Increased Cell Activity Research Articles

MiR-330-5p inhibits intervertebral disk degeneration via targeting CILP

BackgroundIntervertebral disk degeneration (IDD) is caused by nucleus pulposus (NP) degeneration and extracellular matrix (ECM) remodeling and cartilage intermediate layer protein (CILP) expression has been confirmed to be increased in IDD. This study is mainly conducted to clarify the mechanism of CILP in the NP cell degeneration and ECM remodeling in IDD.MethodsCILP expression in the degenerated NP tissues and cells is quantified by quantitative real-time PCR and western blot. CILP function is assessed by cell cycle assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry, β-galactosidase staining, and the detection of ECM-related molecules aggrecan, collagen type I, collagen type II, MMP-3, and MMP-9 expression is accomplished by qRT-PCR. The potential mechanism is authenticated by dual-luciferase reporter gene assay.ResultsCILP was increased in the degenerated NP tissues and cells, and the knockdown of CILP promoted the NP cell cycle, increased cell activity, and repressed cell apoptosis and repressed cell senescence and ECM production. Moreover, miR-330-5p targeted the CILP 3′-untranslated region, and miR-330-5p negatively regulated CILP expression. Moreover, the overexpression of miR-330-5p repressed NP cell degeneration and ECM remodeling to relieve IDD by downregulating CILP.ConclusionMiR-330-5p represses NP cell degeneration and ECM remodeling to ameliorate IDD by downregulating CILP.

Transcriptional Changes in CD16+ Monocytes May Contribute to the Pathogenesis of COVID-19.

The COVID-19 pandemic has caused more than three million deaths globally. The severity of the disease is characterized, in part, by a dysregulated immune response. CD16+ monocytes are innate immune cells involved in inflammatory responses to viral infections, and tissue repair, among other functions. We characterized the transcriptional changes in CD16+ monocytes from PBMC of people with COVID-19, and from healthy individuals using publicly available single cell RNA sequencing data. CD16+ monocytes from people with COVID-19 compared to those from healthy individuals expressed transcriptional changes indicative of increased cell activation, and induction of a migratory phenotype. We also analyzed COVID-19 cases based on severity of the disease and found that mild cases were characterized by upregulation of interferon response and MHC class II related genes, whereas the severe cases had dysregulated expression of mitochondrial and antigen presentation genes, and upregulated inflammatory, cell movement, and apoptotic gene signatures. These results suggest that CD16+ monocytes in people with COVID-19 contribute to a dysregulated host response characterized by decreased antigen presentation, and an elevated inflammatory response with increased monocytic infiltration into tissues. Our results show that there are transcriptomic changes in CD16+ monocytes that may impact the functions of these cells, contributing to the pathogenesis and severity of COVID-19.

Transmigration of Neutrophils From Patients With Familial Mediterranean Fever Causes Increased Cell Activation.

Familial Mediterranean fever (FMF) is caused by pyrin-encoding MEFV gene mutations and characterized by the self-limiting periods of intense inflammation, which are mainly mediated by a massive influx of polymorphonuclear neutrophils (PMNs) into the inflamed sites. Perturbation of actin polymerization by different pathogens was shown to activate the pyrin inflammasome. Our aim was to test whether cytoskeletal dynamics in the absence of pathogens may cause abnormal activation of PMNs from FMF patients. We also aimed to characterize immunophenotypes of circulating neutrophils and their functional activity. Circulating PMNs displayed heterogeneity in terms of cell size, granularity and immunophenotypes. Particularly, PMNs from the patients in acute flares (FMF-A) exhibited a characteristic of aged/activated cells (small cell size and granularity, up-regulated CXCR4), while PMNs form the patients in remission period (FMF-R) displayed mixed fresh/aged cell characteristics (normal cell size and granularity, up-regulated CD11b, CD49d, CXCR4, and CD62L). The findings may suggest that sterile tissue-infiltrated PMNs undergo reverse migration back to bone marrow and may explain why these PMNs do not cause immune-mediated tissue damage. A multidirectional expression of FcγRs on neutrophils during acute flares was also noteworthy: up-regulation of FcγRI and down-regulation of FcγRII/FcγRIII. We also observed spontaneous and fMPL-induced activation of PMNs from the patients after transmigration through inserts as seen by the increased expression of CD11b and intracellular expression of IL-1β. Our study suggests heightened sensitivity of mutated pyrin inflammasome towards cytoskeletal modifications in the absence of pathogens.

