Objective To study the effects of TYRO protein kinase-binding protein (TYROBP) deficiency on learning behavior, glia activation and pro-inflammatory cycokines, and Tau phosphorylation of a new Alzheimer's disease (AD) mouse model carrying a PSEN1 p.G378E mutation.Methods A new AD mouse model carrying PSEN1 p.G378E mutation was built based on our previously found AD family which might be ascribed to the PSEN1 mutation, and then crossed with TYROBP deficient mice to produce the heterozygous hybrid mice (PSEN1^G378E/WT; Tyrobp^+/-) and the homozygous hybrid mice (PSEN1^G378E/G378E; Tyrobp^-/-). Water maze test was used to detect spatial learning and memory ability of mice. After the mice were sacrificed, the hippocampus was excised for further analysis. Immunofluorescence was used to identify the cell that expresses TYROBP and the number of microglia and astrocyte. Western blot was used to detect the expression levels of Tau and phosphorylated Tau (p-Tau), and ELISA to measure the levels of pro-inflammatory cytokines. Results Our results showed that TYROBP specifically expressed in the microglia of mouse hippocampus. Absence of TYROBP in PSEN1^G378E mutation mouse model prevented the deterioration of learning behavior, decreased the numbers of microglia and astrocytes, and the levels of interleukin-6, interleukin-1β and tumor necrosis factor-α in the hippocampus (all P < 0.05). The ratios of AT8/Tau5, PHF1/Tau5, pT181/Tau5, pT231/Tau5 and p-ERK/ERK were all higher in homozygous hybrid mice (PSEN1^G378E/G378E; Tyrobp^-/- mice) compared with PSEN1^G378E/G378E mice (all P < 0.05). Conclusions TYROBP deficiency might play a protective role in the modulation of neuroinflammation of AD. However, the relationship between neuroinflammation processes involving microglia and astrocyte activation, and release of pro-inflammatory cytokines, and p-Tau pathology needs further study.
Mice ; Animals ; Alzheimer Disease/genetics* ; Neuroinflammatory Diseases ; Hippocampus/pathology* ; Mutation ; Cytokines/pharmacology* ; Disease Models, Animal ; tau Proteins/pharmacology* ; Amyloid beta-Peptides/metabolism* ; Adaptor Proteins, Signal Transducing/pharmacology*

BACKGROUNDResolvin D1 (RvD1) is a newly found anti-inflammatory bioactive compound derived from polyunsaturated fatty acids. The current study aimed to explore the protective effect of RvD1 on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) and its possible mechanism.

METHODSBoth in vivo and in vitro studies were conducted. Male BALB/c mice were randomly divided into control group (saline), LPS group (LPS 5 mg/kg), RvD1 group (RvD1 5 μg/kg + LPS 5 mg/kg), and blockage group (Boc-MLP 5 μg/kg + RvD1 5 μg/kg + LPS 5 mg/kg). Boc-MLP is a RvD1 receptor blocker. The mice were intraperitoneally injected with these drugs and recorded for general condition for 48 h, while the blood and kidneys were harvested at 2, 6, 12, 24, and 48 h time points, respectively (n = 6 in each group at each time point). Human proximal tubule epithelial cells (HK-2) were randomly divided into control group (medium only), LPS group (LPS 5 μg/ml), RvD1 group (RvD1 10 ng/ml + LPS 5 μg/ml), and blockage group (Boc-MLP 10 ng/ml + RvD1 10 ng/ml + LPS 5 μg/ml). The cells were harvested for RNA at 2, 4, 6, 12, and 24 h time points, respectively (n = 6 in each group at each time point). Blood creatinine was tested by using an Abbott i-STAT portable blood gas analyzer. Tumor necrosis factor-α (TNF-α) level was detected by ELISA. Kidney pathology was observed under hematoxylin and eosin (HE) staining and transmission electron microscope (TEM). We hired immune-histological staining, Western blotting, and fluorescence quantitative polymerase chain reaction to detect the expression of RvD1 receptor ALX, nuclear factor-kappa B (NF-κB) signaling pathway as well as caspase-3. Kidney apoptosis was evaluated by TUNEL staining.

