Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells / 中国结合医学杂志

OBJECTIVE@#To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages.@*METHODS@#RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured.@*RESULTS@#The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity.@*CONCLUSION@#EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

Nuclear respiratory factor 1 mediates LPS-induced acute lung injury through NF-κB / 生理学报

The purpose of this paper was to study the transcriptional regulation of nuclear respiratory factor 1 (NRF1) on nuclear factor kappa B (NF-κB), a key molecule in lipopolysaccharide (LPS)-induced lung epithelial inflammation, and to clarify the mechanism of NRF1-mediated inflammatory response in lung epithelial cells. In vivo, male BALB/c mice were treated with NRF1 siRNA, followed with LPS (4 mg/kg) or 0.9% saline through respiratory tract, and sacrificed 48 h later. Expression levels of NRF1, NF-κB p65 and its target genes were detected by Western blot and real-time PCR. Nuclear translocation of NRF1 or p65 was measured by immunofluorescent technique. In vitro, L132 cells were transfected with NRF1 siRNA or treated with BAY 11-7082 (5 μmol/L) for 24 h, followed with treatment of 1 mg/L LPS for 6 h. Cells were lysed for detections of NRF1, NF-κB p65 and its target genes as well as the binding sites of NRF1 on RELA (encoding NF-κB p65) promoter by chromatin immunoprecipitation assay (ChIP). Results showed that LPS stimulated NRF1 and NF-κB p65. Pro-inflammatory factors including interleukin-1β (IL-1β) and IL-6 were significantly increased both in vivo and in vitro. Obvious nuclear translocations of NRF1 and p65 were observed in LPS-stimulated lung tissue. Silencing NRF1 resulted in a decrease of p65 and its target genes both in vivo and in vitro. In addition, BAY 11-7082, an inhibitor of NF-κB, significantly repressed the inflammatory responses induced by LPS without affecting NRF1 expression. Furthermore, it was proved that NRF1 had three binding sites on RELA promoter region. In summary, NRF1 is involved in LPS-mediated acute lung injury through the transcriptional regulation on NF-κB p65.

Particulate matter 2.5 triggers airway inflammation and bronchial hyperresponsiveness in mice by activating the SIRT2-p65 pathway / 医学前沿

Exposure to particulate matter 2.5 (PM2.5) potentially triggers airway inflammation by activating nuclear factor-κB (NF-κB). Sirtuin 2 (SIRT2) is a key modulator in inflammation. However, the function and specific mechanisms of SIRT2 in PM2.5-induced airway inflammation are largely understudied. Therefore, this work investigated the mechanisms of SIRT2 in regulating the phosphorylation and acetylation of p65 influenced by PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Results revealed that PM2.5 exposure lowered the expression and activity of SIRT2 in bronchial tissues. Subsequently, SIRT2 impairment promoted the phosphorylation and acetylation of p65 and activated the NF-κB signaling pathway. The activation of p65 triggered airway inflammation, increment of mucus secretion by goblet cells, and acceleration of tracheal stenosis. Meanwhile, p65 phosphorylation and acetylation, airway inflammation, and bronchial hyperresponsiveness were deteriorated in SIRT2 knockout mice exposed to PM2.5. Triptolide (a specific p65 inhibitor) reversed p65 activation and ameliorated PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Our findings provide novel insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure. Triptolide inhibition of p65 phosphorylation and acetylation could be an effective therapeutic approach in averting PM2.5-induced airway inflammation and bronchial hyperresponsiveness.

Effects of leptin-modified human placenta-derived mesenchymal stem cells on angiogenic potential and peripheral inflammation of human umbilical vein endothelial cells (HUVECs) after X-ray radiation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-κB (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/ leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-κB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.

Regulatory effect of Di'ao Xinxuekang on TLR4/MyD88/NF-κB signaling pathway in atherosclerotic rats / 中国中药杂志