Dexmedetomidine exerts a protective effect on ischemic brain injury by inhibiting the P2X7R/NLRP3/Caspase-1 signaling pathway

Dexmedetomidine (Dex) has been suggested to exert a protective function in ischemic brain injury. In this study, we aimed to elucidate the intrinsic mechanisms of Dex in regulating microglia pyroptosis in ischemic brain injury via the purinergic 2X7 receptor (P2X7R)/NLRP3/Caspase-1 signaling pathway. First, permanent middle cerebral artery occlusion (p-MCAO) rat model was established, followed by the measurement of behavioral deficit, neuronal injury, the volume of brain edema and the infarct size. Dex treatment was suggested to alleviate the neurological deficits in p-MCAO rats and reduce the brain water content and infarct size. Additionally, rat microglia were cultured in vitro and a model of oxygen and glucose (OGD) was established. Microglia cell activity and ultrastructure were detected. Dex could increase cell activity and reduce LDH activity, partially reversing the changes in cell morphology. Furthermore, the activation of P2X7R/NLRP3/Caspase-1 pathway was tested. The obtained findings indicated Dex suppressed microglial pyroptosis by inhibiting the P2X7R/NLRP3/Caspase-1 pathway. Inhibition of P2X7R or NLRP3 could inhibit Caspase-1 p10 expression, improve cell activity, and reduce LDH activity. The same result was verified in vivo experiments. This study indicated that Dex inhibited microglia pyroptosis by blocking the P2X7R/NLRP3/Caspase-1 pathway, thus playing a protective role against ischemic brain injury.

Inhibition of GPR4 attenuates SH-SY5Y cell injury in cerebral ischemia/reperfusion via anti-apoptotic pathways.

Cerebral ischemia/reperfusion injury (CIRI) can lead to increased vascular endothelial permeability and blood-brain barrier damage in patients with stroke. G protein-coupled receptor 4 (GPR4) is a functional pH sensor that plays a key role in renal ischemia-reperfusion-induced apoptosis. However, whether GPR4 has a role in cerebral ischemia remains to be further studied. Our study found that after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, the levels of GPR4 and CHOP in SH-SY5Y cells were significantly increased, which was accompanied by a decrease in cell viability, and an increase in LDH release and apoptosis. After knockdown of GPR4 using shRNA, CHOP levels in SH-SY5Y cells were also decreased, which unexpectedly increased cell activity and decreased LDH release and apoptosis rate. Interestingly, CHOP overexpression reversed the effect of GPR4 knockdown, suggesting that OGD/R-induced CIRI may involve endoplasmic reticulum stress-related apoptosis. In conclusion, our study provided a basis for further research on the mechanism of CIRI.

Effect of surface topography on in vitro osteoblast function and mechanical performance of 3D printed titanium.

Critical-sized defects remain a significant challenge in orthopaedics. 3D printed scaffolds are a promising treatment but are still limited due to inconsistent osseous integration. The goal of the study is to understand how changing the surface roughness of 3D printed titanium either by surface treatment or artificially printing rough topography impacts the mechanical and biological properties of 3D printed titanium. Titanium tensile samples and discs were printed via laser powder bed fusion. Roughness was manipulated by post-processing printed samples or by directly printing rough features. Experimental groups in order of increasing surface roughness were Polished, Blasted, As Built, Sprouts, and Rough Sprouts. Tensile behavior of samples showed reduced strength with increasing surface roughness. MC3T3 pre-osteoblasts were seeded on discs and analyzed for cellular proliferation, differentiation, and matrix deposition at 0, 2, and 4 weeks. Printing roughness diminished mechanical properties such as tensile strength and ductility without clear benefit to cell growth. Roughness features were printed on mesoscale, unlike samples in literature in which roughness on microscale demonstrated an increase in cell activity. The data suggest that printing artificial roughness on titanium scaffold is not an effective strategy to promote osseous integration.

Space-velocity thermostatted kinetic theory model of tumor growth.

