Molecular Mechanisms of Oxidative Stress Relief by CAPE in ARPE-19 Cells.

Caffeic acid phenylethyl ester (CAPE) is an antioxidative agent originally derived from propolis. Oxidative stress is a significant pathogenic factor in most retinal diseases. Our previous study revealed that CAPE suppresses mitochondrial ROS production in ARPE-19 cells by regulating UCP2. The present study explores the ability of CAPE to provide longer-term protection to RPE cells and the underlying signal pathways involved. ARPE-19 cells were given CAPE pretreatment followed by t-BHP stimulation. We used in situ live cell staining with CellROX and MitoSOX to measure ROS accumulation; Annexin V-FITC/PI assay to evaluate cell apoptosis; ZO-1 immunostaining to observe tight junction integrity in the cells; RNA-seq to analyze changes in gene expression; q-PCR to validate the RNA-seq data; and Western Blot to examine MAPK signal pathway activation. CAPE significantly reduced both cellular and mitochondria ROS overproduction, restored the loss of ZO-1 expression, and inhibited apoptosis induced by t-BHP stimulation. We also demonstrated that CAPE reverses the overexpression of immediate early genes (IEGs) and activation of the p38-MAPK/CREB signal pathway. Either genetic or chemical deletion of UCP2 largely abolished the protective effects of CAPE. CAPE restrained ROS generation and preserved the tight junction structure of ARPE-19 cells against oxidative stress-induced apoptosis. These effects were mediated via UCP2 regulation of p38/MAPK-CREB-IEGs pathway.

M2 Macrophage-Derived Exosomes Regulate Myocardial Ischemia-Reperfusion And Pyroptosis Via ROS/NLRP3 Pathway.

OBJECTIVE: To evaluate whether M2 macrophage-derived exosomes protect against MI/R injury and reveal the protective mechanism of exosomes [Kourembanas 2015]. METHODS: I/R model injury was induced by temporary left anterior descending coronary artery occlusion in Sprague-Dawley (SD) rats, macrophages isolated from bone marrow-derived macrophages (BMDMs) were induced to M2 polarization, and H9C2 cells subjected to hypoxia/reperfusion (H/R) were used to establish an in vitro model. I/R-induced rats and H/R-induced H9C2 cells were treated with M2-exos in vivo and in vitro, respectively. Masson staining was performed to observe myocardial fibrosis in rats. Immunohistochemical (IHC) staining of myocardial tissues showed the expression of NLRP3 inflammasome activation and pyrolysis. Exosomes derived from IL-4-treated macrophages (M2-exos) were detected by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western bolt. Western bolt was performed to determine the protein level, including NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, cleaved IL-1ß, gasdermin D (GSDMD), and N-terminus of gasdermin D (GSDMD-N). RESULTS: Activity of NLRP3 inflammasome and existence of pyroptosis in the rats subjected to MI/R were significantly higher than those in the control (P < 0.05). Moreover, we confirmed the accumulation of ROS during I/R injury in cardiomyocytes. M2-exos protected against I/R injury and reduced activity of NLRP3 inflammasome and existence of pyroptosis, accompanied with attenuating oxidative stress. In vitro studies showed similar effects, H9c2 cells co-cultured with M2-exos could attenuated H/R-induced cell injury, while M2-exos suppressed the expression of NLRP3 inflammasome and pyroptosis (P < 0.05).

Neuroprotection of retinal cells by Caffeic Acid Phenylethyl Ester（CAPE) is mediated by mitochondrial uncoupling protein UCP2.

