Docetaxel-loaded pH/ROS dual-responsive nanoparticles with self-supplied ROS for inhibiting metastasis and enhancing immunotherapy of breast cancer.

BACKGROUND: Although stimuli-responsive nanoplatforms were developed to deliver immunogenic cell death (ICD) inducers to enhance cancer immunotherapy, the complete release of ICD inducers into the tumor microenvironment (TME) was limited by the inadequate supplementation of endogenous stimulus (e.g., reactive oxygen species (ROS)). To address this issue, we synthesized a self-responsive nanomaterial with self-supplied ROS, which mainly consists of a ROS responsive moiety HPAP and cinnamaldehyde (CA) as the ROS-generating agent. The endogenous ROS can accelerate the degradation of HPAP in materials to release docetaxel (DTX, an ICD inducer). In intracellular acidic environment, the pH-sensitive acetal was cleaved to release CA. The released CA in turn induces the generation of more ROS through mitochondrial damage, resulting in amplified DTX release. Using this self-cycling and self-responsive nanomaterial as a carrier, DTX-loaded pH/ROS dual-responsive nanoparticles (DTX/FA-CA-Oxi-αCD NPs) were fabricated and evaluated in vitro and in vivo. RESULTS: In vitro experiments validated that the NPs could be effectively internalized by FA-overexpressed cells and completely release DTX in acidic and ROS microenvironments to induce ICD effect. These NPs significantly blocked 4T1 cell migration and decreased cell invasion. In vivo experiments demonstrated that the tumor-targeted NPs significantly inhibited tumor growth and blocked tumor metastasis. More importantly, these NPs significantly improved immunotherapy through triggering effector T-cell activation and relieving the immunosuppressive state of the TME. CONCLUSIONS: Our results demonstrated that DTX/FA-CA-Oxi-αCD NPs displayed great potential in preventing tumor metastasis, inhibiting tumor growth, and improving the efficacy of anti-PD-1antibody.

Simultaneous quantitative analysis and in vitro anti-arthritic effects of five polyphenols from Terminalia chebula.

Background: The fruit of Terminalia chebula has been widely used for a thousand years for treating diarrhea, ulcers, and arthritic diseases in Asian countries. However, the active components of this Traditional Chinese medicine and their mechanisms remain unclear, necessitating further investigation. Objectives: To perform simultaneous quantitative analysis of five polyphenols in T. chebula and evaluate their anti-arthritic effects including antioxidant and anti-inflammatory activity in vitro. Materials and methods: Water, 50% water-ethanol, and pure ethanol were used as extract solvents. Quantitative analysis of gallic acid, corilagin, chebulanin, chebulagic acid, and ellagic acid in the three extracts was performed using high-performance liquid chromatography (HPLC). Antioxidant activity was assessed by the 2,2-diphenylpicrylhydrazyl (DPPH) radical-scavenging assay, and anti-inflammatory activity was evaluated by detecting interleukin (IL)-6 and IL-8 expression in IL-1ß-stimulated MH7A cells. Results: The 50% water-ethanol solvent was the optimal solvent yielding the highest total polyphenol content, and the concentrations of chebulanin and chebulagic acid were much higher than those of gallic acid, corilagin, and ellagic acid in the extracts. The DPPH radical-scavenging assay showed that gallic acid and ellagic acid were the strongest antioxidative components, while the other three components showed comparable antioxidative activity. As for the anti-inflammatory effect, chebulanin and chebulagic acid significantly inhibited IL-6 and IL-8 expression at all three concentrations; corilagin and ellagic acid significantly inhibited IL-6 and IL-8 expression at high concentration; and gallic acid could not inhibit IL-8 expression and showed weak inhibition of IL-6 expression in IL-1ß-stimulated MH7A cells. Principal component analysis indicated that chebulanin and chebulagic acid were the main components responsible for the anti-arthritic effects of T. chebula. Conclusion: Our findings highlight the potential anti-arthritic role of chebulanin and chebulagic acid from T. chebula.

Self-Illuminating Triggered Release of Therapeutics from Photocleavable Nanoprodrug for the Targeted Treatment of Breast Cancer.