The aim of this paper was to observe the effect of Di'ao Xinxuekang(DXXK) on TLR4/MyD88/NF-κB signaling pathway in atherosclerotic rats, and to explore its anti-atherosclerotic mechanism. Sixty SD rats were randomly divided into normal group, model group, atorvastatin group(4.0 mg·kg~(-1)), and DXXK groups(100, 30, 10 mg·kg~(-1)), with 10 rats in each group. The atherosclerosis model was induced by high fat diet plus vitamin D_2. Experimental drugs were administered intragastrically once daily for 8 weeks starting from the 9 th week. Biochemical analyzers were used to detect levels of triglyceride(TG), total cholesterol(TC), low-density lipoprotein cholesterol(LDL-C) and high-density lipoprotein cholesterol(HDL-C) in blood lipid. The levels of serum tumor necrosis factor(TNF)-α, interleukin(IL)-6 and IL-1β were detected by ELISA. Pathological changes of aortic tissues were observed by using Sudan Ⅳ and HE staining. The mRNA and protein expressions of TLR4, MyD88 and NF-κB p65 in aortic tissues were detected by RT-PCR and Western blot, respectively. As compared with the model group, TC, TG, and LDL-C levels in serum were significantly decreased, HDL-C content was significantly increased, and levels of TNF-α, IL-6, and IL-1β in serum were significantly decreased in atorvastatin group and DXXK high and middle dose groups. Aortic lesions in atorvastatin group and DXXK group were significantly improved, and the mRNA and protein expressions of TLR4, MyD88, NF-κB p65 in the aorta were decreased. DXXK has a preventive and therapeutic effect on atherosclerosis in rats, and its mechanism may be related to inhibiting inflammatory reaction by regulating TLR4/MyD88/NF-κB signal transduction, thereby inhibiting the progression of atherosclerosis.

Protective effect of safflower yellow injection against rat MIRI by TLR-NF-κB inflammatory pathway / 中国中药杂志

This study was to investigate the mechanism of safflower yellow injection for regulating inflammatory response against myocardial ischemia-reperfusion injury( MIRI) in rats. Male Wistar rats were randomly divided into sham operation group,model group,Hebeishuang group,safflower yellow injection high,medium and low dose groups. MIRI model was established by ligating left anterior descending coronary artery. Myocardial histopathological changes were observed by HE staining; myocardial infarct size was detected by TTC staining; content and changes of tumor necrosis factor-α( TNF-α) and interleukin-6( IL-6),serum creatine kinase( CK),aspartate aminotransferase( AST),and lactate dehydrogenase( LDH) were detected by biochemical method or enzyme-linked immunosorbent assay( ELISA). Western blot assay was used to detect the protein expression of Toll-like receptor 4( TLR4) and nuclear factor-κB( NF-κB p65) in myocardial tissues. The results showed that as compared with the sham operation group,the myocardial arrangement of the model group was disordered,with severe edemain the interstitial,significantly increased area of myocardial infarction,increased activities of AST,CK and LDH in serum,and significantly increased contents of TNF-α and IL-6; the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were also increased. As compared with the model group,the myocardial tissues were arranged neatlyin the Hebeishuang group and safflower yellow injection high,medium and low dose groups; the edema was significantly reduced; the myocardial infarct size was significantly reduced; the serum AST,CK,LDH activity and TNF-α,IL-6 levels were significantly decreased,and the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were decreased. As compared with the Hebeishuang group,the myocardial infarct size was larger in the safflower yellow injection high,medium and low dose groups; the activities of AST,CK and LDH in serum and the contents of TNF-α and IL-6 in serum were higher,but there was no statistically significant difference in the expression levels of TLR4 and NF-κB( p65) protein in tissues. It is suggested that safflower yellow injection has a significant anti-MIRI effect,and its mechanism may be related to the regulation of TLR-NF-κB pathway to inhibit inflammatory response.

Noni (Morinda citrifolia L.) fruit juice delays immunosenescence in the lymphocytes in lymph nodes of old F344 rats / 中西医结合学报

<p><b>OBJECTIVE</b>Aging is associated with the development of diseases because of immunosuppression and altered functioning of the neuroendocrine system. The medicinal properties of Morinda citrifolia L. have been widely exploited for the treatment of age-associated diseases. This study aims to investigate the in vitro and in vivo effects of noni (M. citrifolia) fruit juice (NFJ) on neuro-immunomodulation in the lymph node lymphocytes of F344 rats.</p><p><b>METHODS</b>Lymphocytes isolated from axillary and inguinal lymph nodes of young (3-4 months) and old (18-21 months) rats were treated in vitro with different concentrations (0.0001%, 0.01%, and 1%) of NFJ for a period of 24 h. In the in vivo study, old (16-17 months) male F344 rats were treated with 5 mL/kg body weight of 5%, 10% and 20% of NFJ, twice a day, by oral gavage, and lymph node lymphocytes were isolated after 60 d. Concanavalin A (Con A)-induced lymphocyte proliferation, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production and expression of intracellular markers, such as phospho-extracellular signal-regulated kinase (p-ERK1/2), phospho-cAMP response element-binding protein, phospho-protein kinase B (p-Akt), phospho-tyrosine hydroxylase (p-TH), phospho-nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α (p-IκB-α) and phospho-nuclear factor-κB (p-NF-κB p65 and p50) were examined in the lymphocytes of lymph nodes.</p><p><b>RESULTS</b>NFJ increased Con A-induced lymphocyte proliferation, IL-2 and IFN-γ production, and p-ERK1/2 expression both in vitro and in vivo. In in vivo NFJ-treated old rats, lymph node lymphocytes showed increased expression of p-TH and Akt, nitric oxide production and decreased expression of p-NF-κB p65 and p50.</p><p><b>CONCLUSION</b>These results suggest that the immunostimulatory properties of NFJ are facilitated through intracellular signaling pathways involving ERK1/2, Akt and NF-κB.</p>

