Smooth-Muscle-Cell-Specific Deletion of CD38 Protects Mice from AngII-Induced Abdominal Aortic Aneurysm through Inhibiting Vascular Remodeling.

Abdominal aortic aneurysm (AAA) is a serious vascular disease which is associated with vascular remodeling. CD38 is a main NAD+-consuming enzyme in mammals, and our previous results showed that CD38 plays the important roles in many cardiovascular diseases. However, the role of CD38 in AAA has not been explored. Here, we report that smooth-muscle-cell-specific deletion of CD38 (CD38SKO) significantly reduced the morbidity of AngII-induced AAA in CD38SKOApoe-/- mice, which was accompanied with a increases in the aortic diameter, medial thickness, collagen deposition, and elastin degradation of aortas. In addition, CD38SKO significantly suppressed the AngII-induced decreases in α-SMA, SM22α, and MYH11 expression; the increase in Vimentin expression in VSMCs; and the increase in VCAM-1 expression in smooth muscle cells and macrophage infiltration. Furthermore, we demonstrated that the role of CD38SKO in attenuating AAA was associated with the activation of sirtuin signaling pathways. Therefore, we concluded that CD38 plays a pivotal role in AngII-induced AAA through promoting vascular remodeling, suggesting that CD38 may serve as a potential therapeutic target for the prevention of AAA.

Diagnostic value of sphingolipid metabolism-related genes CD37 and CXCL9 in nonalcoholic fatty liver disease.

The development of nonalcoholic fatty liver disease (NAFLD) has been reported to be caused by sphingolipid family inducing insulin resistance, mitochondrial dysfunction, and inflammation, which can be regulated by multiple sphingolipid metabolic pathways. This study aimed to explore the molecular mechanism of crucial sphingolipid metabolism related genes (SMRGs) in NAFLD. Firstly, the datasets (GSE48452, GSE126848, and GSE63067) from the Gene Expression Omnibus database and sphingolipid metabolism genes (SMGs) from previous research were collected for this study. The differentially expressed genes (DEGs) between different NAFLD and controls were acquired through "limma," and the SMRGs were authenticated via weighted gene co-expression network analysis (WGCNA). After overlapping the DEGs and SMRGs, the causality between the intersection genes (DE-SMRGs) and NAFLD was explored to sort out the candidate biomarkers by Mendelian randomization (MR) study. The receiver operating characteristic (ROC) curves of candidate biomarkers in GSE48452 and GSE126848 were yielded to determine the biomarkers, followed by the nomogram construction and enrichment analysis. Finally, the immune infiltration analysis, the prediction of transcription factors (TFs) and drugs targeting biomarkers were put into effect. A total of 23 DE-SMRGs were acquired based on the differential analysis and weighted gene co-expression network analysis (WGCNA), of which 3 DE-SMRGs (CD37, CXCL9 and IL7R) were picked out for follow-up analysis through univariate and multivariate MR analysis. The values of area under ROC curve of CD37 and CXCL9 were >0.7 in GSE48452 and GSE126848, thereby being regarded as biomarkers, which were mainly enriched in amino acid metabolism. With respect to the Spearman analysis between immune cells and biomarkers, CD37 and CXCL9 were significantly positively associated with M1 macrophages (P < .001), whose proportion was observably higher in NAFLD patients compared with controls. At last, TFs (ZNF460 and ZNF384) of CD37 and CXCL9 and a total of 79 chemical drugs targeting CD37 and CXCL9 were predicted. This study mined the pivotal SMRGs, CD37 and CXCL9, and systematically explored the mechanism of action of both biomarkers based on the public databases, which could tender a fresh reference for the clinical diagnosis and therapy of NAFLD.

TRIM65 knockout inhibits the development of HCC by polarization tumor-associated macrophages towards M1 phenotype via JAK1/STAT1 signaling pathway.