Photocleavable biomaterials and bioconjugates have been widely researched for tissue engineering, cell culture, and therapeutics delivery. However, most in vivo applications of these materials or conjugates require external irradiation, and some of the light sources used such as ultraviolet (UV) light have poor tissue penetration. To address these key limitations, we synthesized a photocleavable nanoprodrug using luminol (a luminescent donor), chlorambucil (CHL, i.e., an antitumor drug with a photocleavable linker), and polyethylene glycol-folic acid conjugates (a targeted moiety) loaded onto polyamidoamine (PAMAM). The synthesized nanoprodrug can smartly release its payloads through photocleavage of photoresponsive linker by UV light, which was produced in situ by reacting luminol with pathological reactive oxygen species (ROS). The luminescence performance and absorption spectrum of this nanoprodrug was characterized in detail. In vitro cellular assays verified that the nanoprodrugs could be efficiently internalized by 4T1 and MDA-MB-231 cells, and the CHL released from the nanoprodrugs could distinctly decrease cell viability through the damage of DNA in cells. In vivo animal experiments demonstrated that the nanoprodrugs were mainly accumulated at tumor sites, and the antitumor drug CHL could be smartly released from the nanoprodrugs through cleavage of photosensitive linkers at a high level of ROS. The released CHL significantly inhibited the growth of tumors without any obvious adverse effects. Our results provide a practicable strategy to expand the in vivo application of photocleavable biomaterials and bioconjugates.

Folic acid-modified ROS-responsive nanoparticles encapsulating luteolin for targeted breast cancer treatment.

Luteolin (Lut) is a natural flavonoid polyphenolic compound with multiple pharmacological activities, such as anti-oxidant, anti-inflammatory, and anti-tumor effects. However, the poor aqueous solubility and low bioactivity of Lut restrict its clinical translation. Herein, we developed a reactive oxygen species (ROS)-responsive nanoplatforms to improve the bioactivity of Lut. Folic acid (FA) was employed to decorate the nanoparticles (NPs) to enhance its targeting ability. The size of Lut-loaded ROS-responsive nanoparticles (Lut/Oxi-αCD NPs) and FA-modified Lut/Oxi-αCD NPs (Lut/FA-Oxi-αCD NPs) is 210.5 ± 6.1 and 196.7 ± 1.8 nm, respectively. Both Lut/Oxi-αCD NPs and Lut/FA-Oxi-αCD NPs have high drug loading (14.83 ± 3.50 and 16.37 ± 1.47%, respectively). In vitro cellular assays verified that these NPs could be efficiently internalized by 4T1 cells and the released Lut from NPs could inhibit tumor cells proliferation significantly. Animal experiments demonstrated that Lut/Oxi-αCD NPs, especially Lut/FA-Oxi-αCD NPs obviously accumulated at tumor sites, and inhibited tumor growth ∼3 times compared to the Lut group. In conclusion, the antitumor efficacy of Lut was dramatically improved by targeting delivery with the ROS-responsive nanoplatforms.

A Novel Cryptococcal Meningitis Therapy: The Combination of Amphotericin B and Posaconazole Promotes the Distribution of Amphotericin B in the Brain Tissue.

The deficient brain tissue distribution of amphotericin B (AMPB) seriously restricts its treatment for the clinical efficacy of cryptococcus neoformans meningitis (CNM). We strive to develop a tactic to increase its concentration in brain tissue. We aimed to investigate whether the combination of AMPB and posaconazole (POS) could be more effective in the treatment of CNM and to elucidate its potential mechanisms. HPLC analysis was used to analyze the concentration of AMPB in mouse serum, brain tissue, and BCECs cells. Schrodinger molecular docking, in vitro plasma balance dialysis, and ultrafiltration analysis were performed to evaluate the combinative effect of AMPB and POS with serum albumin and POS on AMPB plasma protein binding. H&E staining and colonization culture experiment of CN were employed to assess the effect of POS on the efficacy of AMPB. POS + AMPB significantly reduced the concentration of plasma total AMPB and increased its concentration in the brain tissue. However, the P-gp inhibitor zosuquidar, BCRP inhibitor Ko143, and a common inhibitor of both, elacridar, had no significant effect on its concentration. Molecular docking, balance dialysis, and ultrafiltration analysis showed that AMPB and POS had potential binding properties to serum albumin. Meanwhile, 4 and 8 µg/mL POS could significantly increase the concentration of free AMPB in plasma. POS and three inhibitors all had no significant effect on the uptake of AMPB by BCECs, but serum albumin had. The therapeutic effect of CNM in mice was confirmed that AMPB and AMPB+POS could restrain the infiltration of neutrophils and lymphocytes in cortical neurons and improve the bleeding and markedly inhibit the proliferation of CN. Collectively, we propose that POS competitively binds to the plasma protein sites of AMPB, thereby increasing its level in the brain tissue. Meanwhile, POS could enhance the efficacy of AMPB in the treatment of CNM, which may be independent of P-gp and BCRP proteins.

