Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection
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99 GENERAL AND MISCELLANEOUS↗
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Intravenous Delivery of Lung-Targeted Nanofibers for Pulmonary Hypertension in Mice
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Marulanda, Kathleen↗
Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice
Large-scale phosphoproteomics with coverage of over 10,000 sites of phosphorylation have now been routinely achieved with advanced mass spectrometry (MS)-based workflows. However, accurate targeted MS-based quantification of phosphorylation dynamics, an important direction for gaining quantitative understanding of signaling pathways or networks, has been much less investigated. Herein, we report an assessment of the targeted workflow in the context of signal transduction pathways, using the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway as our model. 43 phosphopeptides from the EGFR–MAPK pathway were selected for the study. The recovery and sensitivity of a workflow consisted of two commonly used enrichment methods, immobilized metal affinity chromatography (IMAC) and titanium oxide (TiO2), combined with selected reaction monitoring (SRM)-MS, were evaluated. The recovery of phosphopeptides by IMAC and TiO2 enrichment was quantified to be 38 ± 5% and 58 ± 20%, respectively, based on internal standards. Moreover, both enrichment methods provided comparable sensitivity from 1-100 g starting peptides. Robust quantification was consistently achieved for most targeted phosphopeptides when starting with 25-100 g peptides. However, the numbers of quantified targets significantly dropped when peptide samples were in the 1-25g range. Finally, IMAC-SRM was applied to quantify signaling dynamics of EGFR-MAPK pathway in Hs578T cells following 3 ng/mL EGF treatment. The kinetics of phosphorylation clearly revealed early and late phases of phosphorylation, even for very low abundance proteins. These results demonstrate the feasibility of robust targeted quantification of phosphorylation dynamics for specific pathways, even starting with relatively small amounts of protein.
Sharon, Gil↗
Plumbojarosite Remediation of Soil Affects Lead Speciation and Elemental Interactions in Soil and in Mice Tissues
Lead (Pb) contamination of soils is of global concern due to the devastating impacts of Pb exposure in children. Because early-life exposure to Pb has long-lasting health effects, reducing exposure in children is a critical public health goal that has intensified research on the conversion of soil Pb to low bioavailability phases. Recently, plumbojarosite (PLJ) conversion of highly available soil Pb was found to decrease Pb relative bioavailability (RBA <10%). However, there is sparse information concerning interactions between Pb and other elements when contaminated soil, pre- and post-remediation, is ingested and moves through the gastrointestinal tract (GIT). Addressing this may inform drivers of effective chemical remediation strategies. In this study, we utilize bulk and micro-focused Pb X-ray absorption spectroscopy to probe elemental interactions and Pb speciation in mouse diet, cecum, and feces samples following ingestion of contaminated soils pre- and post-PLJ treatment. RBA of treated soils was less than 1% with PLJ phases transiting the GIT with little absorption. In contrast, Pb associated with organics was predominantly found in the cecum. These results are consistent with transit of insoluble PLJ to feces following ingestion. The expanded understanding of Pb interactions during GIT transit complements our knowledge of elemental interactions with Pb that occur at higher levels of biological organization.
54 ENVIRONMENTAL SCIENCES↗
Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice
Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). Here, we investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-Kras G12D/+ ;LSL-Trp53 R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11 +/+ and Cdh11 –/– mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11 +/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11 +/– and KPC/Cdh11 –/– mice survived significantly longer than KPC/Cdh11 +/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11 +/+ mice and incompletely in KPC/Cdh11 +/– and KPC/Cdh11 –/– mice, whose lesions also contained fewer FOXP3 + cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11 +/+ mice, tumors of KPC/Cdh11 +/– mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11 +/– and KPC/Cdh11 –/– mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11 +/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.
