Protective Role of C3aR (C3a Anaphylatoxin Receptor) Against Atherosclerosis in Atherosclerosis-Prone Mice.

OBJECTIVE: Emerging evidence suggests that C3aR (C3a anaphylatoxin receptor) signaling has protective roles in various inflammatory-related diseases. However, its role in atherosclerosis has been unknown. The purpose of the study was to investigate the possible protective role of C3aR in aortic atherosclerosis and explore molecular and cellular mechanisms involved in the protection. Approach and Results: C3ar-/-/Apoe-/- mice were generated by cross-breeding of atherosclerosis-prone Apoe-/- mice and C3ar-/- mice. C3ar-/-/Apoe-/- mice and Apoe-/- mice (as a control) underwent high-fat diet for 16 weeks were assessed for (1) atherosclerotic plaque burden, (2) aortic tissue inflammation, (3) recruitment of CD11b+ leukocytes into atherosclerotic lesions, and (4) systemic inflammatory responses. Compared with Apoe-/- mice, C3ar-/-/Apoe-/- mice developed more severe atherosclerosis. In addition, C3ar-/-/Apoe-/- mice have increased local production of proinflammatory mediators (eg, CCL2 [chemokine (C-C motif) ligand 2], TNF [tumor necrosis factor]-α) and infiltration of monocyte/macrophage in aortic tissue, and their lesional macrophages displayed an M1-like phenotype. Local pathological changes were associated with enhanced systemic inflammatory responses (ie, elevated plasma levels of CCL2 and TNF-α, increased circulating inflammatory cells). In vitro analyses using peritoneal macrophages showed that C3a stimulation resulted in upregulation of M2-associated signaling and molecules, but suppression of M1-associated signaling and molecules, supporting the roles of C3a/C3aR axis in mediating anti-inflammatory response and promoting M2 macrophage polarization. CONCLUSIONS: Our findings demonstrate a protective role for C3aR in the development of atherosclerosis and suggest that C3aR confers the protection through C3a/C3aR axis-mediated negative regulation of proinflammatory responses and modulation of macrophage toward the anti-inflammatory phenotype.

Endothelial cell-specific anticoagulation reduces inflammation in a mouse model of acute lung injury.

Tissue factor (TF)-dependent coagulation contributes to lung inflammation and the pathogenesis of acute lung injury (ALI). In this study, we explored the roles of targeted endothelial anticoagulation in ALI using two strains of transgenic mice expressing either a membrane-tethered human tissue factor pathway inhibitor (hTFPI) or hirudin fusion protein on CD31+ cells, including vascular endothelial cells (ECs). ALI was induced by intratracheal injection of LPS, and after 24 h the expression of TF and protease-activated receptors (PARs) on EC in lungs were assessed, alongside the extent of inflammation and injury. The expression of TF and PARs on the EC in lungs was upregulated after ALI. In the two strains of transgenic mice, expression of either of hTFPI or hirudin by EC was associated with significant reduction of inflammation, as assessed by the extent of leukocyte infiltration or the levels of proinflammatory cytokines, and promoted survival after LPS-induced ALI. The beneficial outcomes were associated with inhibition of the expression of chemokine CCL2 in lung tissues. The protection observed in the CD31-TFPI-transgenic strain was abolished by injection of an anti-hTFPI antibody, but not by prior engraftment of the transgenic strains with WT bone marrow, confirming that the changes observed were a specific transgenic expression of anticoagulants by EC. These results demonstrate that the inflammation in ALI is TF and thrombin dependent, and that expression of anticoagulants by EC significantly inhibits the development of ALI via repression of leukocyte infiltration, most likely via inhibition of chemokine gradients. These data enhance our understanding of the pathology of ALI and suggest a novel therapeutic strategy for treatment.

Comparative proteomics-network analysis of proteins responsible for ursolic acid-induced cytotoxicity in colorectal cancer cells.

