Inhibition of EphA2 protects against atherosclerosis by synergizing with statins to mitigate macrophage inflammation.

Statins are highly prevalent in patients with coronary artery disease. Statins exert their anti-inflammatory effects on the vascular wall and circulating levels of pro-inflammatory cytokines. However, increasing attention revealed the exacerbation of macrophage inflammation induced by statins, and a clear mechanistic explanation of whether the detrimental effects of statins on macrophage inflammatory phenotypes outweigh the beneficial effects is has not yet been established. Here, RNA-sequencing and RT-qPCR analyses demonstrated that statins significantly upregulated EphA2, Nlrp3, IL-1ß and TNF-α expression in macrophages. Mechanistically, we found that atorvastatin reduced KLF4 binding to the EphA2 promoter using KLF4-chromatin immunoprecipitation, suppressed HDAC11-mediated deacetylation and subsequently led to enhanced EphA2 transcription. The 4D-label-free proteomics analysis further confirmed the upregulated EphA2 and inflammatory signals. Furthermore, the proinflammatory effect of atorvastatin was neutralized by an addition of recombinant Fc-ephrinA1, a selective Eph receptor tyrosine kinase inhibitor (ALW-II-41-27) or EphA2-silencing adenovirus (siEphA2). In vivo, EphA2 was identified a proatherogenic factor and apoE-/- mice placed on a high-fat diet following gastric gavage with atorvastatin exhibited a consistent elevation in EphA2 expression. We further observed that the transfection with siEphA2 in atorvastatin-treated mice significantly attenuated atherosclerotic plaque formation and abrogated statin-orchestrated macrophages proinflammatory genes expression as compared to that in atorvastatin alone. Increased plaque stability index was also observed following the addition of siEphA2, as evidenced by increased collagen and smooth muscle content and diminished lipid accumulation and macrophage infiltration. The data suggest that blockage of EphA2 provides an additional therapeutic benefit for further improving the anti-atherogenic effects of statins.

Fabrication of Tß4-Exosome-releasing artificial stem cells for myocardial infarction therapy by improving coronary collateralization.

Currently, stem cell transplantations in cardiac repair are limited owing to disadvantages, such as immunological rejection and poor cell viability. Although direct injection of exosomes can have a curative effect similar to that of stem cell transplantation, high clearance hinders its application in clinical practice. Previous reports suggested that induction of coronary collateralization can be a desired method of adjunctive therapy for someone who had missed the optimal operation time to attenuate myocardial ischemia. In this study, to mimic the paracrine and biological activity of stem cells, we developed artificial stem cells that can continuously release Tß4-exosomes (Tß4-ASCs) by encapsulating specific exosomes within microspheres using microfluidics technology. The results show that Tß4-ASCs can greatly promote coronary collateralization in the periphery of the myocardial infarcted area, and its therapeutic effect is superior to that of directly injecting the exosomes. In addition, to better understand how it works, we demonstrated that the Tß4-ASC-derived exosomes can enhance the angiogenic capacity of coronary endothelial cells (CAECs) via the miR-17-5p/PHD3/Hif-1α pathway. In brief, as artificial stem cells, Tß4-ASCs can constantly release functional exosomes and stimulate the formation of collateral circulation after myocardial infarction, providing a feasible and alternative method for clinical revascularization.

New Insight into a Fenton-like Reaction Mechanism over Sulfidated ß-FeOOH: Key Role of Sulfidation in Efficient Iron(III) Reduction and Sulfate Radical Generation.