59 BASIC BIOLOGICAL SCIENCES↗
Diphenyl diselenide regulates Nrf2/Keap-1 signaling pathway and counteracts hepatic oxidative stress induced by bisphenol A in male mice
Highlights: • BPA exposure induced hepatic oxidative stress in a sex-dependent manner in mice. • BPA exposure deregulates Nrf2/keap1 pathway in the mouse liver. • (PhSe){sub 2} reversed hepatic oxidative stress caused by BPA in male mice. • (PhSe){sub 2} modulates NrF2/keap1 pathway in male mice exposed to BPA. Bisphenol A (BPA) is a chemical toxicant that has deleterious effects on human. BPA causes oxidative stress in tissues, including the liver. Diphenyl diselenide (PhSe){sub 2} improves the antioxidant response via activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/Kelch-like ECH-associated protein (keap 1) pathway in macrophage cells. In the present study, we investigated whether (PhSe){sub 2} counteracts hepatic oxidative stress induced by BPA in male and female Swiss mice. Three-week-old mice received by the intragastric (i.g.) route BPA (5 mg/kg) from 21st to 60th postnatal day (PND). At PND 61, the mice were treated with (PhSe){sub 2} (1 mg/kg, i.g.) for seven days. Parameters of hepatic damage and oxidative stress were determined in male and female mice. The results show that BPA increased the activity of aspartate aminotransferase in female mice, and in male mice the activity of alanine aminotranseferase was increased. Male and female mice had an increase in fat mass accumulation. Male mice showed an increase in hepatic oxidative damage of proteins and a decrease in non-enzymatic (ascorbic acid and non-protein thiol) and enzymatic (superoxide dismutase) defenses, which are consistent with oxidative stress status. Male mice were more susceptible than female mice to hepatic oxidative stress induced by BPA. BPA decreased Nrf2/Keap1 protein content in male mice. (PhSe){sub 2} reduced hepatic oxidative stress induced by BPA in male mice. Our results demonstrate that male mice were more susceptible to hepatic oxidative stress induced by BPA than female mice. (PhSe){sub 2} regulated Nrf2/Keap-1 signaling pathway and countered hepatic oxidative stress induced by BPA in male mice.
60 APPLIED LIFE SCIENCES↗
Immunological responses against hepatitis B virus in human peripheral blood mononuclear cell-engrafted mice
Highlights: • DKO-NOG mice, which were NOG mice deficient in both MHC class I and II, were established. • Liver damage was reduced, and survival time was prolonged in human PBMC-engrafted DKO-NOG mice. • HBV-specific immune responses were observed in the established DKO-NOG mice. It is well known that immune-mediated virus elimination is necessary for the treatment of HBV infection. Reconstitution of human immune cells in liver chimeric mice is warranted to understand the immunopathogenesis of HBV infection. Here, we report a new immunologically humanized mouse model with a human immune system via reconstitution of immunodeficient NOG-Iaβ/β2 m double KO mice, which are NOG mice that are deficient in both MHC class I and II (DKO-NOG mice), with human HLA-A2-positive peripheral blood mononuclear cells (PBMCs). After injection of PBMCs, the xenogeneic graft-versus-host disease observed in PBMC-engrafted NOG mice was prevented in PBMC-engrafted DKO-NOG mice. Liver damage was reduced, and the survival time was prolonged in human PBMC-engrafted DKO-NOG mice compared to those in the NOG mice. The expression levels of PD-1 and Tim-3 on human T cells from PBMC-engrafted DKO-NOG mice were lower than those from NOG mice. By induction of HBV-specific T cell responses, such as vaccination with HBc-derived, peptide-pulsed DCs, hydrodynamic injection of HBV vector and intrasplenic injection of HepG2.2.15, the number of HBc-derived, peptide-specific CTLs increased in PBMC-engrafted DKO-NOG mice. Moreover, the recombinant HBV vaccine resulted in the production of hepatitis B surface antibody in 50% of the vaccinated mice. The induction of HBV-specific immune responses could be established in the immunologically humanized mice.