Chebulanin exerts its anti-inflammatory and anti-arthritic effects via inhibiting NF-κB and MAPK activation in collagen-induced arthritis mice.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and progressive joint destruction. Chebulanin is a natural polyphenol acid isolated from the traditional Tibetan medicine Terminalia chebula Retz that has previously been reported to possess anti-inflammatory properties. The present study aimed to investigate the anti-inflammatory and anti-arthritic effects of chebulanin and explore its underlying mechanisms in vivo and in vitro using a collagen-induced arthritis (CIA) mouse model and lipopolysaccharide (LPS) stimulated RAW264.7 cell inflammation model. Arthritis severity scores were assessed twice weekly; the levels of cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum were detected using enzyme-linked immunosorbent assay kits; histopathological assessment was performed using micro computed tomography and hematoxylin and eosin staining. Activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were assessed using western blotting. The inhibition of translocation of cytosolic p38 and p65 into the nucleus was observed using immunofluorescence staining and western blotting in vitro. Chebulanin significantly suppressed the progression and development of RA in CIA mice by decreasing the arthritis severity scores, attenuating paw swelling and joint destruction, and reducing the levels of IL-6 and TNF-α significantly (p < 0.05). Furthermore, chebulanin reduced the levels of excised phosphorylated (p)-p38, phosphorylated-c-JUN N-terminal kinase (p-JNK), p-p65 and phosphorylated NF-κB inhibitor alpha (p-IκBα) in CIA mice, but did not affect the level of phosphorylated extracellular-signal-regulated kinase (ERK). In addition, chebulanin could inhibit the nuclear translocation of p38 and p65 in LPS-stimulated macrophages in dose-dependent manner. In conclusion, this study demonstrated that chebulanin exerts anti-inflammatory and anti-arthritic effects by inhibiting the activation of NF-κB and MAPK signaling pathways.

Comparison of cryptococcal meningitis in HIV-negative patients with and without lung infections.

OBJECTIVE: To investigate the clinical features and outcomes of cryptococcal meningitis (CM) in HIV-negative patients with and without lung infections. METHODS: We retrospectively reviewed the medical records of HIV-negative patients with CM admitted to two university hospitals in Southwest China over the past 5 years. RESULTS: Seventy-one patients were included, of whom 35 (49.3%) had lung disease. Compared with patients without lung infection, CM patients with lung infection tended to be male and younger (≤30 years), experienced more fever, less vomiting and fewer central nervous system symptoms; more often had low white blood cell (WBC) counts (<20 × 106/L), and fewer often had ethmoid sinusitis, maxillary sinusitis, paranasal sinusitis, and otitis media. Cryptococcus neoformans isolates from these patients were sensitive to itraconazole, voriconazole, fluconazole, and amphotericin B but resistant to flucytosine. CM patients with lung infection had higher mortality at discharge compared with patients without lung infection (8.6% vs. 0%). Multivariable analyses showed that a WBC count <20 × 106/L was significantly associated with poor treatment outcome (odds ratio 0.01, 95% confidence interval 0-0.83). CONCLUSION: HIV-negative CM patients with lung infections tended to be male and younger. Fever, fewer central nervous system symptoms, and WBC counts <20 × 106/L were characteristic of this patient group.

Therapeutic drug monitoring and safety of voriconazole in elderly patients.