BACKGROUND & AIMS: Tumor-associated macrophages (TAMs) are main components of immune cells in tumor microenvironment (TME), and play a crucial role in tumor progression. Tripartite motif-containing protein 65 (TRIM65) has been associated with tumor progression. However, whether TRIM65 regulate the interaction of tumor cell and TAMs in HCC and the underlying mechanisms remain unknown. In this study, we investigated the role of TRIM65 in TME of HCC and explored its underlying mechanisms. METHODS: The relation of TRIM65 expression level with tumor grades, TNM stages, and worse prognosis of HCC patients was evaluated by bioinformatics analysis, as well as immune infiltration level of macrophages. TRIM65 shRNA was transfected into HepG2 cells, and TRIM65 overexpression plasmid was transfected into Huh7 cells, and the effect of TRIM65 on cell growth was examined by EdU assay. The mouse subcutaneous Hep1-6 tumor-bearing model with WT and TRIM65-/- mice was established to study the role of TRIM65 in HCC. Immunohistochemistry staining, Immunofluorescence staining, qRT-PCR and western blot were performed to evaluate the effect of TRIM65 on TAM infiltration, TAM polarization and JAK1/STAT1 signaling pathway. RESULTS: Bioinformatics analysis revealed that TRIM65 was upregulated in 16 types of cancer especially in HCC, and high level of TRIM65 was strongly correlated with higher tumor grades, TNM stages, and worse prognosis of patients with HCC as well as immune infiltration level of macrophages (M0, M1, and M2). Moreover, we observed that TRIM65 shRNA-mediated TRIM65 knockdown significantly inhibited the HepG2 cells growth while TRIM65 overexpression highly increased the Huh7 cells growth in vitro. TRIM65 knockout significantly inhibited the tumor growth as well as macrophages polarization towards M2 but promoted macrophages polarization towards M1 in vivo. Mechanistically, the results demonstrate that TRIM65 knockout promoted macrophage M1 polarization in conditioned medium-stimulated peritoneal macrophages and in tumor tissues by activating JAK1/STAT1 signaling pathway. CONCLUSIONS: Taken together, our study suggests that tumor cells utilize TRIM65-JAK1/STAT1 axis to inhibit macrophage M1 polarization and promote tumor growth, reveals the role of TRIM65 in TAM-targeting tumor immunotherapy.

The pterostilbene-dihydropyrazole derivative Ptd-1 ameliorates vascular calcification by regulating inflammation.

Vascular calcification is an independent risk factor for cardiovascular disease. However, there is still a lack of adequate treatment. This study aimed to examine the potential of (E)-1-(5-(2-(4-fluorobenzyloxy)Styryl)-4,6-dimethoxyphenyl)-3-methyl-4,5-dihydro-1H-pyrazole-1-yl) ethyl ketone (Ptd-1) to alleviate vascular calcification. ApoE-deficient mice were fed a high-fat diet for 12/16 weeks to induce intimal calcification, and wild-type mice were induced with a combination of nicotine and vitamin D3 to induce medial calcification. Human aortic smooth muscle cells (HASMCs) and aortic osteogenic differentiation were induced in vitro with phosphate. In the mouse model of atherosclerosis, Ptd-1 significantly ameliorated the progression of atherosclerosis and intimal calcification, and there were significant reductions in lipid deposition and calcium salt deposition in the aorta and aortic root. In addition, Ptd-1 significantly improved medial calcification in vivo and osteogenic differentiation in vitro. Mechanistically, Ptd-1 reduced the levels of the inflammatory factors IL-1ß, TNFα and IL-6 in vivo and in vitro. Furthermore, we demonstrated that Ptd-1 could attenuate the expression of p-ERK1/2 and ß-catenin, and that the levels of inflammatory factors were elevated in the presence of ERK1/2 and ß-catenin agonists. Interestingly, we determined that activation of the ERK1/2 pathway promoted ß-catenin expression, which further regulated the IL-6/STAT3 signaling pathway. Ptd-1 blocked ERK1/2 signaling, leading to decreased expression of inflammatory factors, which in turn improved vascular calcification. Taken together, our study reveals that Ptd-1 ameliorates vascular calcification by regulating the production of inflammatory factors, providing new ideas for the treatment of vascular calcification.

