Receptor Protein Levels Research Articles

Tongluo Yishen Decoction Ameliorates Renal Fibrosis via NLRP3-Mediated Pyroptosis In Vivo and In Vitro.

Purpose: In this study, we investigated the mechanism of Tongluo Yishen (TLYS) decoction in more detail, from the perspective of pyroptosis in the unilateral ureteral ligation (UUO) model and the hypoxia-induced renal tubular epithelial (NRK-52E) cell. Method: The UUO model was used, and after 14 days of TLYS intervention, rats were tested for blood creatinine and urea nitrogen, HE staining was used to observe the pathological changes in the kidney, Masson staining was used to assess the degree of interstitial fibrosis, western blot was used to detect the changes of α-smooth muscle actin (α-SMA) protein expression level, immunohistochemistry and western blot detected the changes in protein expression levels of NOD-like receptor protein 3 inflammasome (NLRP3), gasdermin D (GSDMD), cysteinyl aspartate specific proteinase (caspase-1), interleukin 18 (IL-18) and interleukin 1β (I L-1β). A hypoxia model was created using NRK-52E cell, and after different concentrations of TLYS decoction intervention, the changes in the expression levels of pyroptosis were used with immunofluorescence and western blot methods. Results: TLYS decoction improved renal function, delayed the advancement of renal interstitial fibrosis, and inhibited pyroptosis in UUO rats. Furthermore, we observed that TLYS can mitigate hypoxia-induced NRK-52E cell damage via the suppression of the NLRP3-mediated pyroptosis. Conclusion: TLYS decoction exert renoprotective effects by inhibiting NLRP3-mediated pyroptosis.

Effects of NLRP3/Caspase-1 pathway on Banxia Houpu decoction in the intervention of chronic intermittent hypoxia in mice with renal inflammatory injury

Objective: To investigate the effects of Banxia Houpo decoction on the renal NLRP3/Caspase-1/IL-1β signaling pathway in chronic intermittent hypoxia mice. Methods: C57BL/6 mice were randomly divided into 3 groups, normal control group (Control), chronic intermittent hypoxia group (CIH), and Banxia Houpo decoction treatment group (BHD), with 10 mice in each group. Mice in the CIH group and BHD group were placed in a hypoxic chamber. The oxygen volume fraction in the cabin was decreased from 21% to 9% in 90 s, and then oxygen was filled in 90 s to gradually increase the oxygen volume fraction in the cabin to 21%, while the mice in the control group were placed in the cabin and filled with normal air, processing 8 hours per day for 21 days. The mice in BHD group were treated with Banxia Houpu decoction by gavage before entering the cabin every day, and the control group and CIH group were given an equal volume of normal saline. After modeling, the changes of renal function indexes in each group were detected; HE and Masson staining were used to observe the pathological conditions of the kidney; Western blot and immunohistochemical staining were used to detect the protein expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3), aspartate-specific cysteine protein 1(Caspase-1) and interleukine-1beta(IL-1β). Results: Compared with control group, the contents of serum renal functional indexes UA, BUN and SCr in CIH group were increased significantly (P<0.01), and after BHD treatment, they all were decreased significantly compared with CIH group (P<0.01). Compared with control group, the results of HE staining showed that in the CIH group, glomerular endothelial cells were degenerated and necrotic, and vacuoles of different sizes appeared in renal tubular epithelial cells, and a small amount of renal tubular epithelial cells fell off and died. The pathological condition of the BHD group was improved compared with CIH group, the glomerular morphology gradually returned to normal, and a small amount of renal tubular epithelial cells fell off and died. Compared with control group, Masson staining results showed that there was obvious fibrosis around the glomeruli in the CIH group, the fibrosis was significantly reduced in the BHD group. The expression levels of NLRP3, Caspase-1, IL-1β and IL-18 were increased significantly compared with control group (P<0.05 or P<0.01), and immunohistochemical staining showed that NLRP3 was mainly expressed in renal tubular epithelial cells and interstitial macrophages, caspase-1 and IL-1β were mainly found in the cytoplasm of renal tubular epithelial cells. After BHD treatment, the expression levels of each protein were decreased compared with CIH group (P<0.05). Conclusion: Banxia Houpu decoction can reduce the kidney damage by inhibiting the expression of related molecules in the NLRP3/Casapse-1/IL-1β signaling pathway.