BACKGROUND: Data on therapeutic drug monitoring of voriconazole in elderly patients are limited. Impaired liver function and an inflammatory state in elderly individuals are hypothesized to impact the voriconazole serum level. METHODS: A total of 166 adult patients (317 trough concentrations) who underwent voriconazole therapeutic drug monitoring were enrolled. The voriconazole trough concentration, its associated covariates, and its correlation with adverse effects in 73 elderly (≥60 years) patients (116 trough concentrations) were analyzed and compared to those in 93 adult (<60 years) patients. RESULTS: The voriconazole trough concentration was 4.31 ± 3.03 µg/mL (range, 0.4-15.5 µg/mL) in the elderly patients, which was significantly higher than the 3.11 ± 2.13 µg/mL (range, 0.4-14.3 µg/mL) in the adult patients (P = 0.001). The proportion of voriconazole trough concentrations higher than 5 µg/mL was 35.3% in the elderly patients, which was also significantly higher than the 15.4% in the adult patients (P < 0.001). A stepwise multivariable linear regression model showed that procalcitonin and gamma-glutamyl transpeptidase were independently associated factors in the elderly patients (OR = 2.590, 95% confidence interval [CI] = 1.506-3.673, P = 0.001; OR = -0.016, 95% CI = -0.027 to -0.006, P = 0.005). Receiver operating characteristic (ROC) curve analysis indicated that procalcitonin concentrations of ≥1.31 ng/mL increased the incidence of a voriconazole trough concentration higher than 5 µg/mL (95% CI = 0.53-0.87 µg/mL) (P = 0.03). The incidence of decreased albumin concentrations was higher in the elderly cohort than that in the adult cohort independent of the voriconazole trough concentration (P < 0.05). CONCLUSIONS: The voriconazole trough concentrations in the elderly patients were significantly higher than those in the adult patients who received voriconazole therapy and were significantly affected by severe inflammation as evaluated by the procalcitonin concentration. Frequent monitoring of the voriconazole serum concentration and procalcitonin concentration during and after severe inflammation is critical to maintain the voriconazole serum concentration within the therapeutic range.

Ezetimibe for the prevention of cardiovascular disease and all-cause mortality events.

BACKGROUND: Cardiovascular disease (CVD) remains an important cause of mortality and morbidity, and high levels of blood cholesterol are thought to be the major modifiable risk factors for CVD. The use of statins is the preferred treatment strategy for the prevention of CVD, but some people at high-risk for CVD are intolerant to statin therapy or unable to achieve their treatment goals with the maximal recommended doses of statin. Ezetimibe is a selective cholesterol absorption inhibitor, whether it has a positive effect on CVD events remains uncertain. Results from clinical studies are inconsistent and a thorough evaluation of its efficacy and safety for the prevention of CVD and mortality is necessary. OBJECTIVES: To assess the efficacy and safety of ezetimibe for the prevention of CVD and all-cause mortality. SEARCH METHODS: We searched the CENTRAL, MEDLINE, Embase and Web of Science on 27 June 2018, and two clinical trial registry platforms on 11 July 2018. We checked reference lists from primary studies and review articles for additional studies. No language restrictions were applied. SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared ezetimibe versus placebo or ezetimibe plus other lipid-modifying drugs versus other lipid-modifying drugs alone in adults, with or without CVD, and which had a follow-up of at least 12 months. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies for inclusion, extracted data, assessed risk of bias and contacted trialists to obtain missing data. We performed statistical analyses according to the Cochrane Handbook for Systematic Reviews of Interventions and used the GRADE to assess the quality of evidence. MAIN RESULTS: We included 26 RCTs randomising 23,499 participants. All included studies assessed effects of ezetimibe plus other lipid-modifying drugs compared with other lipid-modifying drugs alone or plus placebo. Our findings were driven by the largest study (IMPROVE-IT), which had weights ranging from 41.5% to 98.4% in the different meta-analyses.Ezetimibe with statins probably reduces the risk of major adverse cardiovascular events compared with statins alone (risk ratio (RR) 0.94, 95% confidence interval (CI) 0.90 to 0.98; a decrease from 284/1000 to 267/1000, 95% CI 256 to 278; 21,727 participants; 10 studies; moderate-quality evidence). Trials reporting all-cause mortality used ezetimibe with statin or fenofibrate and found they have little or no effect on this outcome (RR 0.98, 95% CI 0.91 to 1.05; 21,222 participants; 8 studies; high-quality evidence). Adding ezetimibe to statins probably reduces the risk of non-fatal myocardial infarction (MI) (RR 0.88, 95% CI 0.81 to 0.95; a decrease from 105/1000 to 92/1000, 95% CI 85 to 100; 21,145 participants; 6 studies; moderate-quality evidence) and non-fatal stroke (RR 0.83, 95% CI 0.71 to 0.97; a decrease 32/1000 to 27/1000, 95% CI 23 to 31; 21,205 participants; 6 studies; moderate-quality evidence). Trials reporting cardiovascular mortality added ezetimibe to statin or fenofibrate, probably having little or no effect on this outcome (RR 1.00, 95% CI 0.89 to 1.12; 19457 participants; 6 studies; moderate-quality evidence). The need for coronary revascularisation might be reduced by adding ezetimibe to statin (RR 0.94, 95% CI 0.89 to 0.99; a decrease from 196/1000 to 184/1000, 95% 175 to 194; 21,323 participants; 7 studies); however, no difference in coronary revascularisation rate was observed when a sensitivity analysis was limited to studies with a low risk of bias.In terms of safety, adding ezetimibe to statins may make little or no difference in the risk of hepatopathy (RR 1.14, 95% CI 0.96 to 1.35; 20,687 participants; 4 studies; low-quality evidence). It is uncertain whether ezetimibe increase or decrease the risk of myopathy (RR 1.31, 95% CI 0.72 to 2.38; 20,581 participants; 3 studies; very low-quality evidence) and rhabdomyolysis, given the wide CIs and low event rate. Little or no difference in the risk of cancer, gallbladder-related disease and discontinuation due to adverse events were observed between treatment groups. For serum lipids, adding ezetimibe to statin or fenofibrate might further reduce the low-density lipoprotein cholesterol (LDL-C), total cholesterol and triglyceride levels and likely increase the high-density lipoprotein cholesterol levels; however, substantial heterogeneity was detected in most analyses.None of the included studies reported on health-related quality of life. AUTHORS' CONCLUSIONS: Moderate- to high-quality evidence suggests that ezetimibe has modest beneficial effects on the risk of CVD endpoints, primarily driven by a reduction in non-fatal MI and non-fatal stroke, but it has little or no effect on clinical fatal endpoints. The cardiovascular benefit of ezetimibe might involve the reduction of LDL-C, total cholesterol and triglycerides. There is insufficient evidence to determine whether ezetimibe increases the risk of adverse events due to the low and very low quality of the evidence. The evidence for beneficial effects was mainly obtained from individuals with established atherosclerotic cardiovascular disease (ASCVD, predominantly with acute coronary syndrome) administered ezetimibe plus statins. However, there is limited evidence regarding the role of ezetimibe in primary prevention and the effects of ezetimibe monotherapy in the prevention of CVD, and these topics thus requires further investigation.

