Elucidating the Mechanism of Metabolism of Cannabichromene by Human Cytochrome P450s.

Cannabichromene (CBC) is a nonpsychoactive phytocannabinoid well-known for its wide-ranging health advantages. However, there is limited knowledge regarding its human metabolism following CBC consumption. This research aimed to explore the metabolic pathways of CBC by various human liver cytochrome P450 (CYP) enzymes and support the outcomes using in vivo data from mice. The results unveiled two principal CBC metabolites generated by CYPs: 8'-hydroxy-CBC and 6',7'-epoxy-CBC, along with a minor quantity of 1″-hydroxy-CBC. Notably, among the examined CYPs, CYP2C9 demonstrated the highest efficiency in producing these metabolites. Moreover, through a molecular dynamics simulation spanning 1 µs, it was observed that CBC attains stability at the active site of CYP2J2 by forming hydrogen bonds with I487 and N379, facilitated by water molecules, which specifically promotes the hydroxy metabolite's formation. Additionally, the presence of cytochrome P450 reductase (CPR) amplified CBC's binding affinity to CYPs, particularly with CYP2C8 and CYP3A4. Furthermore, the metabolites derived from CBC reduced cytokine levels, such as IL6 and NO, by approximately 50% in microglia cells. This investigation offers valuable insights into the biotransformation of CBC, underscoring the physiological importance and the potential significance of these metabolites.

Multi-Omic blood analysis reveals differences in innate inflammatory sensitivity between species.

Vertebrates differ greatly in responses to pro-inflammatory agonists such as bacterial lipopolysaccharide (LPS), complicating use of animal models to study human sepsis or inflammatory disorders. We compared transcriptomes of resting and LPS-exposed blood from six LPS-sensitive species (rabbit, pig, sheep, cow, chimpanzee, human) and four LPS-resilient species (mice, rats, baboon, rhesus), as well as plasma proteomes and lipidomes. Unexpectedly, at baseline, sensitive species already had enhanced expression of LPS-responsive genes relative to resilient species. After LPS stimulation, maximally different genes in resilient species included genes that detoxify LPS, diminish bacterial growth, discriminate sepsis from SIRS, and play roles in autophagy and apoptosis. The findings reveal the molecular landscape of species differences in inflammation, and may inform better selection of species for pre-clinical models.

Impact of campus living conditions on Chinese medical school students' mental health during the COVID-19 campus lockdown: the chain mediating role of cognitive reappraisal and expression suppression.

Objective: To investigate the effect of changes in campus living conditions related to the Corona Virus Disease 2019 (COVID-19) pandemic on medical school students' mental health status, to explore the mediating role of emotion regulation strategies, and to provide effective suggestions for promoting medical school students' mental health. Methods: A self-report questionnaire, an emotion regulation questionnaire (ERQ), and psychological questionnaires for emergent events of public health (PQEEPH) were used to interview 998 medical school students who experienced campus lockdowns during the COVID-19 pandemic. Results: The mean total PQEEPH score was 3.66 ± 3.06. The degrees of inconvenience in daily life and change in routine and expression suppression as an emotion regulation strategy were significantly positively correlated with all PQEEPH dimensions. Cognitive reappraisal was significantly negatively associated with depression, neurosis, obsessive-compulsive anxiety, and hypochondriasis (ps < 0.05). Cognitive reappraisal and expression suppression demonstrated a chain mediating role between the degree of inconvenience in life and mental health and between the degree of change in routine and mental health (F = 32.883, 41.051, ps < 0.05). Conclusion: Campus lockdown management significantly impacts medical school students' mental health. Extensive use of cognitive reappraisal and expression suppression can reduce students' adverse psychological reactions during campus lockdowns to an extent.

Thermal Annealing Effect on Surface-Enhanced Raman Scattering of Gold Films Deposited on Liquid Substrates.