Neuroprotective effects of an engineered Escherichia coli Nissle 1917 on Parkinson's disease in mice by delivering GLP-1 and modulating gut microbiota.

Considerable evidence suggests that insulin resistance is closely linked to Parkinson's disease (PD), leading to agents aiming at treating diabetes can be regarded as new neuroprotective strategies in PD, notably glucagon-like peptide-1 (GLP-1). However, the extremely short half-life of GLP-1 due to degradation by the ubiquitous proteolytic enzyme limits its clinical application. In this study, we engineered the recombinant integrant probiotic strain Escherichia coli Nissle 1917 (EcN) to create a strain EcN-GLP-1 that effectively delivers the heterologous GLP-1 molecule. Subsequently, we assessed its neuroprotective effects on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD mice. We demonstrated that EcN-GLP-1 treatment could improve motor deficits, increase tyrosine hydroxylase-positive neurons, suppress microglia and astrocyte activation, reduce brain and colon inflammation, and ameliorate colonic barrier function damaged by MPTP induction. Meanwhile, we confirmed that the oral administration of EcN-GLP-1 could restore the disturbance of gut microbiota in the MPTP-induced PD mice, by reducing the relative abundances of Akkermansia and Oscillospira, and increasing the level of Prevotella in the gut. These results support further development of an engineered probiotic platform in which production of GLP-1 for gut-brain disorders, such as PD.

CD38 deficiency promotes skeletal muscle and brown adipose tissue energy expenditure through activating NAD+-Sirt1-PGC1α signaling pathway.

Obesity is a metabolic syndrome characterized by abnormal lipid deposition and energy imbalance. CD38 is a single-chain transmembrane glycoprotein widely expressed in a variety of cell types. The roles of skeletal muscle and brown fat in CD38 deficiency under HFD-induced obesity remain unknown. In this study, we established obesity model with HFD and examined the changes in metabolites with metabonomics. Our results showed that CD38 expression was increased in muscle and brown fat after HFD treatment. Moreover, the results of metabonomics showed that CD38 deficiency significantly altered the metabolites in energy metabolism, cofactor generation, and redox homeostasis. Furthermore, CD38 deficiency reduced the expressions of NADPH oxidase 2 and FASN in mRNA level. We found that the expressions of Sirt1, Sirt3, and PGC1α were upregulated in CD38-deficient muscle tissue. In brown fat, the Sirt1-3, cell death inducing DFFA-like effector A, ELOVL3, and Dio2 expressions were increased in CD38-deficient mice. Our results showed the uncoupling protein 1 expression was upregulated. And NAD+ supplementation increased the expression of Sirt1 and PGC1α after palmitic acid treatment. Taken together, our results demonstrated that the protection of CD38 deficiency on HFD-induced obesity was related to the inhibition of oxidative stress and increasing energy expenditure via activating NAD+/Sirtuins signaling pathways in muscle and brown fat.

LPS inhibits TRIM65 expression in macrophages and C57BL/6J mouse by activating the ERK1/2 signaling pathway.