Ursolic acid is a key active compound present in many medicinal herbs that have been widely used in traditional Chinese medicine for the clinical treatment of various cancers. However, the precise mechanisms of its antitumor activity have been poorly understood. To identify the cellular targets of ursolic acid, two-dimensional gel electrophoresis combined with mass spectrometry was performed in this study, which identified 15 proteins with significantly altered levels in protein expression. This demonstrated that ursolic acid-induced cytotoxicity in colorectal cancer cells involves dysregulation in protein folding, signal transduction, cell proliferation, cell cycle, and apoptosis. Corresponding protein regulation was also confirmed by Western blotting. Furthermore, the study of functional association between these 15 proteins revealed that 10 were closely related in a protein-protein interaction network, whereby the proteins either had a direct interaction with each other or were associated via only one intermediary protein. In this instance, the ATP5B/CALR/HSP90B1/HSPB1/HSPD1-signaling network was revealed as the predominant target which was associated with the majority of the observed protein-protein interactions. As a result, the identified targets may be useful in explaining the anticancer mechanisms of ursolic acid and as potential targets for colorectal cancer therapy.

Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/- mice.

BACKGROUND: Tissue factor (TF) and coagulation proteases are involved in promoting atherosclerosis, but the molecular and cellular bases for their involvement are unknown. METHODS AND RESULTS: We generated a new strain (ApX4) of apolipoprotein E-deficient mice expressing a membrane-tethered human tissue factor pathway inhibitor fusion protein on smooth muscle actin-positive cells, including vascular smooth muscle cells (SMCs). ApX4 mice developed little atherosclerosis on either a normal chow or high-fat diet. Lipid levels were similar to those in parental ApoE(-/-) mice, and there was no detectable difference in systemic (circulating) tissue factor pathway inhibitor levels or activity. The small lipid-rich lesions that developed had markedly reduced leukocyte infiltrates, and in contrast to ApoE(-/-) mice, SMCs did not express macrophage migratory inhibitory factor (MIF), including at sites distant from atheromatous lesions. Low levels of circulating MIF in ApX4 mice normalized to levels seen in ApoE(-/-) mice after injection of an inhibitory anti-human tissue factor pathway inhibitor antibody, which also led to MIF expression by tissue factor-positive medial SMCs. MIF production by SMCs in ApoE(-/-) mice in vitro and in vivo was shown to be dependent on tissue factor and protease-activated receptor signaling, which were inhibited in ApX4 mice. CONCLUSIONS: Our data indicate that tissue factor plays a hitherto unreported role in the generation of MIF by SMCs in atherosclerosis-prone ApoE(-/-) mice, inhibition of which significantly prevents the development of atherosclerosis, through inhibition of leukocyte recruitment. These data significantly enhance our understanding of the pathophysiology of this important pathology and suggest new potential translational strategies to prevent atheroma formation.

Interactions of Bovine Serum Albumin with Anti-Cancer Compounds Using a ProteOn XPR36 Array Biosensor and Molecular Docking.

The aim of the work was to determine the interactions of a set of anti-cancer compounds with bovine serum albumin (BSA) using a ProteOn XPR36 array biosensor and molecular docking studies. The results revealed that a total of six anti-cancer compounds: gallic acid, doxorubicin, acteoside, salvianolic acid B, echinacoside, and vincristine were able to reversibly bind to the immobilized BSA. The sensorgrams of these six compounds were globally fit to a Langmuir 1:1 interaction model for binding kinetics analysis. There were significant differences in their affinity for BSA, with doxorubicin, the weakest binding compound having 1000-fold less affinity than salvianolic acid B, the strongest binding compound. However, compounds with a similar KD often exhibited markedly different kinetics due to the differences in ka and kd. Molecular docking experiments demonstrated that acteoside was partially located within sub-domain IIA of BSA, whereas gallic acid bound to BSA deep within its sub-domain IIIA. In addition, the interactions between these compounds and BSA were dominated by hydrophobic forces and hydrogen bonds. Understanding the detailed information of these anti-cancer compounds can provide important insights into optimizing the interactions and activity of potential compounds during drug development.

Qualitative and Quantitative Analysis of the Major Constituents in Shexiang Tongxin Dropping Pill by HPLC-Q-TOF-MS/MS and UPLC-QqQ-MS/MS.

