Anti-malarial artesunate ameliorates atherosclerosis by modulating arterial inflammatory responses via inhibiting the NF-κB-NLRP3 inflammasome pathway.

Introduction: Chronic inflammation plays a critical role in the pathogenesis of atherosclerosis (AS), and involves a complex interplay between blood components, macrophages, and arterial wall. Therefore, it is valuable in the development of targeted therapies to treat AS. Methods: AS rat model was induced by atherogenic diet plus with lipopolysaccharide (LPS) and then treated by anti-malarial artesunate (Art), a succinate derivative of artemisinin. The arterial morphology was observed after Oil red O, hematoxylin-eosin, and Masson's staining. The arterial protein level was detected by immunohistochemistry or immunofluorescence. The expression level of mRNA was determined by PCR array or real-time PCR. Results: Herein, we showed that Art possessed a dose-dependently protective effect on AS rats. In detail, Art showed a comparable inhibitory effect on arterial plaque and serum lipids compared to those of rosuvastatin (RS), and further showed a better inhibition on arterial lipid deposition and arterial remodeling comprised of arterial wall thicken and vascular collagen deposition, than those of RS. The improvement of Art on AS rats was related to inhibit arterial macrophage recruitment, and inhibit nuclear factor κB (NF-κB)-related excessive arterial inflammatory responses. Critically, Art showed significant inhibition on the NLRP3 inflammasome activation in both arterial wall and arterial macrophages, by down-regulating the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and apoptosis associated speckle-like protein containing CARD (ASC), leading to less production of the NLRP3 inflammasome-derived caspase-1, interleukin-1ß (IL-1ß), IL-18, and subsequent transforming growth factor ß1 (TGF-ß1) in AS rats. Conclusion: We propose that Art is an anti-AS agent acts through modulating the arterial inflammatory responses via inhibiting the NF-κB - NLRP3 inflammasome pathway.

Cardiomyocytic FoxP3 is involved in Parkin-mediated mitophagy during cardiac remodeling and the regulatory role of triptolide.

Rationale: Forkhead/winged helix transcriptional factor P3 (FoxP3) is a well-studied transcription factor that maintains the activity of T cells, but whether cardiomyocytic FoxP3 participates in cardiac remodeling (CR) remains unclear. The present study was to investigate the role of cardiomyocytic FoxP3 in CR from the perspective of mitophagy. Methods: CR was induced by angiotensin II (AngII) in vitro, or by isoproterenol (Iso) in vivo using male C57 mice or FoxP3DTR mice. Histological changes were observed by hematoxylin-eosin and Masson staining. Molecular changes were detected by immunohistochemistry, immunofluorescence, immunoblotting, and real-time PCR. Mitophagy was shaped by transmission electron microscopy and co-localization. The mRNA expression was operated by siRNA or adeno associated virus (AAV). Molecular interactions were detected by co-localization, immunoprecipitation (IP), and chromatin IP. Results: The expression and nuclear translocation of cardiomyocytic FoxP3 were downregulated in CR, while they were upregulated after triptolide (TP) treatment. In left ventricle (LV) remodeling in mice, autophagy was activated continuously in the myocardium, and TP significantly attenuated it. AngII induced massive mitophagy characterized by the activation of autophagy regulatory protein 5 (Atg5)-dependent autophagic flux. Critically, Parkin was identified as the main adaptor mediated myocardial mitophagy and was responsible for the effect of TP. Moreover, FoxP3 was responsible for the downregulation of Parkin and inhibited AngII-induced cardiac mitophagy. We found that mitophagy increased significantly and the inhibition of TP treatment reversed completely in FoxP3-deficient LVs. Mechanistically, FoxP3 interacted with a motif located downstream of the activating transcription 4 (ATF4)-binding motif involved in the promoter of Parkin and hijacked free nuclear ATF4 to decrease Parkin mRNA expression in CR. Conclusion: Cardiomyocytic FoxP3 could negatively regulate Parkin-mediated mitophagy in CR, and restoring cardiomyocytic FoxP3 activity provided a cardioprotective strategy by inhibiting excessive cardiac mitophagy.

Artesunate interrupts the self-transcriptional activation of MarA to inhibit RND family pumps of Escherichia coli.

