Cannabinoid Receptor 2-Centric Molecular Feedback Loop Drives Necroptosis in Diabetic Heart Injuries.

BACKGROUND: Diabetic heart dysfunction is a common complication of diabetes. Cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise time to intervene of particular cell death type remain largely unknown in the diabetic heart. This study aims to identify the particular cell death type that is responsible for diabetic heart dysfunction and to propose a promising therapeutic strategy by intervening in the cell death pathway. METHODS: Type 2 diabetes models were established using db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced mice. The type 1 diabetes model was established in streptozotocin-induced mice. Apoptosis and programmed cell necrosis (necroptosis) were detected in diabetic mouse hearts at different ages. G protein-coupled receptor-targeted drug library was searched to identify potential receptors regulating the key cell death pathway. Pharmacological and genetic approaches that modulate the expression of targets were used. Stable cell lines and a homemade phosphorylation antibody were prepared to conduct mechanistic studies. RESULTS: Necroptosis was activated after apoptosis at later stages of diabetes and was functionally responsible for cardiac dysfunction. Cannabinoid receptor 2 (CB2R) was a key regulator of necroptosis. Mechanically, during normal glucose levels, CB2R inhibited S6 kinase-mediated phosphorylation of BACH2 at serine 520, thereby leading to BACH2 translocation to the nucleus, where BACH2 transcriptionally repressed the necroptosis genes Rip1, Rip3, and Mlkl. Under hyperglycemic conditions, high glucose induced CB2R internalization in a ß-arrestin 2-dependent manner; thereafter, MLKL (mixed lineage kinase domain-like), but not receptor-interacting protein kinase 1 or 3, phosphorylated CB2R at serine 352 and promoted CB2R degradation by ubiquitin modification. Cardiac re-expression of CB2R rescued diabetes-induced cardiomyocyte necroptosis and heart dysfunction, whereas cardiac knockout of Bach2 diminished CB2R-mediated beneficial effects. In human diabetic hearts, both CB2R and BACH2 were negatively associated with diabetes-induced myocardial injuries. CONCLUSIONS: CB2R transcriptionally repressed necroptosis through interaction with BACH2; in turn, MLKL formed a negative feedback to phosphorylate CB2R. Our study provides the integrative view of a novel molecular mechanism loop for regulation of necroptosis centered by CB2R, which represents a promising alternative strategy for controlling diabetic heart dysfunction.

Increased Secreted Frizzled-Related Protein 2 in Hypertension-Induced Left Ventricular Remodeling.

Background: Secreted frizzled-related protein 2 (sFRP2) is involved in various cardiovascular diseases. However, its relevance in left ventricular (LV) remodeling in patients with hypertension (HTN) is obscure. Methods: In this study, 196 patients with HTN were included, 59 with echocardiographic LV remodeling. A total of 100 healthy subjects served as normal controls. The serum-sFRP2 level was measured by enzyme-linked immunosorbent assay (ELISA). Data were collected from medical records for baseline characteristics, biochemistry tests, and echocardiography. Receiver operating characteristic (ROC) curves were used to assess the distinguishing value of sFRP2 for LV remodeling in patients with HTN. Spearman rank correlation analysis was utilized to identify factors correlated with sFRP2. Cardiac sFRP2 was determined by Western blot and quantitative polymerase chain reaction (qPCR). Results: The level of serum-sFRP2 was higher in HTN patients with echocardiographic LV remodeling than their non-remodeling counterparts. ROC analysis showed that the area under the curve (AUC) for sFRP2 in distinguishing echocardiographic LV remodeling in HTN patients was 0.791 (95% confidence interval (CI): 0.714-0.869). The sFRP2 was negatively correlated with LV dimension and positively correlated with relative wall thickness (RWT). The expression of sFRP2 was higher in hypertrophic hearts, which could be reversed by myricetin. Conclusions: The serum level and cardiac sFRP2 increased in the setting of LV remodeling and decreased by myricetin. Serum sFRP2 may be a promising distinguishing factor for LV remodeling in HTN patients.

GADD34-mediated dephosphorylation of eIF2α facilitates pseudorabies virus replication by maintaining de novo protein synthesis.