Oxidative stress due to mitochondrial produced reactive oxygen species is a major cause of damage seen in many retinal degenerative diseases. Caffeic acid phenylethyl ester （CAPE） is protective agent in multiple tissues and is reported to have anti-oxidant properties. Systemically applied CAPE protected retinal ganglion cells from ischemic injury induced by increased intraocular pressure. CAPE provided complete protection for ARPE19 retinal pigment epithelial cells against tert-butyl hydrogen peroxide and reduced both basal and LPS-stimulated ROS production. The major effect of CAPE was mediated by the mitochondrial uncoupling protein UCP2 since both pharmacological inhibition of UCP2 and siRNA-induced knockdown removed the ability of CAPE to block ROS production. Based on common structural features, CAPE may be acting as a mimetic of the natural UCP2 homeostatic regulator 4-hydroxy-2-nonenal. CAPE may provide a valuable tool to treat oxidative stress-related damage in retinal and other degenerative diseases.

Comparison of sequential feeding and continuous feeding on the blood glucose of critically ill patients: a non-inferiority randomized controlled trial.

BACKGROUND: Glucose control is an important aspect in managing critically ill patients. The goal of this study was to compare the effects of sequential feeding (SF) and continuous feeding (CF) on the blood glucose of critically ill patients. METHODS: A non-inferiority randomized controlled trial was adopted in this study. A total of 62 patients who were fed enteral nutritional suspension through gastric tubes were enrolled. After achieving 80% of the nutrition target calories (25 kcal·kg-1·day-1) through CF, the patients were then randomly assigned into SF and CF groups. In the SF group, the feeding/fasting time was reasonably determined according to the circadian rhythm of the human body as laid out in traditional Chinese medicine theory. The total daily dosage of the enteral nutritional suspension was equally distributed among three time periods of 7 to 9 o'clock, 11 to 13 o'clock, and 17 to 19 o'clock. The enteral nutritional suspension in each time period was pumped at a uniform rate within 2 h by an enteral feeding pump. In the CF group, patients received CF at a constant velocity by an enteral feeding pump throughout the study. Blood glucose values at five points (6:00/11:00/15:00/21:00/1:00) were monitored and recorded for seven consecutive days after randomization. Enteral feeding intolerance was also recorded. Non-inferiority testing was adopted in this study, the chi-square test or Fisher test was used for qualitative data, and the Mann-Whitney U test was used for quantitative data to determine differences between groups. In particular, a repeated measure one-way analysis of variance was used to identify whether changes in glucose value variables across the time points were different between the two groups. RESULTS: There were no significant demographic or physiological differences between the SF and CF groups (P > 0.050). The average glucose level in SF was not higher than that in CF (8.8 [7.3-10.3] vs. 10.7 [9.1-12.1] mmol/L, Z = -2.079, P for non-inferiority = 0.019). Hyperglycemia incidence of each patient was more common in the CF group than that in the SF group (38.4 [19.1-63.7]% vs. 11.8 [3.0-36.7]%, Z = -2.213, P = 0.027). Hypoglycemia was not found in either group. Moreover, there was no significant difference during the 7 days in the incidence of feeding intolerance (P > 0.050). CONCLUSIONS: In this non-inferiority study, the average blood glucose in SF was not inferior to that in CF. The feeding intolerance in SF was similar to that in CF. SF may be as safe as CF for critically ill patients. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03439618; https://clinicaltrials.gov/ct2/show/record/NCT03439618.

Thermotherapy for knee osteoarthritis: A protocol for systematic review.

BACKGROUND: Osteoarthritis of the knee is one of the leading causes of pain and disability among adults. Thermotherapy has been widely used to treat knee osteoarthritis. But its efficiency has not been scientifically and methodically evaluated. The aim of this study is to assess the benefits of thermotherapy for people with osteoarthritis of the knee, in terms of pain, stiffness, and physical dysfunction. METHODS: Eight databases will be searched from their inception to September 2020. They are as follows: PubMed, Embase, Cochrane Library, ClinicalTrials.gov, China Knowledge Resource Integrated Database (CNKI), Weipu Database for Chinese Technical Periodicals (VIP), Chinese Biomedical Literature Database (CBM), and Wanfang Database. Two researchers will independently select studies, collect data, and assess the methodology quality by the Cochrane risk of bias tool. RESULTS: The systematic review will provide high-quality evidence to assess the benefits and harms of thermotherapy for people with osteoarthritis of the knee, in terms of pain, stiffness, and dysfunction of knee joint, and quality of life, as well as adverse events. CONCLUSION: The systematic review will provide evidence to assess the effectiveness and safety of thermotherapy for knee osteoarthritis patients. INPLASY REGISTRATION NUMBER: INPLASY202140038.