60 APPLIED LIFE SCIENCES↗
Investigation of TRPV1 loss-of-function phenotypes in TRPV1 Leu206Stop mice generated by N-ethyl-N-nitrosourea mutagenesis
Highlights: • N-ethyl-N-nitrosourea mutagenesis can generate mouse models for pain research. • TRPV1 Leu206Stop mice closely resemble the phenotype of TRPV1 knockout mice. • Mechanical hypersensitivity is not impaired in TRPV1 Leu206Stop mice. N-ethyl-N-nitrosourea (ENU) random mutagenesis was used to generate a mouse model for the analysis of the transient receptor potential vanilloid 1 (TRPV1) cation channel. A transversion from T→A in exon 4 led to a Leu206Stop mutation generating a loss-of-function mutant. The TRPV1 agonist capsaicin was used to analyze functional and nociceptive parameters in vitro and in vivo in TRPV1 Leu206Stop mice and congenic C3HeB/FeJ controls. Capsaicin-induced [Ca{sup 2+}]{sub i} changes in small diameter DRG neurons were significantly diminished in TRPV1 Leu206Stop mice and administration of capsaicin induced neither hypothermia nor nocifensive behaviour in vivo. TRPV1 Leu206Stop mice were tested in the spinal nerve ligation of mononeuropathic pain and developed mechanical hypersensitivity two weeks after nerve injury. In the open field test, a significant increase in spontaneous locomotion was detected in TRPV1 Leu206Stop mice as compared to wildtype controls. TRPV1 knockout mice have been reported to carry a similar phenotype regarding capsaicin-evoked responses in vitro and in vivo. However, in contrast to TRPV1 Leu206Stop mice, TRPV1 knockout mice did not differ in spontaneous locomotion as compared to congenic C57BL/6 mice, suggesting subtle ENU-dependent or independent strain differences between TRPV1 Leu206Stop mice and their wildtype controls. In summary, these data revealed a target-related (i.e. capsaicin-evoked) phenotype of TRPV1 Leu206Stop mice closely resembling that of published TRPV1 knockout mice. However, since ENU-mutant mice are congenic with the mouse strain initially used in random mutagenesis, direct phenotypic comparison with the respective wildtype controls is possible, and the time-consuming backcrossing in lines with targeted mutations is avoided.
60 APPLIED LIFE SCIENCES↗
Role of PKB/SGK-dependent phosphorylation of GSK-3α/β in vascular calcification during cholecalciferol overload in mice
Highlights: • Vascular calcification was blunted in mice with PKB/SGK-resistant Gsk-3α/β. • PKB/SGK-resistant Gsk-3α/β inhibited aortic osteoinduction. • PKB/SGK-resistant Gsk-3α/β blunted Pit1 and Pai1 up-regulation during calcification. • Vascular PKB/SGK-resistant Gsk-3α/β ameliorated aortic ring calcification ex vivo. Medial vascular calcification is a highly regulated process involving osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells. Both, protein kinase B (PKB) and serum- and glucocorticoid-inducible kinase 1 (SGK1) are involved in the intracellular signaling of vascular calcification and both phosphorylate and inactivate glycogen synthase kinase 3 (GSK-3). The present study explored whether PKB/SGK-dependent phosphorylation of GSK-3α/β is involved in vascular calcification. Experiments were performed in Gsk-3α/β double knockin mice lacking functional PKB/SGK phosphorylation sites (gsk-3{sup KI}) and corresponding wild-type mice (gsk-3{sup WT}) following high-dosed cholecalciferol treatment as well as ex vivo in aortic ring explants from gsk-3{sup KI} and gsk-3{sup WT} mice treated without and with phosphate. In gsk-3{sup WT} mice, high-dosed cholecalciferol induced vascular calcification and aortic osteo-/chondrogenic signaling, shown by increased expression of osteogenic markers Msx2, Cbfa1 and tissue-nonspecific alkaline phosphatase (Alpl). All these effects were suppressed in aortic tissue from gsk-3{sup KI} mice. Cholecalciferol decreased aortic Gsk-3α/β phosphorylation (Ser{sup 21/9}) in gsk-3{sup WT} mice, while no phosphorylation was observed in gsk-3{sup KI} mice. Moreover, the mRNA expression of type III sodium-dependent phosphate transporter (Pit1) and plasminogen activator inhibitor 1 (Pai1) was increased following cholecalciferol treatment in aortic tissue of gsk-3{sup WT} mice, effects again blunted in gsk-3{sup KI} mice. In addition, phosphate treatment induced mineral deposition and osteogenic markers expression in aortic ring explants from gsk-3{sup WT} mice, effects reduced in aortic ring explants from gsk-3{sup KI} mice. In conclusion, vascular PKB/SGK-dependent phosphorylation of GSK-3α/β contributes to the osteoinductive signaling leading to vascular calcification.
