PM2.5 exposure induces vascular dysfunction via NO generated by iNOS in lung of ApoE-/- mouse.

PM2.5 exposure exacerbates cardiovascular diseases via oxidative stress and inflammation, the detailed mechanism of which is unclear. In this study, the effects of oxidative stress and inflammation, as well as vascular structure and function were studied by multiple PM2.5 exposure model of ApoE-/- mice. The results indicated that NO produced by iNOS not cNOS might play important roles in inducing vascular dysfunction after PM2.5 exposure. The occurrence order and causality among NO, other oxidative stress indicators and inflammation is explored by single PM2.5 exposure. The results showed that NO generated by iNOS occurred earlier than that of other oxidative stress indicators, which was followed by the increased inflammation. Inhibition of NOS could effectively block the raise of NO, oxidative stress and inflammation after PM2.5 exposure. All in all, we firstly confirmed that NO was the initiation factor of PM2.5 exposure-induced oxidative stress, which led to inflammation and the following vascular dysfunction.

PM2.5 aggravates diabetes via the systemically activated IL-6-mediated STAT3/SOCS3 pathway in rats' liver.

PM2.5 exposure aggravates type 2 diabetes, in which inflammatory factors play an important role. In this study, we aimed to explore the mechanisms responsible for aggravating diabetes after PM2.5 exposure, and study the roles of inflammatory factors in insulin-resistant type 2 diabetes. Our study indicated that short-time PM2.5 exposure enhances insulin resistance in type 2 diabetic rats and significantly raises inflammatory factors, including IL-6, TNF-α, and MCP-1, in lungs. However, we found that of these inflammatory factors only IL-6 levels are elevated in blood, liver, adipose tissue, and macrophages, but not in skeletal muscle. IL-6 induced activation of the STAT3/SOCS3 pathway in liver, but not other downstream pathways including STAT1, ERK1/2, and PI3K. Both STAT3 inhibition and IL-6 neutralization effectively alleviated the disorders of glucose metabolism after PM2.5 exposure. Taken together, this suggests that the systemic increase in IL-6 may play an important role in the deterioration of the type 2 diabetes via IL-6/STAT3/SOCS3 pathway in liver after short-time exposure to PM2.5. Besides, we unexpectedly found a stronger resistance to the PM2.5 exposure-induced increase in IL-6 in skeleton muscle than those of many other tissues.

PM2.5 Exposure Induces More Serious Apoptosis of Cardiomyocytes Mediated by Caspase3 through JNK/ P53 Pathway in Hyperlipidemic Rats.

Exposure to airborne particulate matter with an aerodynamic diameter less than or equivalent to 2.5 microns (PM2.5) easily induces acute myocardial infarction in populations with high-risk cardiovascular diseases such as hyperlipidemia, but its mechanism remains unclear. In this study, hyperlipidemic rats were used to examine the effects of PM2.5 exposure on the cardiovascular system and the mechanism for its induction of cardiovascular events. We found that PM2.5 exposure resulted in bigger changes in the myocardial enzyme profile (cTnI, LDH, CK, CK-MB) in hyperlipidemic rats than that of control rats, as well as a significant increase in the C-reactive protein (CRP) level and a decrease in the superoxide dismutase (SOD) activity. It promoted a hypercoagulable state, significantly increased blood pressure and heart rate, while decreased the variability of heart rate in hyperlipidemic rats. In addition, pathological test showed that PM2.5 exposure more easily deteriorated myocardial injury in hyperlipidemic rats. It upregulated the phosphorylation levels of myocardial c-Jun NH2-terminal kinase (JNK) and P53, resulting in the elevated expression of downstream effector protein Bax and the decreased expression of Bcl-2, and then increased caspase3 level leading to cardiomyocyte apoptosis, while little change of caspase2 was observed. Taken together, PM2.5 exposure induced more serious inflammation and oxidative stress in the circulation system of hyperlipidemic rats, promoted a hypercoagulable state and triggered cardiomyocyte apoptosis, in which JNK/P53 pathway played a key role.

SAK-HV Promotes RAW264.7 cells Migration Mediated by MCP-1 via JNK and NF-κB Pathways.