Shexiang Tongxin dropping pill (STP) is a traditional Chinese medicine formula that consists of total saponins of ginseng, synthetic Calculus bovis, bear gall, Venenum bufonis, borneol and Salvia miltiorrhiza. STP has been widely used in China and Southeast Asia for the treatment of cardiovascular diseases. In this study, a qualitative analytical method using high performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was developed for identification of the major constituents in STP. Based on the retention time and MS spectra, 41 components were identified by comparison with reference compounds and literature data. Moreover, using ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring mode, we quantified 13 of the identified constituents (ginsenoside Rg1, ginsenoside Rk3, cinobufagin, arenobufagin, bufalin, resibufogenin, tanshinone IIA, taurine, tauroursodeoxycholic acid, taurocholic acid, cholic acid, deoxycholic acid, and chenodeoxycholic acid). These results suggest that this new approach is applicable for the routine analysis and quality control of STP products and provides fundamental data for further in vivo pharmacokinetical studies.

Neointimal hyperplasia after endoluminal injury in mice is dependent on tissue factor- and angiopoietin-2 dependent interferon gamma production by fibrocytes and macrophages.

Background: The intimal hyperplasia (IH) and vascular remodelling that follows endovascular injury, for instance after post-angioplasty re-stenosis, results in downstream ischaemia and progressive end organ damage. Interferon gamma (IFNγ) is known to play a critical role in this process. In mouse models we have previously shown that fibrocytes expressing tissue factor (TF) are recruited early to the site of injury. Through thrombin generation and protease activated receptor-1 (PAR-1) activation, fibrocytes secrete angiopoietin-2, stimulate neointimal cell proliferation, inhibit apoptosis and induce CXCL-12 production, all of which contribute to the progressive IH that then develops. In this study we investigated the relationship between TF, angiopoietin-2 and IFNγ. Methods and results: IH developing in carotid arteries of wild-type mice 4 weeks after endoluminal injury contained a significant proportion of IFNγ+ fibrocytes and macrophages, which we show, using a previously defined adoptive transfer model, were derived from circulating CD34+ cells. IH did not develop after injury in IFNγ-deficient mice, except after transplantation of WT bone marrow or adoptive transfer of WT CD34+ cells. In vitro, CD34+ cells isolated from post-injury mice did not express IFNγ, but this was induced when provided with FVIIa and FX, and enhanced when prothrombin was also provided: In both cases IFNγ secretion was TF-dependent and mediated mainly through protease activated PAR-1. IFNγ was predominantly expressed by fibrocytes. In vivo, all IFNγ+ neointimal cells in WT mice co-expressed angiopoietin-2, as did the small numbers of neointimal cells recruited in IFNγ-/- mice. Adoptively transferred WT CD34+ cells treated with either an anti-TIE-2 antibody, or with siRNA against angiopoetin-2 inhibited the expression of IFNγ and the development of IH. Conclusion: TF-dependent angiopoietin-2 production by newly recruited fibrocytes, and to a lesser extent macrophages, switches on IFNγ expression, and this is necessary for the IH to develop. These novel findings enhance our understanding of the pathophysiology of IH and expose potential targets for therapeutic intervention.