Activated macrophages serve a key role in various inflammatory diseases, such as atherosclerosis and septic shock. Tripartite motif-containing protein 65 (TRIM65) has been previously reported to participate in tumor progression and lung inflammation. However, the molecular mechanisms that controls its expression under inflammatory conditions and its consequences in activated macrophages remain poorly understood. The present study first collected the tissues of C57BL/6J mice, smooth muscle cells, macrophages and endothelial cells to determine the expression and distribution of TRIM65 by reverse transcription-quantitative (RT-q) PCR and western blotting. Mouse and human macrophages were treated with LPS and C57BL/6J mice were intraperitoneally injected with LPS followed by isolation of spleen, lung, aorta and bone marrow. Following treatment, TRIM65 mRNA and protein level was examined by RT-qPCR and western blotting. The results showed that TRIM65 was highly expressed in organs of the immune system, such as the spleen, lymph node and thymus, but lowly expressed in heart, liver, brain and kidneys. TRIM65 was also highly expressed in macrophages and endothelial cells. TRIM65 mRNA and protein expression levels were found to be decreased in LPS-treated macrophages in vitro and in tissues isolated from C57BL/6J mice intraperitoneally injected with LPS in vivo. In addition, to identify the signaling pathways by which LPS regulates TRIM65 expression, inhibitors of MAPK and Akt signaling pathways were used to treat macrophages followed by examination the expression of TRIM65 by western blotting. The results demonstrated that LPS-inhibited TRIM65 expression was blocked by treatment with the ERK1/2 inhibitor U0126. Moreover, the RT-qPCR results showed that TRIM65 knockout potentiated LPS-induced expression of inflammatory cytokines in macrophages. Taken together, data from the present study suggest that LPS decreased TRIM65 expression in macrophages and C57BL/6J mouse by activating the ERK1/2 signaling pathway, whilst TRIM65 knockout promoted macrophage activation. This information may facilitate the development of potential therapeutic strategies for the prevention and treatment of inflammatory diseases, such as atherosclerosis.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulated in calcific areas. We further demonstrated TCF21 expression was increased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1ß and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

A review of the signaling pathways of aerobic and anaerobic exercise on atherosclerosis.

Atherosclerosis (AS), a chronic inflammatory vascular disease with lipid metabolism abnormalities, is one of the major pathological bases of coronary heart disease. As people's lifestyles and diets change, the incidence of AS increases yearly. Physical activity and exercise training have recently been identified as effective strategies for lowering cardiovascular disease (CVD) risk. However, the best exercise mode to ameliorate the risk factors related to AS is not clear. The effect of exercise on AS is affected by the type of exercise, intensity, and duration. In particular, aerobic and anaerobic exercise are the two most widely discussed types of exercise. During exercise, the cardiovascular system undergoes physiological changes via various signaling pathways. The review aims to summarize signaling pathways related to AS in two different exercise types and provide new ideas for the prevention and treatment of AS in clinical practice.

An update on the role of TRIM/NLRP3 signaling pathway in atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory disease of large and medium arteries that includes lipid metabolism disorder and recruitment of immune cells to the artery wall. An increasing number of studies have confirmed that inflammasome over-activation is associated with the onset and progression of atherosclerosis. The NLRP3 inflammasome, in particular, has been proven to increase the incidence rate of cardiovascular diseases (CVD) by promoting pro-inflammatory cytokine release and reducing plaque stability. The strict control of inflammasome and prevention of excessive inflammatory reactions have been the research focus of inflammatory diseases. Tripartite motif (TRIM) is a protein family with a conservative structure and rapid evolution. Several studies have demonstrated the TRIM family's regulatory role in mediating inflammation. This review aims to clarify the relationship between TRIMs and NLRP3 inflammasome and provide insights for future research and treatment discovery.

Osteopontin Promotes Macrophage M1 Polarization by Activation of the JAK1/STAT1/HMGB1 Signaling Pathway in Nonalcoholic Fatty Liver Disease.

Background and Aims: Osteopontin (OPN) is reported to be associated with the pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, the function of OPN in NAFLD is still inconclusive. Therefore, our aim in this study was to evaluate the role of OPN in NAFLD and clarify the involved mechanisms. Methods: We analyzed the expression change of OPN in NAFLD by bioinformatic analysis, qRT-PCR, western blotting and immunofluorescence staining. To clarify the role of OPN in NAFLD, the effect of OPN from HepG2 cells on macrophage polarization and the involved mechanisms were examined by FACS and western blotting. Results: OPN was significantly upregulated in NAFLD patients compared with normal volunteers by microarray data, and the high expression of OPN was related with disease stage and progression. OPN level was also significantly increased in liver tissue samples of NAFLD from human and mouse, and in HepG2 cells treated with oleic acid (OA). Furthermore, the supernatants of OPN-treated HepG2 cells promoted the macrophage M1 polarization. Mechanistically, OPN activated the janus kinase 1(JAK1)/signal transducers and activators of transcription 1 (STAT1) signaling pathway in HepG2 cells, and consequently HepG2 cells secreted more high-mobility group box 1 (HMGB1), thereby promoting macrophage M1 polarization. Conclusions: OPN promoted macrophage M1 polarization by increasing JAK1/STAT1-induced HMGB1 secretion in hepatocytes.