Effects of electroacupuncture on inflammatory response of cardiac muscle tissue in mice with acute myocardial ischemia / 中国针灸

<p><b>OBJECTIVE</b>To observe the effects of electroacupuncture (EA) on inflammatory reaction of acute myocardial ischemia (MI) in mice, and to explore its action mechanism.</p><p><b>METHODS</b>Forty adult male C57BL/6 mice were randomly divided into a control group, a sham operation group, a model group and an EA group, 10 mice in each one. The model was established in the model group and EA group by ligating the left anterior descending branch of coronary artery. The mice in the EA group were treated with EA at "Neiguan" (PC 6) with 2 mA of intensity and 2 Hz /100 Hz of frequency; EA was given 30 min per treatment, once a day for totally 5 days. The mice in the control group and model group were treated with immobilization and no EA was given. The mice in the sham operation group were not treated with ligating at the left anterior descending branch of coronary artery, but the remaining procedure was identical to the model group. The electrocardiogram was recorded and △ST was calculated to evaluate the model. TTC and HE staining methods were applied to evaluate the infarct size and pathologic change of myocardial tissue, respectively. Western blot method was applied to test the protein expression levels of tumor necrosis factor-α (TNF-α), nuclear factor-κB p65 (NF-κB p65), interleukin-1β (IL-1β) and interleukin-8 (IL-8).</p><p><b>RESULTS</b>Compared with the sham operation group, the S-T segments in the model group and EA group were increased obviously after modeling (both <0.01), indicating the MI model was established successfully. The TTC and HE staining results indicated, compared with the sham operation group, the model group had larger infarction size (<0.01), more myocardial fibers injury and inflammatory infiltration; compared with the model group, the infarction size of the EA group was significantly reduced (<0.01), and the myocardial fibers injury and inflammatory infiltration were improved. Compared with the control group, the protein expression levels in the sham operation group were similar (all >0.05); compared with the sham operation group, the expression levels of TNF-α, NF-κB p65, IL-1β and IL-8 were significantly increased in the model group (<0.01, <0.05); compared with the model group, the expression levels of TNF-α, NF-κB p65, IL-1β and IL-8 were significantly reduced in the EA group (all <0.05).</p><p><b>CONCLUSION</b>EA might reduce the protein expression levels of TNF-α, NF-κB p65, IL-1β and IL-8 in cardiac muscle tissue to inhibit inflammatory reaction and achieve myocardial protective effect in mice with acute myocardial ischemia.</p>

Inhibitory effect of polyphyllin Ⅰ on the proliferation of prostate cancer PC3 cells via ERK1/2/P65/DNMT1 and its molecular mechanism / 中华男科学杂志

<p><b>Objective</b>To explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.</p><p><b>METHODS</b>We cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.</p><p><b>RESULTS</b>Compared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).</p><p><b>CONCLUSIONS</b>PPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.</p>

CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-κB activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-κB regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into various vascular cell derivatives to study how NF-κB modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating inflammation, survival, vasculogenesis, cell differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon tumor necrosis factor α (TNFα) stimulation. Lastly, further evaluation of gene expression patterns in IκBα knockout vascular cells demonstrated that IκBα acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-κB/RelA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.

Effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by DSS in mice / 中国中药杂志

To observe the effect of total triterpenoids of Chaenomeles speciosa on PPARγ/SIRT1/NF-κBp65 signaling pathway and intestinal mucosal barrier of ulcerative colitis induced by dextran sulfate sodium (DSS) in mice, C57BL/6 mice were randomly divided into normal group, model group, total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) groups and sulfasalazine (250 mg·kg⁻¹) group. The ulcerative colitis (UC) model was induced by orally administering 2.5% DSS to the experimental mice, and the corresponding drugs were given to each group 3 days before the administration with 2.5% DSS. The normal group and the model group were given the equal volume of 0.5% carboxymethyl cellulose sodium solution by gavage continuously for 10 days, q.d. The general conditions of the mice were observed on a daily basis, and the disease activity index (DAI) score was recorded. On the 10th day after the treatment, mice were put to death, the contents of TNF-α, IL-1β, IL-6, IFN-γ, IL-4 and IL-10 in the blood were detected, colon length was measured, colon mucosa damage index (CMDI) score was calculated, and MPO activity detection and histomorphology analysis were conducted. Real-time PCR was applied to detect the mRNA expressions of E-cadherin, occluding,MUC2 and TFF3; the protein expressions of SIRT1, IKKβ, p-IKKβ, IκBα, p-IκBα and cytosol and nucleus PPARγ, NF-κBp65 in intestinal tissue were detected by western blot. The results indicated that total triterpenoids of C. speciosa (50, 100 mg·kg⁻¹) could significantly improve the general conditions of UC mice, reduce the DAI, CMDI and histopathological scores, increase the colon length, reduce the colonic mucosa ulcers, erosion and inflammatory infiltration, restore the normal intestinal mucosal barrier function, reduce the contents of TNF-α, IL-1β, IL-6, IFN-γ, increase the contents of IL-4 and IL-10 in the blood, inhibit MPO activity in colon tissue, up-regulate the mRNA expressions of E-cadherin, occludin, MUC2 and TFF3 in colon tissue, down-regulate the protein expressions of cytosol PPARγ, tissue p-IKKβ, p-IκBα and nucleus NF-κBp65 in the colon tissue, decrease the p-IKKβ/IKKβ and p-IκBα/IκBα ratios, up-regulate the protein expressions of nucleus PPARγ, tissue SIRT1 and cytosol NF-κBp65 (<0.05 or <0.01, respectively), with a dose-effect relationship between the total triterpenoids of C. speciosa treated groups. These findings suggested that total triterpenoids of C. speciosa had a significantly therapeutic effect on UC mice induced by DSS, its mechanism might be related to the regulation of PPARγ/SIRT1/NF-κBp65 signaling pathway, the inhibition of pro-inflammatory factor formation and the up-regulation of protein expression of protective factors.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation / 中国天然药物

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

Inhibition of miR-128 Abates Aβ-Mediated Cytotoxicity by Targeting PPAR-γ via NF-κB Inactivation in Primary Mouse Cortical Neurons and Neuro2a Cells

PURPOSE: Alzheimer's disease (AD) is the sixth most common cause of death in the United States. MicroRNAs have been identified as vital players in neurodegenerative diseases, including AD. microRNA-128 (miR-128) has been shown to be dysregulated in AD. This study aimed to explore the roles and molecular mechanisms of miR-128 in AD progression. MATERIALS AND METHODS: Expression patterns of miR-128 and peroxisome proliferator-activated receptor gamma (PPAR-γ) messenger RNA in clinical samples and cells were measured using RT-qPCR assay. PPAR-γ protein levels were determined by Western blot assay. Cell viability was determined by MTT assay. Cell apoptotic rate was detected by flow cytometry via double-staining of Annexin V-FITC/PI. Caspase 3 and NF-κB activity was determined by a Caspase 3 Activity Assay Kit or NF-κB p65 Transcription Factor Assay Kit, respectively. Bioinformatics prediction and luciferase reporter assay were used to investigate interactions between miR-128 and PPAR-γ 3′UTR. RESULTS: MiR-128 expression was upregulated and PPAR-γ expression was downregulated in plasma from AD patients and amyloid-β (Aβ)-treated primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells. Inhibition of miR-128 decreased Aβ-mediated cytotoxicity through inactivation of NF-κB in MCN and N2a cells. Moreover, PPAR-γ was a target of miR-128. PPAR-γ upregulation attenuated Aβ-mediated cytotoxicity by inactivating NF-κB in MCN and N2a cells. Furthermore, PPAR-γ downregulation was able to abolish the effect of anti-miR-128 on cytotoxicity and NF-κB activity in MCN and N2a cells. CONCLUSION: MiR-128 inhibitor decreased Aβ-mediated cytotoxicity by upregulating PPAR-γ via inactivation of NF-κB in MCN and N2a cells, providing a new potential target in AD treatment.