Qingda granule alleviates cerebral ischemia/reperfusion injury by inhibiting TLR4/NF-κB/NLRP3 signaling in microglia.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingda granule (QDG) is effective for treating hypertension and neuronal damage after cerebral ischemia/reperfusion. However, the anti-neuroinflammatory effect of QDG on injury due to cerebral ischemia/reperfusion is unclear. AIM OF THE STUDY: The objective was to evaluate the effectiveness and action of QDG in treating neuroinflammation resulting from cerebral ischemia/reperfusion-induced injury. MATERIALS AND METHODS: Network pharmacology was used to predict targets and pathways of QDG. An in vivo rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) as well as an in vitro model of LPS-stimulated BV-2 cells were established. Magnetic resonance imaging (MRI) was used to quantify the area of cerebral infarction, with morphological changes in the brain being assessed by histology. Immunohistochemistry (IHC) was used to assess levels of the microglial marker IBA-1 in brain tissue. Bioplex analysis was used to measure TNF-α, IL-1ß, IL-6, and MCP-1 in sera and in BV-2 cell culture supernatants. Simultaneously, mRNA levels of these factors were examined using RT-qPCR analysis. Proteins of the TLR4/NF-κB/NLRP3 axis were examined using IHC in vivo and Western blot in vitro, respectively. While NF-κB translocation was assessed using immunofluorescence. RESULTS: The core targets of QDG included TNF, NF-κB1, MAPK1, MAPK3, JUN, and TLR4. QDG suppressed inflammation via modulation of TLR4/NF-κB signaling. In addition, our in vivo experiments using MCAO/R rats demonstrated the therapeutic effect of QDG in reducing brain tissue infarction, improving neurological function, and ameliorating cerebral histopathological damage. Furthermore, QDG reduced the levels of TNF-α, IL-1ß, IL-6, and MCP-1 in both sera from MCAO/R rats and supernatants from LPS-induced BV-2 cells, along with a reduction in the expression of the microglia biomarker IBA-1, as well as that of TLR4, MyD88, p-IKK, p-IκBα, p-P65, and NLRP3 in MCAO/R rats. In LPS-treated BV-2 cells, QDG downregulated the expression of proinflammatory factors and TLR4/NF-κB/NLRP3 signaling-related proteins. Additionally, QDG reduced translocation of NF-κB to the nucleus in both brains of MCAO/R rats and LPS-induced BV-2 cells. Moreover, the combined treatment of the TLR4 inhibitor TAK242 and QDG significantly reduced the levels of p-P65, NLRP3, and IL-6. CONCLUSIONS: QDG significantly suppressed neuroinflammation by inhibiting the TLR4/NF-κB/NLRP3 axis in microglia. This suggests potential for QDG in treating ischemia stroke.

Protease-activated receptor-2 signalling by tissue factor on dendritic cells suppresses antigen-specific CD4+ T-cell priming.

The precise function of tissue factor (TF) expressed by dendritic cells (DC) is uncertain. As well as initiating thrombin generation it can signal through protease-activated receptor 2 (PAR-2) when complexed with factor VIIa. We investigated the expression and function of TF on mouse bone marrow (BM) -derived DC; 20% of BM-derived DC expressed TF, which did not vary after incubation with lipopolysaccharide (LPS) or dexamethasone (DEX). However, the pro-coagulant activity of DEX-treated DC in recalcified plasma was 30-fold less than LPS-treated DC. In antigen-specific and allogeneic T-cell culture experiments, the TF on DEX-treated DC provided a signal through PAR-2, which contributed to the reduced ability of these cells to stimulate CD4(+) T-cell proliferation and cytokine production. In vivo, an inhibitory anti-TF antibody and a PAR-2 antagonist enhanced antigen-specific priming in two models where antigen was given without adjuvant, with an effect approximately 50% that seen with LPS, suggesting that a similar mechanism was operational physiologically. These data suggest a novel TF and PAR-2-dependent mechanism on DEX-DC in vitro and unprimed DC in vivo that contributes to the low immunogenicity of these cells. Targeting this pathway has the potential to influence antigen-specific CD4(+) T-cell activation.

Immunization with a combination of 2 peptides derived from the C5a receptor significantly reduces early atherosclerotic lesion in Ldlr(tm1Her) Apob(tm2Sgy) J mice.