60 APPLIED LIFE SCIENCES↗
Myeloid sirtuin1 deficiency aggravates hippocampal inflammation in mice fed high-fat diets
Highlights: • Myeloid sirtuin1 deficiency increases insulin resistance in high-fat diet mice. • Loss of myeloid sirtuin1 promotes macrophage infiltration in high-fat diet mice. • Chronic high-fat diet lowers systemic lipoclain-2 levels in myeloid sirtuin1-deficient mice. • Myeloid sirtuin1 deficiency reduces high-fat diet-induced hippocampal inflammation. Chronic low-grade inflammation-induced insulin resistance is associated with neuroinflammation. Myeloid sirtuin1 (SIRT1) deficiency aggravates high-fat diet (HFD)-induced insulin resistance. However, the function of myeloid-specific SIRT1 in the hippocampus of obese mice is largely unknown. To address this question, we fed myeloid SIRT1 knockout (KO) mice a HFD for 40 weeks. We found that HFD-fed SIRT1 KO mice had increased insulin resistance and macrophage infiltration in adipose tissue than wild type (WT) mice. Levels of HFD-induced lipocalin-2 (LCN2) were lower in SIRT1 KO mice than in WT. HFD-induced hippocampal LCN2 expression was lower in HFD-fed SIRT1 KO mice than in WT. Hippocampal acetylation of nuclear factor-κB (NF-κB) and amyloid precursor protein levels were higher in HFD-fed SIRT1 KO mice than in HFD-fed WT mice. Taken together, our results suggest that targeted induction of the anti-inflammatory effects of SIRT1 and LCN2 may help prevent obesity-associated insulin resistance and neuroinflammation.
60 APPLIED LIFE SCIENCES↗
Depletion of Fractalkine ameliorates renal injury and Treg cell apoptosis via the p38MAPK pathway in lupus-prone mice
Highlights: • FKN and p-p38 were increased in the renal of lupus mice. • Anti-FKN antibody and SB203580 ameliorated renal injury in lupus mice. • FKN KD and SB203580 reduced cell apoptosis, and suppressed p38MAPK signal in Treg cells derived from lupus mice spleen. • The p38MAPK activator U-46619 had the opposite effect in these cells. • Depletion of FKN ameliorates renal injury and Treg cell apoptosis via p38MAPK signal in lupus nephritis. Fractalkine (FKN) is a chemokine with several roles, including chemotaxis; adhesion; and immune damage, which also participates in cell inflammation and apoptosis and responds to the pathogenesis of autoimmune diseases. Given the involvement of regulatory T cells (Treg) cells in autoimmune diseases, this study investigated the regulatory mechanism of FKN in renal injury and Treg apoptosis via the p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in lupus-prone mice. Lupus was induced in BALB/c female mice by injection of pristane, followed by isolation of CD4{sup +}CD25{sup +} Treg cells from the spleen of lupus model mice. To deplete FKN, mice received injection of an anti-FKN antibody, and Treg cells were transfected with FKN small-interfering RNA. Lupus mice and Treg cells were treated with the p38MAPK inhibitor SB203580 and activator U-46619, respectively, and urine protein and serum urea nitrogen, creatinine, and autoantibodies were measured and renal histopathological changes analyzed. We determined levels of FKN, phosphorylated p38 (p-p38), and forkhead box P3 (FOXP3) in renal tissue and Treg cells, and analyzed apoptosis rates and levels of key apoptotic factors in Treg cells. The renal FKN and p-p38 levels increased, whereas renal FOXP3 level decreased in lupus-prone mice. Treatment with the anti-FKN antibody and the p38MAPK inhibitor ameliorated proteinuria and renal function, significantly reducing serum autoantibody, renal FKN, and p-p38 levels while increasing renal FOXP3 level in lupus-prone mice. Moreover, FKN knockdown and administration of the p38MAPK inhibitor reduced apoptosis and levels of pro-apoptotic factors, increased levels of anti-apoptotic factors, and suppressed activation of p38MAPK signaling in Treg cells derived from lupus model mice. Furthermore, treatment with the p38MAPK activator U-46619 had the opposite effect on these cells. These data indicated that depletion of FKN ameliorated renal injury and Treg cell apoptosis via inhibition of p38MAPK signaling in lupus nephritis, suggesting that targeting FKN represents a potential therapeutic strategy for treating Lupus nephritis.
