a selective CXCR7 agonist among 168 GPCR targets.

Cadinane Sesquiterpenoids and Their Glycosides from Alangium chinense That Inhibit Spontaneous Calcium Oscillations.

Nine new cadinane sesquiterpenoids, alanenses A-I (1-9), were isolated from the leaves of Alangium chinense together with three previously reported analogues (10-12). The structures of these molecules were elucidated by interpretation of spectroscopic and spectrometric data. Absolute configurations were established by the comparison of experimental and calculated ECD data, chemical degradation studies for sugar moieties, and a single-crystal X-ray diffraction analysis. Compounds 1 and 2 were isolated as racemates, and enantiopurification was achieved by chiral HPLC. Compounds 3-5 are glycosylated cadinanes bearing a ß-d-glucose unit, while compounds 6-9 incorporate a hydroxymethyl group in either the free form or additional ring fusion. The structure of compound 11 was originally misassigned and later revised using additional NMR data. The corrected structure is here supported by X-ray single-crystal analysis. Compounds 1 and 2 inhibit spontaneous calcium channel oscillations at low micromolar concentrations.

Sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA2b) mediates oxidation-induced endoplasmic reticulum stress to regulate neuropathic pain.

BACKGROUND AND PURPOSE: Neuropathic pain is a widespread health problem with limited curative treatment. Decreased sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) expression has been reported in dorsal root ganglion (DRG) of animals suffering from neuropathic pain. We aimed to establish the relationship between SERCA expression and the pain responses and to elucidate the underlying molecular mechanism. EXPERIMENTAL APPROACH: Neuropathic pain was modelled using rat chronic constriction injury (CCI). Ca2+ imaging and current clamp patch-clamp were used to determine cytosolic Ca2+ levels and action potential firing, respectively. Western blots, immunofluorescence staining and qRT-PCR were used to quantitatively assess protein and mRNA expression, respectively. H&E staining and coupled enzyme assays were used to evaluate the nerve injury and SERCA2b activity, respectively. KEY RESULTS: SERCA2b is the predominant SERCA isoform in rat DRG and its expression is decreased after CCI at mRNA, protein and activity levels. Whereas inhibiting SERCA with thapsigargin causes neuronal hyperexcitation, nerve injury, endoplasmic reticulum (ER) stress, satellite glial cell activation and mechanical allodynia, activating SERCA by CDN1163 or overexpressing SERCA2b in DRG after CCI produces long-term relief of mechanical and thermal allodynia accompanied by morphological and functional restoration through alleviation of ER stress. Furthermore, the down-regulation of DRG SERCA2b in CCI rats is caused by increased production of ROS through Sp1-dependent transcriptional inhibition. CONCLUSION AND IMPLICATIONS: Our findings reveal a novel pathway centring around SERCA2b as the key molecule underlying the mechanism of development and maintenance of neuropathic pain, and SERCA2b activators have the potential for therapeutic treatment of neuropathic pain.

Water-soluble alkaloids isolated from Portulaca oleracea L.

Fifteen new water-soluble alkaloids were obtained from the fresh herbs of Portulaca oleracea L. The structures of 15 alkaloids 1-15 were established according to spectroscopic data, and the stereoconfigurations were determined based on experimental and calculated electronic circular dichroism (ECD) data and single crystal X-ray diffraction. Alkaloids 1-15 were found to display good anti-inflammatory activity at 10 µM and could significantly reduce the interleukin-6 (IL-6) and nitric oxide (NO) levels induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages.

BmK NSPK, a Potent Potassium Channel Inhibitor from Scorpion Buthus martensii Karsch, Promotes Neurite Outgrowth via NGF/TrkA Signaling Pathway.