The competition between cancer cells and immune system cells in inhomogeneous conditions is described at cell scale within the framework of the thermostatted kinetic theory. Cell learning is reproduced by increased cell activity during favorable interactions. The cell activity fluctuations are controlled by a thermostat. The direction of cell velocity is changed according to stochastic rules mimicking a dense fluid. We develop a kinetic Monte Carlo algorithm inspired from the direct simulation Monte Carlo (DSMC) method initially used for dilute gases. The simulations generate stochastic trajectories sampling the kinetic equations for the distributions of the different cell types. The evolution of an initially localized tumor is analyzed. Qualitatively different behaviors are observed as the field regulating activity fluctuations decreases. For high field values, i.e. efficient thermalization, cancer is controlled. For small field values, cancer rapidly and monotonously escapes from immunosurveillance. For the critical field value separating these two domains, the 3E's of immunotherapy are reproduced, with an apparent initial elimination of cancer, a long quasi-equilibrium period followed by large fluctuations, and the final escape of cancer, even for a favored production of immune system cells. For field values slightly smaller than the critical value, more regular oscillations of the number of immune system cells are spontaneously observed in agreement with clinical observations. The antagonistic effects that the stimulation of the immune system may have on oncogenesis are reproduced in the model by activity-weighted rate constants for the autocatalytic productions of immune system cells and cancer cells. Local favorable conditions for the launching of the oscillations are met in the fluctuating inhomogeneous system, able to generate a small cluster of immune system cells with larger activities than those of the surrounding cancer cells.

Patients Over Age 40 with Cam-Type FAI Demonstrate Increased Cell Activity in Articular Chondrocytes

Femoroacetabular impingement (FAI) is a frequent cause of hip pain and may lead to secondary osteoarthritis yet little is known about the molecular events linking mechanical hip impingement and articular cartilage degeneration. The purpose of this study is to compare the cell activity and histological appearance of articular chondrocytes harvested from patients with FAI and examine the relationship of patient age. Cartilage biopsies were obtained from 26 donors with FAI who underwent hip arthroscopy for debridement of cam lesions. Quantitative real-time polymerase chain reaction [qRT-PCR] was performed to assess gene expression of markers for extracellular matrix (ECM) synthesis, inflammation, and cellular senescence. Specimens were separated into the senescent or active cell activity groups based on positive red staining by using safranin O/ fast green staining. The age of the donors were then compared and divided into 2 groups: above and below 40 year olds. Student t test was used to compare between the two groups. P < 0.05 was considered significant. Standard deviation was used to indicate the variation within groups. Chondrocytes derived from FAI cartilage in donors younger than 40 years old (mean=32.3 years old, SD = 7.0 years) had significantly less cell activity (p= 0.005) than donors greater than age 40 (mean = 42.1 years old, SD = 10.8). Red positive staining of safranin O was seen in the extracellular matrix or surrounding the cells of the active group. There was no significant difference between the alpha angles of the two groups (p=0.4). Cartilage in cam-type FAI is molecularly and morphologically different between age groups above and below 40 years old. Specimens with primarily active cells were from donors greater than 40 years old and donors younger than 40 years old had senescent cells. There was no significant difference based on alpha angle and gender. Greater understanding of the molecular differences between cartilage in younger and older patients with FAI may improve their surgical management for the future development of novel treatment that attenuate disease progression to OA.

Protective effect of procyanidin A-type dimers against H2O2-induced oxidative stress in prostate DU145 cells through the MAPKs signaling pathway

It has been reported that B-type procyanidins can alleviate oxidative damage of prostatic cells, but there has been limited information on the similar role of A-type procyanidins. This study investigated the protective effect of procyanidin A-type dimers from peanut skin against H2O2-induced oxidative stress damage in prostate cancer DU145 cells. According to the UPLC-Q-TOF-MS/MS analysis and comparison with standards, the fourth fraction of peanut skin procyanidin (PSP-4) was identified as procyanidin A-type dimers, namely, procyanidin A1 and A2. Results revealed that PSP-4 treatment prior H2O2 exposure increased cell activity and attenuated the cell cycle arrest and apoptosis rate. The H2O2-induced increase in intracellular reactive oxygen species (ROS) was remarkably inhibited by PSP-4. PSP-4 treatment enhanced the activity of catalase (CAT) and total super oxide dismutase (T-SOD) and restored glutathione (GSH) content, compared with the H2O2 treatment. Furthermore, the results indicated that PSP-4 protected DU145 cells by attenuating phosphorylation of the mitogen-activated protein kinases (MAPKs), by increasing the Bcl-2/Bax ratio, and by reducing the activation of caspase-3 and caspase-9 by cascade reactions. This study reveals that procyanidin A-type dimers from peanut skin have the potential function in preventing oxidative stress damage of prostatic cells.