Reineckia carnea Alleviates the Production of Inflammatory Cytokines and MUC5AC in Rats with Chronic Obstructive Pulmonary Disease.

Background Reineckia carnea (RC), a perennial evergreen herb which belongs to Reineckia Kunth (Liliaceae), can be used for clearing the lungs and relieving cough, reducing phlegm and anti-inflammatory effects. Moreover, chronic obstructive pulmonary disease (COPD) is characterized by airway and lung inflammation and increased secretion of airway mucus. Therefore, RC has the potential to treat COPD. Methods NR8383 cells were cultured and treated with various concentrations of RC (100 mg/mL, 10 mg/mL, 1 mg/mL, 100 μg/mL, 10 μg/mL, 1 μg/mL, 100 ng/mL, and 10 ng/mL). Cell viability and levels of interleukin (IL)-1β, cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2) in the cell culture supernatant or rat serum were analyzed using CCK-8 and enzyme-linked immunosorbent assay (ELISA), respectively. Sprague Dawley rats were assigned to mock, COPD model, RC (0.67 g/kg, 1.35 g/kg, and 2.7 g/kg), and ambroxol (5.4 mg/kg) groups. Western blot and quantitative polymerase chain reaction (qPCR) analyses were used to evaluate the protein and mRNA expression levels of mucin 5AC (MUC5AC) and Toll-like receptor 4 (TLR4). Results The results showed that Reineckia carnea (RC) extract (RCE) inhibited the proliferation of NR8383 cells and suppressed the production of IL-1β, PGE2, and COX-2 in NR8383 cells. Moreover, RCE decreased the levels of IL-1β, PGE2, and COX-2 in the serum of rats with COPD and alleviated the expression of TLR4 and MUC5AC induced by COPD in rat lung tissue. Conclusion RCE alleviated COPD by inhibiting the expression of COPD-induced inflammatory cytokines and MUC5AC in rats.

Chrysin Attenuates Fructose-Induced Nonalcoholic Fatty Liver in Rats via Antioxidant and Anti-Inflammatory Effects: The Role of Angiotensin-Converting Enzyme 2/Angiotensin (1-7)/Mas Receptor Axis.

Aim Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome, and if untreated, it may propagate into end-stage liver disease. The classical arm of the renin-angiotensin system (RAS) has a fundamental role in triggering oxidative stress and inflammation, which play potential roles in the pathogenesis of NAFLD. However, the nonclassical alternative axis of RAS, angiotensin- (Ang-) converting enzyme 2 (ACE2)/Ang (1-7)/Mas receptor, opposes the actions of the classical arm, mitigates the metabolic dysfunction, and improves hepatic lipid metabolism rendering it a promising protective target against NAFLD. The current study is aimed at investigating the impact of chrysin, a well-known antioxidant flavonoid, on this defensive RAS axis in NAFLD. Methods Rats were randomly distributed and treated daily for eight weeks as follows: the normal control, chrysin control (50 mg/kg, p.o), NAFLD group (received 20% fructose in drinking water), and treated groups (25 and 50 mg/kg chrysin given orally and concomitantly with fructose). Diminazene aceturate (DIZE) (15 mg/kg, s.c.) was used as a reference ACE2 activator. Key Findings. High fructose induced significant weight gain, hepatocyte degeneration with fat accumulation, and inflammatory cell infiltration (as examined by H&E staining). This was accompanied by a substantial increase in liver enzymes, glucose, circulating and hepatic triglycerides, lipid peroxides, inflammatory cytokines, and Ang II (the main component of classical RAS). At the same time, protein levels of ACE2, Ang (1-7), and Mas receptors were markedly reduced. Chrysin (25 and 50 mg/kg) significantly ameliorated these abnormalities, with a prominent effect of the dose of 50 mg/kg over DIZE and the lower dose in improving ACE2, Ang (1-7), and Mas. Significance. Chrysin is a promising efficient protective remedy against NAFLD; mechanisms include the activation of ACE2/Ang (1-7)/Mas axis.