60 APPLIED LIFE SCIENCES↗
Genetic interactions between polycystin-1 and Wwtr1 in osteoblasts define a novel mechanosensing mechanism regulating bone formation in mice
Molecular mechanisms transducing physical forces in the bone microenvironment to regulate bone mass are poorly understood. Here, we used mouse genetics, mechanical loading, and pharmacological approaches to test the possibility that polycystin-1 and Wwtr1 have interdependent mechanosensing functions in osteoblasts. We created and compared the skeletal phenotypes of control Pkd1 flox/+ ;Wwtr1 flox/+ , Pkd1 Oc-cKO , Wwtr1 Oc-cKO , and Pkd1/Wwtr1 Oc-cKO mice to investigate genetic interactions. Consistent with an interaction between polycystins and Wwtr1 in bone in vivo, Pkd1/Wwtr1 Oc-cKO mice exhibited greater reductions of BMD and periosteal MAR than either Wwtr1 Oc-cKO or Pkd1 Oc-cKO mice. Micro-CT 3D image analysis indicated that the reduction in bone mass was due to greater loss in both trabecular bone volume and cortical bone thickness in Pkd1/Wwtr1 Oc-cKO mice compared to either Pkd1 Oc-cKO or Wwtr1 Oc-cKO mice. Pkd1/Wwtr1 Oc-cKO mice also displayed additive reductions in mechanosensing and osteogenic gene expression profiles in bone compared to Pkd1 Oc-cKO or Wwtr1 Oc-cKO mice. Moreover, we found that Pkd1/Wwtr1 Oc-cKO mice exhibited impaired responses to tibia mechanical loading in vivo and attenuation of load-induced mechanosensing gene expression compared to control mice. Finally, control mice treated with a small molecule mechanomimetic, MS2 that activates the polycystin complex resulted in marked increases in femoral BMD and periosteal MAR compared to vehicle control. In contrast, Pkd1/Wwtr1 Oc-cKO mice were resistant to the anabolic effects of MS2. These findings suggest that PC1 and Wwtr1 form an anabolic mechanotransduction signaling complex that mediates mechanical loading responses and serves as a potential novel therapeutic target for treating osteoporosis.
60 APPLIED LIFE SCIENCES↗
Substance P preserves pancreatic β-cells in type 1 and type 2 diabetic mice
Highlights: • Substance P preserved β-cells in nonobese diabetic mice with type 1 diabetes. • Substance P preserved β-cells in db/db mice with type 2 diabetes. • Substance P inhibited the activation of pancreatic stellate cells. Preservation of pancreatic β-cells is required for the development of therapies for type 1 and type 2 diabetes (T1D and T2D, respectively). Our previous study demonstrated that substance P (SP) preserves β-cell populations in mice with streptozotocin-induced T1D. Here, we demonstrated that chronic systemic treatment with SP restored the mass of β-cells both in nonobese diabetic (NOD) mice with T1D or db/db mice with T2D. SP delayed the onset of T1D in NOD mice via immune modulation. SP inhibited immune infiltration into islets and the salivary glands of NOD mice. In db/db mice, SP treatment rescued glucose intolerance. Moreover, SP inhibited apoptosis, as well as the activation of pancreatic stellate cells in pancreatic islets of db/db mice. SP downregulated the number of α-smooth muscle actin (α-SMA) expressing cells in db/db pancreatic islets. Cleaved-caspase-3 expression was reduced in islets of SP-treated db/db mice compared to that in the control. Therefore, these results suggested that SP may preserve pancreatic β-cells through immune modulation and protection from the stimulated activation of pancreatic stellate cells and apoptosis in T1D and T2D, respectively.
60 APPLIED LIFE SCIENCES↗
Deficiency of unc-51 like kinase 1 (Ulk1) protects against mice traumatic brain injury (TBI) by suppression of p38 and JNK pathway
Highlights: • Ulk1-KO mice show reduced inflammation and apoptosis in hippocampus after TBI. • Ulk1-KO mice alleviate glial cells activation and autophagy in hippocampus of mice with TBI. • Ulk1 inhibition reduces inflammatory response, apoptosis and autophagy in LPS-stimulated astrocytes (AST) via p38/JNK pathway. Unc-51 like autophagy activating kinase 1 (Ulk1) is a serine/threonine kinase that plays a key role in regulating autophagy processes. We attempted to investigate the effects of Ulk1 on traumatic brain injury (TBI) progression by using wild type (WT) mice and Ulk1-knockout (KO) mice suffered with or not TBI. The results were verified using LPS-treated primary astrocyte (AST). Here, Ulk1 was over-expressed in hippocampus of WT mice after TBI, as well as in lipopolysaccharide (LPS)-stimulated AST. Ulk1-deletion improved cognitive ability and hippocampus histological changes in TBI mice. Nissl and neuronal nuclei (NeuN) staining indicated that Ulk1-deletion increased the number of surviving neurons in hippocampus of TBI mice. Ulk1-ablation alleviated neuroinflammation, as evidenced by the reduced expression of hippocampus pro-inflammatory cytokines in TBI mice. TBI-induced apoptosis was also ameliorated by Ulk1-ablation, as proved by the reduced number of TUNEL-staining cells, and cleaved Caspase-3 and poly (ADP-ribose) polymerase (PARP) expressions. Moreover, Ulk1-knockout suppressed TBI-stimulated activation of astrocytes and microglia cells. Additionally, hippocampus autophagy induced by TBI was attenuated by Ulk1-knockout. Further, TBI-activated p38/c-Jun N-terminal Kinase (JNK) pathway was repressed by Ulk1-deletion in hippocampus of mice. The findings above were confirmed in LPS-stimulated AST with or without Ulk1 siRNA transfection. Intriguingly, pre-treatment of p38 or JNK activator markedly abolished the anti-inflammation, anti-apoptosis and anti-autophagy effects of Ulk1-knockdown on LPS-incubated AST. In conclusion, our results demonstrated that Ulk1 might be a potential target for developing therapeutic strategy against TBI in future.