We prepare metal films with various thicknesses on liquid substrates by thermal evaporation and investigate the annealing effect on these films. Gold films deposited on a silicone oil surface consist of a large number of branched aggregates, which contains plenty of gold nanoparticles. This characteristic morphology is mainly attributed to the isotropic and free-sustained liquid substrate. Thermal annealing results in the reintegration of nanoparticles; thus, the surface morphology and microstructure of gold films change significantly. The dependence of annealing conditions on the surface-enhanced Raman scattering performance of gold films is studied, in which gold films show favorable Raman activity when annealed at certain annealing temperature and the experimental results are verified by simulation analysis. The study on the optimal annealing temperature of surface-enhanced Raman scattering substrate will pave the way for the potential application of films deposited on liquid surfaces in microfluidics and enhanced Raman detection.

Metabolites of Cannabigerol Generated by Human Cytochrome P450s Are Bioactive.

The phytocannabinoid cannabigerol (CBG) is the central biosynthetic precursor to many cannabinoids, including Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Though the use of CBG has recently witnessed a widespread surge because of its beneficial health effects and lack of psychoactivity, its metabolism by human cytochrome P450s is largely unknown. Herein, we describe comprehensive in vitro and in vivo cytochrome P450 (CYP)-mediated metabolic studies of CBG, ranging from liquid chromatography tandem mass spectrometry-based primary metabolic site determination, synthetic validation, and kinetic behavior using targeted mass spectrometry. These investigations revealed that cyclo-CBG, a recently isolated phytocannabinoid, is the major metabolite that is rapidly formed by selected human cytochrome P450s (CYP2J2, CYP3A4, CYP2D6, CYP2C8, and CYP2C9). Additionally, in vivo studies with mice administered with CBG supported these studies, where cyclo-CBG is the major metabolite as well. Spectroscopic binding studies along with docking and modeling of the CBG molecule near the heme in the active site of P450s confirmed these observations, pointing at the preferred site selectivity of CBG metabolism at the prenyl chain over other positions. Importantly, we found out that CBG and its oxidized CBG metabolites reduced inflammation in BV2 microglial cells stimulated with LPS. Overall, combining enzymological studies, mass spectrometry, and chemical synthesis, we showcase that CBG is rapidly metabolized by human P450s to form oxidized metabolites that are bioactive.

Chronic E-Cigarette Use Impairs Endothelial Function on the Physiological and Cellular Levels.

BACKGROUND: The harmful vascular effects of smoking are well established, but the effects of chronic use of electronic cigarettes (e-cigarettes) on endothelial function are less understood. We hypothesized that e-cigarette use causes changes in blood milieu that impair endothelial function. METHODS: Endothelial function was measured in chronic e-cigarette users, chronic cigarette smokers, and nonusers. We measured effects of participants' sera, or e-cigarette aerosol condensate, on NO and H2O2 release and cell permeability in cultured endothelial cells (ECs). RESULTS: E-cigarette users and smokers had lower flow-mediated dilation (FMD) than nonusers. Sera from e-cigarette users and smokers reduced VEGF (vascular endothelial growth factor)-induced NO secretion by ECs relative to nonuser sera, without significant reduction in endothelial NO synthase mRNA or protein levels. E-cigarette user sera caused increased endothelial release of H2O2, and more permeability than nonuser sera. E-cigarette users and smokers exhibited changes in circulating biomarkers of inflammation, thrombosis, and cell adhesion relative to nonusers, but with distinct profiles. E-cigarette user sera had higher concentrations of the receptor for advanced glycation end products (RAGE) ligands S100A8 and HMGB1 (high mobility group box 1) than smoker and nonuser sera, and receptor for advanced glycation end product inhibition reduced permeability induced by e-cigarette user sera but did not affect NO production. CONCLUSIONS: Chronic vaping and smoking both impair FMD and cause changes in the blood that inhibit endothelial NO release. Vaping, but not smoking, causes changes in the blood that increase microvascular endothelial permeability and may have a vaping-specific effect on intracellular oxidative state. Our results suggest a role for RAGE in e-cigarette-induced changes in endothelial function.