Viruses have evolved multiple strategies to manipulate their host's translational machinery for the synthesis of viral proteins. A common viral target is the alpha subunit of eukaryotic initiation factor 2 (eIF2α). In this study, we show that global protein synthesis was increased but the eIF2α phosphorylation level was markedly decreased in porcine kidney 15 (PK15) cells infected with pseudorabies virus (PRV), a swine herpesvirus. An increase in the eIF2α phosphorylation level by salubrinal treatment or transfection of constructs expressing wild-type eIF2α or an eIF2α phosphomimetic [eIF2α(S51D)] attenuated global protein synthesis and suppressed PRV replication. To explore the mechanism involved in the inhibition of eIF2α phosphorylation during PRV infection, we examined the phosphorylation status of protein kinase R-like endoplasmic reticulum kinase (PERK) and double-stranded RNA-dependent protein kinase R (PKR), two kinases that regulate eIF2α phosphorylation during infection with numerous viruses. We found that the level of neither phosphorylated (p)-PERK nor p-PKR was altered in PRV-infected cells or the lungs of infected mice. However, the expression of growth arrest and DNA damage-inducible protein 34 (GADD34), which promotes eIF2α dephosphorylation by recruiting protein phosphatase 1 (PP1), was significantly induced both in vivo and in vitro. Knockdown of GADD34 and inhibition of PP1 activity by okadaic acid treatment led to increased eIF2α phosphorylation but significantly suppressed global protein synthesis and inhibited PRV replication. Collectively, these results demonstrated that PRV induces GADD34 expression to promote eIF2α dephosphorylation, thereby maintaining de novo protein synthesis and facilitating viral replication.

Comparative pathogenicity of two subtypes (hepatitis-type and pancreatitis-type) of duck hepatitis A virus type 1 in experimentally infected Muscovy ducklings.

Duck hepatitis A virus type 1 (DHAV-1) causes acute hepatitis with high morbidity and mortality in ducklings of the genera Cairina and Anas and is characterized by ecchymotic haemorrhage and necrosis of the liver surface. Since September 2011, a new subtype of DHAV-1 (named pancreatitis-type DHAV-1) has been isolated. This new subtype is characterized by yellowish or haemorrhagic pancreatitis, but with no significant pathological changes in the liver. To further investigate the difference in pathogenicity between hepatitis-type DHAV-1 and pancreatitis-type DHAV-1, we infected Muscovy ducklings with a hepatitis-type DHAV-1 strain, FZ86, or a pancreatitis-type DHAV-1 strain, MPZJ1206, and then compared the resulting gross lesions, histopathological changes, viral distribution and cellular apoptosis in the liver and pancreas of Muscovy ducklings. The results suggested that FZ86 induced a more efficient viral propagation in the liver than MPZJ1206, and the gross and histopathological lesions were also limited to the liver. However, MPZJ1206 induced more effective viral replication in the pancreas than FZ86. The MPZJ1206-infected Muscovy ducklings showed an obviously yellowed and haemorrhagic pancreas, but with no significant pathological changes in the liver. Furthermore, FZ86 induced notable hepatocyte apoptosis and increased the expression of caspase-3 in the liver, whereas MPZJ1206 caused apoptosis in a large number of acinar epithelial cells and elevated the expression of caspase-3 in the pancreas. Taken together, these results demonstrated that pancreatitis-type DHAV-1 has many new pathogenic features which distinguish it from the hepatitis-type DHAV-1. RESEARCH HIGHLIGHTS Pancreatitis-type DHAV-1 (MPZJ1206) was characterized by pancreatic haemorrhage and yellow discolouration, but with no obvious haemorrhage and necrosis in the liver. Pancreatitis-type DHAV-1 (MPZJ1206) exhibits many new pathogenic features which distinguish it from the hepatitis-type DHAV-1 (FZ86).

Role of GPER on proliferation, migration and invasion in ligand-independent manner in human ovarian cancer cell line SKOV3.

G protein-coupled estrogen receptor (GPER) is identified as a critical estrogen receptor, in addition to the classical estrogen receptors ERα and ERß. In ERα-negative ovarian cancer cells, our previous studies have found that estrogen stimulated cell proliferation and metastasis via GPER. However, the ligand-independent function of GPER in ovarian cancer cells is still not clear. Herein, we describe that GPER has a co-expression with ERα and ERß, which are first determined in SKOV3 ovarian cancer cell line. In the absence of estrogen, GPER depletion by specific siRNA inhibits the proliferation, migration and invasion of SKOV3 cells. Whereas abrogation of ERα or ERß by specific antagonist MPP and PHTPP has the opposite effects for stimulation of cell growth. Markedly, GPER knockdown attenuates MPP or PHTPP-induced cell proliferation, migration and invasion. Furthermore, GPER modulates protein expression of the cell cycle critical components, c-fos and cyclin D1 and factors for cancer cell invasion and metastasis, matrix metalloproteinase 2 (MMP-2) and MMP-9. These findings establish that GPER ligand-independently stimulates the proliferation, migration and invasion of SKOV3 cells. Knockdown of GPER attenuates the progression of ovarian cancer that caused by functional loss of ERα or ERß. Targeting GPER provides new aspect as a potential therapeutic strategy in ovarian cancer.

