Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis Through miR-3546/SOX4/Survivin Axis in Rat Thy-1 Nephritis

Background/Aims: The activation of complement system and the formation of C5b-9 complex have been confirmed in the glomeruli of patients with mesangioproliferative glomerulonephritis (MsPGN). However, the role and mechanism of C5b-9-induced injury in glomerular mesangial cell (GMC) are poorly understood. Rat Thy-1N is an animal model for studying MsPGN. It has been revealed that the attack of C5b-9 to the GMC in rat Thy-1N is sublytic, and sublytic C5b-9 can cause GMC apoptosis, but the underlying mechanism is not fully elucidated. To explore the role and regulatory mechanism of C5b-9 in MsPGN lesion, we used rat Thy-1N model and first detected the change of microRNA (miRNA) profiles both in Thy-1N rat renal tissues (in vivo) and in the cultured GMCs with sublytic C5b-9 stimulation (in vitro). Then we determined the effect of miR-3546, which increased both in vivo and in vitro, on GMC apoptosis upon sublytic C5b-9 as well as the involved mechanism. Methods: Rat Thy-1N model was established and GMCs were treated with sublytic C5b-9. The rat renal cortex and the stimulated GMCs were obtained for miRNA microarray detection. Subsequently, the increased miRNAs were verified by real-time PCR. Meanwhile, to ascertain the ability of some miRNAs to upregulate cleaved caspase 3 and induce GMC apoptosis, the corresponding miRNA mimics were transfected into GMCs, followed by western blotting (WB) and flow cytometry mesurement. Thereafter, the miR-3546-targeted gene (SOX4) was predicted using bioinformatics approaches, and SOX4 expression in Thy-1N tissues and in the GMCs upon sublytic C5b-9 stimulation or miR-3546 mimic/inhibitor transfection were detected using real-time PCR and WB. To prove that miR-3546 can affect SOX4 gene transcription and SOX4 can regulate survivin expression, dual luciferase reporter assay, real-time PCR, WB and chromatin immunoprecipitation (ChIP) assays were performed. Furthermore, the role of miR-3546/SOX4/survivin axis in the GMC apoptosis induced by sublytic C5b-9 was examined using WB and flow cytometry. Results: Compared with normal renal tissues and untreated GMCs, there were 43 and 62 upregulated miRNAs (> 2-fold) in Thy-1N tissues and sublytic C5b-9-stimulated GMCs respectively. A total of 17 miRNAs were increased both in vivo and in vitro, 11 of which were validated by real-time PCR. Among them, miR-3546 could markedly promote GMC apoptosis and inhibit SOX4 or survivin expression in response to sublytic C5b-9, and either SOX4 or survivin overexpression markedly rescued the GMC apoptosis mediated by miR-3546 mimic. Additionally, SOX4 overexpression could reverse the survivin suppression by miR-3546 mimic, and SOX4 could bind to survivin promoter (-1,278 to -853 nt) and activate survivin gene transcription. Conclusion: MiR-3546/ SOX4/survivin axis has a promoting role in the GMC apoptosis triggered by sublytic C5b-9, and our findings may provide a new insight into the pathogenesis of rat Thy-1N and human MsPGN

α-Cyperone Confers Antidepressant-Like Effects in Mice Neuroplasticity Enhancement by SIRT3/ROS Mediated NLRP3 Inflammasome Deactivation

alpha-Cyperone (Cy) is a major active compound of Cyperus rotundus that has various pharmacological activities. But whether Cy possesses antidepressant effect is unknown. In this study, we exposed mice to chronic unpredictable mild stress (CUMS) with or without intervention with Cy. Our results showed that Cy significantly improved the depressive phenotypes in sucrose preference test, tail suspension test and forced swimming test. Meanwhile, increased SIRT3 expression, reduced ROS production and activated NF-kappa B signal were detected in the hippocampus of mice. NLRP3 inflammasome related proteins including NLRP3, ASC, Caspase-1, IL-1 beta, IL-18 and GSDMD-N were downregulated after Cy administration. Synaptic proteins including Synapsin-1 and PSD-95 and dendritic spine density were improved after Cy treatment. Moreover, the protective effects of Cy in CUMS mice were compromised when co-administrated with SIRT3 inhibitor 3-TYP. Taken together, these findings suggested that Cy has therapeutic potential for treating depression and that this antidepressant effect may be attributed to SIRT3 stimulated neuroplasticity enhancement by suppressing NLRP3 inflammasome.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Anti-Depressive Effectiveness of Baicalin In Vitro and In Vivo