N-Oleoyl dopamine induces IL-10 via central nervous system TRPV1 and improves endotoxemia and sepsis outcomes.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) participates in thermosensation and inflammatory pain, but its immunomodulatory mechanisms remain enigmatic. N-Oleoyl dopamine (OLDA), an endovanilloid and endocannabinoid, is a TRPV1 agonist that is produced in the central nervous system and the peripheral nervous system. We studied the anti-inflammatory effects and TRPV1-dependent mechanisms of OLDA in models of inflammation and sepsis. METHODS: Mice were challenged intratracheally or intravenously with LPS, or intratracheally with S. aureus to induce pneumonia and sepsis, and then were treated intravenously with OLDA. Endpoints included plasma cytokines, leukocyte activation marker expression, mouse sepsis scores, lung histopathology, and bacterial counts. The role of TRPV1 in the effects of OLDA was determined using Trpv1-/- mice, and mice with TRPV1 knockdown pan-neuronally, in peripheral nervous system neurons, or in myeloid cells. Circulating monocytes/macrophages were depleted using clodronate to determine their role in the anti-inflammatory effects of OLDA in endotoxemic mice. Levels of exogenous OLDA, and of endovanilloids and endocannabinoids, at baseline and in endotoxemic mice, were determined by LC-MS/MS. RESULTS: OLDA administration caused an early anti-inflammatory response in endotoxemic and septic mice with high serum levels of IL-10 and decreased levels of pro-inflammatory cytokines. OLDA also reduced lung injury and improved mouse sepsis scores. Blood and lung bacterial counts were comparable between OLDA- and carrier-treated mice with S. aureus pneumonia. OLDA's effects were reversed in mice with pan-neuronal TRPV1 knockdown, but not with TRPV1 knockdown in peripheral nervous system neurons or myeloid cells. Depletion of monocytes/macrophages reversed the IL-10 upregulation by OLDA in endotoxemic mice. Brain and blood levels of endovanilloids and endocannabinoids were increased in endotoxemic mice. CONCLUSIONS: OLDA has strong anti-inflammatory actions in mice with endotoxemia or S. aureus pneumonia. Prior studies focused on the role of peripheral nervous system TRPV1 in modulating inflammation and pneumonia. Our results suggest that TRPV1-expressing central nervous system neurons also regulate inflammatory responses to endotoxemia and infection. Our study reveals a neuro-immune reflex that during acute inflammation is engaged proximally by OLDA acting on neuronal TRPV1, and through a multicellular network that requires circulating monocytes/macrophages, leads to the systemic production of IL-10.

ERK1/2 Has Divergent Roles in LPS-Induced Microvascular Endothelial Cell Cytokine Production and Permeability.

ABSTRACT: Endothelial cells play a major role in inflammatory responses to infection and sterile injury. Endothelial cells express Toll-like receptor 4 (TLR4) and are activated by LPS to express inflammatory cytokines/chemokines, and to undergo functional changes, including increased permeability. The extracellular signal-regulated kinase 1/2 (ERK1/2) mediates pro-inflammatory signaling in monocytes and macrophages, but the role of ERK1/2 in LPS-induced activation of microvascular endothelial cells has not been defined. We therefore studied the role of ERK1/2 in LPS-induced inflammatory activation and permeability of primary human lung microvascular endothelial cells (HMVEC). Inhibition of ERK1/2 augmented LPS-induced IL-6 and vascular cell adhesion protein (VCAM-1) production by HMVEC. ERK1/2 siRNA knockdown also augmented IL-6 production by LPS-treated HMVEC. Conversely, ERK1/2 inhibition abrogated permeability and restored cell-cell junctions of LPS-treated HMVEC. Consistent with the previously described pro-inflammatory role for ERK1/2 in leukocytes, inhibition of ERK1/2 reduced LPS-induced cytokine/chemokine production by primary human monocytes. Our study identifies a complex role for ERK1/2 in TLR4-activation of HMVEC, independent of myeloid differentiation primary response gene (MyD88) and TIR domain-containing adaptor inducing IFN-ß (TRIF) signaling pathways. The activation of ERK1/2 limits LPS-induced IL-6 production by HMVEC, while at the same time promoting HMVEC permeability. Conversely, ERK1/2 activation promotes IL-6 production by human monocytes. Our results suggest that ERK1/2 may play an important role in the nuanced regulation of endothelial cell inflammation and vascular permeability in sepsis and injury.