A novel estrogen receptor GPER mediates proliferation induced by 17ß-estradiol and selective GPER agonist G-1 in estrogen receptor α (ERα)-negative ovarian cancer cells.

G protein-coupled estrogen receptor (GPER) is recently identified as a membrane-associated estrogen receptor that mediates non-genomic effects of estrogen. Our previous immunohistochemistry study found an association between GPER and the proliferation of epithelial ovarian cancer. However, the contributions and mechanisms of GPER in the proliferation of ovarian cancers are not clear. We have examined the role of GPER in estrogen receptor α (ERα)-negative/GPER positive OVCAR5 ovarian cancer cell line. MTT assay was used to detect cell proliferation. BrdU incorporation assay was used to measure the cells in S-phase. Protein expression of marker genes of proliferation, cell cycle and apoptosis were examined by Western blot. The results showed that 17ß-estradiol and selective GPER agonist G-1 stimulated the proliferation of OVCAR5 cells and increased the cells in S-phase. Both ligands upregulated the protein levels of c-fos and cyclin D1. Small interfering RNA targeting GPER or G protein inhibitor pertussin toxin (PTX) inhibited basal cell proliferation and attenuated 17ß-estradiol- or G-1-induced cell proliferation. GPER mediated cell growth was also associated with the apoptosis of OVCAR5 cells. These findings suggest that GPER has an important function in the proliferation of ovarian cancer cells lacking ERα. GPER might be a promising therapeutic target in ovarian cancer.

Targeting RXFP1 by Ligustilide: A novel therapeutic approach for alcoholic hepatic steatosis.

BACKGROUND: Ligustilide (Lig) is the main active ingredient of Umbelliferae Angelicae Sinensis Radix (Chinese Angelica) and Chuanxiong Rhizoma (Sichuan lovase rhizome). Lig possesses various pharmacological properties and could treat obesity by regulating energy metabolism. However, the impact and regulatory mechanism of Lig on alcoholic hepatic steatosis remains unclear. PURPOSE: This study aimed to explore the therapeutic effect of Lig on alcoholic hepatic steatosis and its related pharmacological mechanism. RESULTS: With chronic and binge ethanol feeding, liver tissue damage and lipid accumulation in mice suffering alcoholic hepatic steatosis were significantly improved after Lig treatment. Lig effectively regulated the expression levels of lipid metabolism-related proteins in alcoholic hepatic steatosis. In addition, Lig reduced RXFP1 expression, inhibited the activation of NLRP3 inflammasome, and blocked NET formation. Lig reduced the infiltration of immune cells to the liver and the further prevented the occurrence of alcohol-stimulated inflammatory response in liver. Lig significantly regulated lipid accumulation in alcohol exposed AML12 cells via modulating PPARα and SREBP1. In MPMs, Lig decreased the expression of RXFP1, inhibited the activation of NLRP3 in macrophages stimulated by LPS/ATP, and slowed down the occurrence of inflammatory response. CONCLUSION: Lig sustained lipid metabolism homeostasis in alcoholic hepatic steatosis, through inhibiting the activation of NLRP3 inflammasomes and the formation of NETs, especially targeting RXFP1 in macrophages.

The novel estrogen receptor GPER regulates the migration and invasion of ovarian cancer cells.

G protein-coupled estrogen receptor (GPER) was identified as a new member of the estrogen receptor family in recent years. It has become apparent that GPER mediates the non-genomic signaling of 17ß-estradiol (E2) in a variety of estrogen-related cancers. Our previous study has found that GPER was overexpressed in human epithelial ovarian cancer and was positively correlated with the expression of matrix metalloproteinase 9 (MMP-9), which suggested GPER might promote the metastasis of ovarian cancer. However, the mechanisms underlying GPER-dependent metastasis of ovarian cancer are still not clear. In the present study, estrogen receptor α (ERα)-negative/GPER-positive OVCAR5 ovarian cancer cell line was used to investigate the role of GPER in the migration and invasion of ovarian cancer. Wound healing assay and transwell matrigel invasion assay were performed to determine the potentials of cell migration and invasion, respectively. The production and activity of MMP-9 in OVCAR5 cells were examined by Western blot and gelatin zymography analysis. The results showed that E2 and selective GPER agonist G-1 increased cell motility and invasiveness, and upregulated the production and proteolytic activity of MMP-9 in OVCAR5 cells. Small interfering RNA (siRNA) targeting GPER and G protein inhibitor pertussin toxin (PTX) inhibited the migration and invasion of OVCAR5 cells, and also reduced the expression and activity of MMP-9. Our data suggested that GPER promoted the migration and invasion of ovarian cancer cells by increasing the expression and activity of MMP-9. GPER might play an important role in the progression of ovarian cancer.