Effect of autophagy inhibitor chloroquine on acute alcoholinduced liver disease / 中国应用生理学杂志

OBJECTIVES@#To investigate the role of autophagy inhibitor chloroquine (CQ) in acute ethanol-induced liver injury and its mechenism.@*METHODS@#Twenty-one C57BL/6 male mice were randomly divided into three groups:control group, ethanol group, CQ + ethanol group (=7). Mice in ethanol group were administered 33% (v/v) ethanol at a dose of 4.5 g/kg body weight. Ethanol-induced liver steatosis in each group was detected by hematoxylin and eosin staining. Hepatic lipid accumulation was detected by staining with Oil red O. Hepatic tissue triglyceride (TG) levels, serum aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were determined by biochemical assays. Protein expression of microtubule-associated protein 1 light chain 3(LC3) and nuclear factorκB p65(NF-κB p65) were measured by Western blot and immunofluorescence. Pro-inflammatory factors tumor necrosis factor-α(TNF-α)、interleukin 6(IL-6) were detected by ELISA.@*RESULTS@#Compared with control group, ethanol induced liver injury proved by accumulation of hepatic lipids, TG levels, AST and ALT activities were significantly increased by ethanol, protein expression of LC3-Ⅱ was also markedly increased by ethanol. Compared with ethanol group, addition of CQ increased furtherthe level of LC3-Ⅱexpression, and TG amount, serum AST and ALT activities, and the expression of NF-κB p65, TNF-αand IL-6.@*CONCLUSIONS@#Acute ethanol-intake could induce liver steatosis and inflammation, and autophagy inhibitor CQ exacerbatedethanol-induced liver injury, suggested that autophagy might be protective effect in acute ethanol-induced liver disease.

Salvianolic acid A attenuates ischemia reperfusion induced rat brain damage by protecting the blood brain barrier through MMP-9 inhibition and anti-inflammation / 中国天然药物

Salvianolic acid A (SAA) is a water-soluble component from the root of Salvia Miltiorrhiza Bge, a traditional Chinese medicine, which has been used for the treatment of cerebrovascular diseases for centuries. The present study aimed to determine the brain protective effects of SAA against cerebral ischemia reperfusion injury in rats, and to figure out whether SAA could protect the blood brain barrier (BBB) through matrix metallopeptidase 9 (MMP-9) inhibition. A focal cerebral ischemia reperfusion model was induced by middle cerebral artery occlusion (MCAO) for 1.5-h followed by 24-h reperfusion. SAA was administered intravenously at doses of 5, 10, and 20 mg·kg. SAA significantly reduced the infarct volumes and neurological deficit scores. Immunohistochemical analyses showed that SAA treatments could also improve the morphology of neurons in hippocampus CA1 and CA3 regions and increase the number of neurons. Western blotting analyses showed that SAA downregulated the levels of MMP-9 and upregulated the levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) to attenuate BBB injury. SAA treatment significantly prevented MMP-9-induced degradation of ZO-1, claudin-5 and occludin proteins. SAA also prevented cerebral NF-κB p65 activation and reduced inflammation response. Our results suggested that SAA could be a promising agent to attenuate cerebral ischemia reperfusion injury through MMP-9 inhibition and anti-inflammation activities.

Effects of salidroside on the secretion of inflammatory mediators induced by lipopolysaccharide in murine macrophage cell line J774.1 / 生理学报