Activation of CB1R Promotes Lipopolysaccharide-Induced IL-10 Secretion by Monocytic Myeloid-Derived Suppressive Cells and Reduces Acute Inflammation and Organ Injury.

Cannabis sativa and its principal components, Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol, are increasingly being used to treat a variety of medical problems, including inflammatory conditions. Although studies suggest that the endocannabinoid system has immunomodulatory properties, there remains a paucity of information on the effects of cannabinoids on immunity and on outcomes of infection and injury. We investigated the effects and mechanism(s) of action of cannabinoid receptor agonists, including Δ9-THC, on inflammation and organ injury in endotoxemic mice. Administration of Δ9-THC caused a dramatic early upregulation of plasma IL-10 levels, reduced plasma IL-6 and CCL-2 levels, led to better clinical status, and attenuated organ injury in endotoxemic mice. The anti-inflammatory effects of Δ9-THC in endotoxemic mice were reversed by a cannabinoid receptor type 1 (CB1R) inverse agonist (SR141716), and by clodronate-induced myeloid-cell depletion, but not by genetic invalidation or blockade of other putative Δ9-THC receptors, including cannabinoid receptor type 2, TRPV1, GPR18, GPR55, and GPR119. Although Δ9-THC administration reduced the activation of several spleen immune cell subsets, the anti-inflammatory effects of Δ9-THC were preserved in splenectomized endotoxemic mice. Finally, using IL-10-GFP reporter mice, we showed that blood monocytic myeloid-derived suppressive cells mediate the Δ9-THC-induced early rise in circulating IL-10. These results indicate that Δ9-THC potently induces IL-10, while reducing proinflammatory cytokines, chemokines, and related organ injury in endotoxemic mice via the activation of CB1R. These data have implications for acute and chronic conditions that are driven by dysregulated inflammation, such as sepsis, and raise the possibility that CB1R-signaling may constitute a novel target for inflammatory disorders.

Deposition of Ag Films on Liquid Substrates via Thermal Evaporation for Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS) substrates were prepared by depositing Ag atoms on liquid surfaces via thermal evaporation at room temperature. These free-sustained substrates result in the formation of uniform Ag films, in which ramified Ag aggregates consist of substantial Ag nanoclusters with narrow gaps of several nanometers in between. SERS spectra of rhodamine 6G were investigated for this substrate to evaluate the SERS performance of this characteristic film morphology, and the results indicated that the SERS intensity from the closely-packed Ag nanostructures and small intervals were significantly enhanced. The dependence of SERS enhancement on the film thickness, nanoparticle size, and gap width was studied. An analytical model was proposed to simulate the electric field distribution during SERS detection, and the results validated the experimental observations.

Surface enhanced Raman scattering substrates prepared by thermal evaporation on liquid surfaces.