Resistance-Nodulation-Division (RND) family pumps are responsible for producing multidrug resistance in Escherichia coli; however, there has been little study of targeted inhibitors of RNDs. In the present study, we investigated the inhibition of RND pumps by artesunate (AS) in E. coli, and further investigated the mechanism with respect to MarA, a regulator of RNDs. Although AS had no direct antibacterial effect, it showed a synergistic effect in combination with ß-lactams against E. coli ATCC35218 in vitro and in vivo, suggesting it possesses antibacterial enhancement activity. Notably, AS, alone or in combination with ß-lactams, downregulated the mRNA expression levels of marA, soxS, and rob, known as the marA-soxS-rob regulon, which then decreased the expression levels of RNDs, thereby increased ampicillin accumulation within ATCC35218. Using gene-deletion strains, we found that the antibacterial sensitization effect of AS persisted in wildtype bacteria, but was completely lost in the strain lacking marA, and decreased in the strain lacking soxS or rob, suggesting marA plays a crucial role in the sensitization of AS. Critically, we showed that AS inhibited the binding of MarA to the promoter of marA itself, not acrB, resulting in decreased mRNA expression of both acrB and marA. Mechanistically, we found AS directly bound to the central cavity of MarA through the R59 and K62 residues, and thus altered the charge distribution of MarA to interrupt the recognition between MarA and its promoter. We concluded that AS interrupts the self-transcriptional activation of MarA, thereby inhibits MarA-dependent mRNA expression of marA, acrAB, and tolC, and also certain other RNDs and regulatory genes related to MarA. Therefore, AS is a novel inhibitor of RND pumps that acts on the regulator MarA.

Magnesium isoglycyrrhizinate ameliorates radiation-induced pulmonary fibrosis by inhibiting fibroblast differentiation via the p38MAPK/Akt/Nox4 pathway.

Radiation-induced pulmonary fibrosis (RIPF) is a severe complication in patients treated with thoracic irradiation. Until now, there are no effective therapeutic drugs for RIPF. In the present study, we attempted to evaluate the effect of Magnesium isoglycyrrhizinate (MgIG) on RIPF, and to further explore the underlying mechanisms. We found that MgIG treatment markedly improved radiation-induced lung pathological changes, reduced collagen deposition, and decreased the transforming growth factor beta1 (TGF-ß1) elevation induced by irradiation. In addition, MgIG treatment significantly relieved oxidative damage of pulmonary fibrosis in mice characterized by increased antioxidant factors expression and reduced oxidative factors expression. And, MgIG treatment also significantly reduced the production of intracellular reactive oxygen species (ROS) in vitro. Interestingly, administration of MgIG achieved lower expression levels of Nox4, and phosphorylation of p38MAPK and Akt in vivo and in vitro. Furthermore, treatment with MgIG notably reduced the expression levels of myofibroblast markers, Nox4, and phosphorylation of p38MAPK and Akt both in vivo and in vitro. More importantly, the inhibitory effects of MgIG on fibroblast differentiation were enhanced when the p38MAPK/Akt/Nox4 pathway was inhibited using their respective antagonists or Nox4 siRNA in vitro. Taken together, these findings suggested that MgIG could attenuate RIPF partly by inhibiting fibroblast differentiation, which was closely related to modulation of the p38MAPK/Akt/Nox4 pathway.

Dual Role of Triptolide in Interrupting the NLRP3 Inflammasome Pathway to Attenuate Cardiac Fibrosis.

In a previous paper, we reported that triptolide (TP), a commonly used immunomodulator, could attenuate cardiac hypertrophy. This present study aimed to further explore the inhibition of cardiac fibrosis by TP and the possible mechanism from the perspective of the NOD-like receptor protein 3 (NLRP3) inflammasome. Hematoxylin-eosin and Masson's staining, immunohistochemistry, and immunofluorescence were performed to observe cardiac fibrotic changes in mice and mouse cardiac fibroblasts (CFs). The Western blot, colocalization, and immunoprecipitation were applied to detect protein expression and interactions. Results suggested that TP dose-dependently inhibited cardiac fibrosis induced by isoproterenol and collagen production of CFs induced by angiotensin II. TP exhibited an antifibrotic effect via inhibiting activation of the NLRP3 inflammasome, which sequentially decreased IL-1ß maturation, myeloid differentiation factor 88 (MyD88)-related phosphorylation of c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase 1/2 (ERK1/2), and TGF-ß1/Smad signaling, and ultimately resulted in less collagen production. Moreover, TP showed no antifibrotic effect in Nlrp3-knockout CFs. Notably, TP inhibited the expression of NLRP3 and apoptosis-associated speck-like proteins containing a caspase recruitment domain (ASC) as well as inflammasome assembly, by interrupting the NLRP3-ASC interaction to inhibit inflammasome activation. Finally, TP indeed inhibited the NLRP3-TGFß1-Smad pathway in vivo. Conclusively, TP was found to play a dual role in interrupting the activation of the NLRP3 inflammasome to attenuate cardiac fibrosis.