To investigate the effect of salidroside (Sal) on the inflammatory activation of lipopolysaccharide (LPS)-induced murine macrophage cell line J774.1 and its possible mechanism, the cells were treated with PBS, LPS (0.5 µg/mL) or different doses of Sal (5, 25, 125 µg/mL) + LPS (0.5 µg/mL). CCK-8 colorimetric method was used to detect the cell activity. The enzyme-linked immunosorbent assay (ELISA) was used to detect the contents of TNF-α, MCP-1 and MIP-2 in the supernatant, and the content of NO in the supernatant was determined by nitrate reductase method. The expression levels of iNOS mRNA was detected by RT-PCR. Western blot was used to detect the expression levels of iNOS protein in cytoplasm and NF-kappaB/p65 (NF-κB/p65) protein in both cytoplasm and nucleus, and DNA binding activity of NF-κB/p65 was detected by using TransAMTM NF-κB/p65 activity assay kit. The results showed that the treatment with 0.5 µg/mL LPS and different doses of Sal (5, 25, 125 µg/mL) for 12 h had no effect on cell viability. Compared with LPS stimulation group, pretreatment with Sal significantly reduced the contents of TNF-α, MCP-1, MIP-2 and NO in culture supernatant induced by LPS in a dose dependent manner (P < 0.05), downregulated the expression levels of iNOS mRNA and protein (P < 0.05), decreased the expression level of NF-κB/p65 protein in nucleus (P < 0.05) while accordingly increased that in cytoplasm (P < 0.05), and decreased DNA binding activity of NF-κB/p65 in a dose dependent manner (P < 0.05). The results suggested that Sal pretreatment can reduce macrophage inflammatory activation induced by LPS, and the mechanism may be through the LPS/TLR4/NF-κB signaling pathway, thereby reducing the excessive expression and secretion of inflammatory mediators and cytokines.

Huaiqihuang Granules () reduce proteinuria by enhancing nephrin expression and regulating necrosis factor κB signaling pathway in adriamycin-induced nephropathy / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effects of Huaiqihuang Granules (, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy (ADRN) in rats and its underlying mechanisms.</p><p><b>METHODS</b>Rats with ADRN were divided into four groups: the sham group, the model group (distilled water), the low-dose HQH-treated (2 g/kg) group, and the high-dose HQH-treated (4 g/kg) group. Body weight and 24-h urinary protein (Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers.</p><p><b>RESULTS</b>HQH ameliorated the rat's general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα (IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α (TNF-α), phosphorylated nuclear factor κB p65 (p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis.</p><p><b>CONCLUSION</b>HQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.</p>

Aggressive Supratentorial Ependymoma, RELA Fusion-Positive with Extracranial Metastasis: A Case Report

Ependymoma is the third most common pediatric primary brain tumor. Ependymomas are categorized according to their locations and genetic abnormalities, and these two parameters are important prognostic factors for patient outcome. For supratentorial (ST) ependymomas, RELA fusion-positive ependymomas show a more aggressive behavior than YAP1 fusion-positive ependymomas. Extracranial metastases of intra-axial neuroepithelial tumors are extremely rare. In this paper, we report a case of aggressive anaplastic ependymoma arising in the right frontoparietal lobe, which had genetically 1q25 gain, CDKN2A homozygous deletion, and L1CAM overexpression. The patient was a 10-year-old boy who underwent four times of tumor removal and seven times of gamma knife surgery. Metastatic loci were scalp and temporalis muscle overlying primary operation site, lung, liver, buttock, bone, and mediastinal lymph nodes. He had the malignancy for 10 years and died. This tumor is a representative case of RELA fusion-positive ST ependymoma, showing aggressive behavior.

Estudos da resposta estringente de Bacillus subtilis e busca por pequenas moléculas moduladoras de RelA / Studies of the stringent response of Bacillus subtilis and search for small molecules capable of modulating RelA activity