An Empirical Study on Physical Subhealth Risk Perception: A Physical Examination Data of Tertiary Grade-A Hospitals in Anhui Province, China.

Background: Physical subhealth directly correlates to people's work effectiveness and quality of life, so subhealth prevention has become an urgent medical problem. Methods: A random sampling method was used to conduct a questionnaire survey of physical examinees from June to September, 2019. In total, 770 people participated in our study. The Pearson correlation and multiple stepwise regression analysis were used to explore the relationship among demographic variables, physical subhealth, and risk perception. Also, this study used a two-way interaction moderated multiple regression approach to examine the moderating effects of demographic variables on physical subhealth and risk perception. Results: The risk perception level was negatively associated with physical health. Age, education level, and subhealth proportion in the work unit all significantly and positively influence physical health, whereas living place, subhealth duration, and marital status negatively influence physical health. Living place, average annual household income, number of employees in the work unit, and subhealth proportion in the work unit significantly and positively influence the risk perception, and only age negatively influences the risk perception. The number of children had a moderating effect on physical subhealth and risk perception (Interaction coefficient α = -0.3, P < 0.05). Conclusions: To achieve the overall improvement of public health, relevant management departments can provide targeted interventions for the public with different levels of risk perception. Also, the physical subhealth of the public can be addressed by encouraging the public to attach importance to education, improving the public living environment to build a livable city, strengthening psychological guidance and intervention for couples heading toward divorce to reduce the divorce rate, focusing on the health of work unit employees and regularly organizing employees to attend medical checkups, and actively responding to the national policy of family planning.

A matter of the beehive sound: Can honey bees alert the pollution out of their hives?

Honey bees (Apis spp.) are often used as biological indicators of environmental changes. Recently, bees have been explored to monitor air contaminants by listening to the beehive sound. The beehive sound is believed to encode information on bee responses to chemicals outside their hives. Here we conducted an experiment to address this. First, we randomly fed colonies with pure syrup (PS), acetone-laced syrup (AS), or ethyl acetate-laced syrup (ES) in front of the beehives and collect the beehive sound. Based on the audio data, we build machine learning (ML) models to identify the types of syrup. The result shows that ML models achieved over 90% accuracy for identifying syrup types, indicating that the bees inside their hives emitted the sound associated with the chemicals outside their hives. Then, we sequentially fed the colonies in the order of PS, ES, and AS (the first session) and again in the reverse order (the second session), but did not remove the accumulated ES or AS in the alternative feeding experiment. Based on the audio data, the identification accuracy of both ES and AS by the ML model built on the randomly feeding experiment was different, indicating that the accumulated chemical residuals could confuse the ML models. Therefore, the beehive sound-based environmental monitoring should simultaneously consider the responses of bees to the chemicals outside their hives and their responses to those accumulated inside their hives, which may act simultaneously.

Carnosic acid suppressed the formation of NETs in alcoholic hepatosteatosis based on P2X7R-NLRP3 axis.

BACKGROUND: Alcoholic liver disease (ALD) is accompanied by a disruption of lipid metabolism and an inflammatory response in the liver during the process of disease. Carnosic acid (CA), a natural diterpene extracted from Rosmarinus officinalis (rosemary) and Salvia officinalis (sage), has more pharmacological activities, which is known to be useful in the treatment of obesity and acts by regulating energy metabolism. However, the role and regulation mechanism of CA against ALD remain unclear. HYPOTHESIS: We hypothesized that CA might improve alcoholic-induced hepatosteatosis. STUDY DESIGN AND METHODS: The alcoholic liver disease model was established a mouse chronic ethanol feeding by Lieber-DeCarli control liquid feed (10 d) plus a single binge with or without CA administration. AML12 cells were exposed to ethanol for 24 h. Murine peritoneal macrophages (MPM) were stimulated with LPS and ATP. RESULTS: CA ameliorated lipid accumulation in the liver of mice in the NIAAA model, acting by inhibiting the expression of genes related to lipid synthesis. CA reduced alcohol-induced immune cell infiltration in the liver, and inhibited the activation of P2X7R-NLRP3 inflammasome, meanwhile blocked the formation of NETs in mouse livers tissue. In AML12 cells, CA attenuated the lipid accumulation triggered by ethanol stimulation, which was achieved by inhibiting the expression of SREBP1 and CA reduced the release of inflammatory factor IL-1ß by inhibiting the activation of P2X7R-NLRP3. In MPM, IL-1ß and HMGB1 were reduced after LPS/ATP stimulation in CA-treated cells and supernatant. CONCLUSIONS: CA attenuated alcohol-induced fat accumulation, suppressed the formation of NETs based on P2X7R-NLRP3 axis in mouse livers. Our data indicated that CA exerted hepatoprotective effects, which might be a promising candidate.