Sulfidation can greatly improve the efficiency of utilization of reducing equivalents for contaminant removal; however, whether this method benefits Fenton-like reactions or not and the possible mechanism are not well understood. In this study, we revealed that surface sulfidation can greatly promote the heterogeneous Fenton activity of ß-FeOOH (Fe3S4@ß-FeOOH) by 40 times, in which not only the •OH formation was enhanced but also SO4•- as a new oxidation species was generated. Moreover, their contribution to metronidazole (MTZ) degradation was 52.5 and 37.1%, respectively. In comparison, almost no HO2•/O2•- was detected in the Fe3S4@ß-FeOOH/H2O2 system. These results were different from some previously reported Fenton counterparts. Based on the characterization and probe experiments, sulfur species, including S2-, S0, and Sn2-, as an electron donor and electron shuttle were responsible for efficient conversion of Fe(III) into Fe(II) other than via the Haber-Weiss mechanism, leading to excellent •OH generation via a Fenton-like mechanism. Most importantly, HSO5- can be generated from SO32- oxidized by •OH, and its scission into SO4•- was not dependent on the extra electric potential or Fe-O2-S(IV) intermediate. These findings provided new insight for utilizing sulfidation to improve the activity of iron-based Fenton catalysts.

C3/C3aR inhibition alleviates GMH-IVH-induced hydrocephalus by preventing microglia-astrocyte interactions in neonatal rats.

Activation of microglia and astrocytes following germinal matrix hemorrhage and intraventricular hemorrhage (GMH-IVH) plays a detrimental role in posthemorrhagic hydrocephalus (PHH). It is still unclear whether or how an interaction occurs between microglia and astrocytes in PHH. Here, we investigated the role of the C3/C3aR pathway in microglia and astrocyte interactions and whether C3/C3aR-targeted inhibition could alleviate PHH following GMH-IVH. A total of 152 Sprague-Dawley rats at postnatal day seven (P7) were enrolled in the study, and collagenase VII was used to induce GMH-IVH. Minocycline (45 mg/kg) was administered to inhibit microglial activation. Complement C3a peptide and C3aR antagonist (SB 290157, 10 mg/kg) were used to regulate the C3/C3aR pathway. As a result, the data demonstrated that periventricular C3aR+/Iba-1+ microglia and C3+/GFAP+ astrocytes were significantly increased in GMH-IVH pups at 28 days after surgery. Intranasal C3a peptide upregulated C3aR expression in microglia. Inhibition of microglia by minocycline decreased both C3+/GFAP+ astrocytes and the colocalization volume of Iba-1 and GFAP. In addition, intraperitoneally injected C3aRA alleviated the periventricular colocalization volume of microglia and astrocytes. Compared with vehicle-treated pups, the protein level of IL-1ß, IL-6 and TNF-α in cerebral spinal fluid and brain tissue at 28 days following GMH-IVH were reduced in C3aRA-treated pups. Moreover, hydrocephalus was alleviated, and long-term cognitive ability were improved in the C3aRA-treated group. Our data presented simultaneous periventricular astrogliosis and microgliosis of pups following GMH-IVH and proved their potential interaction through the C3/C3aR pathway, indicating C3aRA as a potential pharmacological treatment of PHH in neonates.

Reduced SULT2B1b expression alleviates ox-LDL-induced inflammation by upregulating miR-148-3P via inhibiting the IKKß/NF-κB pathway in macrophages.

Atherosclerosis is a lipid-driven chronic inflammatory disease in which lipid-laden macrophage foam cells lead to inflamed lesions in arteries. Previous studies have proven that sulfotransferase 2B1b (SULT2B1b) has several roles in the regulation of lipid metabolism and the inflammatory response. However, little is known about the functions of SULT2B1b in ox-LDL-induced inflammation in macrophages. In this study, after treatment with either ox-LDL alone or combined with transfection of siRNAs targeting SULT2B1b, IL-6, TNF-α, NF-κB, IKKß and IκB mRNA and protein expression were determined in Raw264.7 cells by real-time PCR and Western blot, respectively. The proliferative capacity was determined by EdU staining and Cell Counting Kit-8. Our data demonstrated that SULT2B1b knockdown could reduce phosphorylated NF-κB levels and downregulate IKKß protein levels. Additionally, IκB levels were increased and the proliferation of ox-LDL stimulated cells was inhibited after SULT2B1b silencing. Downregulation of SULT2B1b expression was found to upregulate miR-148a-3p expression by microarray assay, while IKKß was a miR-148a-3p target gene. Our study suggests that SULT2B1b knockdown could promote miR148a-3p expression and inhibit activation of the IKKß/NF-κB signalling pathway, which suppressed the inflammatory response in macrophages. Therefore, targeting the SULT2B1b gene might be potentially beneficial for atherosclerosis prevention by decreasing the inflammatory response.