Scorpion toxins represent a variety of tools to explore molecular mechanisms and cellular signaling pathways of many biological functions. These toxins are also promising lead compounds for developing treatments for many neurological diseases. In the current study, we purified a new scorpion toxin designated as BmK NSPK (Buthus martensii Karsch neurite-stimulating peptide targeting Kv channels) from the BmK venom. The primary structure was determined using Edman degradation. BmK NSPK directly inhibited outward K+ current without affecting sodium channel activities, depolarized membrane, and increased spontaneous calcium oscillation in spinal cord neurons (SCNs) at low nanomolar concentrations. BmK NSPK produced a nonmonotonic increase on the neurite extension that peaked at ~10 nM. Mechanistic studies demonstrated that BmK NSPK increased the release of nerve growth factor (NGF). The tyrosine kinases A (TrkA) receptor inhibitor, GW 441756, eliminated the BmK NSPK-induced neurite outgrowth. BmK NSPK also increased phosphorylation levels of protein kinase B (Akt) that is the downstream regulator of TrkA receptors. These data demonstrate that BmK NSPK is a new voltage-gated potassium (Kv) channel inhibitor that augments neurite extension via NGF/TrkA signaling pathway. Kv channels may represent molecular targets to modulate SCN development and regeneration and to develop the treatments for spinal cord injury.

New phenylpropanoid-substituted and benzyl-substituted flavonols from Alangium chinense.

Two previously undescribed flavonols with phenylpropanoid or benzyl substitution, named alangsine A (1), and alangsine B (2), together with four known compounds (3-6) were isolated from the leaves of Alangium chinense. Alangsine A was a racemic mixture, which was further separated into two enantiomers via high-performance liquid chromatography on a chiral column. The absolute configurations of the enantiomer pairs were deduced from the circular dichroism (CD) spectra. The activity of the isolated compounds towards neuronal excitability was examined.

Activation of voltage-gated sodium channels by BmK NT1 augments NMDA receptor function through Src family kinase signaling pathway in primary cerebellar granule cell cultures.

Voltage-gated sodium channels (VGSCs) are responsible for the generation and propagation of action potentials in excitable cells and are the molecular targets of an array of neurotoxins. BmK NT1, an α-scorpion toxin obtained from the scorpion Buthus martensii Karsch (BmK), produces neurotoxicity that is associated with extracellular Ca2+ influx through Na+-Ca2+ exchangers, N-methyl-d-aspartic acid (NMDA) receptors, and L-type Ca2+ channels in cultured cerebellar granule cells (CGCs). In the present study, we demonstrated that BmK NT1 triggered concentration-dependent release of excitatory neurotransmitters, glutamate and aspartate; both effects were eliminated by VGSC blocker, tetrodotoxin. More importantly, we demonstrated that a threshold concentration of BmK NT1 that produced marginal Ca2+ influx and neuronal death augmented glutamate-induced Ca2+ elevation and neuronal death in CGCs. BmK NT1-augmented glutamate-induced Ca2+ influx and neuronal death were suppressed by tetrodotoxin and MK-801 suggesting that the augmentation was through activation of VGSCs and NMDA receptors. Consistently, BmK NT1 also enhanced NMDA-induced Ca2+ influx. Further mechanistic investigations demonstrated that BmK NT1 increased the expression level of NMDA receptors on the plasma membrane and increased the phosphorylation level of NR2B at Tyr1472. Src family kinase inhibitor, 1-tert-butyl-3-(4-chlorophenyl)pyrazolo[3,4-d]pyrimidin-4-yl]amine (PP2), but not the inactive analogue, 4-amino-1-phenylpyrazolo[3,4-d]pyrimidine (PP3), eliminated BmK NT1-triggered NR2B phosphorylation, NMDA receptor trafficking, as well as BmK NT1-augmented NMDA Ca2+ response and neuronal death. Considered together, these data demonstrated that both presynaptic (excitatory amino acid release) and postsynaptic mechanisms (augmentation of NMDA receptor function) are critical for VGSC activation-induced neurotoxicity in primary CGC cultures.

Gut microbiota remodeling reverses aging-associated inflammation and dysregulation of systemic bile acid homeostasis in mice sex-specifically.