The Release of Exosomes from Astrocytes in Response to Fatty Acid Alters the Metabolism of Proopiomelanocortin (POMC) Neurons

Exosomes are extracellular micro-vesicles with a complex content that include a vast heterogeneity of molecules such as growth factors, cytokines and RNAs, including micro-RNAs (mi-RNAs) that affect target cells. Neurons are influenced by neighboring astrocytes through the release of exosomes amongst other signals. We postulated that the metabolic status is communicated by hypothalamic astrocytes via exosomes to neighboring POMC neurons to modify their functions in the promotion of energy expenditure and satiety. With this aim, cultures of primary hypothalamic astrocytes were treated with both palmitic or oleic acid (PA or OA; 0.5 mM) for 24 hours. Exosomes were isolated and purified from the media culture and used to treat (1.25 or 2.50 µg/mL) a POMC neuronal cell line for 24 hours. Exosomes released in response to PA (E-PA) or (E-OA) did not change the expression of the markers of inflammation [interleukin (IL)-6] and Endoplasmic Reticle stress (CHOP) comparing to exosomes released in response to vehicle (E-V) or with no exosomes (control). Next-Generation miRNA sequencing analysis was performed to reveal modifications in miRNAs contained in exosomes. The content of miR-199a-3p and miR-145-5p was higher in E-PA compared to E-V. POMC neurons transfected with a mimetic of miR-199a-3p (1.5 pmol) increased insulin-like growth factor 1 receptor (IGF1r) protein levels (p &lt; 0.05), as well as POMC expression (ns). Moreover, levels of mTOR and p70S6k, reported targets of miR-199a-3p, were decreased (p &lt; 0.05, both). Mimetic overexpression of miR- 145-5p reduced POMC expression (p &lt; 0.001) and protein levels of insulin receptor substrate 1 (IRS1; p &lt; 0.001), which is a known target of this miRNA. To determine modifications in cellular metabolism in the POMC neurons in response to exosomal astrocytes, Seahorse Cell Mito Stress test was performed. The mitochondrial spare respiratory capacity of neurons was increased (p &lt; 0.0001) in response to both doses (1.25 or 2.50 µg/mL) of E-PA and E-OA, with the maximal respiration (p &lt; 0.0001) also increasing with E-PA or E-OA (both doses) compared to E-V or control. Our results suggest that astrocytes may communicate directly with neurons via exosomes, and that the exosomes content is modulated by the source cell in response to the nutritional environment. The messages contained in exosomes from astrocytes can directly modulate the levels of cellular receptors and factors involved in cell proliferation, protection, metabolism, and nutrient sensing. Specific miRNAs participate in these processes as well as on the neuropeptide expression in targeted neurons. In addition, cellular respiration is affected in POMC neurons treated with exosomes from astrocytes in response to fatty acid in a way that suggests that cells are preparing for a possible respiratory stress by increasing their spare respiratory capacity and maximal respiration.

Synergic action of L-acetylcarnitine and L-methylfolate in Mouse Models of Stress-Related Disorders and Human iPSC-Derived Dopaminergic Neurons.

The epigenetic agents, L-acetylcarnitine (LAC) and L-methylfolate (MF) are putative candidates as add-on drugs in depression. We evaluated the effect of a combined treatment with LAC and MF in two different paradigms of chronic stress in mice and in human inducible pluripotent stem cells (hiPSCs) differentiated into dopaminergic neurons. Two groups of mice were exposed to chronic unpredictable stress (CUS) for 28 days or chronic restraint stress (CRS) for 21 day, and LAC (30 or 100 mg/kg) and/or MF (0.75 or 3 mg/kg) were administered i.p. once a day for 14 days, starting from the last week of stress. In both stress paradigms, LAC and MF acted synergistically in reducing the immobility time in the forced swim test and enhancing BDNF protein levels in the frontal cortex and hippocampus. In addition, LAC and MF acted synergistically in enhancing type-2 metabotropic glutamate receptor (mGlu2) protein levels in the hippocampus of mice exposed to CRS. Interestingly, CRS mice treated with MF showed an up-regulation of NFκB p65, which is a substrate for LAC-induced acetylation. We could also demonstrate a synergism between LAC and MF in cultured hiPSCs differentiated into dopamine neurons, by measuring dendrite length and number, and area of the cell soma after 3 days of drug exposure. These findings support the combined use of LAC and MF in the treatment of MDD and other stress-related disorders.

Reduced insulin signaling and high glucagon in early insulin resistance impaired fast-fed regulation of renal gluconeogenesis via insulin receptor substrate.