Elevated insulin-like growth factor 2 mRNA binding protein 1 levels predict a poor prognosis in patients with breast carcinoma using an integrated multi-omics data analysis.

Background: Insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) controls the cytoplasmic fate of certain mRNAs and is hypothesized to predict a poor patient prognosis in several malignant tumors. However, the prognostic relevance of IGF2BP1 in breast cancer remains debatable. Methods: We interrogated large publicly available datasets from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and cBioportal databases to analyze the genetic alterations in the expression levels of IGF2BP1 in patients with invasive breast carcinoma (BRCA), and to discern the prognostic value of IGF2BP1 in BRCA. We applied Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genome (KEGG), and gene set enrichment analysis (GSEA) to uncover a functional association between IGF2BP1 and BRCA using differentially expressed genes (DEGs), and we screened genes and proteins related to BRCA. Results: We determined that both genetic alterations in IGF2BP1 (approximately 10%) and an increase in IGF2BP1 mRNA levels were related to certain cancer subtypes and an unfavorable prognosis in BRCA patients, and we then established an OS nomogram upon our multivariate regression model. The DEGs and IGF2BP1-correlated genes/proteins that implied the involvement of cornification, keratinization, drug/xenobiotic metabolism by cytochrome P450, chemical carcinogenesis, cell interactions, and cell adhesion to the extracellular matrix (ECM) pathways with respect to the prognostic relevance of IGF2BP1. Conclusion: In summary, our results indicated that both genetic alterations in IGF2BP1 and increased levels of IGF2BP1 mRNA and protein predict a poor patient prognosis in BRCA patients.

MEK1/2 inhibitor inhibits neointima formation by activating miR-126-3p/ C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C motif chemokine receptor 4 (CXCR4) axis.

Endothelial dysfunction is an initial and essential step in vascular-remodeling diseases, including atherosclerosis and neointima formation. During vascular remodeling, activated endothelial cells can release pro-inflammatory factors that promote phenotypic switching of vascular smooth muscle cells (VSMCs) to the proliferative phenotype. We previously reported that MEK1/2 inhibitor, U0126, has a protective effect on the development of atherosclerosis and vascular calcification. However, the effect of MEK1/2 inhibitors on neointimal formation and the underlying mechanism is not fully understood. We determined that MEK1/2 inhibitor reduced carotid artery ligation-induced neointimal formation, while increased collagen and elastin levels and vascular integrality. Mechanistically, MEK1/2 inhibitor or ERK1/2 siRNA increased miR-126-3p level in endothelial cells, thereby inhibiting expression of regular of G-protein signaling 16 (RGS16), a miR-126-3p target gene, to activate the C-X-C motif chemokine ligand 12 (CXCL12)/C-X-C motif chemokine receptor 4 (CXCR4) signaling pathway. Accordingly, miR-126-3p was also increased by U0126 in serum and carotid artery. RGS16 was inhibited while CXCR4 and CXCL12 was increased by U0126 in neointimal areas, especially in the endothelium. Moreover, similar results were observed in atherosclerotic plaques of high-fat diet-fed apolipoprotein E deficiency (apoE-/-) mice. In addition, vascular cell adhesion molecule 1 (VCAM-1), another miR-126-3p target gene, was reduced by U0126 in the neointimal areas, resulting reduced monocytes/macrophages accumulation. Taken together, our results indicate that MEK1/2 inhibitor can reduce neointima formation by activating endothelial miR-126-3p production to facilitate endothelium repair while reduce monocyte adhesion/infiltration.

Identification and Verification of Potential Core Genes in Pediatric Septic Shock.