[Research progress on pharmacokinetics and pharmacological activities of artesunate].

Artesunate (AS), a famous derivative of the artemisinin, is the basic treatment globally for mild to severe malaria infection due to the prominent advantages such as high efficiency, fast effect, low toxicity and not easy to produce resistance. More and more research reports have shown that AS and its active metabolites dihydroartemisinin (DHA) had various bioactivities in addition to antimalarial activity, attracting researchers to further study its new pharmacological effects in order to explore new use of the old drug. A comprehensive understanding of the pharmacokinetic characteristics of AS will be conducive to the further development of new pharmacological actions and clinical application of AS. Therefore, this paper would review the absorption, distribution, metabolism and excretion of AS in vivo, as well as the pharmacokinetics characteristics of AS and DHA after clinical administration of AS by intravenous (IV), intramuscular (IM), oral or rectal routes. The in vivo process and pharmacokinetic parameters of AS and DHA were compared between healthy volunteers, malaria patients, and special populations (children, women). Meanwhile, the research progress on pharmacological effects of AS and active metabolite DHA such as anti-tumor, anti-inflammatory, anti septic, antiangiogenic, anti-fibrosis and immunoregulation activities would be also reviewed, hoping to provide a theoretical basis for the further development and utilization of AS and its metabolites.

Artesunate Attenuates Pro-Inflammatory Cytokine Release from Macrophages by Inhibiting TLR4-Mediated Autophagic Activation via the TRAF6-Beclin1-PI3KC3 Pathway.

BACKGROUND/AIMS: Lipopolysaccharide (LPS) plays a critical role in excessive inflammatory cytokine production during sepsis. Previously, artesunate (AS) was reported to protect septic mice by reducing LPS-induced pro-inflammatory cytokine release. In the present study, the possible mechanism of the anti-inflammatory effect of AS was further investigated. METHODS: An enzyme-linked immunosorbent assay was used to detect TNF-α and IL-6 release from macrophages. Specific small interfering RNAs (siRNAs) were used to knockdown the mRNA expression of target genes. Transmission electron microscopy and laser confocal microscopy were used to observe changes in autophagy. Western blotting was performed to detect the protein levels of tumor necrosis factor receptor-associated factor6 (TRAF6), Beclin1, phosphatidylinositol 3-kinase class III (PI3KC3), autophagy-related protein 5 (ATG5), and sequestosome 1. Immunoprecipitation (IP) and fluorescent co-localization were used to detect the interactions between TRAF6-Beclin1 and Beclin1-PI3KC3, and the ubiquitination of Beclin1. RESULTS: AS inhibited TNF-α and IL-6 release from RAW264.7 cells, mouse bone marrow-derived monocytes (BMDMs) and peritoneal macrophages (PMs) induced by LPS. However, the inhibition by AS of LPS-induced cytokine release decreased when autophagy was inhibited using 3-MA, bafilomycin A1, or a siRNA targeting the Atg5 gene. Notably, AS showed an inhibition of LPS-induced autophagic activation not degradation. Whereas, these effects of AS were lost in macrophages lacking TLR4 and decreased in macrophages with down-regulated TRAF6, indicating that AS inhibited LPS-induced cytokine release and autophagic activation via TLR4-TRAF6 signaling. Western blotting results showed AS could reduce the levels of TRAF6, Beclin1, and PI3KC3. Importantly, the IP results showed AS only inhibited K63-linked ubiquitylation not total ubiquitylation of Beclin1 by acting on TRAF6. This interrupted the TRAF6-Beclin1 interaction and subsequent the formation of Beclin1- PI3KC3 core complex of the PI3K-III complex. CONCLUSION: AS inhibited LPS-induced cytokine release from macrophages by inhibiting autophagic activation. This effect was tightly related to blockade of the TRAF6-Beclin1-PI3KC3 pathway via decreasing K63-linked ubiquitination of Beclin1 and then interrupting the formation of Beclin1-PI3KC3 core complex of the PI3K-III complex. Our findings reveal the mechanism of AS's anti-inflammatory effect and is significant for future targeted investigations of sepsis treatment.