Aging is usually characterized with inflammation and disordered bile acids (BAs) homeostasis, as well as gut dysbiosis. The pathophysiological changes during aging are also sexual specific. However, it remains unclear about the modulating process among gut microbiota, BA metabolism, and inflammation during aging. In this study, we established a direct link between gut microbiota and BA profile changes in the liver, serum, and four intestinal segments of both sexes during aging and gut microbiota remodeling by co-housing old mice with young ones. We found aging reduced Actinobacteria in male mice but increased Firmicutes in female mice. Among the top 10 altered genera with aging, 4 genera changed oppositely between male and female mice, and most of the changes were reversed by co-housing in both sexes. Gut microbiota remodeling by co-housing partly rescued the systemically dysregulated BA homeostasis induced by aging in a sex- and tissue-specific manner. Aging had greater impacts on hepatic BA profile in females, but intestinal BA profile in males. In addition, aging increased hepatic and colonic deoxycholic acid in male mice, but reduced them in females. Moreover, muricholic acids shifted markedly in the intestine, especially in old male mice, and partially reversed by co-housing. Notably, the ratios of primary to secondary BAs in the liver, serum, and all four intestinal segments were increased in old mice and reduced by co-housing in both sexes. Together, the presented data revealed that sex divergent changes of gut microbiota and BA profile in multiple body compartments during aging and gut microbiota remodeling, highlighting the sex-specific prevention and treatment of aging-related disorders by targeting gut microbiota-regulated BA metabolism should particularly be given more attention.

Variations of Gut Microbiome Profile Under Different Storage Conditions and Preservation Periods: A Multi-Dimensional Evaluation.

Gut dysbiosis is heavily involved in the development of various human diseases. There are thousands of publications per year for investigating the role of gut microbiota in diseases. However, emerging evidence has indicated the frequent data inconsistency between different studies, which is largely overlooked. There are many factors that can cause data variation and inconsistency during the process of microbiota study, in particular, sample storage conditions and sequencing process. Here, we systemically evaluated the impacts of six fecal sample storage conditions (three non-commercial storage protocols, -80°C, -80°C with 70% ethanol (ET_-80°C), 4°C with 70% ethanol (ET_4°C), and three commercial storage reagents, OMNIgeneGUT OMR-200 (GT) and MGIEasy (MGIE) at room temperature, and Longsee at 4°C (LS) on gut microbiome profile based on 16S rRNA gene sequencing. In addition, we also investigated the impacts of storage periods (1 and 2 weeks, or 6 months) and sequencing platform on microbiome profile. The efficacy of storage conditions was evaluated by DNA yield and quality, α and ß diversity, relative abundance of the dominant and functional bacteria associated with short-chain fatty acid (SCFA) production, and BAs metabolism. Our current study suggested that -80°C was acceptable for fecal sample storage, and the addition of 70% ethanol had some benefits in maintaining the microbial community structure. Meanwhile, we found that samples in ET_4°C and GT reagents were comparable, both of them introduced some biases in α or ß diversity, and the relative abundance of functional bacteria. Samples stored in MGIE reagent resulted in the least variation, whereas the most obvious variations were introduced by LS reagents. In addition, our results indicated that variations caused by storage condition were larger than that of storage time and sequencing platform. Collectively, our study provided a multi-dimensional evaluation on the impacts of storage conditions, storage time periods, and sequencing platform on gut microbial profile.

BmK NSP, a new sodium channel activator from Buthus martensii Karsch, promotes neurite outgrowth in primary cultured spinal cord neurons.

Scorpion venom is a rich source of bioactive compounds that affect neuronal excitability by modulating the activities of various channels/receptors. In the current study, guided by a Ca2+ mobilization assay, we purified a new neuroactive peptide designated as BmK NSP (Buthus martensii Karsch neurite-stimulating peptide, MW: 7064.30 Da). The primary structure of BmK NSP was determined by Edman degradation. BmK NSP concentration-dependently elevated intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 4.18 µM in primary cultured spinal cord neurons (SCNs). Depletion of extracellular Ca2+ abolished BmK NSP-triggered Ca2+ response. Moreover, we demonstrated that BmK NSP-induced Ca2+ response was partially suppressed by the inhibitors of L-type Ca2+ channels, Na+-Ca2+ exchangers and NMDA receptors and was abolished by voltage-gated sodium channel (VGSC) blocker, tetrodotoxin. Whole-cell patch clamp recording demonstrated that BmK NSP delayed VGSC inactivation (EC50 = 1.10 µM) in SCNs. BmK NSP enhanced neurite outgrowth in a non-monotonic manner that peaked at ~30 nM in SCNs. BmK NSP-promoted neurite outgrowth was suppressed by the inhibitors of L-type Ca2+ channels, NMDA receptors, and VGSCs. Considered together, these data demonstrate that BmK NSP is a new α-scorpion toxin that enhances neurite outgrowth through main routes of Ca2+ influx. Modulation of VGSC activity by α-scorpion toxin might represent a novel strategy to regulate the neurogenesis in SCNs.