Gluconeogenesis is one of the key processes through which the kidney contributes to glucose homeostasis. Urinary exosomes (uE) have been used to study renal gene regulation noninvasively in humans and rodents. Recently, we demonstrated fast-fed regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme for gluconeogenesis, in human uE. The regulation was impaired in subjects with early insulin resistance. Here, we studied primary human proximal tubule cells (hPT) and human uE to elucidate a potential link between insulin resistance and fast-fed regulation of renal PEPCK. We demonstrate that fasted hPTs had higher PEPCK and insulin receptor substrate-2 (IRS2) mRNA and protein levels, relative to fed cells. The fast-fed regulation was, however, attenuated in insulin receptor knockdown (IRKO) hPTs. The IRKO was confirmed by the blunted insulin-induced response on PEPCK, PGC1α, p-IR, and p-AKT expression in IRKO cells. Exosomes secreted by the wild-type or IRKO hPT showed similar regulation to the respective hPT. Similarly, in human uE, the relative abundance of IRS-2 mRNA (to IRS1) was higher in the fasted state relative to the fed condition. However, the fast-fed difference was absent in subjects with early insulin resistance. These subjects had higher circulating glucagon levels relative to subjects with optimal insulin sensitivity. Furthermore, in hPT cells, glucagon significantly induced PEPCK and IRS2 gene, and gluconeogenesis. IR knockdown in hPT cells further increased the gene expression levels. Together the data suggest that reduced insulin sensitivity and high glucagon in early insulin resistance may impair renal gluconeogenesis via IRS2 regulation.

Genistein accelerates glucose catabolism via activation the GPER-mediated cAMP/PKA-AMPK signaling pathway in broiler chickens

Genistein, the most abundance of phytoestrogens in soybeans, has beneficial effects in regulating metabolism-related disease; however, there is few available literatures about whether genistein regulates glucose metabolism that in turn affects the lipid accumulation in animals or humans. The current study showed that genistein promoted glucose uptake by enhancing glucose transporter-2 (GLUT2) protein level; and it also increased the activity of phosphofructokinase-1 (PFK) and pyruvate dehydrogenase (PDH), and the mRNA level of succinate dehydrogenase (SDH) both in broiler chickens or hepatocytes. Moreover, genistein obviously increased the p-LKB1 and p-AMPKα protein levels both in vivo and in vitro. Furthermore, the enhancement of genistein on glucose uptake and catabolism were reversed in hepatocytes pre-treated with AMPK inhibitor Compound C, and the increasing of genistein on the p-LKB1 and p-AMPKα protein levels were also reversed in hepatocytes pre-treated with PKA inhibitor H89. Importantly, the results showed that genistein simultaneously increased the estrogen receptor β (ERβ) and G protein-coupled estrogen receptor (GPER) protein levels, but the elevation effect of genistein on cAMP content was completely reversed in hepatocytes pre-treated with GPER antagonist G15, rather than ERβ inhibitor PHTPP. Meanwhile, the increasing of p-LKB1 and p-AMPKα protein levels induced by genistein were also reversed in hepatocytes pre-treated with G15. Collectively, our data demonstrated that genistein improves glucose metabolism via activating the GPER-mediated cAMP/PKA-AMPK signaling pathway. These findings provide theoretical basis for genistein as a promising nutritional supplemental to alleviate metabolism disorders and related diseases in animals or even humans.

Interference of P2X4 receptor expression in tumor-associated macrophages suppresses migration and invasion of glioma cells

To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells. C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment. The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA(P < 0.01)and protein (P < 0.01, P < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P < 0.05). Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.

IL-6 could regulate Th17/Treg cells to restore the local microenvironment of endometriosis

IntroductionIn this study, we studied anti-IL-6 antibodies to treat endometriosis (EMS) by balancing Th17/Treg cells, providing a new basis for EMS research and treatment.Material and methodsThe rat EMS model was constructed to study the effect of anti-IL-6 on EMS. HE staining was performed to observe the lesions of the endometrium in rats. We have used anti-IL-6 to treat EMS rats. Then ELISA was used to measure the inflammatory factors IL-6, TGF-β and hormone estrogen levels in the rat serum. qRT-PCR and Western blot were used to measure estrogen receptor protein and apoptosis protein levels. Th17/Treg and CD163+/CD86+ ratios were determined by flow cytometry. CD34+ and CD163+ were measured by immunohistochemistry. TUNEL was performed to detect apoptosis in EMS lesions.ResultsThe elevated levels of IL-6, TGF-β and estrogen in EMS rats indicated successful modeling. The ratio of Th17/Treg was imbalanced and raised. The ratio of Th17/Treg in anti-IL-6 rats recovered. The expression of FOXP3 in anti-IL-6 rats increased, and ROR-γt expression was suppressed. Anti-IL-6 could increase the apoptosis of the cells in the lesion. The size of the lesion was reduced. The expression of VEGF in the microenvironment of the lesions of EMS rats decreased.ConclusionsIL-6 regulated Th17/Treg cells to restore the local microenvironment of EMS. Anti-IL-6 could regulate the immune microenvironment of EMS rats to alleviate the progression of EMS disease.