Luminous exothermic hollow optical elements for enhancement of biofilm growth and activity.

In this work, we present a luminous-exothermic hollow optical element (LEHOE) that performs spectral beam splitting in the visible spectral range for the enhancement of biofilm growth and activity. The LEHOE is composed of a four-layer structure with a fiber core (air), cladding (SiO2), coating I (LaB6 film), and coating II (SiO2-Agarose-Medium film). To clarify the physical, optical and photothermal conversion properties of the LEHOE, we determined the surface morphology and composition of the coating materials, and examined the luminous intensity and heating rate at the LEHOE surface. The biofilm activity on the biocompatible LEHOE is far greater than that of commercial fibers, and the biofilm weight on the LEHOE is 4.5 × that of the uncoated hollow optical element.

High-sensitivity four-layer polymer fiber-optic evanescent wave sensor.

We present a novel four-layer structure consisting of bottom, second, third, and surface layers in the sensing region, for a D-shaped step-index fiber-optic evanescent wave (FOEW) sensor. To reduce the background noise, the surface of the longitudinal section in the D-shaped region is coated with a light-absorbing film. We check the morphologies of the second and surface layers, examine the refractive indices (RIs) of the third and surface layers, and analyze the composition of the surface layer. We also investigate the effects of the thicknesses and RIs of the third and surface layers and the LA film on the light transmission and sensitivity of the FOEW sensors. The results highlight the very good sensitivity of the proposed FOEW sensor with a four-layer structure, which reached -0.077 (µg/l)-1 in the detection of the target antibody; the sensitivity of the novel FOEW sensor was 7.60 and 1.52 times better than that of a conventional sensor with a core-cladding structure and an FOEW sensor with a three-layer structure doped with GeO2. The applications of this high-sensitivity FOEW sensor can be extended to biodefense, disease diagnosis, and biomedical and biochemical analysis.

Systematic investigation on the turning point of over-inflammation to immunosuppression in CLP mice model and their characteristics.

Immunosuppression is involved in refractory innate and adaptive immune responses and is considered to be the predominant driving force for mortality in sepsis. The cecal ligation and puncture (CLP) model is regarded as a golden standard model for sepsis study, but the turning point of over-inflammation to immunosuppression was reported differently. Herein, systematic investigation on the turning point of over-inflammation to immunosuppression in CLP mice model was carried out. The results showed only the mortality of mice challenged with of Pseudomonas aeruginosa on Day 1 not other days after the surgery was higher than that of other mice with Sham surgery, suggesting Day 1 after the CLP surgery might be the turning point. There was very low mortality even without death in Sham mice but the mortality was 80% after mice were challenged with 2.5×107, 5.0×106 and 1.0×106CFU/10g of Pseudomonas aeruginosa, further demonstrating Day 1 after the CLP surgery was the turning point. And, CLP mice presented low levels of pro-inflammatory and anti-inflammatory cytokines, and high bacterial loads on Day 1. Additionally, the amounts and proportion of blood cells and monocytes significantly changed, too. In conclusion, Day 1 after the CLP surgery was the turning point of over-inflammation to immunosuppression, and low levels of cytokines and high bacterial loads were the characteristics of this model on Day 1, which is significant for pharmacological investigation on sepsis.

Artesunate attenuated progression of atherosclerosis lesion formation alone or combined with rosuvastatin through inhibition of pro-inflammatory cytokines and pro-inflammatory chemokines.