Expression of Vav1-Myo1F Fusion Affects T-Cell Differentiation and Induces T-Cell Lymphoma

Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T cell lymphomas not otherwise specified (PTCL, NOS) account for &amp;gt;45% diagnoses, show limited response to intensified chemotherapy treatment and are associated with dismal survival. Genomic studies from our group have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS. Interestingly, mutation co-occurrence analysis in AITL and PTCL, NOS showed significant mutual exclusivity of VAV1 genomic alterations and the highly prevalent RHOA mutations (p-value 0.0142), supporting a common mechanism of action. Gene fusions involving VAV1 are characterized by the substitution of their auto-inhibitory C-terminal SH3 domains by different domains from their fusion partners, leading to increased activation of VAV1-dependent signaling pathways. Among them, the VAV1-MYO1F fusion shows the strongest increase in VAV1 activity and activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and nuclear factor of activated T-cells (NFAT) pathways. To study the role of VAV1-MYO1F, we engineered a conditional knockin mouse that expresses the Vav1-myo1f fusion in CD4+ T-cells. Expression of Vav1-myo1f in CD4 T cells induces cell activation and alterations in T-cell specification, associated with up-regulation of master transcription factors involved in helper T-cell cell differentiation. Moreover, Vav1-Myo1f increased CD4+ T-cell survival upon cytokine withdrawal and enhanced Vav1 phosphorylation and activation of the MAPK pathway, resulting in increased cell activation and proliferation both in vivo and in vitro in response to TCR engagement. Notably, expression of Vav1-Myo1f fusion in CD4+ T-cells is sufficient to induce development of fatal malignant lymphomas with a latency of 6-14 months. Histological examination showed disrupted splenic architecture associated with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS phenotype. Similar results were obtained in a genetic model that combined the expression of Vav1-myo1f with the deletion of the Tet2 epigenetic regulator. In both genetic models, tumor cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features including increased expression of the transcription factors Gata3 and c-Maf and the IL4 and Il10 cytokines. Overall, these results demonstrate a direct oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, associated with deregulation of T-cell specification and of signaling programs critical for the control of T-cell proliferation. Disclosures Palomero: Kura Onclology: Research Funding.

Abstract PO-41: Expression of Vav1-Myo1F fusion affects T-cell differentiation and induces T-cell lymphoma

Abstract Peripheral T-cell lymphomas (PTCL) are highly aggressive, malignant hematologic tumors that arise from clonal proliferation of mature T cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphomas not otherwise specified (PTCL, NOS) account for &amp;gt;45% diagnoses, show limited response to intensified chemotherapy treatment, and are associated with dismal survival. Genomic studies from our group have uncovered recurrent mutations and novel cancer-associated gene fusions involving the guanine nucleotide exchange factor VAV1 in AITL and PTCL, NOS. Interestingly, mutation co-occurrence analysis in AITL and PTCL, NOS showed significant mutual exclusivity of VAV1 genomic alterations and the highly prevalent RHOA mutations (p-value 0.0142), supporting a common mechanism of action. Gene fusions involving VAV1 are characterized by the substitution of their auto-inhibitory C-terminal SH3 domains by different domains from their fusion partners, leading to increased activation of VAV1-dependent signaling pathways. Among them, the VAV1-MYO1F fusion shows the strongest increase in VAV1 activity and activation of the mitogen-activated protein kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor of activated T-cells (NFAT) pathways. To study the role of VAV1-MYO1F, we engineered a conditional knock-in mouse that expresses the Vav1-myo1f fusion in CD4+ T-cells. Expression of Vav1-myo1f in CD4 T cells induces cell activation and alterations in T-cell specification, associated with upregulation of master transcription factors involved in helper T-cell cell differentiation. Moreover, Vav1-Myo1f increased CD4+ T-cell survival upon cytokine withdrawal and enhanced Vav1 phosphorylation and activation of the MAPK pathway, resulting in increased cell activation and proliferation both in vivo and in vitro in response to TCR engagement. Notably, expression of Vav1-Myo1f fusion in CD4+ T cells is sufficient to induce development of fatal malignant lymphomas with a latency of 6-14 months. Histologic examination showed disrupted splenic architecture associated with clonal expansion of CD4+ cells indicative of T-cell lymphoma with PTCL, NOS phenotype. Similar results were obtained in a genetic model that combined the expression of Vav1-myo1f with the deletion of the Tet2 epigenetic regulator. In both genetic models, tumor cells specifically present a memory cell-associated immunophenotype (CD44+ CD62L-) and characteristic Th2-like features including increased expression of the transcription factors Gata3 and c-Maf and the IL4 and Il10 cytokines. Overall, these results demonstrate a direct oncogenic role for Vav1-Myo1f in the pathogenesis of PTCL, associated with deregulation of T-cell specification and of signaling programs critical for the control of T-cell proliferation. Citation Format: Jose Rodriguez Cortes, Robert Albero, Ioan Filip, Juan Angel Patino, Anisha R. Cooke, Wen-Hsuan Lin, Anouchka P. Laurent, Bobby B. Shih, Aidan S. Quinn, Raul Rabadan, Adolfo Ferrando, Teresa Palomero. Expression of Vav1-Myo1F fusion affects T-cell differentiation and induces T-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-41.