Macrophage migration plays an essential role in immune system and is also involved in many pathological situations. However, the regulatory mechanism of macrophage migration remains to be elucidated due to its diverse responses to various stimuli. SAK-HV, a multifunctional protein possessing thrombolytic and lipid-lowering activity, can selectively induce the macrophage proliferation. Here, we reported SAK-HV significantly triggered RAW264.7 cells migration through its functional domain of SAK-mutant by activating both c-jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) pathways. Meanwhile, SAK-HV upregulated the expression of some effector proteins, among which only the expression of Monocyte chemoattractant protein-1 (MCP-1) was inhibited by the blockade of JNK and NF-κB pathways. Further research showed that MCP-1 promoted migration ultimately by interacting with Chemokine (C-C motif) Receptor 2 (CCR2) in an autocrine manner. In summary, SAK-HV induced RAW264.7 cells migration through its SAK-mutant domain, during which MCP-1 chemokine mediated by JNK and NF-κB pathways played a key role. These results revealed a novel effect of SAK-HV on modulating macrophage migration and also deepened the understanding of its pharmacodynamics.

A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats.

BACKGROUND: Current options to treat clinical relapse in inflammatory central nervous system (CNS) conditions such as cerebral ischemia-reperfusion injury are limited, and agents that are more effective are required. Disruption of the blood-brain barrier is an early feature of lesion formation that correlates with clinical exacerbation and facilitates the entry of inflammatory medium and inflammatory cells. Interleukin-1 receptor antagonist (IL-1RA) is a naturally occurring anti-inflammatory antagonist of the interleukin-1 (IL-1) family. The broad-spectrum anti-inflammatory effects of IL-1RA have been investigated against various forms of neuroinflammation. However, the effect of IL-1RA on blood-brain barrier disruption following ischemia-reperfusion has not been reported. METHODS: In this study, we investigated the effects of IL-1RA and a novel protein (IL-1RA-PEP) that was fused to IL-1RA with a cell penetrating peptide, on blood-brain barrier integrity, in male rats subjected to transient middle cerebral artery occlusion. RESULTS: After intravenous administration, IL-1RA-PEP (50 mg/kg) penetrated cerebral tissues more effectively than IL-1RA. Moreover, it preserved blood-brain barrier integrity, attenuated changes in expression and localization of tight junction proteins and matrix metalloproteinases, and enhanced angiogenesis in ischemic brain tissue. Further study suggested that the effects of IL-1RA-PEP on preserving blood-brain barrier integrity might be closely correlated with the p65/NF-κB pathway, as evidenced by the effects of the inhibitor JSH-23. CONCLUSIONS: Collectively, our results demonstrated that IL-1RA-PEP could effectively penetrate the brain of rats with middle cerebral artery occlusion and ameliorate blood-brain barrier disruption. This finding might represent its novel therapeutic potential in the treatment of the cerebral ischemia-reperfusion injury.

A novel IL-1RA-PEP fusion protein with enhanced brain penetration ameliorates cerebral ischemia-reperfusion injury by inhibition of oxidative stress and neuroinflammation.

Neuroinflammation and oxidative stress are involved in cerebral ischemia-reperfusion, in which Interleukin 1 (IL-1), as an effective intervention target, is implicated. Interleukin-1 receptor antagonist (IL-1RA) is the natural inhibitor of IL-1, but blood-brain barrier (BBB) limits the brain penetration of intravenously administered IL-1RA, thereby restricting its therapeutic effect against neuroinflammation. In this study, we evaluated the potential effects of anti-inflammation and anti-oxidative stress of a novel protein IL-1RA-PEP, which fused IL-1RA with a cell penetrating peptide (CPP). Studies were carried out in transient middle cerebral artery occlusion (MCAO) in rats and oxygen glucose deprivation/reoxygenation (OGD/R) in primary cortical neurons. In MCAO rat model, IL-1RA-PEP (50mg/kg) injected i.v., penetrated BBB effectively, and alleviated brain infarction, cerebral edema, neurological deficit score and motor performance as well as inhibited the inflammatory cytokines expression. Furthermore, our results firstly showed that IL-1RA-PEP also regulated the oxidases expression, decreased the levels of NO, MDA and ROS. In addition, the inhibitory effects of IL-1RA-PEP on oxidative stress and inflammation were confirmed in rat cortical neurons induced by OGD/R, it reduced ROS, IL-6 and TNF-α. Further study showed that the effects of IL-1RA-PEP were closely associated with the NF-κB and p38 pathways which were proved respectively by their inhibitors JSH-23 and SB203580. Our results indicated that IL-1RA-PEP could effectively penetrate the brain of MCAO rats, alleviated the cerebral ischemia reperfusion injury by inhibiting neuroinflammation and oxidative stress, showing a great clinical potential for stroke.