Association between HLA-DP Gene Polymorphisms and Cervical Cancer Risk: A Meta-Analysis.

Objective. We aimed to derive a more precise estimation of the associations between human leukocyte antigens DP (HLA-DP) gene polymorphisms and cervical cancer risk by meta-analysis. Methods. PubMed, EMBASE, ScienceDirect, Web of Science, Chinese National Knowledge Infrastructure (CNKI), and Wanfang databases were systematically searched to identify studies investigating the relationship between HLA-DP gene polymorphisms and cervical cancer. The associations between them were evaluated by pooled OR and 95% CI. Results. A total of 11 studies including 5008 cases and 9322 controls with 11 HLA-DP alleles were included in the current meta-analysis. Results. The results showed that HLA-DPB1⁎03:01 was significantly associated with an increased risk of cervical cancer (OR=1.252, 95%CI: 1.116-1.403, Pz=0.001), while HLA-DPB1⁎04:02 and HLA-DP rs3117027 G allele were significantly associated with a decreased risk of cervical cancer (OR=0.744, 95%CI: 0.652-0.848, Pz=0.001; OR=0.790, 95%CI: 0.745-0.837, Pz=0.001), and HLA-DP rs9277535 G allele was significantly associated with a decreased risk of cervical cancer in Asia (OR=0.802, 95%CI: 0.753-0.855, Pz=0.001). Subgroup analyses based on race system showed that HLA-DPB1⁎13:01 was significantly associated with an increased risk of cervical cancer in Asia (OR=1.834, 95%CI: 1.107-3.039, Pz=0.019). No significant association was established for the HLA-DP following alleles: DPB1⁎02:01, DPB1⁎02:02, DPB1⁎04:01, DPB1⁎05:01, rs4282438, and rs3077. Conclusion. HLA-DP gene polymorphisms (HLA-DPB1⁎03:01, DPB1⁎04:02, DPB1⁎13:01, rs9277535, and rs3117027) were significantly associated with cervical cancer.

Astragalus polysaccharides protect against dextran sulfate sodium-induced colitis by inhibiting NF-κВ activation.