Xiaoyaosan (Tiaogan-Liqi therapy) protects peritoneal macrophages from corticosterone-induced stress by regulating the interaction between glucocorticoid receptor and ABCA1.

BackgroundPrevious studies have reported that Xiaoyaosan (XYS), Tiaogan-Liqi therapy, has a protective function in depressive disorder, and can regulate body weight and corticosterone (CORT) level. However, little is known about the effect of XYS in treating atherosclerosis. This study aimed to explore the influence XYS on macrophage foam cell formation and related mechanism.MethodsRat peritoneal macrophages (PMs) were separated and stimulated with CORT and oxidized low density lipoprotein (ox-LDL). The serum was obtained from rats treated with different doses of XYS and was added into the medium for macrophages. Then, the cell activity and lipid content of PMs were measured through Cell Counting Kit-8 (CCK-8) assay and oil red staining, respectively. The expressions of glucocorticoid receptor (GR), ATP binding cassette subfamily A member 1 (ABCA1), and heat shock protein 90 (HSP90) were detected. In addition, overexpression of GR and ABCA1 was performed and the effect on XYS treatment was subsequently assessed.ResultsThe CCK-8 assay showed the serum increased cell activity of CORT-induced stress PMs in a XYS dose-dependent manner. Oil red staining and enzyme-linked immunosorbent assay (ELISA) showed that the serum decreased lipids of PMs. In the XYS treated groups, HSP90 protein was decreased and protein levels of ABCA1 and GR were increased in cytoplasm, while GR protein in nucleus was decreased. Co-immunoprecipitation (Co-IP) assay indicated GR might interact with HSP90 and be involved with the function of XYS. Furthermore, overexpression of GR attenuated the protective function of XYS on CORT-induced stress in PMs, while overexpression of ABCA1 enhanced that.ConclusionsThis study denoted that XYS could protect PMs from CORT-induced stress by regulating the interaction of GR and ABCA1, which might contribute to the treatment of atherosclerosis.

Anxiety and depression: A matter of stiffness?

Cells react to stress by the universal responses of “fluidization” or “reinforcement” (stiffening) of the cytoplasm, through dramatic re-arrangements of the cytoskeleton. Here it is suggested that, at a supracellular level, the brain exhibits such a fundamental behavior as part of its complex response to stress: it is hypothesized that the soft gel formed by brain cell cytoskeletons and the surrounding extracellular matrix (the “cytoskeletons-matrix system”) undergoes transitions either to sol (fluidization) or stiff gel (reinforcement) as a very fundamental and evolutionarily conserved brain response to stress, alongside more sophisticated neural pathways. Sol state corresponds to increased cell activity (a sort of “fight or flight” response), whereas stiff gel state corresponds to inactivity (an “immobility” strategy).Psychological stress, through simple stress signals such as pH changes, would lead to an initial tissue fluidization in key regions of the brain, followed, if the stress stimuli persist, by reinforcement (slow formation of actomyosin stress fibers and matrix stiffening).It is also hypothesized that the cytoskeletons-matrix system is one of the biological correlates of so-called “background feelings”, i.e conscious feelings built on inner chemical-physical states of the body. Optimal dynamics of the cytoskeletons-matrix system would contribute to a core feeling of well-being, while shifts towards fluidization (activation) or stiffening (inactivation) would contribute to background feelings at the basis of anxiety and stress-induced depression, respectively.It is suggested that the cytoskeletons-matrix system behaves as a “self-organized critical system”, anxiety and depression arising whenever the system is driven too far from the optimal critical point.Finally, some application hints from the proposed ideas are given.