OBJECTIVE: The goal of this study was to assess whether immunization of Ldlr(tm1Her) Apob(tm2Sgy) J mice with 2 peptides located at the N-terminus of the C5a receptor (C5aR), either alone or in combination, is effective in reducing atherosclerotic lesions. METHODS AND RESULTS: Five- to 6-week-old female Ldlr(tm1Her)Apob(tm2Sgy) J mice were immunized using a repetitive immunization multiple sites strategy with keyhole limpet hemocyanin-conjugated peptides derived from the C5aR, either alone (designated as C5aR-P1 [aa 1-21] and C5aR-P2 [aa 19-31]) or in combination (designated as C5aR-P1+C5aR-P2). Mice were fed a high-fat diet for 10 weeks. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants. Immunization of Ldlr(tm1Her)Apob(tm2Sgy) J mice with these peptides elicited high concentrations of antibodies against each peptide. Immunization with the single peptide inhibited plaque development. Combined inoculation with C5aR-P1+C5aR-P2 had an additive effect on reducing the lesion in the aorta sinus and descending aortas when compared with controls. This effect correlated with cellular infiltration and cytokine/chemokine secretion in the serum or in stimulated spleen cells as well as specific cellular immune responses when compared with controls. CONCLUSIONS: Immunization of mice with C5aR-P1 and C5aR-P2, either alone or in combination, was effective in reducing early atherosclerotic lesion development. The combined peptide is more potential than either epitope alone to reduce atherosclerotic lesion formation through the induction of a specific Treg cell response as well as blockage of monocyte differentiation into macrophages.

Inhibition of thrombin receptor signaling on α-smooth muscle actin(+) CD34(+) progenitors leads to repair after murine immune vascular injury.

OBJECTIVE: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). METHODS AND RESULTS: BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. CONCLUSIONS: Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.

Liensinine improves AngII-induced vascular remodeling via MAPK/TGF-ß1/Smad2/3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Liensinine(Lien, C37H42N2O6) is an alkaloid compound from plumula nelumbinis that demonstrates an antihypertensive effect. The protective effects of Lien on target organs during hypertension are still unclear. AIM OF THE STUDY: This study aimed to understand the mechanism of Lien during the treatment of hypertension, with emphasis on vascular protection. MATERIALS AND METHODS: Lien was extracted and isolated from plumula nelumbinis for further study. In vivo model of Ang II-induced hypertension, non-invasive sphygmomanometer was used to detect the blood pressure in and out of the context of Lien intervention. Ultrasound was used to detect the abdominal aorta pulse wave and media thickness of hypertensive mice, and RNA sequencing was used to detect the differential genes and pathways of blood vessels. The intersection of Lien and MAPK protein molecules was detected by molecular interconnecting technique. The pathological conditions of abdominal aorta vessels of mice were observed by HE staining. The expression of PCNA, α-SMA, Collagen Type Ⅰ and Collagen Type Ⅲ proteins were detected by IHC. The collagen expression in the abdominal aorta was detected by Sirius red staining. The MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA was detected by Western blot. In vitro, MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA were detected by Western blot, and the expression of α-SMA was detected by immunofluorescence; ELISA was used to detect the effect of ERK/MAPK inhibitor PD98059 on Ang Ⅱ-induced TGF-ß1secrete; and the detection TGF-ß1and α-SMA protein expression by Western blot; Western blot was used to detect the effect of ERK/MAPK stimulant12-O-tetradecanoyl phorbol-13-acetate (TPA) on the protein expression of TGF-ß1 and α-SMA. RESULTS: Lien displayed an antihypertensive effect on Ang Ⅱ-induced hypertension, reducing the pulse wave conduction velocity of the abdominal aorta and the thickness of the abdominal aorta vessel wall, ultimately improving the pathological state of blood vessels. RNA sequencing further indicated that the differential pathways expressed in the abdominal aorta of hypertensive mice were enriched in proliferation-related markers compared with the Control group. The profile of differentially expressed pathways was ultimately reversed by Lien. Particularly, MAPK protein demonstrated good binding with the Lien molecule. In vivo, Lien inhibited Ang Ⅱ-induced abdominal aorta wall thickening, reduced collagen deposition in the ventral aortic vessel, and prevented the occurrence of vascular remodeling by inhibiting MAPK/TGF-ß1/Smad2/3 signaling activation. In addition, Lien inhibited the activation of Ang II-induced MAPK and TGF-ß1/Smad2/3 signaling, attenuating the expression of PCNA and inhibiting the reduction of α-SMA, collectively playing a role in the inhibition of Ang Ⅱ-induced hypertensive vascular remodeling. PD98059 alone could inhibit Ang Ⅱ-induced elevation of TGF-ß1 and the decrease of α-SMA expression. Further, PD98059 combined with Lien had no discrepancy with the inhibitors alone. Simultaneously TPA alone could significantly increase the expression of TGF-ß1 and decrease the expression of α-SMA. Further, Lien could inhibit the effect of TPA. CONCLUSION: This study helped clarify the protective mechanism of Lien during hypertension, elucidating its role as an inhibitor of vascular remodeling and providing an experimental basis for the research and development of novel antihypertensive therapies.