Seja no meio ambiente, dentro de um hospedeiro ou em outro habitat, bactérias estarão frequentemente enfrentando condições adversas, como exposição a compostos antibacterianos ou carência nutricional. Em situações como essas, as bactérias são capazes de ativar a chamada resposta estringente, modulada pelo alarmônio (p)ppGpp. O acúmulo de (p)ppGpp promove a inibição da transcrição de rRNAs e tRNAs e a supressão do processo de tradução, e a ativação de operons de biossíntese de aminoácidos. Sabe-se também hoje que a resposta estringente está relacionada a outras importantes carências nutricionais em Escherichia coli, como a falta de ácidos graxos, porém não se sabe se o mesmo ocorre em Bacillus subtilis ou em outras Grampositivas. (p)ppGpp atua também direta e indiretamente em vários outros processos celulares, como motilidade, resistência a antibióticos, virulência e persistência, indicando que (p)ppGpp é um regulador central que integra informação metabólica e respostas adaptativas. O presente trabalho buscou estudar a correlação da resposta estringente de B. subtilis com a carência de ácidos graxos e a busca por pequenas moléculas capazes de modular RelA (a principal proteína envolvida na síntese de (p)ppGpp) e impedir o acúmulo de (p)ppGpp. Para a indução da carência de ácidos graxos, foram utilizadas duas estratégias; uso da droga Cerulenina (inibidor de FabF) e mutantes condicionais no gene FabF. Observou-se que mutantes incapazes de ativar a resposta estringente (cepa ppGpp(0) ou RelAD264G) apresentaram grande perda de viabilidade celular durante a carência de ácidos graxos, ao passo que a cepa selvagem manteve sua viabilidade celular. A causa da morte se deu majoritariamente devido ao colapso do potencial de membrana. Apesar de não termos observado aumento de (p)ppGpp nas células selvagens durante a carência de ácidos graxos, observou-se uma redução da razão GTP/ATP, ao passo que na cepa ppGpp(0), a razão GTP/ATP aumentou, devido ao acúmulo de GTP. O uso da droga decoinina, capaz de reduzir os níveis intracelulares de GTP, resgatou parcialmente a viabilidade da cepa e impediu a perda do potencial de membrana, indicando que os níveis de GTP são importantes durante a carência de ácidos graxos em B. subtilis. Para a triagem de pequenas moléculas inibidoras do acúmulo de (p)ppGpp, foi utilizada uma biblioteca de 2320 diferentes compostos químicos, e buscou-se drogas capazes de reverter o fenótipo de crescimento lento de cepas de B. subtilis que acumulam (p)ppGpp (via mutação pontual; mutante RelAH77A e via tratamento com o indutor hidroxamato de arginina) em meio rico. A primeira etapa selecionou 40 moléculas capazes de resgatar o crescimento de células tratadas com arginina-hidroxamato, porém apenas uma, salicilanilida, foi capaz de também resgatar o crescimento da cepa RelAH77A. Todavia, apesar de ser capaz de acelerar o crescimento de B. subtilis esse efeito é limitado. Diversos análogos de salicilanilida foram testados, porém não apresentaram efeito superior a salicilanilida para a reversão do fenótipo de crescimento lento de B. subtilis. Em adição, a droga não foi capaz de aumentar a sensibilidade dos organismos a diversos antibióticos testados, e aparentemente é incapaz de alterar os níveis internos de (p)ppGpp, porém é capaz de causar alterações nos níveis de ATP. Logo, acredita-se que o efeito observado para o crescimento das células seja devido a efeitos indiretos, possivelmente envolvendo alteração de outros nucleotídeos fosforilados

In the environment, inside a host or other habitat, bacteria will always face adverse conditions, as for example exposure to antimicrobials or starvation. In situations like those, bacteria activate the stringent response, modulated by the alarmone (p)ppGpp. (p)ppGpp accumulation promotes inhibition of rRNA and tRNA transcription and suppression of translational process, at the same time that it activates several amino acid biosynthesis operons. It is known also that the stringent response it is related to other starvation stress in Escherichia coli, like lack of fatty acids, but there is no knowledge if the same occurs for Bacillus subtilis or other gram-positive bacteria. ppGpp acts directly and indirectly affecting several other cellular process, as motility, resistance to antibiotics, virulence and persistence, indicating that (p)ppGpp is a central regulator that integrates metabolic information and adaptive responses. This work aimed to study the correlation between the stringent response in B. subtilis with fatty acid starvation, and search for small moleculas capable of modulating RelA (the main enzyme responsible for ppGpp synthesis) and stop (p)ppGpp production. For fatty acid starvation induction, two strategies were used; use of the drug Cerulenin (inhibitor of the FabF protein) and conditional mutants of the FabF gene. We observed that mutants incapable of activating the stringent response (strains ppGpp(0) ou RelAD264G) presented great loss of viability during fatty acid starvation, whereas the wild-type strain keeps its viability. The main cause of death is due membrane rupture in some cells, but mainly due to membrane potential collapse. Although we did not observed increase of (p)ppGpp in wild-type strains during fatty acid starvation, we observed reduction in GTP/ATP ratios, a hallmark of (p)ppGpp production in gram-positive bacteria. In the strain ppGpp(0) GTP/ATP ratio increased, mainly due to GTP increase. Using the drug decoyinine, capable of reducing GTP levels, partially recued viability and protects cells of losing its membrane potential, indicating that GTP levels plays an important role during fatty acid starvation in B. subtilis. For the screening of small molecules capable of inhibit (p)ppGpp production, a library of 2320 different chemical compounds were used, and we looked for drugs capable of reverting the slow growth phenotype of B. subtilis strains with (p)ppGpp accumulation (using a mutant RelAH77A; and using a stringent response inductor, arginine hidroxamate). The first step selected for 40 molecules capable of rescuing the growth of cells treated with arginine hidroxamate, but only one drug, salicilanilyde could also rescue the growth of the strain RelAH77A. Although capable of rescuing growth of B. subtilis that accumulates (p)ppGpp, this rescue is limited. Several analogues of salicilanilyde were tested, but none were stronger than salicilanilyde itself in rescuing growth of slow growing strains of B. subtilis. In addition, the drug was not capable of increasing antibiotic sensibility and it is incapable of changing intracellular (p)ppGpp levels, but it does shifts ATP levels. Therefore, we believe that the observed effects of salicilanilyde is due indirect action, probably involving other phosphorylated nucleotides, rather than modifying (p)ppGpp levels