SULT2B1b inhibits reverse cholesterol transport and promotes cholesterol accumulation and inflammation in lymphocytes from AMI patients with low LDL-C levels.

The current main treatment for coronary artery disease (CAD) is to reduce low-density lipoprotein cholesterol (LDL-C) by statins, which could decrease the incidence of major adverse cardiovascular events (MACEs) by 30%. However, many residual risks still remain. To clarify the mechanism involved, we studied patients with acute myocardial infarction (AMI) with low LDL-C levels. Lymphocytes were isolated, and it was found that despite no difference in plasma LDL-C level, the lymphocyte cholesterol content was higher in AMI patient than those in non-CAD patients; thus, the decrease in intracellular cholesterol content was inconsistent with that in the plasma. Additionally, [3H]-cholesterol efflux rates were lower and mRNA levels of the inflammatory factors tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) higher in AMI lymphocytes. It was found that sulphotransferase 2B1b (SULT2B1b) expression was higher in AMI lymphocytes. Further research using Jurkat T lymphocytes confirmed that SULT2B1b knockdown increased cholesterol efflux capacity and decreased mRNA levels of TNF-α and IFN-γ by increasing liver X receptor (LXR)-ß levels. Furthermore, the degree of CpG island methylation in the SULT2B1b promoter was reduced in cells from AMI patients. In conclusion, SULT2B1b up-regulation due to hypomethylation of its promoter promotes cholesterol accumulation and inflammation by inhibiting LXR-ß in lymphocytes of AMI patients with low LDL-C levels. Therefore, reducing intracellular cholesterol is also important as plasma cholesterol levels. Therapeutic approaches to decrease SULT2B1b expression might be potentially beneficial for CAD prevention by decreasing intracellular cholesterol.

Human Hair Keratin Hydrogels Alleviate Rebleeding after Intracerebral Hemorrhage in a Rat Model.

Surgery is an important therapeutic strategy for intracerebral hemorrhage (ICH) in the clinic and is theoretically beneficial for the outcome of ICH by decreasing hematoma, reducing nervous tissue damage, and removing harmful chemicals. However, the outcome of ICH surgery is always unsatisfactory due to postoperative rebleeding. We hypothesized that the injection of hemostatic agents in situ after aspiration surgery could immediately activate hemostasis once rebleeding occurs. Therefore, keratin hydrogels (K-gels) were easily prepared as a hemostatic material via a rehydration method and had a porous structure. Collagenase was injected into the basal lamina to mimic ICH rebleeding, and the K-gels were then injected into the same injured site after 2 h for hemostatic therapy. The hematoma volume was significantly reduced by K-gel treatment, indicating that in situ infusion of the K-gels inhibited hematoma enlargement when rebleeding occurred. Moreover, brain damage, including cell apoptosis, neuroinflammatory reactions, and neurological deficits, was also relieved after K-gel treatment. These results suggested that in situ injection of the K-gels into the hematoma area after ICH surgery improves the therapeutic outcome by stopping postoperative rebleeding. K-gels have great potential for clinical hemostatic application because of their excellent hemostatic properties and biocompatibility.

PSRC1 overexpression attenuates atherosclerosis progression in apoE-/- mice by modulating cholesterol transportation and inflammation.