We present an effective surface-enhancement Raman scattering (SERS) substrate enabled by depositing metallic film on a liquid surface at room temperature. Thermal evaporation is used to deposit Au atoms on silicone oil surface and then form quasi-continuous films. Due to the isotropic characteristics of the liquid surface, this film consists of substantial nanoparticles with uniform diameter, which is different from films fabricated on solid substrates and can be served as an applicable substrate for SERS detection. With the assistance of this substrate, SERS signals of rhodamine 6G were significantly enhanced, the dependence between SERS spectra and film thickness was investigated. Analytical simulation results confirm the experimental observations and the superiorities of our proposed method for preparation of SERS substrate. This work provides a potential application of metallic film deposition on free-sustained surface and holds promise as an efficient sensor in rapid trace detection of small molecule analytes.

N-Arachidonoyl Dopamine Modulates Acute Systemic Inflammation via Nonhematopoietic TRPV1.

N-Arachidonoyl dopamine (NADA) is an endogenous lipid that potently activates the transient receptor potential vanilloid 1 (TRPV1), which mediates pain and thermosensation. NADA is also an agonist of cannabinoid receptors 1 and 2. We have reported that NADA reduces the activation of cultured human endothelial cells by LPS and TNF-α. Thus far, in vivo studies using NADA have focused on its neurologic and behavioral roles. In this article, we show that NADA potently decreases in vivo systemic inflammatory responses and levels of the coagulation intermediary plasminogen activator inhibitor 1 in three mouse models of inflammation: LPS, bacterial lipopeptide, and polymicrobial intra-abdominal sepsis. We also found that the administration of NADA increases survival in endotoxemic mice. Additionally, NADA reduces blood levels of the neuropeptide calcitonin gene-related peptide but increases the neuropeptide substance P in LPS-treated mice. We demonstrate that the anti-inflammatory effects of NADA are mediated by TRPV1 expressed by nonhematopoietic cells and provide data suggesting that neuronal TRPV1 may mediate NADA's anti-inflammatory effects. These results indicate that NADA has novel TRPV1-dependent anti-inflammatory properties and suggest that the endovanilloid system might be targeted therapeutically in acute inflammation.

One-pot synthesis of Mn-doped TiO2 grown on graphene and the mechanism for removal of Cr(VI) and Cr(III).

Mn-doped TiO2 grown on reduced graphene oxide(rGO) was synthesized by one-pot hydrothermal method and the photocatalytic removal of Cr by the material was investigated under sunlight. The materials were characterized by a combination of scanning electron microscopy, transmission electron microscopy, X-ray diffraction, thermogravimetric analysis, Brunauer-Emmett-Teller method, UV-vis diffuse reflectance spectra, photoluminescence spectra, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy. Cr(total) removal efficiency of the material is 97.32% in 30min and 99.02% in 60min under sunlight irradiation, as the initial concentration of Cr(VI) is 20mg/L. The high photocatalytic activity under visible light is considered mainly due to the Mn-doping, and rGO plays an important role in the synergetic effect of adsorption and photocatalysis to sustain the high efficient removal of Cr(VI) and Cr(III). Cr(VI) adsorbed on the surface of rGO is reduced to Cr(III) by photo electrons which are transported through rGO, and the reaction product Cr(III) continues to be adsorbed. The process contributes to the release of abundant photocatalytic sites of Mn-TiO2 and improves photocatalytic efficiency. The excellent adsorption and photocatalytic effect with the explanation of the synergetic mechanism are very useful not only for fundamental research but also for the potential practical applications.

TGF-ß/SMAD Pathway and Its Regulation in Hepatic Fibrosis.