Protections of transcription factor BACH2 and natural product myricetin against pathological cardiac hypertrophy and dysfunction.

Pathological hypertrophic myocardium under consistent adverse stimuli eventually can cause heart failure. This study aims to explore the role of BACH2, a member of the basic region leucine zipper transcription factor family, in cardiac hypertrophy and failure. Transverse aortic constriction surgery was operated to induce cardiac hypertrophy and failure in mice. BACH2 was overexpressed in mice through tail vein injection of AAV9-Bach2. Mice with systemic or cardiac-specific knockdown of Bach2 were adopted. Neonatal rat ventricular myocytes (NRVMs) were isolated and infected with lentivirus to overexpress Bach2 or transfected with siRNA to knock down Bach2. Our data showed that overexpression of BACH2 ameliorated TAC-induced cardiac hypertrophy and failure in mice and decreased isoproterenol (ISO)-triggered myocyte hypertrophy in NRVMs. Systemic or cardiac-specific knockdown of Bach2 worsened the cardiac hypertrophy and failure phenotype in mice. Further assays showed that BACH2 bound to the promotor region of Akap6 at the -600 to -587 site and repressed its expression, which functioned as a crucial scaffold for cardiac hypertrophy and failure signaling pathways. Small molecular natural product library screening suggested that myricetin could up-regulate expression of Bach2 and simultaneously suppress the transcriptional levels of hypertrophic marker genes Bnp and Myh7. Further studies showed that myricetin exerted a BACH2-dependent protective effect against cardiac hypertrophy in vivo and in vitro. Taken together, our findings demonstrated that BACH2 plays a crucial role in the regulation of cardiac hypertrophy and failure and can be a potential therapeutic target in the future.

Subhealth Risk Perception Scale: Development and Validation of a New Measure.

BACKGROUND: To develop an individual's physical subhealth risk perception scale and evaluate its reliability and validity, so as to provide a measurement tool for individual physical health risk. METHODS: A questionnaire on the perception risk of physical subhealth was developed. Using a random sampling method, 785 people in the Anhui provincial physical examination centre were selected as the research participants. Of the questionnaires returned, 770 were valid, giving an effective rate of 98%. Firstly, the Pearson correlation coefficient method was used to study the correlation of 35 items in the initial scale, and then, polychoric factor structure analysis was carried out by using the Pratt D matrix to optimize the item structure. The Cronbach'α coefficient method was used to test the internal consistency reliability, and a structural equation model was used to explore the construct validity of the scale. The discriminant validity of the scale was obtained by factor analysis. A general linear model was used to analyse the relationship between the clinical manifestations of physical subhealth and the level of risk perception, and the convergent validity of the scale was evaluated. RESULTS: All the data of 35 items were significantly correlated at the 0.01 level. The correlation coefficients between a1 and a2, a3 and a4, b1 and b2, b2 and b3, c4 and c5, c5 and c6, c6 and c7, c8 and c9, d1 and d2, d2 and d3, e5 and e6, g1 and g2, g2 and g3, and g2 and g4 were greater than 0.6. The items with correlation coefficients greater than 0.6 were reduced by a Pratt D matrix. The resulting physical subhealth risk perception scale covers five factors with a total of 18 items. The Cronbach'α coefficient of the scale was 0.889, and the Cronbach'α coefficients of the five factors F1-F5 were 0.780, 0.825, 0.801, 0.736, and 0.704, respectively. Structural equation model analysis showed that χ 2/df = 3.43, p < 0.001, RMSEA = 0.08, GFI = 0.88, NFI = 0.84, AGFI = 0.84, and CFI = 0.88. Factor analysis showed that factors F1-F5 had significant correlations (p < 0.01), and the correlation coefficients were less than the corresponding square root value of AVE. Based on the subhealth clinical manifestations of the participants, the general linear model was used to explore the convergent validity of the scale, and the results indicated that the scale passed the convergent validity test. CONCLUSIONS: We propose a physical subhealth risk perception scale amounting to 18 items, which includes five dimensions: health knowledge (2 items), risk perception (5 items), trust selection (4 items), information channel (4 items), and social groups (3 items). The reliability and validity of the physical subhealth risk perception scale are acceptable. Applying the scale into practice has potential to improve the overall public health level.