BACKGROUND: Septic shock is a frequent and costly problem among patients in the pediatric intensive care unit (PICU) and is associated with high mortality and devastating survivor morbidity. In this study, we aimed to screen candidate biomarkers and potential therapeutic targets for septic shock. METHODS: GSE26440 dataset was downloaded from Gene Expression Omnibus (GEO), including 32 normal controls and 98 children with septic shock RNA samples from whole blood. The pathways and functional annotations of differentially expressed genes (DEGs) in the two types of samples were examined by GO and KEGG pathway enrichment analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool. Protein-protein interactions (PPI) of the above-described DEGs were investigated using the Search Tool for the Retrieval of Interacting Genes (STRING) and Hub gene identification was performed by the plug-in cytoHubba in Cytoscape software. RESULTS: A total of 140 genes were identified as DEGs, of which 98 genes were up-regulated and 42 genes were down-regulated. GO function analysis showed that DEGs were significantly enriched in biological processes, including immune response, leukocyte activation involved in immune response, and so on. The top hub genes, namely MMP9, CEACAM8, ARG1, MCEMP1, LCN2, RETN, S100A12, GPR97, and TRAT1 were recognized from the protein-protein interaction (PPI) network. Furthermore, qRT-PCR results demonstrated that the mRNA level of MMP9, CEACAM8, ARG1, MCEMP1, LCN2, RETN, and S100A12 was elevated while GPR97 was decreased in involved mouse and human models. However, TRAT1 expression is species-dependent which was decreased in the mouse septic shock model but elevated in the human LPS-treated macrophages model. CONCLUSION: Taken together, the identification and validation of several novel hub genes, especially GPR97 and TRAT1, deepen our comprehension of the molecular mechanisms of septic shock progression. These genes may be therapeutic molecular targets or diagnostic biomarkers in patients with septic shock.

TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages through JNK signaling pathway.

Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. TRIM59 has been showed to participate in many pathological processes, such as inflammation, cytotoxicity and tumorigenesis. However, the molecular mechanisms controlling its expression in activated macrophages are not fully understood. Here we report that TRIM59 expression is regulated by Sp1 and Nrf1 in LPS-activated macrophages. TRIM59 is highly expressed in macrophages, and markedly decreased by LPS stimuli in vivo and in vitro. TRIM59 promoter activity is also significantly suppressed by LPS and further analysis demonstrated that Sp1 and Nrf1 directly bound to the proximal promoter of TRIM59 gene. LPS treatment significantly decreased Sp1 expression, nuclear translocation and reduced its binding to the promoter, whereas increased Nrf1 expression, nuclear translocation and enhanced its binding to the promoter. Moreover, LPS-decreased TRIM59 expression was reversed by JNK inhibitor. Finally, TRIM59 level is significantly decreased during atherosclerosis progression. Taken together, our results demonstrated that TRIM59 expression was precisely regulated by Sp1 and Nrf1 in LPS-activated macrophages, which may be dependent on the activation of JNK signaling pathway and TRIM59 may be a potential therapeutic target for inflammatory diseases such as atherosclerosis.

Shikonin exerts antitumor activity by causing mitochondrial dysfunction in hepatocellular carcinoma through PKM2-AMPK-PGC1α signaling pathway.

Shikonin, a naphthoquinone derivative isolated from the root of Lithospermum erythrorhizon, exhibits broad-spectrum antitumor activity via different molecular mechanisms. In this study, we investigated the effect of shikonin on mitochondrial dysfunction in hepatocellular carcinoma (HCC). Our results showed that shikonin inhibited the proliferation, migration, and invasiveness of HCCLM3 cells, and promoted cell apoptosis in a dose-dependent manner. More importantly, shikonin affected mitochondrial function by disrupting mitochondrial membrane potential and oxidative stress (OS) status. Furthermore, shikonin decreased the oxygen consumption rate of HCCLM3 cells, as well as the levels of ATP and metabolites involved in the tricarboxylic acid cycle (TCA cycle). We also investigated the molecular mechanisms underlying the regulation of mitochondrial function by shikonin as an inhibitor of PKM2. Shikonin decreased the expression of PKM2 in the mitochondria and affected other metabolic pathways (AMPK and PGC1α pathways), which aggravated the oxidative stress and nutrient deficiency. Our results indicate a novel role of shikonin in triggering mitochondria dysfunction via the PKM2-AMPK-PGC1α signaling pathway and provide a promising therapeutic approach for the treatment of HCC.