p-cresol mas não p-cresil sulfato estimulam a produção de MCP-1 via NF-κB p65 em células vasculares musculares lisas humanas / p-cresol but not p-cresyl sulfate stimulate MCP-1 production via NF-κB p65 in human vascular smooth muscle cells

RESUMO Introdução: p-cresol (PC) e p-cresil sulfato (PCS) são responsáveis por muitas das consequências clínicas uremia, tais como a aterosclerose em pacientes com Doença Renal Crônica (DRC). Objetivos: No presente trabalho, investigamos in vitro o impacto de PC e PCS na expressão da quimiocina monocyte chemoattractant protein-1 (MCP-1) via NF-kappa B (NF-κB) p65 em VSMC. Métodos: O PCS foi sintetizado por sulfatação do PC. As VSMC foram extraídas por digestão enzimática da veia do cordão umbilical e caracterizadas por imunofluorescência através do anticorpo α-actina. As células foram tratadas com PC e PCS em suas concentrações normal (n), urêmica (u) e urêmica máxima (m). A viabilidade celular foi avaliada pelo ensaio de MTT. A expressão de MCP-1 foi investigada por ELISA em sobrenadantes de células após o tratamento com as toxinas, com ou sem o inibidor de NF-κB p65. Resultados: Não houve diferença significativa na viabilidade das células após o tratamento com toxinas para todas as concentrações testadas. Houve um aumento significativo na expressão de MCP-1 em células tratadas com PCu e PCm (p < 0,001) e PCSn, PCSu e PCSm (p < 0,001), em comparação com o controle. Quando as VSMC foram tratadas com o inibidor de NF-κB p65 mais PCu e PCm, houve uma diminuição significativa na produção de MCP-1 (p < 0,005). Este efeito não foi observado com PCS. Conclusões: VSMC estão envolvidas na formação da lesão aterosclerótica e produção de MCP-1, o que contribui para o início da resposta inflamatória. Os nossos resultados sugerem que a PC medeia a produção de MCP-1 em VSMC, provavelmente através da via NF-κB p65 e que PCS atue através de uma subunidade diferente da via, uma vez que o inibidor da porção p65 não foi capaz de inibir a produção de MCP-1.

ABSTRACT Introduction: p-cresol (PC) and p-cresyl sulfate (PCS) are responsible for many of the uremia clinical consequences, such as atherosclerosis in Chronic Kidney Disease (CKD) patients. Objectives: We investigate the in vitro impact of PC and PCS on monocyte chemoattractant protein-1 (MCP-1) expression via NF-kappa B (NF-κB) p65 in VSMC. Methods: PCS was synthesized by PC sulfatation. VSMC were extracted by enzymatic digestion of umbilical cord vein and characterized by immunofluorescence against α-actin antibody. The cells were treated with PC and PCS at their normal (n), uremic (u) and maximum uremic concentrations (m). Cell viability was assessed by MTT. MCP-1 expression was investigated by ELISA in cells supernatants after toxins treatment with or without the NF-κB p65 inhibitor. Results: There was no significant difference in cell viability after toxins treatment for all concentrations tested. There was a significant increase in MCP-1 expression in cells treated with PCu and PCm (p < 0.001) and PCSn, PCSu and PCSm (p < 0.001), compared with the control. When VSMC were treated with the NF-κB p65 inhibitor plus PCu and PCm, there was a significant decrease in MCP-1 production (p < 0.005). This effect was not observed with PCS. Conclusions: VSMC are involved in atherosclerosis lesion formation and production of MCP-1, which contributes to the inflammatory response initiation. Our results suggest that PC mediates MCP-1 production in VSMC, probably through NF-κB p65 pathway, although we hypothesize that PCS acts through a different subunit pathway since NF-κB p65 inhibitor was not able to inhibit MCP-1 production.