LINC01006 and miR-3199 Serve as Novel Markers of Poor Prognosis in Colon Cancer and Regulate Cell Proliferation, Migration and Invasion.

PURPOSE: Colon cancer is the most commonly diagnosed gastrointestinal cancer. This research intended to evaluate the prognostic values of LINC01006 and miR-3199 for colon cancer and their effects on cell physiology. PATIENTS AND METHODS: LINC01006 and miR-3199 expression levels were determined by RT-qPCR. Patients' 5-year cumulative survival rate was analyzed by Kaplan-Meier curves with the Log rank test. Chi-square test and multivariate Cox regression analysis were used to access the clinical significance. CCK-8 assay, transwell assay, and TUNEL assays were used to monitor the change of cell proliferation, invasion, migration, and apoptosis. RESULTS: The expression level of LINC01006 was increased while miR-3199 was decreased in colon tissues and cells compared to normal ones. This dysregulated expression was correlated with T stage (P = 0.002) and N stage (P = 0.009). High LINC01006 level (HR = 4.048, 95%: 1.502-10.911, P = 0.006) or low miR-3199 level (HR = 3.421, 95% CI: 1.254-9.330, P = 0.016) was outstanding for predicting poor prognosis in patients with colon cancer. Downregulation of LINC01006 reduced cell proliferation, invasion, and migration but induced cell apoptosis (P < 0.05). CONCLUSION: LINC01006 knockdown showed anti-proliferative, anti-metastatic, and apoptotic-induced effects on colon cancer cells. This study contributes to research on promising prognostic biomarkers of colon cancer and might give way to further investigation of alternative tumor targets.

Manipulation of tissue factor-mediated basal PAR-2 signalling on macrophages determines sensitivity for IFNγ responsiveness and significantly modifies the phenotype of murine DTH.

Background: Tissue factor (TF) generates proteases that can signal through PAR-1 and PAR-2. We have previously demonstrated PAR-1 signalling primes innate myeloid cells to be exquisitely sensitive to interferon-gamma (IFNγ). In this work we explored how TF mediated PAR-2 signalling modulated responsiveness to IFNγ and investigated the interplay between PAR-1/-2 signalling on macrophages. Methodology: We characterised how TF through PAR-2 influenced IFNγ sensitivity in vitro using PCR and flow cytometry. and how it influenced oxazolone-induced delayed type hypersensitivity (DTH) responses in vivo. We investigated how basal signalling through PAR-2 influenced PAR-1 signalling using a combination of TF-inhibitors and PAR-1 &-2 agonists and antagonists. Finally, we investigated whether this system could be targeted therapeutically using 3-mercaptopropionyl-F-Cha-Cha-RKPNDK (3-MP), which has actions on both PAR-1 and -2. Results: TF delivered a basal signal through PAR-2 that upregulated SOCS3 expression and blunted M1 polarisation after IFNγ stimulation, opposing the priming achieved by signalling through PAR-1. PAR-1 and -2 agonists or antagonists could be used in combination to modify this basal signal in vitro and in vivo. 3-MP, by virtue of its PAR-2 agonist properties was superior to agents with only PAR-1 antagonist properties at reducing M1 polarisation induced by IFNγ and suppressing DTH. Tethering a myristoyl electrostatic switch almost completely abolished the DTH response. Conclusions: TF-mediated signalling through PARs-1 and -2 act in a homeostatic way to determine how myeloid cells respond to IFNγ. 3-MP, an agent that simultaneously inhibits PAR-1 whilst delivering a PAR-2 signal, can almost completely abolish immune responses dependent on M1 polarisation, particularly if potency is enhanced by targeting to cell membranes; this has potential therapeutic potential in multiple diseases.

Therapeutic Effects of Different Animal Bile Powders on Lipid Metabolism Disorders and Their Composition Analysis.