Astragalus polysaccharide (APS) is a bioactive extract of Astragalus membranaceus (AM), which possess a wide range of medicinal benefits, including anti-inflammatory, anti-oxidative, anti-tumor and anti-diabetic effects. The present work evaluated the therapeutic effect of APS and its potential mechanisms in a mouse model of dextran sulfate sodium (DSS)-induced colitis. The APS treatment led to significant improvements in colitis disease activity index (DAI) and histological scores, as well as significantly increased weight and colon length in mice as compared to the control group. Mechanically, reduced NF-κВ DNA phosphorylation activity and downregulated TNF-α, IL-1ß, IL-6, IL-17 expressions and myeloperoxidase (MPO) activity were associated with improvement in colitis observed in APS-treated mice. These findings suggest that APS may represent a natural therapeutic approach for treating inflammatory bowel disease, such as ulcerative colitis.

Astragalus polysaccharide ameliorates ionizing radiation-induced oxidative stress in mice.

Radioprotective compounds from plant resources may represent safe and cost-effective prophylactic and therapeutic agents. This study was designed to investigate the protective effect of polysaccharide derived from the dried roots of the Astragalus spp. (APS) against ionizing radiation (IR) injury in liver and to explore its role in radiation-induced oxidative stress using a mouse model. Prior to (60)Co γ-irradiation (5Gy, single dose), mice received 7 days of APS at low, mid and high doses (50, 100 or 200mg/kg/day, respectively; n=6 each group), vehicle alone (5mL normal saline orally/daily; n=6). A non-irradiated control group (n=6) received the 7-day distilled water regimen only. At 24h post-irradiation, the APS pre-treated mice showed significantly decreased alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase levels, and NF-κB expression. All APS-treated mice also showed attenuation of the IR-induced increase in thiobarbituric acid reactive substance and resolution of the IR-induced decreases in superoxide dismutase, catalase and glutathione activities (all p<0.05). High dose APS pre-treatment led to remarkably less morphologic features of IR-induced hepatic and pulmonary injury. Thus, APS exerts protective effects against IR-induced injury in liver in mice, and the related molecular mechanism may involve suppressing the radiation-induced oxidative stress reaction.

Increased IL-8 production in human bronchial epithelial cells after exposure to azithromycin-pretreated Pseudomonas aeruginosa in vitro.

Although Pseudomonas aeruginosa is not typically susceptible to azithromycin (AZM) in in vitro tests, AZM improves the clinical outcome in patients with chronic respiratory infections, in which both the modulation of the host immune system and of bacterial virulence by AZM are thought to play an important role. However, there is currently little direct evidence showing the impact of bacteria pretreated with AZM on epithelial cells, which represents the first barrier to infecting P. aeruginosa. In this study, we pretreated P. aeruginosa with AZM and subsequently infected human bronchial epithelial cells (HBEs) in the absence of AZM. The results showed that AZM-pretreated P. aeruginosa (PAO1 and six different clinical isolates) significantly stimulated HBE cells to release IL-8, a crucial pro-inflammatory cytokine. This effect was not observed in a P. aeruginosa PAO1 mutant strain unable to produce the type III secretion system effector gene pcrV (strain PW4017). Our results suggest that AZM-pretreated P. aeruginosa could indirectly exacerbate pro-inflammation by inducing IL-8 production in HBEs.

Bioactivity-guided fractionation of physical fatigue-attenuating components from Rubus parvifolius L.

Alleviation of fatigue has been emerging as a serious issue that requires urgent attention. Health professionals and sports physiologists have been looking for active natural products and synthetic compounds to overcome fatigue in humans. This study was designed to define the anti-fatigue property of Rubus parvifolius L. (RPL) by characterization of active constituents using a mouse forced swimming test model. Four RPL fractions with different polarities containing anti-fatigue activity were sequentially isolated from the n-butanol RPL extract, followed by elution of 50% ethanol-water fraction from D101 macroporous resin chromatography to obtain nigaichigoside F1, suavissimoside R1 and coreanoside F1. Active constituents of the 50% ethanol-water eluate of RPL were total saponins. The fractions were examined based on the effect on weight-loaded swimming capacity of mice. Serum levels of urea nitrogen (SUN), triglyceride fatty acids (TG), lactate dehydrogenase (LDH), lactic acid (LA), ammonia and hepatic glycogen (HG) were also examined for potential mechanisms underlying the anti-fatigue effect of RPL extracts. During the experiment, two inflammatory markers, interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in serum, were measured. We found that total saponins from RPL possess potent capabilities to alleviate mouse fatigue induced by forced swimming and that nigaichigoside F1 was responsible for the pharmacological effect. The underlying mechanisms include delays of SUN and LA accumulation, a decrease in TG level by increasing fat consumption, increases in HG and LDH so that lactic acid accumulation and ammonia in the muscle were reduced, and suppression of increased immune activation and inflammatory cytokine production. Our findings will be helpful for functional identification of novel anti-fatigue components from natural medicinal herbs.