Baicalin (BA), a major polyphenol compound isolated from the extracts of Scutellaria radix, has been previously reported to ameliorate depressive-like behaviors in mice with chronic unpredictable mild stress (CUMS). However, its underlying antidepressant mechanisms remain unclear. This study was designed to confirm the antidepressant-like effects of BA on CUMS induced behavioral abnormalities in mice, and sought to explore the pharmacological mechanisms in vivo and in vitro. The CUMS procedure was carried out to induce depression in mice. Afterwards, the tail suspension test (TST), forced swim test (FST), and open field test (OFT) were performed within 24 h, then sucrose preference test (SPT) was conducted. Additionally, PC12 cells were pretreated with BA for 2 h, then further stimulated with corticosterone for 24 h. The levels of Interleukin-1β (IL-1β), IL-6 and Tumor Necrosis Factor-α (TNF-α) in serum, hippocampus homogenate and cell culture medium were determined using the enzyme-linked immunosorbent assay (ELISA) method. The protein expressions of inhibition of high mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) pathways in hippocampus and PC12 cells were detected. Our results showed that CUMS-treated mice presented notable depressive-like symptoms, such as decreased sucrose consumption, increased FST and TST immobility time. While BA (25, 50 mg/kg) significantly attenuated these changes. Besides, BA treatment considerably inhibited inflammatory cytokinesl (IL-1β, IL-6, TNF-α) levels in serum, hippocampus homogenate and cell culture medium. Western blot analysis indicated that BA inhibited the expressions of HMGB1, TLR4, and p-NF-κBp65 both in vivo and in vitro. In conclusion, the present study confirmed that BA possessed efficient antidepressant effects on depression, which was possibly related to the inhibition of HMGB1/TLR4/NF-κB pathways

Disrupted Timing of MET Signaling Derails the Developmental Maturation of Cortical Circuits and Leads to Altered Behavior in Mice

The molecular regulation of the temporal dynamics of circuit maturation is a key contributor to the emergence of normal structure-function relations. Developmental control of cortical MET receptor tyrosine kinase, expressed early postnatally in subpopulations of excitatory neurons, has a pronounced impact on the timing of glutamatergic synapse maturation and critical period plasticity. Here, we show that using a controllable overexpression (cto-Met) transgenic mouse, extending the duration of MET signaling after endogenous Met is switched off leads to altered molecular constitution of synaptic proteins, persistent activation of small GTPases Cdc42 and Rac1, and sustained inhibitory phosphorylation of cofilin. These molecular changes are accompanied by an increase in the density of immature dendritic spines, impaired cortical circuit maturation of prefrontal cortex layer 5 projection neurons, and altered laminar excitatory connectivity. Two photon in vivo imaging of dendritic spines reveals that cto-Met enhances de novo spine formation while inhibiting spine elimination. Extending MET signaling for two weeks in developing cortical circuits leads to pronounced repetitive activity and impaired social interactions in adult mice. Collectively, our data revealed that temporally controlled MET signaling as a critical mechanism for controlling cortical circuit development and emergence of normal behavior

Neuroprotective Effect of Echinacoside in Subacute Mouse Model of Parkinson’s Disease