Identification of Differential Expression Genes between Volume and Pressure Overloaded Hearts Based on Bioinformatics Analysis.

Volume overload (VO) and pressure overload (PO) are two common pathophysiological conditions associated with cardiac disease. VO, in particular, often occurs in a number of diseases, and no clinically meaningful molecular marker has yet been established. We intend to find the main differential gene expression using bioinformatics analysis. GSE97363 and GSE52796 are the two gene expression array datasets related with VO and PO, respectively. The LIMMA algorithm was used to identify differentially expressed genes (DEGs) of VO and PO. The DEGs were divided into three groups and subjected to functional enrichment analysis, which comprised GO analysis, KEGG analysis, and the protein-protein interaction (PPI) network. To validate the sequencing data, cardiomyocytes from AR and TAC mouse models were used to extract RNA for qRT-PCR. The three genes with random absolute values of LogFC and indicators of heart failure (natriuretic peptide B, NPPB) were detected: carboxylesterase 1D (CES1D), whirlin (WHRN), and WNK lysine deficient protein kinase 2 (WNK2). The DEGs in VO and PO were determined to be 2761 and 1093, respectively, in this study. Following the intersection, 305 genes were obtained, 255 of which expressed the opposing regulation and 50 of which expressed the same regulation. According to the GO and pathway enrichment studies, DEGs with opposing regulation are mostly common in fatty acid degradation, propanoate metabolism, and other signaling pathways. Finally, we used Cytoscape's three techniques to identify six hub genes by intersecting 255 with the opposite expression and constructing a PPI network. Peroxisome proliferator-activated receptor (PPARα), acyl-CoA dehydrogenase medium chain (ACADM), patatin-like phospholipase domain containing 2 (PNPLA2), isocitrate dehydrogenase 3 (IDH3), heat shock protein family D member 1 (HSPD1), and dihydrolipoamide S-acetyltransferase (DLAT) were identified as six potential genes. Furthermore, we predict that the hub genes PPARα, ACADM, and PNPLA2 regulate VO myocardial changes via fatty acid metabolism and acyl-Coa dehydrogenase activity, and that these genes could be employed as basic biomarkers for VO diagnosis and treatment.

Modulation of interleukin-36 based inflammatory feedback loop through the hepatocyte-derived IL-36R-P2X7R axis improves steatosis in alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Interleukin-36 is induced by proinflammatory cytokines and promotes inflammatory responses, creating an IL-36-based inflammation loop. Although hepatocytes, produce IL-36 responses to drug-induced liver injury, little is known about the mechanistic role of IL-36 signalling during the progression of alcoholic steatohepatitis (ASH). Regarding IL-36/IL-36R and P2X7R coregulating the inflammatory response, we elucidated that modulation of IL-36R-P2X7R-TLR axis affected hepatocyte steatosis as well as the IL-36-based inflammatory feedback loop that accompanies the onset of ASH. EXPERIMENTAL APPROACH: C57BL/6J mice were subjected to either chronic-plus-binge ethanol feeding or acute gavage with multiple doses of ethanol to establish ASH, followed by pharmacological inhibition or genetic silencing of IL-36R and P2X7R. AML12 cells or mouse primary hepatocytes were stimulated with alcohol, LPS plus ATP or Poly(I:C) plus ATP, followed by silencing of IL-36γ, IL-36R or P2X7R. KEY RESULTS: P2X7R and IL-36R deficiency blocked the inflammatory loop, specifically initiated by IL-36 cytokines, in hepatocytes of mice suffering from ASH. Pharmacological inhibition to P2X7R or IL-36R alleviated lipid accumulation and inflammatory response in ASH. IL-36R was indispensable for P2X7R modulated NLRP3 inflammasome activation in ASH, and IL-36 led to a vicious cycle of P2X7R-driven inflammation in alcohol-treated hepatocytes. TLR ligands promoted IL-36γ production in hepatocytes, based on synergism with P2X7R. CONCLUSIONS AND IMPLICATIONS: Blockade of IL-36 based inflammatory feedback loop, via IL-36R-P2X7R-TLRs-modulated NLRP3 inflammasome activation, circumvented steatosis and inflammation that accompanies the onset of ASH, suggesting that targeting IL-36 can serve as a novel therapeutic approach to combat ASH.