Luteolin Induced Hippocampal Neuronal Pyroptosis Inhibition by Regulation of miR-124-3p/TNF-α/TRAF6 Axis in Mice Affected by Breast-Cancer-Related Depression.

Background Breast-cancer-related depression (BCRD) is associated with an increased mortality rate among breast cancer (BC) survivors. Luteolin has many pharmacological effects, particularly in the treatment of BC. In this study, we aimed to explore the anti-BCRD activity of luteolin and its underlying functional mechanism. Methods A BCRD mouse model was induced by injecting 4T1 cells and corticosterone (COR). Behavioral test, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Nissl staining, immunofluorescence, reverse-transcription quantitative PCR (RT-qPCR), and western blotting were used to study the effect of luteolin in mice with BCRD in vivo. A COR-induced neuron injury model was established in HT-22 cells in vitro. The role of miR-124-3p in the anti-BCRD effects of luteolin was studied using a miR-124-3p inhibitor. Results Luteolin significantly reduced the size and weight of the tumor, increased the mice entry frequency in the symmetrical sector, and reduced the duration of immobility in the tail suspension and forced swimming tests of mice affected by BCRD. Simultaneously, apoptosis of hippocampal neurons was inhibited, and the number of Nissl bodies increased with luteolin treatment. In addition, luteolin resulted in the upregulation of miR-124-3p expression in the hippocampus and downregulated the expression of tumor necrosis factor-α (TNF-α) and TNF receptor-associated factor 6 (TRAF6), as well as lowered the phosphorylation levels of nuclear factor-kappa B (NF-κB) and IkappaB (IκB). Luteolin also inhibited pyroptosis of hippocampal neurons in mice affected by BCRD, as revealed by the low protein levels of NOD-like receptor protein 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), interleukin (IL)-1β, and IL-18. However, the miR-124-3p inhibitor significantly reversed the therapeutic effect of luteolin on COR-induced HT-22 cells. Conclusion Our study demonstrated that the anti-BCRD function of luteolin was mediated by regulating the miR-124-3p/TNF-α/TRAF6-related pathway and inhibiting neuronal cell pyroptosis and subsequent inflammation. Therefore, luteolin may be a potential drug candidate in the treatments of BCRD.

Increased expression of prokineticin 2 and its receptor in endometrium of recurrent implantation failure patients decreased the expression of MMP9 important for decidualization

BackgroundStudies have shown that abnormalities in the decidualization process were closely related to recurrent implantation failure (RIF). Prokineticin 2 (PK2) is a secreted protein with angiogenic and tissue remodeling functions but its role in the endometrium is unknown.MethodsPK2 levels and its receptor PKR1 mRNA and protein levels in mid-secretory endometrium from normal and RIF women were examined by real-time PCR and western blotting, respectively. The effects of PK2 were evaluated by overexpressed PK2 in immortalized endometrial T-HESC cells using lentivirus vector and found different expression of Matrix metalloproteinase 9(MMP9) and lncRNA LUCAT1 by RNA-sequencing. The ability of PK2 to regulate LUCAT1 and MMP9 was verified in endometrial cells by real-time PCR and western blotting.ResultsUsing endometrial biopsies from normal and RIF patients, we found increased expression of PK2, together with its receptor PKR1 in RIF patients. We then overexpressed PK2 in immortalized endometrial T-HESC cells using lentivirus vector and found decreased expression of Matrix metalloproteinase 9(MMP9), and increased expression of lncRNA LUCAT1. We verified the ability of PK2 to stimulate LUCAT1 and decrease MMP9 in endometrial cells. We further demonstrated that increased expression of a long noncoding RNA LUCAT1 and decreased expression of MMP9 in endometrial biopsies of patients with RIF. Thus, we highlighted the important role of PK2 and its receptor PKR1 in decidualization and RIF.ConclusionProkineticin 2 and its receptor are important in endometrium decidualization. PK2 may affect endometrial decidualization through the LUCAT1- MMP9 pathway, thereby affecting embryo implantation.