AIMS: Human genome-wide association studies (GWAS) have found that proline/serine-rich coiled-coil 1 (PSRC1) encodes a protein that is associated with serum lipid levels and coronary artery disease. In addition, our previous study showed that the cholesterol efflux capacity is decreased in macrophages following a treatment silencing Psrc1, indicating that PSRC1 has anti-atherosclerotic effects. However, the role of PSRC1 in the development of atherosclerosis is unknown. This study aims to explore the effect of PSRC1 on atherosclerosis and its underlying mechanisms. METHOD AND RESULTS: A recombinant adenovirus expressing Psrc1 (Ad-PSRC1) was constructed and transfected in RAW264.7 cells as well as injected intravenously into apoE-/- mice. The in vitro study showed that PSRC1 overexpression reduced the cellular cholesterol content, increased the cholesterol efflux capacity and inhibited foam cell formation by upregulating the expression of peroxisome proliferator-activated receptor γ (PPAR-γ) and liver X receptor α (LXR-α), which are key cholesterol transportation-related proteins. Infecting apoE-/- mice with Ad-PSRC1 inhibited the development of atherosclerotic lesions and enhanced atherosclerotic plaque stability. Consistent with these results, PSRC1 overexpression in apoE-/- mice decreased the plasma levels of TC, TG, LDL-C, TNF-α, IL-1ß and IL-6, increased the plasma HDL-C levels and improved HDL function. Similarly, the PPAR-γ and LXR-α expression levels were upregulated in the liver and in peritoneal macrophages of PSRC1-overexpressing apoE-/- mice. Finally, the liver and peritoneal macrophages of apoE-/- mice displayed elevated expression of ß-catenin, which is a direct downstream gene of PSRC1 and an upstream gene of PPAR-γ and LXR-α, but decreased activity of nuclear transcription factor (NF-κB), which acts as a key gene in the regulation of inflammation. CONCLUSIONS: PSRC1 protects against the development of atherosclerosis and enhances the stability of plaques by modulating cholesterol transportation and inflammation in macrophages and the liver of apoE-/- mice.

Mechanically induced autophagy is associated with ATP metabolism and cellular viability in osteocytes in vitro.

Both mechanical loading and intracellular autophagy play important roles in bone homeostasis; however, their relationship remains largely unexplored. The objectives of this study were to determine whether osteocytes undergo autophagy upon fluid shear stress (FSS) loading and to determine the correlation between mechanically induced autophagy and ATP metabolism. Autophagic vacuoles were observed by transmission electron microscopy (TEM) in osteocyte-like MLO-Y4 cells subjected to FSS. Increased autophagic flux was further confirmed by the increased amount of the LC3-II isoform and the degradation of p62. Fluorescent puncta distributed in the cytoplasm were observed in the GFP-LC3 transformed cells subjected to FSS. Furthermore, FSS-induced ATP release and synthesis in osteocytes were attenuated by inhibiting autophagy with 3-MA. After FSS exposure, a high ratio of cell death was observed in cultures pretreated with 3-MA, an autophagy inhibitor, with no significantly different Caspase 3/7 activity. Our results indicated that FSS induces protective autophagy in osteocytes and that mechanically induced autophagy is associated with ATP metabolism and osteocyte survival. From the clinical perspective, it may be possible to enhance skeletal cell survival with drugs that modulate the autophagic state, and the autophagy-related pathway could be a potential target for the prevention of ageing-related bone disorders.

In situ hydrogels enhancing postoperative functional recovery by reducing iron overload after intracerebral haemorrhage.

The role of surgery for most patients with spontaneous intracerebral haemorrhage (ICH) remains controversial due to the continuous occurrence of postoperative iron overload induced by low clot clearance rate. In this study, human hair keratose hydrogel (KG) loading with minocycline hydrochloride (MH) were prepared to reduce iron overload for the improvement of the postoperative functional recovery after ICH aspiration surgery. Hemoglobin-induced iron accumulation in rat primary neuronal culture was delayed by the adsorptive capacity of blank KG, while MH-loaded KG displayed a stronger and more thorough cytoprotective effect than blank KG due to the combined effect of absorptive action to iron and sustained release of the iron chelator. Moreover, high iron-chelating efficiency in the hematoma region supplied by MH-loaded KG significantly reduced dose strength of iron chelator. In situ injection of KG with different MH loadings (2, 20, and 200µg) into the hematoma region after aspiration surgery showed a stronger effect on the reduction of ICH-induced iron accumulation, edema, and neurological deficits in rats compared to the postoperative intraperitoneal administration of MH (approximately 15mg). These results suggested that the in situ KG not only could effectively reduce the ICH postoperative iron overload and improve the postoperative functional recovery via the iron adsorption and sustained release of MH, but also has great potential to reduce the systemic adverse effects by decreasing the dose strength of iron chelator.