Inhibition of HMGB1/TLR4 Signaling Pathway by Digitoflavone: A Potential Therapeutic Role in Alcohol-Associated Liver Disease.

Digitoflavone (DG) is a natural flavonoid abundant in many fruits, vegetables, and medicinal plants. We investigated whether DG inhibits lipid accumulation and inflammatory responses in alcoholic liver disease (ALD) in vivo and in vitro. The mouse ALD model was established by chronically feeding male C57BL/6 mice an ethanol-containing Lieber-DeCarli liquid diet. In vitro, mouse peritoneal macrophages (MPMs) and mouse bone marrow-derived macrophages (BMDMs) were stimulated with LPS/ATP, whereas HepG2 cells and mouse primary hepatocytes were treated with ethanol. DG reduced the serum levels of transaminase and serum and hepatic levels of triglycerides and malondialdehyde in ALD mice. DG downregulated SREBP1 and its target genes and upregulated PPARα and its target genes in the liver of mice with ALD. DG inhibited TLR4-mediated NLRP3 inflammasome activation, consequently reversing the inflammatory response, including the production of HMGB1, IL-1ß, and IL-36γ, as well as the infiltration of macrophages and neutrophils. DG blocked NLRP3/ASC/caspase-1 inflammasome activation and HMGB1 release in LPS/ATP-stimulated MPMs. When Tlr4 was knocked in LPS/ATP-stimulated BMDMs, HMGB1 production and release were blocked, and NLRP3-mediated cleavage and release of IL-1ß was suppressed in Hmgb1-silenced BMDMs. DG amplified these inhibitory effects in Tlr4 or Hmgb1 knockdown BMDMs. In ethanol-exposed hepatocytes, DG reduced lipogenesis and promoted lipid oxidation by inhibiting the HMGB1-TLR4 signaling pathway while suppressing the inflammatory response induced by ethanol exposure. Our data demonstrated that DG inhibited the occurrence of lipid accumulation and the inflammatory response via the HMGB1-TLR4 axis, underscoring a promising approach and utility of DG for the treatment of ALD.

[Endovascular management of symptomatic carotid stenosis combined with kinking].

OBJECTIVE: To study the necessity, feasibility, security of carotid angioplasty and stenting (CAS) for symptomatic carotid stenosis combined with kinking. METHODS: Twelve patients with symptomatic carotid stenosis and kinking demonstrated by digital subtraction angiography (DSA) received CAS from December 2003 to December 2009. There were 9 male and 3 female patients, age ranged from 59 to 77 years (mean 69.3 years). All the patients' clinical, imaging, intervention and follow up data were collected and analyzed. RESULTS: All CAS procedures were successfully performed with 14 self-expandable stents placed. The mean degree of stenosis was reduced from 85.6% before stenting to 11.2% after stenting, the angle of kinking, according to Metz' category, were improved from less than 90° to more than 120° in each case. No perioperative procedure related stroke and transient ischemic attack (TIA) occurred. The clinical symptoms and signs of cerebral ischemia were improved or disappeared for all patients. During follow-up of these 12 patients for 6 to 72 months, one patient experienced ipsilateral carotid territory TIA and another patient experienced contralateral carotid territory TIA. DSA follow up of 5 patients demonstrated 1 case with in-stent restenosis and arterial kinking remote to the stent of internal carotid artery. CAS were performed again and CT angiography follow up demonstrated no kinking and restenosis 2 years after the intervention. Duplex scan of the other 7 patients demonstrated neither kinking nor restenosis. CONCLUSIONS: CAS seems to be feasible and safe for the patients with symptomatic kinking and stenosis, and maybe helpful to lower the risk of cerebral ischemia, but further study is needed.

Inverse Associations Between Circulating Secreted Frizzled Related Protein 2 (sFRP2) and Cardiometabolic Risk Factors.