BACKGROUNDS: Inflammation plays an important role in all stages of atherosclerosis, but little is known about the therapeutic effects of quenching inflammation in atherosclerotic lesions formation. PURPOSE: Herein, the effect of artesunate, a derivant from artemisinin from the traditional Chinese herb sweet wormwood, could attenuate the progression of atherosclerosis lesion formation alone or combined with rosuvastatin in Western-type diet (WD) fed ApoE(-/-) mice, and explored its possible mechanisms. METHODS: The methods such as ELISA for plasma lipids and cytokines analyses, qRT-PCR and western blot for mRNA and protein expressions, and MTT assay for human umbilical vein endothelial cells (HUVECs) viability were used for in vivo and in vitro experiments. RESULTS: Artesunate could attenuate the progression of atherosclerosis lesion formation alone or combined with rosuvastatin in WD fed ApoE(-/-) mice without changes in food uptake, body weight and plasma lipids level, but with a significant reduction of pro-inflammatory cytokine, such as TNF-α and IL-6. Furthermore, artesunate could down-regulate the pro-inflammatory chemokines such as IL-8 and MCP-1 in aorta of mice. Besides, artesunate didn't influence IL-8 and MCP-1 secretion in HUVECs up-regulated by TNF-α, but inhibited IL-8 and MCP-1 secretion up-regulated by LPS. CONCLUSION: AS attenuated progression of atherosclerosis lesion formation alone or combined with rosuvastatin through anti-inflammatory effect, resulting in down-regulation of TNF-α and IL-6, and further down-regulating IL-8 and MCP-1 expressions in aorta of WD fed ApoE(-/-) mice. Rosuvastatin combined with artesunate could more effectively attenuate the progression of atherosclerosis lesions than when treated by one of them, demonstrating that lipid-lowering agents combined with anti-inflammatory agents could provide the greater benefit for cardiovascular disease patients. Artesunate is worth further investigating as a candidate drug for the treatment of atherosclerosis.

Artesunate ameliorates severe acute pancreatitis (SAP) in rats by inhibiting expression of pro-inflammatory cytokines and Toll-like receptor 4.

Severe acute pancreatitis (SAP) is a severe clinical condition with significant morbidity and mortality. Multiple organs dysfunction (MOD) is the leading cause of SAP-related death. The over-release of pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α is the underlying mechanism of MOD; however, there is no effective agent against the inflammation. Herein, artesunate (AS) was found to increase the survival of SAP rats significantly when injected with 3.5% sodium taurocholate into the biliopancreatic duct in a retrograde direction, improving their pancreatic pathology and decreasing serum amylase and pancreatic lipase activities along with substantially reduced pancreatic IL-1ß and IL-6 release. In vitro, AS-pretreatment strongly inhibited IL-1ß and IL-6 release and their mRNA expressions in the pancreatic acinar cells treated with lipopolysaccharide (LPS) but exerted little effect on TNF-α release. Additionally, AS reduced the mRNA expressions of Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) p65 as well as their protein expressions in the pancreatic acinar cells. In conclusion, our results demonstrated that AS could significantly protect SAP rats, and this protection was related to the reduction of digestive enzyme activities and pro-inflammatory cytokine expressions via inhibition of TLR4/NF-κB signaling pathway. Therefore, AS may be considered as a potential therapeutic agent against SAP.

TLR9-ERK-mTOR signaling is critical for autophagic cell death induced by CpG oligodeoxynucleotide 107 combined with irradiation in glioma cells.

Synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODN) function as potential radiosensitizers for glioma treatment, although the underlying mechanism is unclear. It was observed that CpG ODN107, when combined with irradiation, did not induce apoptosis. Herein, the effect of CpG ODN107 + irradiation on autophagy and the related signaling pathways was investigated. In vitro, CpG ODN107 + irradiation induced autophagosome formation, increased the ratio of LC3 II/LC3 I, beclin 1 and decreased p62 expression in U87 cells. Meanwhile, CpG ODN107 also increased LC3 II/LC3 I expression in U251 and CHG-5 cells. In vivo, CpG ODN107 combined with local radiotherapy induced autophagosome formation in orthotopic transplantation tumor. Investigation of the molecular mechanisms demonstrated that CpG ODN107 + irradiation increased the levels of TLR9 and p-ERK, and decreased the level of p-mTOR in glioma cells. Further, TLR9-specific siRNA could affect the expressions of p-ERK and autophagy-related proteins in glioma cells. Taken together, CpG ODN107 combined with irradiation could induce autophagic cell death, and this effect was closely related to the TLR9-ERK-mTOR signaling pathway in glioma cells, providing new insights into the investigation mechanism of CpG ODN.

Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides.

Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

Development of oral dispersible tablets containing prednisolone nanoparticles for the management of pediatric asthma.