Abstract 114: The Epigenetic Enzyme KMT2A/MLL1 Is A Driver Of Coronavirus Associated Coagulopathy

Objectives: Coronavirus associated coagulopathy (CAC) is postulated to be driven by systemic macrophage activation after SARS-CoV-2 infection and presents with elevated risk of thrombogenesis and hyperfibrinolysis. Previous work shows that the histone methyltransferase KMT2A/MLL1 is a key mediator of inflammatory signaling in monocytes and macrophages (Mo/Mϕs). In this study, we sought to identify the regulation of factors important in CAC by MLL1. Methods: Mice with myeloid specific knockout of MLL1 (Cre+) and littermate controls (Cre-) underwent intranasal inoculation of 2 x 10 5 pfu of the murine coronavirus MHVA59, an established model which phenocopies SARS-CoV-2 infection. Splenic Mϕs (surrogate for circulating Mo/Mϕs) were isolated and RNA and protein levels of urokinase (Plau; profibrinolytic), urokinase receptor (Plaur; profibrinolytic), and tissue factor (F3/TF; procoagulant) were analyzed using qRT-PCR and ELISA, respectively. Thromboelastography (TEG) on whole blood and urokinase activity assays from mouse plasma were performed. Urokinase and TF activity assays were performed on plasma from human samples. Results: RNA (top panel) and protein (bottom) levels of Plau, Plaur, and F3 were suppressed in the Splenic Mϕs harvested from sham (intranasal PBS) and infected Cre+ animals (white bars) compared to Splenic Mϕs harvested from Cre- animals (blue bars; Fig. 1A). Cre- mice displayed a shortened R-time (reaction time) as measured by TEG (Fig. 1B) and elevated plasma urokinase activity levels (not shown). Hospitalized COVID-positive patients (hCOV+) displayed elevated plasma urokinase and TF activity levels (Fig. 1C). Conclusions: We identify a role for MLL1 for basal expression and for coronavirus-mediated induction of factors important for fibrinolysis and coagulation in murine Mo/Mϕs and in driving coagulopathy. Our results suggest that MLL1 blockade may be an attractive strategy to combat coronavirus associated coagulopathy.

Hempseed (Cannabis sativa) Peptide H3 (IGFLIIWV) Exerts Cholesterol-Lowering Effects in Human Hepatic Cell Line.

Hempseed (Cannabis sativa) protein is an important source of bioactive peptides. H3 (IGFLIIWV), a transepithelial transported intestinal peptide obtained from the hydrolysis of hempseed protein with pepsin, carries out antioxidant and anti-inflammatory activities in HepG2 cells. In this study, the main aim was to assess its hypocholesterolemic effects at a cellular level and the mechanisms behind this health-promoting activity. The results showed that peptide H3 inhibited the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in vitro in a dose-dependent manner with an IC50 value of 59 μM. Furthermore, the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, followed by the increase of low-density lipoprotein (LDL) receptor (LDLR) protein levels, was observed in human hepatic HepG2 cells treated with peptide H3 at 25 µM. Meanwhile, peptide H3 regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Consequently, the augmentation of the LDLR localized on the cellular membranes led to the improved ability of HepG2 cells to uptake extracellular LDL with a positive effect on cholesterol levels. Unlike the complete hempseed hydrolysate (HP), peptide H3 can reduce the proprotein convertase subtilisin/kexin 9 (PCSK9) protein levels and its secretion in the extracellular environment via the decrease of hepatic nuclear factor 1-α (HNF1-α). Considering all these evidences, H3 may represent a new bioactive peptide to be used for the development of dietary supplements and/or peptidomimetics for cardiovascular disease (CVD) prevention.

Oxytocin inhibits hindpaw hyperalgesia induced by orofacial inflammation combined with stress.