CD38 Deficiency Promotes Inflammatory Response through Activating Sirt1/NF-κB-Mediated Inhibition of TLR2 Expression in Macrophages.

CD38 was first identified as a lymphocyte-specific antigen and then has been found to be widely expressed in a variety of cell types. The functions of CD38 are involved in numerous biological processes including immune responses. Here, we showed the downregulations of both TLR2 mRNA and protein in macrophages from CD38-/- mice and in CD38 knockdown RAW264.7 cells. Several NF-κB-binding motifs in the promoter region of the TLR2 gene were identified by the bioinformatics analysis and were confirmed by the luciferase activity assay with the different truncated TLR2 promoters. CD38 deficiency resulted in the reduction of NF-κB p65 and acetyl-NF-κB p65 (Ac-p65) levels as determined by Western blot. The expression of Sirt1 did not change, but an increased activity of Sirt1 was observed in CD38-deficient macrophages. Inhibition of the Sirt1/NF-κB signaling pathway resulted in downregulation of TLR2 expression in RAW264.7 cells. However, re-expression of CD38 in the knockdown clones reversed the effect on Sirt1/NF-κB/TLR2 signaling, which is NAD-dependent. Moreover, the inflammatory cytokines including G-CSF, IL-1alpha, IL-6, MCP-1, MIP-1alpha, and RANTES were increased in CD38 knockdown RAW264.7 cells. Taken together, our data demonstrated that CD38 deficiency enhances inflammatory response in macrophages, and the mechanism may be partly associated with increased Sirt1 activity, which promoted NF-κB deacetylation and then inhibited expression of the TLR2 gene. Obviously, our study may provide an insight into the molecular mechanisms in CD38-mediated inflammation.

Expression profiling of TRIM protein family in THP1-derived macrophages following TLR stimulation.

Activated macrophages play an important role in many inflammatory diseases including septic shock and atherosclerosis. However, the molecular mechanisms limiting macrophage activation are not completely understood. Members of the tripartite motif (TRIM) family have recently emerged as important players in innate immunity and antivirus. Here, we systematically analyzed mRNA expressions of representative TRIM molecules in human THP1-derived macrophages activated by different toll-like receptor (TLR) ligands. Twenty-nine TRIM members were highly induced (>3 fold) by one or more TLR ligands, among which 19 of them belong to TRIM C-IV subgroup. Besides TRIM21, TRIM22 and TRIM38 were shown to be upregulated by TLR3 and TLR4 ligands as previous reported, we identified a novel group of TRIM genes (TRIM14, 15, 31, 34, 43, 48, 49, 51 and 61) that were significantly up-regulated by TLR3 and TLR4 ligands. In contrast, the expression of TRIM59 was down-regulated by TLR3 and TLR4 ligands in both human and mouse macrophages. The alternations of the TRIM proteins were confirmed by Western blot. Finally, overexpression of TRIM59 significantly suppressed LPS-induced macrophage activation, whereas siRNA-mediated knockdown of TRIM59 enhanced LPS-induced macrophage activation. Taken together, the study provided an insight into the TLR ligands-induced expressions of TRIM family in macrophages.