Looped host defense peptide CLP-19 binds to microtubules and inhibits surface expression of TLR4 on mouse macrophages.

The looped host defense peptide CLP-19 is derived from a highly functional core region of the Limulus anti-LPS factor and exerts robust anti-LPS activity by directly interacting with LPS in the extracellular space. We previously showed that prophylactic administration of CLP-19 even 20 h prior to LPS challenge might significantly increase the survival rate in a lethal endotoxin shock mouse model. Such an effect may be associated with immune regulation of CLP-19. To investigate the underlying mechanisms, peptide affinity chromatography, immunofluorescence, and Western blotting procedures were used to identify α- and ß-tubulin as direct and specific binding partners of CLP-19 in the mouse macrophage cell line RAW 264.7. Bioinformatic analysis using the AutoDock Vina molecular docking and PyMOL molecular graphics system predicted that CLP-19 would bind to the functional residues of both α- and ß-tubulin and would be located within the groove of microtubules. Tubulin polymerization assay revealed that CLP-19 might induce polymerization of microtubules and prevent depolymerization. The immunoregulatory effect of CLP-19 involving microtubules was investigated by flow cytometry, immunofluorescence, and Western blotting, which showed that CLP-19 prophylactic treatment of RAW 264.7 cells significantly inhibited LPS-induced surface expression of TLR4. Taken together, these results suggest that CLP-19 binding to microtubules disrupts the dynamic equilibrium of microtubules, reducing the efficacy of microtubule-dependent vesicular transport that would otherwise translocate TLR4 from the endoplasmic reticulum to the cell surface.

Characterization of the antibacterial activity and the chemical components of the volatile oil of the leaves of Rubus parvifolius L.

Rubus parvifolius L. (Rp) is a medicinal herb that possesses antibacterial activity. In this study, we extracted the volatile oil from the leaves of Rp to assess its antibacterial activity and analyze its chemical composition. A uniform distribution design was used to optimize the extraction procedure, which yielded 0.36% (w/w) of light yellowish oil from the water extract of Rp leaves. We found that the extracted oil effectively inhibited the growth of a wide range of Gram positive and negative bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanii, Bacillus cloacae, and Klebsiella pneumoniae. We further analyzed the components contained in the hydro-distillated Rp volatile oil by gas chromatography-mass spectroscopy. Twenty nine compounds were identified, including 4-hydroxy-3-methoxystyrene (66%), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (10%) and 4-tert-butylbenzoic acid (2%). Our results suggest that one or multiple constituents contained in Rp volatile oil may account for its antibacterial activity.

Correlation between low-level expression of the tumor suppressor gene TAp73 and the chemoresistance of human glioma stem cells.

OBJECTIVE: Glioma stem cells (GSCs) are regarded as the root of glioma growth and recurrence. Chemoresistance is one of the characteristics of GSCs that increases the difficulties in eradicating the cells by anticancer drugs. PURPOSE: The objective of this study is to investigate the correlation between expression of the tumor suppressor gene TAp73 and the chemoresistance of human GSCs. METHODS: MTT and tumor sphere formation assays were used to analyze the chemoresistance phenotype of GSCs derived from primary human glioma specimens under cisplatin exposure. Reverse transcription real-time PCR was applied for assaying mRNA levels of TAp73. Protein levels of TAp73, p21, Bax, and cleared caspase 3 were assayed by western blot. Cell apoptosis was analyzed by flow cytometry after the annexin V fluorescence staining. RESULTS: GSCs exhibited increased chemoresistance compared to differentiated glioma cells (DGCs) derived from the same tumor specimen. The expression of TAp73 was lower in GSCs and was not sensitive to cisplatin treatment as compared to DGCs. Overexpression of TAp73 by transfection increased the apoptosis of GSCs in the presence of cisplatin and reduced the chemoresistance of GSC. TAp73 knockdown by siRNA in DGCs reduced cisplatin-induced apoptosis and increased the resistance to cisplatin. CONCLUSION: These findings indicate that TAp73 silencing is hallmark of GSC to maintain their chemoresistance phenotype. Thus, targeting TAp73 may provide a novel strategy to eradicating GSCs.

Cyclic Limulus anti-lipopolysaccharide (LPS) factor-derived peptide CLP-19 antagonizes LPS function by blocking binding to LPS binding protein.