60 APPLIED LIFE SCIENCES↗
Lentivirus-mediated CTRP6 silencing ameliorates diet-induced obesity in mice
Highlights: • The knockdown of CTRP6 reduced both body weight and white adipose tissue of mice. • CTRP6 knockdown decreased the serum TG and improved the insulin sensitivity in mice. • The knockdown of CTRP6 promoted brown adipogenesis both in mice and in adipocytes. • CTRP6 knockdown activated p38MAPK but inhibited Hh signaling pathway in adipocytes. The C1q/TNF-related protein 6 (CTRP6) is an adipokine involved in diverse biological processes. Formerly, we identified that CTRP6 regulates adipocyte differentiation, fatty acid oxidation and triglyceride accumulation in vitro. However, the effects of CTRP6 on adiposity in vivo have not yet been defined. This study aimed to confirm the involvement of CTRP6 in adipose accumulation and brown adipogenesis by intraperitoneal injection of the CTRP6-shRNA lentivirus into mice (CL mice). CL mice were significantly thinner than the control mice after feeding with a high fat diet (HFD), independent of food intake quantity. These HFD-fed CL mice displayed lower white and brown adipocyte sizes, and serum leptin levels, but an increase in serum adiponectin and insulin sensitivity relative to control mice. Additionally, the brown fat markers, such as UCP1, PRDM16, PGC1α and Cidea were found to be upregulated in the white and brown adipose tissue of the CL mice. These markers were also upregulated in a primary culture of mouse white and brown adipocytes treated with the CTRP6-shRNA lentivirus. Mechanistically, the knockdown of CTRP6 increased p38MAPK phosphorylation, but decreased expression of proteins involved in the Hedgehog signaling pathway (Sufu, Gli2 and Gli3). CTRP6 knockdown also upregulated expression of mitochondrial metabolic factors NRF-1, TFAM, CPT1 and Cyt C. Data from the current study show that CTRP6 knockdown protects against diet-induced obesity and promotes brown adipogenesis by the p38MAPK/Hh signaling pathway in conjunction with the upregulation of brown fat markers and mitochondrial metabolic factors.
60 APPLIED LIFE SCIENCES↗
NLRP1 deficiency attenuates diabetic retinopathy (DR) in mice through suppressing inflammation response
Highlights: • NLRP1 expression was increased in retina of diabetic mice. • NLRP1 deletion attenuates STZ-induced diabetic retinopathy. • Knockout of NLRP1 reduces inflammation and alleviates diabetic retinopathy in STZ mice. • Suppression of NLRP1 restrains inflammation in high fructose-incubated retinal ganglion cells. Diabetic retinopathy (DR) is the common cause of diabetic vascular complications. The NOD-like receptor (NLR) family, pyrin domain containing 1 (NLRP1), also known as NALP1, inflammasome is the first member of the NLR family to be discovered, playing an important role in inflammatory response. However, its effect on DR development has not been reported. In the study, the wild type (WT) and NLRP1{sup −/−} mice were injected with streptozotocin (STZ) to induce DR. The results indicated that NLRP1{sup −/−} significantly increased bodyweight reduction and decreased blood glucose levels induced by STZ. WT/DR mice exhibited higher levels of NLRP1 in retinas. NLRP1{sup −/−} ameliorated retinal abnormalities in DR mice using H&E staining. In addition, attenuated avascular areas and neovascular tufts were also observed in NLRP1{sup −/−}/DR mice. The levels of pro-inflammatory cytokines in serum and retinas were highly induced in WT/DR mice, whereas being markedly reduced by NLRP1{sup −/−}. In addition, vascular endothelial growth factor (VEGF) and Iba1 expressions induced by STZ in serum or retinas were significantly down-regulated in NLRP1{sup −/−}/DR mice. Consistently, NLRP1{sup −/−} attenuated ASC and Caspase-1 expressions in retinas of DR mice. Compared to WT/DR group, NLRP1{sup −/−} markedly decreased retina p-nuclear factor-κB (NF-κB), interleukin-1β (IL-1β) and IL-18 levels. And similar results were confirmed in vitro that suppressing NLRP1/ASC inflammasome ameliorated inflammatory response in fructose-treated retinal ganglion cells. The results above indicated that the modulation of NLRP1 inflammasome might be a promising strategy for DR therapy.