Oxytocin (OT) is recognized as a critical neuropeptide in pain-related disorders. Chronic pain caused by the comorbidity of temporomandibular disorder (TMD) and fibromyalgia syndrome (FMS) is common, but whether OT plays an analgesic role in the comorbidity of TMD and FMS is unknown. Female rats with masseter muscle inflammation combined with 3-day forced swim (FS) stress developed somatic hypersensitivity, which modeled the comorbidity of TMD and FMS. Using this model, the effects of spinal OT administration on mechanical allodynia and thermal hyperalgesia in hindpaws were examined. Furthermore, the protein levels of OT receptors and 5-HT2A receptors in the L4–L5 spinal dorsal horn were analyzed by Western blot. The OT receptor antagonist atosiban and 5-HT2A receptor antagonist ritanserin were intrathecally injected prior to OT injection in the separate groups. Intrathecal injection of 0.125 μg and 0.5 μg OT attenuated the hindpaw hyperalgesia. The expression of OT receptors and 5-HT2A receptors in the L4–L5 spinal dorsal horn significantly increased following intrathecal injection of 0.5 μg OT. Intrathecal administration of either the OT receptor antagonist atosiban or 5-HT2A receptor antagonist ritanserin blocked the analgesic effect of OT. These results suggest that OT may inhibit hindpaw hyperalgesia evoked by orofacial inflammation combined with stress through OT receptors and/or 5-HT2A receptors, thus providing a therapeutic prospect for drugs targeting the OT system and for patients with comorbidity of TMD and FMS.

Correlation Between Plasma Proteomics and Adverse Outcomes Among Older Men With Chronic Coronary Syndrome.

BackgroundChronic coronary syndrome (CCS) is a newly proposed concept and is hallmarked by more long-term major adverse cardiovascular events (MACEs), calling for accurate prognostic biomarkers for initial risk stratification.MethodsData-independent acquisition liquid chromatography tandem mass spectrometry (DIA LC-MS/MS) quantitative proteomics was performed on 38 patients with CCS; 19 in the CCS events group and 19 in the non-events group as the controls. We also developed a machine-learning-based pipeline to identify proteins as potential biomarkers and validated the target proteins by enzyme-linked immunosorbent assay in an independent prospective cohort.ResultsFifty-seven differentially expressed proteins were identified by quantitative proteomics and three final biomarkers were preliminarily selected from the machine-learning-based pipeline. Further validation with the prospective cohort showed that endothelial protein C receptor (EPCR) and cholesteryl ester transfer protein (CETP) levels at admission were significantly higher in the CCS events group than they were in the non-events group, whereas the carboxypeptidase B2 (CPB2) level was similar in the two groups. In the Cox survival analysis, EPCR and CETP were independent risk factors for MACEs. We constructed a new prognostic model by combining the Framingham coronary heart disease (CHD) risk model with EPCR and CETP levels. This new model significantly improved the C-statistics for MACE prediction compared with that of the Framingham CHD risk model alone.ConclusionPlasma proteomics was used to find biomarkers of predicting MACEs in patients with CCS. EPCR and CETP were identified as promising prognostic biomarkers for CCS.

Membrane-Free Stem Cell Extract Enhances Blood-Brain Barrier Integrity by Suppressing NF-κB-Mediated Activation of NLRP3 Inflammasome in Mice with Ischemic Stroke.

Membrane-free stem cell extract (MFSCE) of human adipose tissues possesses various biological activities. However, the effects of MFSCE on blood–brain barrier dysfunction and brain damage are unknown. In this study, we determined the role of MFSCE in an ischemic stroke mouse model. Mice were treated with MFSCE once daily for 4 days and 1 h before ischemic damage. Experimental ischemia was induced by photothrombosis. Pretreatment with MFSCE reduced infarct volume and edema and improved neurological, as well as motor functions. Evans blue leakage and water content in the brain tissue were reduced by MFSCE pretreatment relative to those in the vehicle group. MFSCE increased the expression of the tight junction proteins zonula occludens 1 and claudin-5, as well as vascular endothelial-cadherin, but decreased that of matrix metalloproteinase 9. Notably, MFSCE treatment decreased cell death and the level of NOD-like receptor protein 3 inflammasome, consistent with the downregulated expression of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 in the ischemic brain. These effects might have occurred via the suppression of the expression of Toll-like receptor 4 and activation of nuclear factor-κB. The results highlighted the potential of MFSCE treatment as a novel and preventive strategy for patients at a high risk of ischemic stroke.