Hawthorn (Crataegus pinnatifida Bunge) leave flavonoids attenuate atherosclerosis development in apoE knock-out mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Hawthorn (Crataegus pinnatifida Bunge) leave have been used to treat cardiovascular diseases in China and Europe. Hawthorn leave flavonoids (HLF) are the main part of extraction. Whether hawthorn leave flavonoids could attenuate the development of atherosclerosis and the possible mechanism remain unknown. MATERIALS AND METHODS: High-fat diet (HFD) mixed with HLF at concentrations of 5mg/kg and 20mg/kg were administered to apolipoprotein E (apoE) knock out mice. 16 weeks later, mouse serum was collected to determine the lipid profile while the mouse aorta dissected was prepared to measure the lesion area. Hepatic mRNA of genes involved in lipid metabolism were determined. Peritoneal macrophages were collected to study the impact of HLF on cholesterol efflux, formation of foam cell and the expression of ATP binding cassette transporter A1 (ABCA1). Besides, in vivo reverse cholesterol transport (RCT) was conducted. RESULTS: HLF attenuated the development of atherosclerosis that the mean atherosclerotic lesion area in en face aortas was reduced by 23.1% (P<0.05). In mice fed with 20mg/kg HLF, Total cholesterol (TC) level was decreased by 18.6% and very low density lipoprotein cholesterol plus low density lipoprotein cholesterol (VLDLc+LDLc) level were decreased by 23.1% whereas high density lipoprotein cholesterol (HDLc) and triglyceride (TG) levels were similar compared to that of the control group. Peroxisome proliferator activated receptor alpha (PPARα) mRNA was increased by 31.2% (P<0.05) and 60.9% (P<0.05) in mice fed with 5mg/kg and 20mg/kg HLF respectively. Sterol regulatory element binding protein-1c (SREBP-1c) was decreased by 59.3% in the group of 20mg/kg. Carnitine palmitoyl transferase 1 (CPT-1) mRNA level of 20mg/kg group was induced 66.7% (P<0.05). Superoxide dismutase 1 and 2 (SOD1 and SOD2) mRNA were induced 25.4% (P<0.05) and 71.4% (P<0.05) while induced by 36.3% (P<0.05) and 73.2% (P<0.05) in group of 20mg/kg. Glutathione peroxidase 3 (Gpx3) mRNA in the group of 20mg/kg was induced by 96.7% (P<0.05). Hepatic hydroxymethylglutaryl CoA reductase (HMG-CoAR) expression was as same level as the control group while LDL receptor (LDLR) mRNA and protein were induced by 84.2% (P<0.05) and 98.8% (P<0.05) in group of 20mg/kg. HLF inhibit the formation of foam cell by 27.9% (P<0.05) in the dosage of 25µg/ml, and 33.3% (P<0.05) in the dosage of 50µg/ml. HLF increased the reverse cholesterol transport (RCT) in vivo. DISCUSSION AND CONCLUSION: Hawthorn leave flavonoids can slow down the development of atherosclerosis in apoE knockout mice via induced expression of genes involved in antioxidant activities, inhibition of the foam cell formation and promotion of RCT in vivo, which implies the potential use in the prevention of atherosclerosis.

Activation of PPARγ does not contribute to macrophage ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI.

Activation of macrophage ABCA1/G1 expression and cholesterol efflux is believed one of the mechanisms by which PPARγ inhibits atherosclerosis. PPARγ can also activate CD36 expression, a receptor for oxLDL, which may supply LXR ligands to activate LXR-ABCA1/G1 pathways. However, the controversial effects of PPARγ on ABCA1 expression have been reported. In this study, we used peritoneal macrophages isolated from wild type and CD36 deficient (CD36-/-) mice to clarify if PPARγ ligands can influence ABCA1 expression by CD36 function. We found that CD36 deficiency had no effect on cholesterol efflux and ABCA1/ABCG1 expression at basal levels. In both cell types, PPARγ ligands (15d-PGJ2, troglitazone and pioglitazone) reduced ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI, with most by troglitazone. LXR ligand-induced ABCA1 expression and cholesterol efflux was attenuated by PPARγ ligands. Associated with decreased ABCA1 protein levels, ABCA1 mRNA and promoter activity were reduced by PPARγ ligands. Furthermore, high expressing PPARγ reduced ABCA1 expression and LXR-activated ABCA1 promoter in a CD36-independent manner. In contrast, ABCG1 expression was induced by PPARγ ligands while inhibited by PPARγ inactivation. Taken together, our study suggests that enhancement of macrophage cholesterol metabolism by PPARγ is not contributed by activating ABCA1 expression and ABCA1-mediated cholesterol efflux to apoAI, which is not involved by CD36 expression either.