OBJECTIVE: To compare the therapeutic effect of different animal bile powders on lipid metabolism disorders induced by high-fat diet in rats, and analyze the bioactive components of each animal bile powder. METHODS: Sixty Sprague-Dawley rats were randomly divided into 6 groups (n=10): normal diet control group, high-fat diet model group, high-fat diet groups orally treated with bear, pig, cow and chicken bile powders, respectively. Serum biochemical markers from the abdominal aorta in each group were analyzed. Changes in the body weight and liver weight were recorded. Pathohistological changes in the livers were examined. High performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry was used to determine the composition of bioactive components in each animal bile powder. RESULTS: Treatment with different types of animal bile powders had different inhibitory effects on high-fat diet-induced increase of body weight and/or liver weight in rats, most notably in bear and pig bile powders (P<0.05). High-fat diet induced lipid metabolism disorder in rats, which could be reversed by treatment with all kinds of bile powders. Bear bile and chicken bile showed the most potent therapeutic effect against lipid metabolism disorder. Cow and bear bile effectively alleviated high-fat diet induced liver enlargement and discoloration, hepatocyte swelling, infiltration of inflammatory cells and formation of lipid vacuoles. Bioactive component analysis revealed that there were significant differences in the relative content of taurocholic acid, taurodeoxycholic acid and ursodeoxycholic acid among different types of animal bile. Interestingly, a unique component with molecular weight of 496.2738 Da, whose function has not yet been reported, was identified only in bear bile powder. CONCLUSIONS: Different animal bile powders had varying therapeutic effect against lipid metabolism disorders induced by high-fat diet, and bear bile powder demonstrated the most effective benefits. Bioactive compositions were different in different types of animal bile with a novel compound identified only in bear bile powder.

Qingda granule alleviate angiotensin â ±-induced hypertensive renal injury by suppressing oxidative stress and inflammation through NOX1 and NF-κB pathways.

Hypertension has become one of the important diseases harmful to human health. In China, Qingda granule (QDG) has been used to treat hypertension for decades. Previous studies by our team have shown that oxidative stress may be one of the pathways through which QDG inhibits hypertension-induced organs injury. However, the specific molecular mechanism of its anti-hypotension and renal oxidative stress response were unclearly. This study investigated QDG's potential protective mechanism against hypertension-induced renal injury. Mice were infused with Angiotensin Ⅱ (Ang Ⅱ, 500 ng/kg/min) or equivalent saline solution (Control) and administered oral QDG (1.145 g/kg/day) or saline for four weeks. QDG treatment mitigated the elevated blood pressure and reduced renal pathological changes induced by Ang Ⅱ. As per the RNA sequencing results, QDG affects oxidative stress signaling. In agreement with these findings, QDG significantly attenuated the Ang Ⅱ-induced increase in Nitrogen oxides 1 (NOX1) and reactive oxygen species and the decrease in superoxide dismutase in renal tissue. Additionally, QDG significantly inhibited Interleukin 6 (IL-6), Tumor necrosis factor α (TNF-α), and Interleukin 1ß (IL-1ß) expression in renal tissues and blocked the phosphorylation of P65 (NF-κB subunit) and IκB. These results were confirmed in vitro. Overall, QDG reduced Ang Ⅱ-induced elevated blood pressure and renal injury by inhibiting oxidative stress and inflammation caused by NOX1 and NF-κB pathways. The results of this study provide an experimental basis for the clinical application of QDG, and to open up a new direction for the clinical treatment of hypertension.

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice.

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

PAR-1 signaling on macrophages is required for effective in vivo delayed-type hypersensitivity responses.