SAK-HV Triggered a Short-period Lipid-lowering Biotherapy Based on the Energy Model of Liver Proliferation via a Novel Pathway.

The accumulations of excess lipids within liver and serum are defined as non-alcoholic fatty liver disease (NAFLD) and hyperlipemia respectively. Both of them are components of metabolic syndrome that greatly threaten human health. Here, a recombinant fusion protein (SAK-HV) effectively treated NAFLD and hyperlipemia in high-fat-fed ApoE-/- mice, quails and rats within just 14 days. Its triglyceride and cholesterol-lowering effects were significantly better than that of atorvastatin during the observation period. We explored the lipid-lowering mechanism of SAK-HV by the hepatic transcriptome analysis and serials of experiments both in vivo and in vitro. Unexpectedly, SAK-HV triggered a moderate energy and material-consuming liver proliferation to dramatically decrease the lipids from both serum and liver. We provided the first evidence that PGC-1α mediated the hepatic synthesis of female hormones during liver proliferation, and proposed the complement system-induced PGC-1α-estrogen axis via the novel STAT3-C/EBPß-PGC-1α pathway in liver as a new energy model for liver proliferation. In this model, PGC-1α ignited and fueled hepatocyte activation as an "igniter"; PGC-1α-induced estrogen augmented the energy supply of PGC-1α as an "ignition amplifier", then triggered the hepatocyte state transition from activation to proliferation as a "starter", causing triglyceride and cholesterol-lowering effects via PPARα-mediated fatty acid oxidation and LDLr-mediated cholesterol uptake, respectively. Collectively, the SAK-HV-triggered distinctive lipid-lowering strategy based on the new energy model of liver proliferation has potential as a novel short-period biotherapy against NAFLD and hyperlipemia.

SAK-HV Decreases the Self-Ubiquitination of MEKK1 to Promote Macrophage Proliferation via MAPK/ERK and JNK Pathways.

SAK-HV is an anti-atherosclerosis recombinant fusion protein developed by our lab. Our study determined that SAK-HV promoted macrophage proliferation, of which the mechanism was explored by both RAW264.7 cells and primary macrophages. Mass spectrometric analysis and co-immunoprecipitation were combined to screen the SAK-HV-interacting proteins in RAW264.7 cells. Confocal microscopy was adopted to detect the localization of SAK-HV in cells. The results indicated that SAK-HV triggered macrophage proliferation via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) pathways by its SAK-mutant functional domain. We screened out Uba1 as the SAK-HV-interacting protein in the RAW264.7 cells and discovered their co-localization in the cytoplasm and nucleus. Inhibiting Uba1 significantly decreased the SAK-HV-induced macrophage proliferation. Thus, we postulated an attractive model of ubiquitination, in which the interactions between Uba1 and specific E2 enzymes are blocked by its interaction with SAK-HV. Based on this model, we detected the decreased self-ubiquitination of MEKK1 after SAK-HV treatment and concluded that SAK-HV inhibits the self-ubiquitination of MEKK1 via its SAK-mutant functional domain to activate MAPK/ERK and JNK pathways, promoting macrophage proliferation. This conclusion highly supported our hypothesized model of ubiquitination at the level of Uba1, which may represent a novel paradigm to promote macrophage proliferation by using the E1 enzyme (Uba1) as a switch.

Inhibition of serine proteases by a new class of cyclosulfamide-based carbamylating agents.

A new class of carbamylating agents based on the cyclosulfamide scaffold is reported. These compounds were found to be efficient time-dependent inhibitors of human neutrophil elastase (HNE). Exploitation of the three sites of diversity present in the cyclosulfamide scaffold yielded compounds which inhibited HNE but not proteinase 3 (PR 3) or bovine trypsin. The findings reported herein suggest that the introduction of appropriate recognition elements into the cyclosulfamide scaffold may lead to highly selective agents of potential value in the design of activity-based probes suitable for investigating proteases associated with the pathogenesis of chronic obstructive pulmonary disease.

Serendipitous discovery of an unexpected rearrangement leads to two new classes of potential protease inhibitors.