Feather keratin hydrogel for wound repair: Preparation, healing effect and biocompatibility evaluation.

Keratins are highly attractive for wound healing due to their inherent bioactivity, biocompatibility and physical properties. However, nearly all wound healing studies have focused on human hair keratins, and the wound-repair effects and in vivo biocompatibilities of feather keratins are not clear. Feather keratins are derived from chicken feathers, which are considered to be the major waste in the poultry industry, and the quality of feather keratin is easier to control than that of human hair keratin due to human hair perming and colouring-dyeing. Thus, we extracted keratins from chicken feathers, and a feather keratin hydrogel was then prepared and used to test the in vivo wound-healing properties and biocompatibility. The results indicated that feather keratins displayed wound-healing and biodegradation properties similar to those of human hair keratins and were also highly compatible with those of the tissue and devoid of immunogenicity and systematic toxicity. Collectively, these results suggested that feather keratin hydrogel could be used for biomedical applications, particularly effective wound healing.

Lck Inhibits Heat Shock Protein 65-Mediated Reverse Cholesterol Transport in T Cells.

Previously, we reported that heat shock protein (HSP)65 impairs the effects of high-density lipoprotein on macrophages. We also showed that immune response activation adversely affects reverse cholesterol transport (RCT). In this study, we investigated the effects of the Src family kinase lymphocyte-specific protein tyrosine kinase (Lck) and elucidated the mechanism underlying HSP65-regulated cholesterol efflux in T cells. We evaluated cell proliferation, Lck expression, and inflammatory cytokine production in Jurkat cells and CD4+ T cells. HSP65-mediated inhibition of RCT was assessed by evaluating ABCA1, ABCG1, SR-BI, PPAR-γ, and liver X receptor-α expression. A dose-dependent relationship was found between the levels of these proteins and the suppression of cholesterol efflux. Stimulation of Lck-silenced T cells with ionomycin resulted in a decrease in intracellular calcium levels. Treatment of Jurkat cells with PP2, an inhibitor of Src family kinase, inhibited calcium-induced, but not PMA-induced, ERK phosphorylation. NF-κB activation in response to PMA was minimally inhibited in cells stimulated with PP2. HSP65 failed to trigger downstream ERK or JNK phosphorylation or to activate NF-κB or protein kinase C-γ in Lck-silenced cells. Additionally, elevation of intracellular calcium was also impaired. However, HSP65 significantly enhanced cholesterol efflux and decreased cellular cholesterol content by inducing the expression of cholesterol transport proteins in Lck-silenced cells. The treatment of Jurkat cells with PP2 also inhibited cell proliferation and promoted RCT. In conclusion, Lck is a key molecule in the TCR signaling cascade that inhibits cholesterol efflux and upregulates intracellular cholesterol ester content in T cells. Our results demonstrate that the immune response plays a previously unrecognized role in RCT.

Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

The co-infection of porcine reproductive respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) is quite common in clinical settings and no effective treatment to the co-infection is available. In this study, we established the porcine alveolar macrophages (PAM) cells model co-infected with PRRSV/PCV2 with modification in vitro, and investigated the antiviral activity of Matrine on this cell model and further evaluated the effect of Matrine on virus-induced TLR3,4/NF-κB/TNF-α pathway. The results demonstrated PAM cells inoculated with PRRSV followed by PCV2 2 h later enhanced PRRSV and PCV2 replications. Matrine treatment suppressed both PRRSV and PCV2 infection at 12 h post infection. Furthermore, PRRSV/PCV2 co- infection induced IκBα degradation and phosphorylation as well as the translocation of NF-κB from the cytoplasm to the nucleus indicating that PRRSV/PCV2 co-infection induced NF-κB activation. Matrine treatment significantly down-regulated the expression of TLR3, TLR4 and TNF-α although it, to some extent, suppressed p-IκBα expression, suggesting that TLR3,4/NF-κB/TNF-α pathway play an important role of Matrine in combating PRRSV/PCV2 co-infection. It is concluded that Matrine possesses activity against PRRSV/PCV2 co-infection in vitro and suppression of the TLR3,4/NF-κB/TNF-α pathway as an important underlying molecular mechanism. These findings warrant Matrine to be further explored for its antiviral activity in clinical settings.