Delayed-type hypersensitivity (DTH) responses underpin chronic inflammation. Using a model of oxazolone-induced dermatitis and a combination of transgenic mice, adoptive cell transfer, and selective agonists/antagonists against protease activated receptors, we show that that PAR-1 signaling on macrophages by thrombin is required for effective granuloma formation. Using BM-derived macrophages (BMMs) in vitro, we show that thrombin signaling induced (a) downregulation of cell membrane reverse cholesterol transporter ABCA1 and (b) increased expression of IFNγ receptor and enhanced co-localization within increased areas of cholesterol-rich membrane microdomains. These two key phenotypic changes combined to make thrombin-primed BMMs sensitive to M1 polarization by 1000-fold less IFNγ, compared to resting BMMs. We confirm that changes in ABCA1 expression were directly responsible for the exquisite sensitivity to IFNγ in vitro and for the impact on granuloma formation in vivo. These data indicate that PAR-1 signaling plays a hitherto unrecognized and critical role in DTH responses.

Cardiac biomarkers, cardiac injury, and comorbidities associated with severe illness and mortality in coronavirus disease 2019 (COVID-19): A systematic review and meta-analysis.

AIMS: To explore the correlation between cardiac-related comorbidities, cardiac biomarkers, acute myocardial injury, and severity level, outcomes in COVID-19 patients. METHOD: Pubmed, Web of Science, Embase, CNKI, VIP, Wanfang, Cochrane Library databases, medRxiv, and Sinomed were reviewed systemically. Various types of clinical research reporting cardiac-related comorbidities, cardiac biomarkers including lactate dehydrogenase (LDH), troponin I (TnI), high sensitivity troponin I (hs-TnI), creatine kinase (CK), creatine kinase-MB (CK-MB), myoglobin (Myo), N-terminal pro-b-type natriuretic peptide (NT-proBNP) and acute cardiac injury grouped by severity of COVID-19 were included. Outcome measures were events and total sample size for comorbidities, acute cardiac injury, and laboratory parameters of these biomarkers. The study was performed with Stata version 15.1. RESULTS: Seventy studies, with a total of 15,354 cases were identified. The results showed that COVID-19's severity was related to cardiovascular disease. Similar odds ratios (ORs) were achieved in hypertension except for severe versus critical group (OR = 1.406; 95% CI, 0.942-2.097; p = .095). The relative risk (RR) of acute cardiac injury is 7.01 (95% CI, 5.64-8.71) in non-survivor cases. When compared with the different severity of cardiac biomarkers, the pool OR of CK, CK-MB, TnI, Myo and LDH were 2.683 (95% CI, 0.83-8.671; p = .106; I2 = 0%), 2.263 (95% CI, 0.939-5.457; p = .069), 1.242 (95% CI, 0.628-2.457; p = .534), 1.756 (95% CI, 0.608-5.071; p = .298; I2 = 42.3%), 1.387 (95% CI, 0.707-2.721;  p = .341; I2 = 0%) in the critical versus severe group, whose trends were not similar to other groups. The standard mean differences (SMD) of CK and TnI in the critical versus severe group were 0.09 (95% CI, -0.33 to 0.50; p = .685; I2 = 65.2%), 0.478 (95% CI, -0.183 to 1.138; p = .156; I2 = 76.7%), which means no difference was observed in the serum level of these indicators. CONCLUSION: Most of the findings clearly indicate that hypertension, cardiovascular disease, acute cardiac injury, and related laboratory indicators are associated with the severity of COVID-19. What is now needed are cross-national prospectively designed observational or clinical trials that will help improve the certainty of the available evidence and treatment decisions for patients.

Comprehensive chemical study on different organs of cultivated and wild Sarcandra glabra using ultra-high performance liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS).

To illuminate the similarities and differences between wild and cultivated Sarcandra glabra (S. glabra), we performed a comprehensively study on 26 batches of cultivated S. glabra and 2 batches of wild S. glabra. Chemical constituents and distribution characteristics of roots, stems and leaves in both wild and cultivated S. glabra were investigated through UHPLC-TOF-MS method. The result revealed that there were significant differences between roots, stems and leaves in S. glabra. And the chemical contents in the root part were less or even absence than those in leaf and stem, which suggested the root organ could be excluded as medicine. Meanwhile, the chemical contents of stems and leaves in cultivated S. glabra was sightly higher than that of wild samples. Therefore, cultivated S. glabra may have a high potential for substitution of wild S. glabra without affecting its pharmaceutical properties. In summary, our study could provide important information to the molecular basis for quality control of S. glabra.