Protective Effect of Pravastatin on Myocardial Ischemia Reperfusion Injury by Regulation of the miR-93/Nrf2/ARE Signal Pathway.

PurposeThis research intended to study the mechanism of pravastatin in myocardial ischemia reperfusion (I/R) injury.Patients and MethodsAltogether 70 male rats were selected and grouped into Sham operation group (Sham group), ischemia reperfusion group (I/R group), pravastatin pretreatment group (I/R+P group), I/R+miR-93-mimics, I/R+P+miR-93-mimics, I/R+Nrf2 siRNA, and I/R+P+Nrf2 siRNA group. The myocardial function of each group was detected.ResultsMyocardial I/R injury could lead to abnormal myocardial enzyme activity, inflammatory reaction and oxidative stress. However, pravastatin could significantly inhibit the activity of myocardial enzymes, alleviate inflammatory reaction and inhibit oxidative stress reaction, thus playing a protective role. Furthermore, cell experiments showed that pravastatin can alleviate the injury of H9C2 myocardial cells caused by I/R, inhibit the apoptosis of myocardial cells, and lead to a significant reduction in pro-apoptotic genes Bax, caspase-3 and caspase-9 transcription levels, an obvious increase in anti-apoptotic gene Bcl-2, and an increase in cell activity. After I/R induced injury, miR-93 level was significantly up-regulated and Nrf2 level was down-regulated. Over-expression of miR-93 or inhibition of Nrf2 expression would lead to further aggravation of I/R myocardial injury, increase the apoptosis rate of cells and decrease the activity of myocardial cells. Pravastatin administration could inhibit miR-93, activate and promote Nrf2 in myocardial tissue, and promote protein expression of downstream regulatory genes HO-1 and NQO1. In the I/R model, pravastatin was given. Over-expression of miR-93 or silencing Nrf2 could reverse the therapeutic effect of pravastatin on I/R.ConclusionPravastatin acts as a protector on myocardial ischemia reperfusion injury by regulating miR-93/Nrf2/ARE signaling pathway.

Resveratrol protects against apoptosis induced by interleukin-1β in nucleus pulposus cells via activating mTOR/caspase-3 and GSK-3β/caspase-3 pathways.

Objective: The purpose of the present study was to investigate the specific downstream signaling pathway mediated by PI3K/Akt in resveratrol (RES) anti-apoptosis of nucleus pulposus cells (NPCs).Materials and methods: Human NPCs were cultured and divided into six groups. Interleukin (IL)-1β was used to induce apoptosis and RES to inhibit apoptosis. Fluorescence-activated cell sorting (FACS) analysis was used to test apoptotic incidence of NPCs, cell counting kit-8 (CCK-8) assay was performed to detect cell viability, The expression level of caspase-3 mRNA was detected by RT-qPCR, and protein levels were determined by Western blot.Results: Flow cytometry analysis showed that IL-1β increased the apoptosis rate of NPCs in each group, and RES significantly decreased the apoptosis rate, while rapamycin (RAPA) and SB216763 inhibited the effect of RES and increased the apoptosis rate again. Similarly, CCK-8 showed that IL-1β decreased activity of NPCs in each group, while RES increased cell activity, RAPA and SB216763 inhibited the effect of RES and decreased cell activity. RT-qPCR results showed IL-1β significantly increased the level of caspase-3 expression, but it was significantly decreased by using RES, RAPA and SB216763 respectively attenuated effects of RES. Western blot results showed that activated caspase-3 was inhibited by RES effect, and was up-regulated again after the addition of RAPA and SB216763. In addition, p-mTOR and p-GSK-3β were up-regulated by RES and down-regulated by RAPA and SB216763.Conclusion: RES can inhibit apoptosis induced by IL-1β in human NPCs. PI3K/Akt/mTOR/caspase-3 and PI3K/Akt/GSK-3β/caspase-3 pathways are potential mechanisms underlying this process.

LncRNA SNHG12 Improves Cerebral Ischemic-reperfusion Injury by Activating SIRT1/FOXO3a Pathway through I nhibition of Autophagy and Oxidative Stress.