Transforming growth factor-beta1 (TGF-ß1), a key member in the TGF-ß superfamily, plays a critical role in the development of hepatic fibrosis. Its expression is consistently elevated in affected organs, which correlates with increased extracellular matrix deposition. SMAD proteins have been studied extensively as pivotal intracellular effectors of TGF-ß1, acting as transcription factors. In the context of hepatic fibrosis, SMAD3 and SMAD4 are pro-fibrotic, whereas SMAD2 and SMAD7 are protective. Deletion of SMAD3 inhibits type I collagen expression and blocks epithelial-myofibroblast transition. In contrast, disruption of SMAD2 upregulates type I collagen expression. SMAD4 plays an essential role in fibrosis disease by enhancing SMAD3 responsive promoter activity, whereas SMAD7 negatively mediates SMAD3-induced fibrogenesis. Accumulating evidence suggests that divergent miRNAs participate in the liver fibrotic process, which partially regulates members of the TGF-ß/SMAD signaling pathway. In this review, we focus on the TGF-ß/SMAD and other relative signaling pathways, and discussed the role and molecular mechanisms of TGF-ß/SMAD in the pathogenesis of hepatic fibrosis. Moreover, we address the possibility of novel therapeutic approaches to hepatic fibrosis by targeting to TGF-ß/SMAD signaling.

Smad2 increases the apoptosis of activated human hepatic stellate cells induced by TRAIL.

The activation of hepatic stellate cells (HSCs) plays a critical role in the development of liver fibrosis. The induction of apoptosis in activated HSCs during the recovery phase of hepatic fibrosis represents a potential anti-fibrotic therapy. We have previously shown that Smad2 protects against hepatic fibrogenesis; however, the role of Smad2 in the regulation of activated HSC apoptosis remains unknown. We hypothesized that Smad2 regulates the apoptosis of activated HSCs, leading to the resolution of liver fibrosis. To test this hypothesis, the livers of rats were harvested at 0 and 4 weeks after hepatic fibrosis was established by CCl4 injection. Furthermore, TGF-ß1-activated HSCs were treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following the silencing or overexpression of Smad2. Both the phosphorylation of Smad2 and TRAIL were detected in fibrotic liver tissues. The results of TUNEL and α-SMA double-staining showed an increase in the apoptosis of activated HSCs during the spontaneous recovery phase. The knockdown of Smad2 reduced TRAIL-induced apoptosis in TGF-ß1-activated human LX-2 cells and resulted in an increased expression of α-SMA and collagen I (Col. I). In contrast, the overexpression of Smad2 increased TRAIL-induced HSC apoptosis and reduced the expression of α-SMA and Col. I. The mechanisms underlying these findings were associated with the Smad2-mediated down-regulation of X-linked inhibitor of apoptosis protein (XIAP), resulting in enhanced caspase-3 activity and apoptosis. In conclusion, Smad2 enhances TRAIL-induced apoptosis in activated HSCs, which facilitates the resolution of hepatic fibrosis.

Silent information regulator 1 (SIRT1) ameliorates liver fibrosis via promoting activated stellate cell apoptosis and reversion.

SIRT1 (silent information regulator 1), a conserved NAD+-dependent histone deacetylase, is closely related with various biological processes. Moreover, the important role of SIRT1 in alcoholic liver disease, nonalcoholic fatty liver and HCC had been widely reported. Recently, a novel role of SIRT1 was uncovered in organ fibrosis diseases. Here, we investigated the inhibitory effect of SIRT1 in liver fibrogenesis. SIRT1 protein was dramatically decreased in CCl4-treated mice livers. Stimulation of LX-2 cells with TGF-ß1 also resulted in a significant suppression of SIRT1 protein. Nevertheless, TGF-ß1-induced LX-2 cell activation was inhibited by SIRT1 plasmid, and this was accompanied by up-regulation of cell apoptosis-related proteins. Overexpression of SIRT1 also attenuated TGF-ß1-induced expression of myofibroblast markers α-SMA and COL1a. However, the important characteristic of the recovery of liver fibrosis is not only the apoptosis of activated stellate cells but also the reversal of the myofibroblast-like phenotype to a quiescent-like phenotype. Restoration of SIRT1 protein was observed in the in vivo spontaneously liver fibrosis reversion model and in vitro MDI (isobutylmethylxanthine, dexamethasone, and insulin)-induced reversed stellate cells, and forced expression of SIRT1 also promoted the reversal of activated stellate cells. Furthermore, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was increased in liver fibrosis. RNAi-mediated suppression of MALAT1 resulted in a decrease of myofibroblast markers and restoration of SIRT1 protein. These observations suggested that SIRT1 contributed to apoptosis and reversion of activated LX-2 cells and SIRT1 might be regulated by MALAT1 in liver fibrosis. Therefore, SIRT1 could be considered as a valuable therapeutic target for translational studies of liver fibrosis.