60 APPLIED LIFE SCIENCES↗
Augmented neutrophil extracellular traps formation promotes atherosclerosis development in socially defeated apoE −/− mice
Highlights: • Repeated social defeat exaggerates plaque size in apoE{sup −/−} mice. • Proportion of circulating neutrophils is significantly increased in defeated mice. • In vivo NETs formation is exaggerated within the atherosclerotic lesions in defeated mice. • DNase I treatment abrogates pro-atherogenic effect of RSD exposure. • In vitro NETs formation is exaggerated in BM PMN-MDSC from defeated mice. Depression is an independent risk factor of cardiovascular disease (CVD); however, the causal association remains undefined. We exposed mice to repeated social defeat (RSD) to precipitate depressive-like behaviors, and investigated the effects on atherosclerosis. Eight-week-old male apoE{sup −/−} mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days and fed a high-cholesterol diet (HCD) for 6 weeks. The social interaction ratio and immobility time showed dramatic social avoidance before and after HCD feeding. Defeated mice showed higher increase in atherosclerotic lesion areas in the aortic root and entire aorta than control mice. Mean blood pressure and lipid profile were equivalent in both groups. While Ly-6G- and Mac3-positive areas in the aortic root were comparable between the groups, citrullinated histone H3 (Cit-H3)- and myeloperoxidase (MPO)-positive areas, markers of neutrophil extracellular traps (NETs), were significantly increased in the defeated mice. Treatment with DNase I completely diminished the exaggerated atherosclerosis. The proportion of peripheral blood polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC), but not of inflammatory monocytes, was markedly increased. Moreover, in vitro NETs formation from bone marrow (BM) PMN-MDSC was markedly augmented, accompanied by higher expression of Nox2 gene and reactive oxygen species. Our findings demonstrate that exposure to RSD promotes atherosclerosis by augmenting NETs formation within the plaque. This provides new insight into the underlying mechanism of depression-related CVD.
60 APPLIED LIFE SCIENCES↗
IL-21 alleviates allergic asthma in DOCK8-knockout mice
Highlights: • DOCK8-KO mice induced by 5 μg of OVA showed markedly allergic asthma. • Nasal administration of rmIL-21 alleviates allergic asthma in KO-OVA mice. • IL-21 administration reduced IgE production in DOCK8-KO mice. Patients with DOCK8 deficiency are at increased susceptibility to develop allergic diseases such as food allergy and asthma. Here, we aimed to analyze the pathogenesis of asthma in DOCK8-deficient patients. In our mouse model, DOCK8-knockout (KO) mice sensitized with low-dose OVA were challenged with 1.5% OVA to induce allergic asthma. As compared to that in WT mice, remarkable airway hyperresponsiveness was observed in KO mice. Increased inflammatory cells and eosinophils infiltrated in airway lumen in KO mice especially around bronchi. KO mice showed higher levels of serum IgE and OVA-specific IgE and significantly elevated IgE-producing B cells in blood and in spleen. Surprisingly, nasal administration with rmIL-21 significantly reduced the airway hyperresponsiveness, inflammatory infiltration, as well as the serum IgE and IgE-producing B cells. DOCK8-knockout mice are susceptible to low-dose OVA induced allergic airway inflammation and airway hyperresponsiveness. Supplementary nasal administration of rmIL-21 alleviates allergic asthma in this mouse model.
60 APPLIED LIFE SCIENCES↗