Hollow mesoporous silica nanoparticles facilitated drug delivery via cascade pH stimuli in tumor microenvironment for tumor therapy.

To efficiently deliver anti-cancer drug to tumor site and reduce its toxic side effects on normal tissues, a polyethylene glycol (PEG) shielding and tumor microenvironment triggering cascade pH-responsive hollow mesoporous silica nanoparticles (HMSNs) drug delivery system was fabricated. 3-(3, 4-dihydroxyphenyl) propionic acid (DHPA) functionalized beta-cyclodextrin (ß-CD) was grafted onto the surfaces of HMSNs via boronic acid-catechol ester bonds. Then, PEG conjugated adamantane (Ada) was anchored on HMSNs-ß-CD nanocarrier via host-gust interaction. Various techniques proved the successful fabrication of the system. The in vitro tests confirmed that the system was biocompatible. After the system permeating into tumor via enhanced permeability and retention (EPR) effect, the benzoic-imine bonds between the PEG and Ada were cleaved under weak acid condition in tumor microenvironment (pH 6.8), while the dissociated PEG protective layer facilitating cellular uptake of HMSNs system. Subsequently, the boronic acid-catechol ester bonds linkers further hydrolyzed under even low endosomal pH (4.5-6.5) condition for intracellular drug delivery, leading to efficient cell apoptosis. The in vivo results demonstrated that drug loaded HMSNs significantly inhibited tumor growth while only with minimal toxic side effects. The strategy provides new insight into the development of new generation of drug delivery carriers triggering by tumor microenvironment.

Up-regulation of miR-138 inhibits hypoxia-induced cardiomyocyte apoptosis via down-regulating lipocalin-2 expression.

Hypoxia-induced cardiomyocyte apoptosis contributes significantly to the development of numerous cardiac diseases, such as ischemic heart disease, heart failure, etc. Promoting cell survival by inhibiting apoptosis is one of the available strategies to attenuate cardiac dysfunction caused by cardiomyocyte loss. Previous studies have been demonstrated that miR-138 and lipocalin-2 (Lcn2) play important roles in cardiomyocyte apoptosis and survival. We presently determined whether Lcn2 is a target gene of miR-138 involved in hypoxia-induced cardiomyocyte apoptosis. Firstly, mimics of miR-138 were transfected into HL-1 cells to investigate its effect on cell apoptosis. Using 3-(4,5-dimethyl-thiazol-2-y1) 2,5-diphenyl tetrazolium bromide (MTT) and Annexin V-FITC/PI flow cytometer assays, over-expression of miR-138 significantly enhanced the cell growth and significantly attenuated the cell apoptosis in hypoxic conditions. Dual-luciferase reporter gene and western blot results confirmed Lcn2 was a direct target of miR-138. Then, the recombinant plasmid, pcDNA3.1/Lcn2 was transfected into the HL-1 cells that over-expressed miR-138. We further observed that the over-expression of Lcn2 diminished the protection of miR-138 over-expression from hypoxia-induced cell survival and apoptosis. In conclusion, our study demonstrated that up-regulation of miR-138 inhibits the hypoxia-induced cardiomyocyte apoptosis via down-regulating the pro-apoptotic gene expression of Lcn2.

SAP: structure, function, and its roles in immune-related diseases.