Background: Secreted frizzled-related protein 2 (sFRP2) plays an important role in metabolic syndrome and cardiovascular diseases (CVDs); However, its relevance with cardiometabolic diseases remains to be elucidated. We aimed to determine the serum levels of sFRP2 in patients at different stages of heart failure (HF) with or without type 2 diabetes mellitus (T2DM), and assess the correlation between circulating sFRP2 levels and cardiometabolic risk factors. Methods: In this study, serum samples from 277 patients visiting Zhongshan Hospital affiliated to Fudan University were collected. These patients were clinically diagnosed and categorized as five groups, including the control group, pre-clinical HF group, pre-clinical HF+T2DM group, HF group and HF+T2DM group. Serum sFRP2 levels were measured with enzyme-linked immunosorbent assay (ELISA) tests and the clinical characteristics of each patient were recorded. Spearman rank correlation analysis and multiple stepwise linear regression analysis were conducted. Univariate and multivariate logistic regression analysis were performed to screen risk factors for HF in patients with CVDs. Results: Serum sFRP2 levels were significantly lower in the HF+T2DM group compared with the other four groups. Spearman rank correlation analysis showed that sFRP2 was negatively correlated with parameters including patients' age, fasting plasma glucose (FPG), glycated hemoglobin A1c (HbA1c), cardiac troponin T (cTNT), N-terminal pro-B-type natriuretic peptide (NT-proBNP), high-sensitivity C-reactive protein (hs-CRP), left atrial dimension (LAD) and left ventricular posterior wall (LVPW), and positively correlated with hemoglobin, estimated glomerular filtration rate (eGFR), albumin, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and left ventricular ejection fraction (LVEF). However, in multiple regression analysis, significant associations with ln(sFRP2) were observed only in FPG, hs-CRP and LAD. Higher serum sFRP2 was significantly linked to lower odds of HF in patients with CVDs. Conclusion: sFRP2 progressively decreased when glucose homeostasis and cardiac function deteriorated. sFRP2 acted as a risk factor for HF in patients with CVDs, especially in those with concomitant T2DM.

[Endovascular treatment of ruptured and unruptured intracranial very small aneurysms].

OBJECTIVE: To evaluate the feasibility and safety of endovascular treatment of ruptured and unruptured intracranial very small aneurysms (< or = 3 mm in maximal diameter). METHODS: Forty-eight intracranial very small aneurysms in 44 patients treated with endovascular therapy from June 2001 to August 2009 were reviewed retrospectively in clinical, imaging, interventional and follow-up data. Among 44 patients, there were 20 males and 24 females with a mean age of 57.8 years old. The Hunt-Kosnik grade was as follows: Grade 0 (n = 11); Grades I & II (n = 23); Grades III & IV (n = 9); and ungraded (n = 1). The sizes of 48 aneurysms were not more than 3 mm in maximal diameter. The locations of aneurysms were as follows: ACoA (n = 11), MCA (n = 8), PCoA (n = 14), ICA (n = 12), pericallosal artery (n = 1), VA (n = 1) and PICA (n = 1). Thirty-nine aneurysms were embolized with coil, of which 13 with stent assistance and 6 by balloon remodeling technique. The other 9 aneurysms underwent sole stent placement in parent artery. RESULTS: Among 39 coiling aneurysms, 100% occlusion was achieved in 9 aneurysms, 90% in 20, 80% in 9 and less than 80% in 1 respectively. Only one aneurysm ruptured during coiling. Two patients had transient hemiparesis and one patient had ataxia caused by bilateral cerebellar infarction postoperatively. All patients were clinically followed up for 4-90 months and no recurrent hemorrhage occurred. Thirteen patients received repeat angiography at 4-72 months post-treatment. And no radiological re-growth was detected. CONCLUSION: Endovascular treatment of ruptured and unruptured intracranial very small aneurysms seems to be technically feasible, relatively safe and practically effective.

[Endovascular treatment of micro-intracranial aneurysms: adverse factors of micro-catheterization and its relevant managements].

OBJECTIVES: To investigate adverse factors that may hinder successful placement and stabilization of the microcatheter during endovascular therapy of micro-intracranial aneurysms (≤ 3 mm in maximum diameter), and to explore the relevant managements. METHODS: Forty-six patients with fifty-one micro-intracranial aneurysms treated by endovascular therapy from June 2001 to October 2009 were retrospectively analyzed for their intervention data. RESULTS: Adverse factors of optimal micro-catheterization mainly included, tortuosity of the proximal vessels (PVs) and the parent artery (PA), relative large gap in diameter among the PVs, the PA and the microcatheter, relative large divergence in direction among the PVs, the PA and the aneurysm dome, and stent deployed in the PA. CONCLUSIONS: Carefully considering the direction of the PVs and the PA, the aneurysm's location and dome orientation, choosing the microcatheter and microwire after balancing among their physical properties, as well as utilizing balloon and/or stent assistance, can facilitate micro-catheterization during endovascular treatment of micro-intracranial aneurysms.