Nutritional digestion and absorption, metabolism fates alteration was associated with intestinal function improvement by dietary threonine in juvenile grass carp (Ctenopharyngodon Idella)

This study was designed to examine the effects of threonine (Thr) on digestion and absorption, serum biochemical parameters, genes expression of Thr metabolism related enzymes and intestinal mucins as well as potential regulation in juveniles grass carp (Ctenopharyngodon idella). Juveniles (9.53 ± 0.02 g) were reared for eight weeks with graded Thr levels (3.99, 7.70, 10.72, 14.10, 17.96 and 21.66 g/kg). Results showed that, comparing with Thr-insufficient diet (3.99 g/kg), optimal dietary Thr group (14.10 g/kg) significantly (P < 0.05) increased the digestive enzymes (trypsin, lipase and amylase) in the hepatopancreas and intestine, absorptive related enzymes Na+, K+-ATPase (NKA), alkaline phosphatase (AKP), creatine kinase (CK) and gamma-glutamyl transpeptidase (γ-GT) in intestines and contents of total protein (TP), albumin (ALB) and globulin (GLB) in the serum of juvenile grass carp. The notable decreases of serum total cholesterol (TC) content and activities of aspartate transaminase (AST) as well as alanine transaminase (ALT) were observed in juveniles fed 14.10 g/kg Thr diet (P < 0.05). Moreover, L-threonine dehydrogenase (tdh) was the main responsible for Thr catabolism enzyme with highly expressed in the hepatopancreas, which could be upregulated by increment dietary Thr in both hepatopancreas and intestines significantly (P < 0.05); L-threonine ammonia-lyase (thal) mRNA abundance was upregulated by dietary Thr in the hepatopancreas but not intestines; L-threonine aldolase (tha) mRNA abundance was not affected by dietary Thr in hepatopancreas and intestines (P > 0.05). Besides, the optimal Thr level promoted intestinal mucin synthesis involved enzyme N-acetyl-galactosaminyltransferase 2 (galnt2), mucin2 (muc-2) and muc-3 mRNA transcript abundances as well as phosphorylated protein levels of epidermal growth factor receptor (EGFR) (Tyr992), extracellular regulating kinase 1/2 (ERK1/2) (Thr202/Tyr204) and transcription factor SP1 (Thr453), thus enhancing the intestinal function. Additionally, the optimal dietary Thr could reverse the intestinal villus morphology structure destroyed with villus twist and exfoliation by dietary Thr deficiency as well as fusion by excess level of dietary Thr, which was in accordance with the improvement of intestinal function.

In Vitro and In Vivo Effects of the Urokinase Plasminogen Activator Inhibitor WX-340 on Anaplastic Thyroid Cancer Cell Lines.

Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.

Apolipoprotein E Isoform-specific changes related to stress and trauma exposure

Post-Traumatic Stress Disorder (PTSD) is a highly prevalent mental health disorder. Due to the high level of variability in susceptibility and severity, PTSD therapies are still insufficient. In addition to environmental exposures, genetic risks play a prominent role and one such factor is apolipoprotein E. The protein (apoE) is functionally involved in cholesterol transport and metabolism and exists as 3 major isoforms in humans: E2, E3, and E4. To model the role of apolipoprotein E isoform in stress-related changes in behavior and cognition, female and male mice (3–5 months of age) expressing E2, E3, or E4 were used. Mice were either placed into control groups or exposed to chronic variable stress (CVS), which has been shown to induce PTSD-like behavioral and neuroendocrine changes. E2 mice showed a unique response to CVS compared to E3 and E4 mice that included impaired spatial learning and memory, increased adrenal gland weight, and no increase in glucocorticoid receptor protein levels (normalized to apoE levels). In addition, the cholesterol metabolite 7-ketocholesterol was elevated in the cortex after CVS in E3 and E4, but not E2 female mice. E2 confers unique changes in behavioral, cognitive, and biomarker profiles after stress exposure and identify 7-ketocholesterol as a possible novel biomarker of the traumatic stress response. We further explored the relationship between E2 and PTSD in an understudied population by genotyping 102 patients of Cambodian and Vietnamese ethnicity. E2 carriers demonstrated a higher odds ratio of having a PTSD diagnosis compared to E3/E3 carriers, supporting that the E2 genotype is associated with PTSD diagnosis after trauma exposure in this population.