Objective. To study the protective effect of Echinacoside for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced dopaminergic (DA) neurons injury in the subacute mouse model of Parkinson’s disease (PD) and to explore its mechanism of action. Methods. We chose 10 weeks of healthy wild type C57BL/6 male mice, hypodermic MPTP 30 mg/kg/day, five days, to prepare PD subacute mouse model. Behavior indexes of open field test and pole test were applied to examine the function of ECH to PD subacute mice model of PD sample action. The effects of ECH on dopaminergic neurons and astrocyte were examined using Immunohistochemistry including tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP) expression. The total numbers of TH-positive neurons and GFAP-positive cells in the substantia nigra pars compacts (SNpc) and ventral tegmental area (VTA) were obtained stereologically using the optical fractionator method. Enzyme-linked immunosorbent assay (ELISA) method was used to detect the inflammatory cytokines in the serum, including TNF-α (Ttumor necrosis factor alpha) and IFN-γ (interferon gamma). Protein expressions of ionized calcium binding adaptor molecule 1 (IBA-1), TNF-α, Cleaved caspase-3, glial derived neurotrophic factor (GDNF), and phosphorylated and total extracellular signal-regulated kinase (p-ERK and ERK) in the anatomical region of substantia nigra (SN) were tested by protein immunoblot method (i.e., Western blotting). Results. ECH reversed the reduction of total distance in open field test in MPTP-induced PD model mice (P < 0.01), shortened the return time and total time of PD subacute model mice in pole test (P < 0.01, P < 0.05), significantly reversed the reduction of TH positive neurons induced by MPTP (P < 0.05), and reduced the activation of astrocytes (P < 0.05). Meanwhile, ECH significantly inhibited the expression of IBA-1, Cleaved caspase-3, and TNF-α in midbrain of MPTP model mice (P < 0.05, P < 0.05, and P < 0.05) and upregulated the expression of GDNF (P < 0.05). And ECH lowered the level of TNF-α and IFN-γ in serum (P < 0.05, P < 0.05). Conclusion. ECH has protective effects on the MPTP subacute model mice, its mechanism may be through inhibiting activation of microglia and astrocytes, reducing inflammatory reaction and promoting the secretion of neurotrophic factors, and eventually resulting in the reduction of the DA neurons apoptosis

Effect of Wenshen-Yanggan Decoction on Movement Disorder and Substantia Nigra Dopaminergic Neurons in Mice with Chronic Parkinson’s Disease

This study aimed to explore the protective effects of Wenshen-Yanggan decoction on dopaminergic (DA) neuron injury in a rotenone-induced mouse model with chronic Parkinson’s disease (PD) and explore its mechanism of action. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure the content of six main components in the Wenshen-Yanggan decoction. The chronic PD mouse model was established by treating 10-month-old healthy wild C57BL/6 male mice with rotenone 30 mg/kg/day for 28 days in succession. The pole test and rotarod test were applied to detect the rescue effect of Wenshen-Yanggan decoction in high, medium, and low dosages, respectively, on PD-like behaviors in mice with chronic PD. The protective effect of Wenshen-Yanggan decoction on the mesencephalic nigrostriatal DA neuron injury was determined employing tyrosine hydroxylase (TH) immunofluorescence staining. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the inflammatory cytokines in serum, including TNF-α (tumor necrosis factor-alpha), IFN-γ (interferon gamma), NF-κB (nuclear factor kappa-B), and IL-1β (interleukin-1 beta). Western blotting was performed to quantify the expression of phosphorylated c-Jun N-terminal kinase (p-JNK), cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), and NF-κB in the brain. Our results showed that the Wenshen-Yanggan decoction in high, medium, and low dosages reduced the turning time of mice P<0.01,P<0.01, and P<0.05. The high and medium dosages shortened the total climbing time of PD mice in the pole test P<0.01 and P<0.05. Meanwhile, the high, medium, and low dosages increased the rod-standing time of PD mice in the rotarod test P<0.01,P<0.05, and P<0.05. Besides, the decoction reversed the decrease in TH-positive neurons induced by rotenone, upregulated TH protein expression, and downregulated the α-syn expression in the PD model. Moreover, the decoction in high dosage significantly inhibited the expression of p-JNK, cleaved caspase-3, and NF-κB in the midbrain of PD mice P<0.05,P<0.05, and P<0.01, upregulated the expression of Bcl-2 P<0.05, and decreased the content of TNF-α, IFN-γ, NF-κB, and IL-1β in the serum P<0.001,P<0.001,P<0.001, and P<0.001. Taken together, the Wenshen-Yanggan decoction could protect mice from rotenone-induced chronic PD, which might be related to the reduction of the DA neuron apoptosis via suppressing the inflammatory reaction and the neuronal apoptosis pathway