RESULTSRvD1 receptor ALX was detected on renal tubular epithelials. Kaplan-Meier analysis indicated that RvD1 improved 48 h animal survival (80%) compared with LPS group (40%) and RvD1 blockage group (60%), while RvD1 also ameliorated kidney pathological injury in HE staining and TEM scan. After LPS stimulation, the mRNA expression of toll-like receptor 4, myeloid differentiation factor 88, and TNF-α in both mice kidneys and HK-2 cells were all up-regulated, while RvD1 substantially inhibited the up-regulation of these genes. Western blotting showed that the phosphorylated-IκB/IκB ratio in LPS group was significantly higher than that in the control group, which was inhibited in the RvD1 group. RvD1 could inhibit the up-regulation of cleaved-caspase-3 protein stimulated by LPS, which was prohibited in RvD1 blockage group. RvD1 group also had a lower proportion of apoptotic nuclei in mice kidney by TUNEL staining compared with LPS group.

CONCLUSIONIn LPS-induced AKI, RvD1 could decrease TNF-α level, ameliorate kidney pathological injury, protect kidney function, and improve animal survival by down-regulating NF-κB inflammatory signal as well as inhibiting renal cell apoptosis.

Acute Kidney Injury ; chemically induced ; prevention & control ; Adaptor Proteins, Signal Transducing ; analysis ; Animals ; Apoptosis ; drug effects ; Docosahexaenoic Acids ; pharmacology ; Down-Regulation ; Kidney ; drug effects ; pathology ; Lipopolysaccharides ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; antagonists & inhibitors ; Tumor Necrosis Factor-alpha ; analysis

OBJECTIVETo investigate the effect of angiotension II (AngII) on the activation of NLRP3 inflammasome and the expression of interleukin-1β (IL-1β) in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs cultured in vitro were treated with different concentrations of AngII for varying lengths of time to determine the optimal concentration and time for AngII exposure. To test the impact of different agents on the effect of AngII exposure, HUVECs were pretreated with AngII receptor blocker losartan, NAD(P)H inhibitor DPI and H(2)O(2) scavenger CAT, caspase 1 inhibitor YVAD, or NLRP3 siRNA for silencing NLRP3, and the protein levels of NOX4, NLRP3, caspase-1 and IL-1β in HUVECs were analyzed by Western blotting.

RESULTSAngII treatment at the optimal concentration (10(-9) mol/L) for 12 h significantly increased the protein levels of NOX4, NLRP3, caspase1 and IL-1β in HUVECs. Pretreatment with losartan, DPI, CAT, YVAD, or NLRP3 siRNA all attenuated the effects of AngII on the cells.

CONCLUSIONAngII can induce vascular inflammation by promoting the production of reactive oxygen species and activating NLRP3 inflammasome to increase the protein expression of IL-1β in HUVECs.

Adaptor Proteins, Signal Transducing ; pharmacology ; Angiotensin II ; pharmacology ; Blotting, Western ; Carrier Proteins ; metabolism ; Caspase 1 ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Hydrogen Peroxide ; Inflammasomes ; metabolism ; Interleukin-1beta ; metabolism ; NADPH Oxidase 4 ; NADPH Oxidases ; metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein ; RNA, Small Interfering ; Reactive Oxygen Species ; metabolism