Serum amyloid P component (SAP), also known as pentraxin-2, is a member of the pentraxin protein family with an established relationship to the immune response. In the last century, SAP has been used as a diagnostic marker in amyloidosis diagnosis and patient follow-up. SAP has been thought to have potential for treating and curing amyloidosis and fibrosis diseases. More recently, it has been shown that SAP may serve as both a diagnostic marker and a therapeutic target for many immune-related diseases, such as cardiovascular, pulmonary, nephritic, neurological and autoimmune diseases. In the cardiovascular system, SAP has been defined as the culprit in amyloidosis in the heart. SAP may also exert a protective role during the early stage of atherosclerosis and myocardial fibrosis. In noncardiovascular system diseases, SAP is being developed for the treatment of pulmonary fibrosis. In this review, we summarize SAP history, structure, and its roles in immune-related diseases in different systems with emphasis on the cardiovascular system.

Heat shock protein 65 promotes atherosclerosis through impairing the properties of high density lipoprotein.

AIM: To explicit whether the functions of high density lipoprotein (HDL) are impaired in murine atherosclerosis by subcutaneous immunization with recombinant mycobacterial heat shock protein 65 (HSP65). METHODS: C57BL/6 mice were fed a normal chow-diet with non immunization as normal group. ApoE knockout (ApoE(-/-)) mice on high-fat diet were randomly divided into three groups (n = 8) and immunized subcutaneously with different concentrations of HSP65 or phosphate-buffered saline (PBS). All animals were treated for 16 weeks. Reverse cholesterol efflux, the anti-oxidant and anti-inflammatory functions of HDL were assayed. Hepatocytes and peritoneal macrophages were isolated to examine the expression of cholesterol transport regulating proteins, including SR-B1, ABCA1, ABCG1, PPAR-γ and LXR-α. RESULTS: In HSP65-immunized mice, paraoxonase1 (PON1) activity and the expression of IL-10 were reduced, while High-density lipoprotein inflammatory index (HII), myeloperoxidase (MPO) activity, and the expression of IFN-γ were elevated gradually. The MPO/PON1 ratio amount was significantly higher in HSP65-immunized group than in normal or PBS-immunized group. In addition, compared with normal or PBS-immunized group, cholesterol efflux rate and the expression of regulating proteins were markedly decreased in HSP65-immunized group. The mice immunized with HSP65 developed significantly larger aorta atherosclerotic plaques when compared with PBS-treated littermates. The high MPO/PON1 ratio was correlated with HII, cholesterol efflux rate and atherosclerotic plaques. CONCLUSIONS: This study demonstrates that subcutaneous immunization with HSP65 impairs the properties of HDL, which may contribute to its important pathogenic role of HSP65 in atherogenesis. Also, MPO/PON1 ratio may be a predictor of AS.

Curcumin inhibits monocyte chemoattractant protein-1 expression and enhances cholesterol efflux by suppressing the c-Jun N-terminal kinase pathway in macrophage.

OBJECTIVE: To investigate the effect of curcumin on monocyte chemoattractant protein 1 (MCP-1) production and reverse cholesterol transport (RCT) in macrophage induced by oxidation low-density lipoprotein (ox-LDL), and to identify the signal pathways involved. METHODS: The macrophages were treated with ox-LDL and various concentrations of curcumin simultaneously. The MCP-1 expression was measured by enzyme-linked immunosorbent assay. The apoAI-mediated cholesterol efflux was measured by (3)H-cholesterol-labeled counting radioactivity. The activation of intracellular signaling pathways was studied by Western blotting. RESULTS: Curcumin decreased the production of MCP-1 induced by ox-LDL in macrophages. MCP-1 expression was restrained by the inhibition of c-Jun N-terminal kinase (JNK) pathway (SP600125) and NF-κB pathway (BAY11-7082). Curcumin suppressed the phosphorylation of JNK and activation of NF-κB. Curcumin also enhanced RCT via up-regulating the expression of liver X receptor alpha (LXRα), ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI). Additionally, the inhibition of JNK (SP600125) increased cholesterol efflux and increased the expression of ABCA1 and SR-BI, but had no effect on LXRα. CONCLUSION: Curcumin suppresses MCP-1 production induced by ox-LDL via the JNK pathway and NK-κB pathway, while enhances cholesterol efflux in macrophage via suppressing the JNK pathway and activating the LXR-ABCA1/SR-BI pathway, which indicate that the vascular protective effect of curcumin is related to anti-inflammation and anti-atherosclerosis.