The purpose of the present study was to develop oral dispersible tablets containing prednisolone (PDS)-loaded chitosan nanoparticles using microcrystalline cellulose (MCC 101), lactose, and croscarmellose sodium (CCS). The PDS-loaded chitosan nanoparticles were formulated by ionotropic external gelation technique in order to enhance the solubility of PDS in salivary pH. Prepared nanoparticles were used for the development of oral fast disintegrating tablets by direct compression method. The prepared tablets were evaluated for disintegration time (DT), in vitro drug release (DR), thickness, weight variation, drug content uniformity, friability, and hardness. The effect of concentrations of the dependent variables (MCC, lactose, CCS) on DT and in vitro DR was studied. Fast disintegrating tablets of PDS can be prepared by using MCC, CCS, and lactose with enhanced solubility of PDS. The minimum DT was found to be 15 seconds, and the maximum DR within 30 minutes was 98.50%. All independent variables selected for the study were statistically significant. Oral fast disintegrating tablets containing PDS nanoparticles could be the better choice for the pediatric patients that would result in better patient compliance. From this study, it can be concluded that fast disintegrating tablets could be a potential drug delivery technology for the management of asthma in pediatrics.

Pharmacokinetic Profile and Acute Toxicological Properties of a Novel Radiosensitizer Cytosine-Phosphate-Guanosine Oligodeoxynucleotide 107 in Mice Following Intravenous and Orthotopic Administration.

The synthetic cytosine-phosphate-guanosine oligodeoxynucleotide 107 (CpG ODN107) is a novel radiosensitizer for glioma treatment. However, the information related to its pharmacokinetics and toxicity remains unclear. Therefore, the plasma pharmacokinetics, distribution, elimination, and acute toxicity of CpG ODN107 in mice were investigated in the present experiments. The results from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that the plasma elimination half-life (t1/2ß) of CpG ODN107 in BALB/c mice varied slightly with the dose, and it was 0.65, 0.49, and 0.50 h at the intravenous doses of 2.5, 5, and 10 mg/kg, respectively. CpG ODN107 rapidly and widely distributed in organs/tissues, except the brain and testes. The highest concentrations were found in the liver (28.6% of the administered dose after 0.5 h) and the kidneys (5.7% of the administered dose after 1 h). CpG ODN107 (0.3, 3, and 30 µg/mL) could highly bind to human and mouse plasma proteins in vitro. CpG ODN107 in the forms of prototype was excreted in urine (1.79%) and feces (0.91%), and its shortened metabolites were excreted in urine (2.1%) and feces (2.2%) within the first 24 h. The mice in vivo optical image showed CpG ODN107 labeled with Alexa Fluor 680 fluorochrome (AF680) accumulated in the brain after orthotopic injection, eliminated very slowly, and excreted in urine compared with poly T labeled with AF680. The median lethal dose (LD50) of CpG ODN107 was 75.7 mg/kg for mice; this dose only could produce apparent spleen and liver damage, in line with the distribution features of CpG ODN. In conclusion, our present pharmacokinetic and toxicity investigation will provide helpful information to further pharmacodynamic and pharmacokinetic research of CpG ODN107 and other oligodeoxynucleotide drugs in the future.

The direct anti-MRSA effect of emodin via damaging cell membrane.

Methicillin-resistant Staphylococcus aureus (MRSA) has become an important bacterium for nosocomial infection. Only a few antibiotics can be effective against MRSA. Therefore, searching for new drugs against MRSA is important. Herein, anti-MRSA activities of emodin and its mechanisms were investigated. Firstly, in vitro antimicrobial activity was investigated by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-growth curve, and multipassage resistance testing was performed. Secondly, protection of emodin on mice survival and blood bacterial load in mice challenged with lethal or sublethal dose of MRSA were investigated. Subsequently, the influences of emodin on the bacterial morphology, messenger RNA (mRNA) expressions related to cell wall synthesis and lysis, ß-lactamase activity, drug accumulation, membrane fluidity, and integrity were performed to investigate its mechanisms. Lastly, in vitro cytotoxicity assay were performed using the 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method. The results showed MICs and MBCs of emodin against MRSA252 and 36 clinical MRSA strains were among 2-8 and 4-32 µg/mL, respectively. There was no MIC increase for emodin during 20 passages. In vivo, emodin dose-dependently protected mice challenged with lethal dose of MRSA and decreased bacterial load in mice challenged with sublethal dose of MRSA. Morphology observation showed emodin might disrupt cell wall and membrane of MRSA. Although emodin had no influence on genes related to cell wall synthesis and lysis as well as ß-lactamase activity and drug accumulation, emodin reduced membrane fluidity and disrupted membrane integrity. Based on the fact that emodin had no significant cytotoxicity against mammalian cells, it could be further investigated as a membrane-damage bactericide against MRSA in the future.