OBJECTIVE: To study the effect of IAA/HRP on the proliferation and apoptosis of human SACC-83 cells in vitro,and its molecular mechanism. METHOD: The rate of proliferation inhibition was determined by CCK-8 assay, the apoptosis-related gene Caspase-3 and Livinα mRNA and protein expression levels were measured by real-time PCR and Western blot. RESULT: IAA/HRP could inhibit proliferation of SACC-83, which depending on the time and dosage (P < 0.05); can-up-regulate the Caspase-3 mRNA and protein expression levels and reduction of the mRNA and protein of the Livina expression, (both P < 0.05). CONCLUSION IAA/HRP can inhibit the proliferation and induce the apoptosis of SACC-83 cells, which may due to its regulation of the expression of Caspase-3 and Livinα mRNA expression.
Adaptor Proteins, Signal Transducing ; metabolism ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Indoleacetic Acids ; pharmacology ; Inhibitor of Apoptosis Proteins ; metabolism ; Neoplasm Proteins ; metabolism

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.
Actins ; metabolism ; Adaptor Proteins, Signal Transducing ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; physiology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Cell Line, Tumor ; Connective Tissue Growth Factor ; genetics ; metabolism ; pharmacology ; Cytochalasin D ; pharmacology ; Fatty Acids, Nonesterified ; pharmacology ; HEK293 Cells ; Humans ; Immunohistochemistry ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; Palmitic Acid ; pharmacology ; Phosphoproteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Rats ; Recombinant Proteins ; genetics ; metabolism ; pharmacology ; Thiazolidines ; pharmacology

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; HEK293 Cells ; Heat-Shock Proteins ; chemistry ; metabolism ; Humans ; Lysine ; metabolism ; Mice ; Neurons ; metabolism ; pathology ; Oxidopamine ; pharmacology ; Parkinson Disease ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; drug effects ; Sequestosome-1 Protein ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects

OBJECTIVETo investigate the effects of small interfering RNA (siRNA) targeting YAP on the proliferation and apoptosis of human periodontal ligament stem cells (hPDLSCs).

METHODSSynthesized sequences of siRNA were transfected into hPDLSCs by Lipofectamine™ 2000. The expression of YAP was identified by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. Proliferation activity was detected by using cell counting kit-8 (CCK-8). Changes in the cell cycle and apoptosis rate were detected by using flow cytometry. Results were analyzed by using SPSS 19.0, and P < 0.05 was considered statistically significant.

RESULTSExpression of YAP mRNA and protein were significantly downregulated after 48 h of transfection (P < 0.001). No obvious difference was found in the expression levels of YAP protein between 48 and 72 h, thus indicating that siRNA could inhibit the expression of YAP persistently and effectively. Proliferation activity was inhibited, and apoptosis rate was increased. Cell cycle was changed as the proportion of G₁and S phases increased (P < 0.01) and G₂ phase decreased (P < 0.05).

CONCLUSIONKnocking down YAP gene by siRNA could inhibit proliferation activity, induce apoptosis, and change the cell cycle of hPDLSCs. Thus, YAP could regulate the proliferation and apoptosis of hPDLSCs.

Adaptor Proteins, Signal Transducing ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Down-Regulation ; Humans ; Periodontal Ligament ; drug effects ; Phosphoproteins ; RNA, Messenger ; RNA, Small Interfering ; pharmacology ; Stem Cells ; drug effects ; Transfection

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.
8,11,14-Eicosatrienoic Acid ; analogs & derivatives ; metabolism ; pharmacology ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Apoptosis ; drug effects ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Atherosclerosis ; genetics ; metabolism ; pathology ; Catalase ; genetics ; metabolism ; Cytochrome P-450 CYP2C8 ; genetics ; metabolism ; Gene Expression Regulation ; Heme Oxygenase-1 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Models, Biological ; NADPH Oxidase 2 ; NADPH Oxidases ; genetics ; metabolism ; NF-E2-Related Factor 2 ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Reactive Oxygen Species ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism ; pharmacology

BACKGROUNDBisdemethoxycurcumin (BDMC) is an active component of curcumin and a chemotherapeutic agent, which has been suggested to inhibit tumor growth, invasion and metastasis in multiple cancers. But its contribution and mechanism of action in invasion and metastasis of non-small cell lung cancer (NSCLC) are not very clear. Therefore, we tried to study the effects of BDMC on regulation of epithelial-to-mesenchymal transition (EMT), which is closely linked to tumor cell invasion and metastasis.