Ischemia/reperfusion (I/R) injury involves complex biological processes and molecular mechanisms such as autophagy. Oxidative stress plays a critical role in the pathogenesis of I/R injury. LncRNAs are the regulatory factor of cerebral I/R injury. This study constructs cerebral I/R model to investigate role of autophagy and oxidative stress in cerebral I/R injury and the underline regulatory mechanism of SIRT1/ FOXO3a pathway. In this study, lncRNA SNHG12 and FOXO3a expression was up-regulated and SIRT1 expression was down-regulated in HT22 cells of I/R model. Overexpression of lncRNA SNHG12 significantly increased the cell viability and inhibited cerebral ischemicreperfusion injury induced by I/Rthrough inhibition of autophagy. In addition, the transfected p-SIRT1 significantly suppressed the release of LDH and SOD compared with cells co-transfected with SIRT1 and FOXO3a group and cells induced by I/R and transfected with p-SNHG12 group and overexpression of cells co-transfected with SIRT1 and FOXO3 further decreased the I/R induced release of ROS and MDA. In conclusion, lncRNA SNHG12 increased cell activity and inhibited oxidative stress through inhibition of SIRT1/FOXO3a signaling-mediated autophagy in HT22 cells of I/R model. This study might provide new potential therapeutic targets for further investigating the mechanisms in cerebral I/R injury and provide.

4464 Effect of Surface Topography on In Vitro and Mechanical Performance of 3D Printed Titanium

OBJECTIVES/GOALS: The goal of the study is to understand how changing the surface roughness of 3D printed Titanium either by processing printed samples or artificially printing rough topography impacts the mechanical and biological properties of the Titanium. METHODS/STUDY POPULATION: Titanium dog bones and discs were printed via laser powder bed fusion. groups were defined as 1. polished, 2.blasted, 4.as built, 4.sprouts and 5.rough sprouts. Roughness was measured with line measurement using a confocal microscope. Tensile testing of dog bones produced stress strain curves. MC3T3 preosteoblast were seeded on discs. Samples were analyzed at 0, 2, and 4 weeks. A cell viability assay and confocal fluorescent microscopy assessed cell growth. Alkaline Phosphatase (ALP) assay and Quantitative Polymerase Chain Reaction (qPCR) examined cell differentiation. Extracellular matrix (ECM) was stained for collagen and calcium. Scanning Electron Microcopy (SEM) was done on sputter coated discs. RESULTS/ANTICIPATED RESULTS: Measured roughness defined by Rz, maximum peak to valley distance of the sample profile ranged from 2.6-65.1 µm. The addition of printed roughness in the sprouts and rough sprouts group significantly diminished ductility resulting in early strain to failure during tensile testing. Cells adhered and proliferated on discs regardless of roughness group. There was no statistical difference in ALP activity, but qPCR showed that rough groups (sprouts and rough sprouts) had diminished Osteocalcin gene expression at week 2 and 4. The ECM in the rough groups was more resistant to repeated washes and was more extensive with SEM. DISCUSSION/SIGNIFICANCE OF IMPACT: Printing roughness diminished mechanical properties without clear benefit to cell growth. Roughness features were on mesoscale, unlike samples in literature on microscale that increase cell activity. Printed topography may aid in implant fixation and not osseous integration as hypothesized. CONFLICT OF INTEREST DESCRIPTION: Dr. Samual Adams, Dr. Ken Gall and Cambre Kelly own stock and/or stock options in restor3d, Inc.

1325-P: Study on the Mechanism of Activating Macrophages TGR5 on Glucose Metabolism during Pregnancy