Emodin is identified as the active component of ether extracts from Rhizoma Polygoni Cuspidati, for anti-MRSA activity.

This study investigated the anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity and chemical compositions of ether extracts from Rhizoma Polygoni Cuspidati (ET-RPC). Significant anti-MRSA activities of ET-RPC against MRSA252 and MRSA clinical strains were tested in in vitro antibacterial experiments, such as inhibition zone diameter test, minimal inhibitory concentration test, and dynamic bacterial growth assay. Subsequently, 7 major compounds of ET-RPC were purified and identified as polydatin, resveratrol-4-O-d-(6'-galloyl)-glucopyranoside, resveratrol, torachryson-8-O-glucoside, emodin-8-O-glucoside, 6-hydroxy-emodin, and emodin using liquid chromatography - electrospray ionization - tandem mass spectrometry. After investigation of anti-MRSA activities of the 7 major compounds, only emodin had significant anti-MRSA activity. Further, transmission electron microscopy was used to observe morphological changes in the cell wall of MRSA252, and the result revealed that emodin could damage the integrity of cell wall, leading to loss of intracellular components. In summary, our results showed ET-RPC could significantly inhibit bacterial growth of MRSA strains. Emodin was identified as the major compound with anti-MRSA activity; this activity was related to destruction of the integrity of the cell wall and cell membrane.

The radiosensitizing effect of CpG ODN107 on human glioma cells is tightly related to its antiangiogenic activity via suppression of HIF-1α/VEGF pathway.

Malignant glioma displays invasive growth and is difficult to be completely excised; surgery combined with subsequent radiotherapy is a standard treatment for patients. CpG oligodeoxynucleotides (CpG ODN) can enhance radiotherapeutic effect in some tumors. Angiogenesis is crucial for tumor progression and metastasis. Anti-angiogenic strategy thus may be effective for tumor treatment. Herein, the antiangiogenic activity and radiosensitizing effect of CpG ODN107 on glioma were investigated. Our results showed that the growth of glioma cell line U87 was significantly inhibited by CpG ODN107 (10µg/ml) in combination with irradiation (5Gy) in vitro. In orthotopic implantation model of nude mice, the survival rate of mice significantly increased after treatment with CpG ODN107 (0.083mg/kg) in combination with radiotherapy (10Gy) as compared with treatment with local radiotherapy alone. CpG ODN107 in combination with radiotherapy significantly decreased microvessel density (MVD), VEGF level and HIF-1α expression in orthotopic implantation glioma. In conclusion, CpG ODN107 significantly increased the radiosensitivity of U87 human glioma cells in vitro and in vivo. The radiosensitizing effect of CpG ODN 107 is tightly related to its anti-angiogenic activity via suppression of HIF-1α/VEGF pathway.

Artesunate has its enhancement on antibacterial activity of ß-lactams via increasing the antibiotic accumulation within methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) has now emerged as a predominant and serious pathogen because of its resistance to a large group of antibiotics, leading to high morbidity and mortality. Therefore, to develop new agents against resistance is urgently required. Previously, artesunate (AS) was found to enhance the antibacterial effect of ß-lactams against MRSA. In this study, AS was first found to increase the accumulation of antibiotics (daunorubicin and oxacillin) within MRSA by laser confocal microscopy and liquid chromatography-tandem MS method, suggesting the increased antibiotics accumulation might be related to the enhancement of AS on antibiotics. Furthermore, AS was found not to destroy the cell structure of MRSA by transmission electron microscope. AS was found to inhibit gene expressions of important efflux pumps such as NorA, NorB and NorC, but not MepA, SepA and MdeA. In conclusion, our results showed that AS was capable of enhancing the antibacterial activity of ß-lactams via increasing antibiotic accumulations within MRSA through inhibiting gene expressions of efflux pumps such as NorA, NorB and NorC, but did not destroy the cell structure of MRSA. AS could be further investigated as a candidate drug for treatment of MRSA infection.