METHODSIn this study, we first induced transforming growth factor-β1 (TGF-β1) mediated EMT in highly metastatic lung cancer 95D cells. Thereafter, we studied the effects of BDMC on invasion and migration of 95D cells. In addition, EMT markers expressions were also analyzed by western blot and immunofluorescence assays. The contribution of Wnt inhibitory factor-1 (WIF-1) in regulating BDMC effects on TGF-β1 induced EMT were further analyzed by its overexpression and small interfering RNA knockdown studies.

RESULTSIt was observed that BDMC inhibited the TGF-β1 induced EMT in 95D cells. Furthermore, it also inhibited the Wnt signaling pathway by upregulating WIF-1 protein expression. In addition, WIF-1 manipulation studies further revealed that WIF-1 is a central molecule mediating BDMC response towards TGF-β1 induced EMT by regulating cell invasion and migration.

CONCLUSIONSOur study concluded that BDMC effects on TGF-β1 induced EMT in NSCLC are mediated through WIF-1 and elucidated a novel mechanism of EMT regulation by BDMC.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Blotting, Western ; Cell Line, Tumor ; Cell Movement ; drug effects ; genetics ; Curcumin ; analogs & derivatives ; pharmacology ; Epithelial-Mesenchymal Transition ; drug effects ; genetics ; Humans ; Lung Neoplasms ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of everolimus(RAD001)combined with all-trans retinoid acid(ATRA) on drug resistance of ATRA-resistance acute promyelocytic leukemia(APL) cell line NB4-R1 and its molecular mechanism.

METHODSAPL NB4-R1 cells were treated with different concentrations of RAD001(1 nmol/L, 10 nmol/L and 100 nmol/L) with ATRA(1μmol/L) for 24, 48 and 72 h, respectively. The differentiation of NB4-R1 cells was analyzed by flow cytometry with CD11b staining and nitro blue tetrozolium(NBT) reduction test. Cell cycle was detected by cell cycle staining kit and apoptosis was detected by flow cytometry with Annexin V/PI staining. Protein expressions of LC-3II, PML-RARα, P-P70S6K and P-4E-BP1 were determined by Western blotting.

RESULTSRAD001 combined with ATRA significantly induced NB4-R1 cells differentiation, but RAD001 or ATRA alone did not enhance NB4-R1 differentiation. The co-treatment induced accumulation of cells in G1 phase and decreased the proportion of cells in S phase. The combined treatment had no effect on cell apoptosis. The differentiation rate of NB4-R1 cells in 100 nmol/L RAD001, 1μmol/L ATRA, RAD001 combined with ATRA and control groups was(2.29±0.57)%,(17.06±2.65)%,(54.47±4.91)% and(2.54±0.53)%, respectively; the proportion of cells in G1 phase was(35.20±11.97)%,(33.54±6.25)%,(53.70±8.73)% and(27.40±6.01)%, respectively; cells apoptosis rate was(2.30±0.14)%,(2.25±0.21)%,(2.40±0.28)% and(1.95±0.07)%, respectively. The combination of RAD001 with ATRA significantly inhibited mTOR signaling downstream proteins P-P70S6K, P-4E-BP1 and enhanced autophagy-related protein LC3-II and Beclin 1. The co-treatment also induced degradation of fusion protein PML-RARα.

CONCLUSIONRAD001 combined with ATRA can induce cell differentiation, inhibit cell cycle, resulting the reverse of drug resistance in NB4-R1 cells, which is associated with increase of autophagy level and degradation of PML-RARα.

Adaptor Proteins, Signal Transducing ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle ; Cell Differentiation ; Cell Line, Tumor ; drug effects ; Drug Resistance, Neoplasm ; Everolimus ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Oncogene Proteins, Fusion ; metabolism ; Phosphoproteins ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; Tretinoin ; metabolism