Extracellular signal-regulated kinase 5 promotes acute cellular and systemic inflammation.

Inflammatory critical illness is a syndrome that is characterized by acute inflammation and organ injury, and it is triggered by infections and noninfectious tissue injury, both of which activate innate immune receptors and pathways. Although reports suggest an anti-inflammatory role for the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 5 (ERK5), we previously found that ERK5 mediates proinflammatory responses in primary human cells in response to stimulation of Toll-like receptor 2 (TLR2). We inhibited the kinase activities and reduced the abundances of ERK5 and MEK5, a MAPK kinase directly upstream of ERK5, in primary human vascular endothelial cells and monocytes, and found that ERK5 promoted inflammation induced by a broad range of microbial TLR agonists and by the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). Furthermore, we found that inhibitors of MEK5 or ERK5 reduced the plasma concentrations of proinflammatory cytokines in mice challenged with TLR ligands or heat-killed Staphylococcus aureus, as well as in mice that underwent sterile lung ischemia-reperfusion injury. Finally, we found that inhibition of ERK5 protected endotoxemic mice from death. Together, our studies support a proinflammatory role for ERK5 in primary human endothelial cells and monocytes, and suggest that ERK5 is a potential therapeutic target in diverse disorders that cause inflammatory critical illness.

Study of Bone Marrow and Embryonic Stem Cell-Derived Human Mesenchymal Stem Cells for Treatment of Escherichia coli Endotoxin-Induced Acute Lung Injury in Mice.

UNLABELLED: : Mesenchymal stem cells (MSCs) can be derived from multiple tissue sources. However, the optimal source of MSCs for cell-based therapy for acute lung injury (ALI) is unclear. In the present experiments, we studied bone marrow (BM)-derived and embryonic stem cell-derived human MSC (ES-MSCs) as a therapeutic agent in Escherichia coli endotoxin-induced ALI in mice. We hypothesized that ES-MSCs would be more potent than BM-MSCs owing to its more primitive source of origin. ALI was induced by the intratracheal instillation of endotoxin at 4 mg/kg into 10-12-week-old C57BL/6 mice with or without BM-MSCs, ES-MSCs, or normal human lung fibroblasts as a cellular control. Compared with the endotoxin-injured mice at 48 hours, the administration of ES-MSCs provided results similar to those of BM-MSCs, significantly reducing the influx of white blood cells and neutrophils and decreasing the secretion of the inflammatory cytokines, macrophage inflammatory protein-2 and tumor necrosis factor-α, in the injured alveolus. BM-MSCs also reduced extravascular lung water, a measure of pulmonary edema, by 60% and the total protein levels, a measure of lung permeability, by 66%. However, surprisingly, ES-MSCs did not have these protective effects, which was partially explained by the increased secretion of matrix metallopeptidase 9 by ES-MSCs, an enzyme known to increase lung protein permeability. In conclusion, both BM-MSCs and ES-MSCs markedly decreased endotoxin-induced inflammation. However, ES-MSCs did not show any beneficial effect on reducing pulmonary edema and lung protein permeability compared with BM-MSCs, suggesting that not all MSCs behave in a similar fashion. Our results highlight the need perhaps for a disease-specific potency assay for MSCs. SIGNIFICANCE: To determine the optimal source of mesenchymal stem cells (MSCs) for cell-based therapy for acute lung injury, bone marrow (BM)- and embryonic stem cell-derived human MSC (ES-MSCs) were compared as therapeutic agents for Escherichia coli endotoxin-induced lung injury in mice. ES-MSCs behaved similarly to BM-MSCs by markedly decreasing the inflammatory response induced by endotoxin. However, unlike BM-MSCs, ES-MSCs provided no protective effects against increasing lung water and protein permeability, in part because of an increase in expression of matrix metallopeptidase 9 by ES-MSCs. In patients with acute respiratory distress syndrome, impaired alveolar fluid clearance (i.e., no resolution of pulmonary edema fluid) has been associated with higher mortality rates. Although ES-MSCs might ultimately be found to have properties superior to those of BM-MSCs, such as for immunomodulation, these results highlight the need for a disease-specific potency assay for stem cell-based therapy.