Inflammation and septic shock due to endotoxins from Gram-negative bacteria infection continue to pose significant challenges to human healthcare. It is, therefore, necessary to develop therapeutic strategies targeting endotoxins, such as lipopolysaccharide (LPS), to prevent their potentially systemic effects. Pathogenesis due to Gram-negative bacteria involves LPS binding to the host LPS-binding protein (LBP), causing detrimental downstream signaling cascades. Our previous study showed that CLP-19, a synthetic peptide derived from the Limulus anti-LPS factor (LALF), could effectively neutralize LPS toxicity; however, the detailed mechanisms underlying this anti-LPS effect remained unexplained. Thus, we carried out investigations to determine how the CLP-19 neutralizes LPS toxicity. CLP-19 was found to block LPS binding to LBP in a dose-dependent manner, as evidenced by competitive enzyme-linked immunosorbent assay (ELISA). In peripheral blood mononuclear cells, CLP-19 blocked LPS-induced phosphorylation of mitogen activated protein kinase (MAPK) signaling proteins p38, extracellular signal-regulating kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK)1/2. Furthermore, CLP-19 potency in LPS antagonism in vitro and in vivo was directly associated with its ability to block the LPS-LBP interaction. Taken together, the results suggested that CLP-19's inhibitory effect on LPS-LBP binding and on the subsequent MAPK pathway signaling may be responsible for its anti-LPS mechanism. This peptide appears to represent a potential therapeutic agent for clinical treatment of sepsis.

Dexmedetomidine provides renoprotection against ischemia-reperfusion injury in mice.

INTRODUCTION: Acute kidney injury following surgery incurs significant mortality with no proven preventative therapy. We investigated whether the α2 adrenoceptor agonist dexmedetomidine (Dex) provides protection against ischemia-reperfusion induced kidney injury in vitro and in vivo. METHODS: In vitro, a stabilised cell line of human kidney proximal tubular cells (HK2) was exposed to culture medium deprived of oxygen and glucose. Dex decreased HK2 cell death in a dose-dependent manner, an effect attenuated by the α2 adrenoceptor antagonist atipamezole, and likely transduced by phosphatidylinositol 3-kinase (PI3K-Akt) signaling. In vivo C57BL/6J mice received Dex (25 µg/kg, intraperitoneal (i.p.)) 30 minutes before or after either bilateral renal pedicle clamping for 25 minutes or right renal pedicle clamping for 40 minutes and left nephrectomy. RESULTS: Pre- or post-treatment with Dex provided cytoprotection, improved tubular architecture and function following renal ischemia. Consistent with this cytoprotection, dexmedetomidine reduced plasma high-mobility group protein B1 (HMGB-1) elevation when given prior to or after kidney ischemia-reperfusion; pretreatment also decreased toll-like receptor 4 (TLR4) expression in tubular cells. Dex treatment provided long-term functional renoprotection, and even increased survival following nephrectomy. CONCLUSIONS: Our data suggest that Dex likely activates cell survival signal pAKT via α2 adrenoceptors to reduce cell death and HMGB1 release and subsequently inhibits TLR4 signaling to provide reno-protection.

Lipopolysaccharide (LPS) detoxification of analogue peptides derived from limulus anti-LPS factor.

Lipopolysaccharide (LPS) plays a critical role in the pathogenesis of sepsis due to gram-negative bacterial infections. Therefore, LPS-neutralizing molecules could have important clinical applications. Our previous work showed, CLP19, an analogue peptide derived from limulus anti-LPS factor (LALF), possessed the capacity to neutralize LPS and thereby inhibit the LPS-induced responses. However, potential cytotoxicity of CLP19 was also found, especially when added to human red blood cells. Accordingly we further developed two peptides (designated as CLP19-1 and CLP19-2) by single- and double-point amino acid substitution of CLP19, respectively, in order to reduce its toxicity and meanwhile retain the anti-LPS activity. In this study, the LPS-detoxifying effectiveness of these peptides was evaluated both in vitro and in vivo. CLP19-1 was found to dose-dependently neutralize LPS in vitro, with significantly lower hemolysis of red blood cells as compared with CLP19. Further in vivo tests verified that CLP19-1 exerted significant protective effects on mice against LPS, characterized by significantly improved survival, decreasing of tumor necrosis factor alpha (TNF-α) serum level and alleviation of tissue injury. Our work indicates that CLP19-1 is worthy of further study as potential anti-LPS agents for the management of sepsis.