Purpose: To explore a new mechanism of improving glucose homeostasis in pregnancy through activation of Takeda G protein-coupled receptor 5 (TGR5) in bone marrow derived macrophages (BMDM) from gestational diabetes mellitus (GDM) model mouse and observation the changes of macrophages migration, inflammatory factors and chemokines expression and secretion, and the expression of Proprotein Convertase 1/3 (PC1/3). Methods: INT-777 was used as TGR5 agonist and LPS was used as inducer for macrophage polarization to M1. Primary bone marrow cells were extracted from GDM model mouse and were induced into macrophages. The macrophages activity and migration were detected by cck-8 tests and Transwell assay respectively when macrophages were treated with INT-777. The expression and secretion levels of inflammatory factors and chemokines in macrophages were detected by real-time PCR, cytometric bead array and ELISA, and the expression levels of PC1/3 was detected by Western blot when macrophages were treated both with INT-777 and lipopolysaccharide (LPS). Results: TGR5 activation of macrophages by INT-777 increased cell activity (P &amp;lt; 0.01) and decreased cell migration (P &amp;lt; 0.001). Moreover, the mRNA levels of inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2, CCL4, CCL5 and CCL7 mRNA were significantly decreased (All P &amp;lt; 0.05), the secretion levels of inflammatory cytokines and chemokines including IL-6, TNF-α, MCP-1, CCL2, CCL3 and CCL4 were significantly decreased (All P &amp;lt; 0.05) and the expression of PC1/3 was increased (P &amp;lt; 0.05) in INT-777 + LPS treated macrophages, compared with LPS treated macrophages only. Conclusions: Activation of TGR5 in BMDM can effectively inhibit the macrophage polarization to M1, including inhibiting inflammatory factors expression and secretion. Moreover, activation of TGR5 can inhibit macrophage chemokine expression and secretion, cell migration and can upregulate PC1/3 expression. Disclosure J. Li: None. F. Liu: None. Funding National Natural Science Foundation of China (81770802)

Preprotection of Tea Polysaccharides with Different Molecular Weights Can Reduce the Adhesion between Renal Epithelial Cells and Nano-Calcium Oxalate Crystals.

Crystal adhesion is an important link in the formation of kidney stones. This study investigated and compared the adhesion differences between nano-calcium oxalate monohydrate (COM) and human renal proximal tubule epithelial (HK-2) cells before and after treatment with tea polysaccharides (TPSs) TPS0, TPS1, TPS2, and TPS3 with molecular weights of 10.88, 8.16, 4.82, and 2.31 kDa, respectively. TPS treatment effectively reduced the damage of COM to HK-2 cells, thereby resulting in increased cell activity, decreased release of lactate dehydrogenase, cell morphology recovery, decreased level of reactive oxygen species, increased mitochondrial membrane potential, increased lysosomal integrity, decreased expression of adhesion molecule osteopontin and eversion of phosphatidylserine, and decreased crystal adhesion. Among the TPSs, TPS2 with moderate molecular weight had the best protective effect on cells and the strongest effect on the inhibition of crystal adhesion. Thus, TPS2 may be a potential anticalculus drug.

TSP1 ameliorates age-related macular degeneration by regulating the STAT3-iNOS signaling pathway

Age-related macular degeneration is a progressive ocular disease that is the leading cause of vision loss among elderly. AMD usually is divided into two types: wet and dry AMD, which is linked with inflammation. Choroidal Neovascularization (CNV) formation or wet AMD is also associated with oxidative stress. Previously, TSP1 has been shown to have a significant alleviating effect on CNV in TSP1 knockout (TSP1−/−) mice. However, the mechanism by which TSP1 ameliorates CNV remains unclear. Here we report that TSP1 reduces nitric oxide production to prevent cells from forming tubes formation and reduced the levels of vascular endothelial growth factor (VEGF) and lipid peroxides (LPO) during oxidative stress. We measured RF/6A cell viability by CCK-8 assay and apoptosis by flow cytometry. RF/6A cell were transfected with TSP1 and STAT3 overexpression, and then the mRNA and protein levels of TSP1 and also the signal pathways were detected by qRT-PCR and Western blot analysis. Migration assays were performed using a transwell system. Co-Immunoprecipitation was used to analyze the binding relationship between CD47 and SHP-2. The results show that overexpression of TSP1 alleviated the damage of oxidative stress to RF/6A cells including increased cell activity and migration, decreased apoptosis and reduced migration compared to the control group. SHP-2 was activated by TSP1 through its receptor CD47 and STAT3 phosphorylation was reduced by activation of SHP-2, thereby blocking STAT3-iNOS pathway and reducing NO concentration in RF/6A cells ultimately protecting them from oxidative stress. Finally, the CNV mice model confirmed that TSP1 overexpression could protect the mice against CNV in vivo, modified the antioxidants levels and decreased the expression of TNF-α and IL-6 under laser irradiation. These results indicate a potential mechanism of TSP1 to slow down formation of CNV in wet AMD, which may bring hope for new treatment strategies.