The pathogenesis of a range of human diseases arises from the aberrant activity of proteolytic enzymes. Agents capable of selectively modulating the activity of these enzymes are of potential therapeutic value. Thus, there is a continuing need for the design of scaffolds that can be used in the development of new classes of protease inhibitors. We describe herein the serendipitous discovery of an unexpected rearrangement that leads to the formation of two novel templates that can be used in the design of protease inhibitors.

Potent inhibition of human leukocyte elastase by 1,2,5-thiadiazolidin-3-one 1,1 dioxide-based sulfonamide derivatives.

The design, synthesis, and in vitro biochemical evaluation of a class of mechanism-based inhibitors of human leukocyte elastase (HLE) that incorporate in their structure a 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold with appropriate recognition and reactivity elements appended to it is described. The synthesized compounds were found to be efficient, time-dependent inhibitors of HLE. The interaction of the inhibitors with HLE is postulated to lead to the formation of a highly reactive N-sulfonyl imine (a Michael acceptor) that arises from an enzyme-induced sulfonamide fragmentation cascade. Subsequent reaction ultimately leads to the formation of a relatively stable acyl enzyme. The results cited herein demonstrate convincingly the superiority of the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold over other scaffolds (e.g., saccharin) in the design of inhibitors of (chymo)trypsin-like serine proteases.

Mechanism-based inactivation of human leukocyte elastase via an enzyme-induced sulfonamide fragmentation process.

We describe herein the design and in vitro biochemical evaluation of a novel class of mechanism-based inhibitors of human leukocyte elastase (HLE) that inactivate the enzyme via an unprecedented enzyme-induced sulfonamide fragmentation cascade. The inhibitors incorporate in their structure an appropriately functionalized saccharin scaffold. Furthermore, the inactivation of the enzyme by these inhibitors was found to be time-dependent and to involve the active site. Biochemical, HPLC, and mass spectrometric studies show that the interaction of these inhibitors with HLE results in the formation of a stable acyl complex and is accompanied by the release of (L) phenylalanine methyl ester. The data are consistent with initial formation of a Michaelis-Menten complex and subsequent formation of a tetrahedral intermediate with the active site serine (Ser(195)). Collapse of the tetrahedral intermediate with tandem fragmentation results in the formation of a highly reactive conjugated sulfonyl imine which can either react with water to form a stable acyl enzyme and/or undergo a Michael addition reaction with an active site nucleophilic residue (His(57)). It is also demonstrated herein that this class of compounds can be used in the design of inhibitors of serine proteases having either a neutral or basic primary substrate specificity. Thus, the results suggest that these inhibitors constitute a potential general class of mechanism-based inhibitors of (chymo)trypsin-like serine proteases.

Potential protease inhibitors based on a functionalized cyclic sulfamide scaffold.

Exploratory studies related to the design and synthesis of functionalized cyclic sulfamides (I) as potential inhibitors of proteolytic enzymes were carried out. The structural motif and three diversity sites embodied in the scaffold render it amenable to combinatorial parallel synthesis and the facile generation of lead discovery prospecting libraries. The scaffold was readily assembled starting with (DL) serine methyl ester, and a series of compounds was generated and screened against human leukocyte elastase. Modification of the P(1) recognition element, believed to be accommodated at the primary specificity site (S(1) subsite) of the enzyme, yielded compounds that inhibited the enzyme by an apparent hyperbolic partial mixed-type inhibition.

Design, synthesis, and in vitro evaluation of inhibitors of human leukocyte elastase based on a functionalized cyclic sulfamide scaffold.

The design of novel functionalized templates capable of binding to the active site of serine proteases could potentially lead to the development of potent and highly selective non-covalent inhibitors of these enzymes. Using the elastase-turkey ovomucoid inhibitor complex and insights gained from earlier work based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold (I), a surrogate cyclosulfamide scaffold (II) was used for the first time in the design of reversible inhibitors of human leukocyte elastase. Compounds 7 and 8 were found to be micromolar reversible inhibitors of the enzyme.

Noncovalent inhibitors of human leukocyte elastase based on the 4-imidazolidinone scaffold.

A central problem associated with the design of enzyme inhibitors in general, and serine protease inhibitors in particular, is the identification of templates capable of binding to the active site of an enzyme in a predictable and substrate-like fashion, orienting appended recognition elements in a correct spatial relationship so that favorable binding interactions with multiple sites are achieved. Described herein for the first time is the design of noncovalent inhibitors of human leukocyte elastase that employs a functionalized 4-imidazolidinone scaffold.