Smad2 protects against TGF-ß1/Smad3-mediated collagen synthesis in human hepatic stellate cells during hepatic fibrosis.

With structural similarity but functional diversity, Smad2 and Smad3 interact with each other to mediate transforming growth factor-ß (TGF-ß)-triggered signaling transduction. However, in the hepatic fibrosis, the detailed roles of R-Smads, and interaction between Smad2 and Smad3 are still undefined. In this setting, we established a rat model of CCl4-induced hepatic fibrosis in vivo and TGF-ß1-treated hepatic stellate cell model in vitro to detect whether Smad2 and Smad3 play distinct roles in mediating liver fibrogenesis. Results indicated that both phosphorylation of Smad2 and Smad3 were detected in the hepatic stellate cells of liver fibrotic tissues and cells. Furthermore, In vitro data demonstrated that knockdown of Smad2 in human hepatic stellate cells increased expression of collagen I (Col.I), tissue inhibitor of metalloproteinase-1 (TIMP-1) whereas decreasing expression of the matrix metalloproteinases-2(MMP-2) in presence of TGF-ß1 compared with control group. In contrast, knockdown of Smad3 significantly reduced TGF-ß1-induced Col.I production. These findings were further evident by the results that overexpression of Smad2 attenuated the expression of Col.I and TIMP-1, but enhanced MMP-2 whereas overexpression of Smad3 showed the opposite effect. Furthermore, Smad2 suppressed the phosphorylation and nuclear translocation of Smad3, which may protect against Smad3-mediated fibrotic response. Collectively, Smad2 may be a potential therapeutic target for the treatment of hepatic fibrosis.

Inhibitory effects of long noncoding RNA MEG3 on hepatic stellate cells activation and liver fibrogenesis.

Long noncoding RNAs (lncRNAs) are being increasingly recognized as major players in governing fundamental biological processes through diverse mechanisms. Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes a lncRNA correlated with several human cancers. Recently, the methylation-dependent downregulation of MEG3 has been described in liver cancers. However, its biological functional role in liver fibrosis remains unknown. In our study, MEG3 levels were remarkably decreased in CCl4-induced mouse liver fibrosis models and human fibrotic livers as demonstrated by real-time quantitative PCR. Moreover, the expression of MEG3 was downregulated in human hepatic stellate cell lines LX-2 cells in response to transforming growth factor-ß1 (TGF-ß1) stimulation in dose and time-dependent manner. Enforced expression of MEG3 in LX-2 cells inhibited TGF-ß1-induced cell proliferation, while promoting cell apoptosis. In addition, hypermethylation of MEG3 promoter was identified by methylation-specific PCR and MEG3 expression was robustly increased by the inhibition of methylation with either 5-aza-2-deoxycytidine (5-azadC), or siRNA to DNA methyltransferase 1 (DNMT1) in TGF-ß1-induced LX-2 cells. More importantly, overexpression of MEG3 could activate p53 and mediate cytochrome c release, subsequently leading to caspase-3-dependent apoptosis in TGF-ß1-treated LX-2 cells. These findings suggested that MEG3 may play an important role in stellate cell activation and liver fibrosis progression and act as a novel potential therapeutic target for liver fibrosis.