IL-36α Exerts Proinflammatory Effects in Aspergillus fumigatus Keratitis of Mice Through the Pathway of IL-36α/IL-36R/NF-κB.

Purpose: To explore the role of IL-36α in corneas infected by Aspergillus fumigatus. Methods: The experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1ß, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry. Results: IL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1ß, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1ß, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra. Conclusions: IL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1ß, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.

Changes in corneal innervation and pain responses in fungal keratitis.

AIM: To characterize changes in the cornea nerve and pain responses in fungal keratitis (FK). METHODS: A retrospective analysis of in vivo confocal microscopy images of 11 FK corneas was performed, and the results were compared with those for 11 normal corneas. Subbasal corneal nerves were analyzed for total nerve number, main nerve trunk number, branching patterns and tortuosity. C57BL/6 mice were infected with Aspergillus fumigatus. Disease severity was determined through clinical scoring and slit lamp photography. Corneas were harvested at 1, 3, 5, and 7d post infection (p.i.) and assessed for ß III tubulin. Corneal mechanical sensitivity thresholds were detected by von Frey test. ß-endorphin (ß-EP) and µ receptor protein expression was detected through Western blotting. RESULTS: Total nerve number, main nerve trunk number, and nerve branching were significantly lower in FK patients than in controls, but tortuosity was not significantly different. In infected mice, subbasal nerve density decreased from 1d p.i., reaching a minimum at 5d p.i. Clinical scores rose at 1d p.i., peaked at 3d p.i., and decreased at 5d p.i. Mechanical sensitivity thresholds showed the same trends. ß-EP and µ receptor protein expression increased after infection. CONCLUSION: Corneal nerve density is lower in FK patients and Aspergillus fumigatus-infected mice than in controls. Pain sensitivity decreases with postinfection corneal ulcer aggravation. ß-EP and µ receptor proteins are both upregulated in infected mouse corneas.

6-Gingerol protects cardiocytes H9c2 against hypoxia-induced injury by suppressing BNIP3 expression.

Background: Cardiomyocytes loss is the predominant pathogenic characteristic in the hypoxia-induced injury. Meanwhile, it has been corroborated that Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) provokes apoptosis and autophagy. For moderating cardiomyocytes loss, we initially probed the cyto-protection effects of 6-Gingerol (6 G), meanwhile, its potential mechanisms associated with BNIP3 were elucidated in our studies. Methods: We pretreated cardiomyocytes H9c2 cells with 6 G at different concentrations (0-100 µM) before exposure to hypoxia. Thereafter, the cell viability, lactate dehydrogenase (LDH), apoptosis and protein expression were respectively assessed using cell counting kit-8 and methyl thiazolyl tetrazolium (MTT) assay, LDH assay kit, Annexin V-fluorescein isothiocyannate/propidium iodide (Annexin V-FITC/PI) apoptosis detection kit and Western blotting analysis. In addition, we also analyzed BNIP3 level after treatment. Moreover, we enforced the exogenous overexpression of BNIP3 and then evaluated the cell viability, apoptosis, and protein level again. Results: In our present work, we observed that the cell viability was promoted by 6 G in the hypoxia-induced H9c2 cells in a dose-dependent manner. Moreover, hypoxia-induced LDH release, apoptosis and autophagy were inhibited by 6 G pretreatment through promoting phosphorylation of PI3K, AKT and mTOR. Remarkably, accumulation of BNIP3 protein was significantly reduced by 6 G in hypoxia-induced H9c2 cells. Mechanistically, 6 G initiated the phosphorylated expression of PI3K, AKT and mTOR by down-regulating BNIP3 with reducing cardiomyocytes apoptosis and autophagy. Conclusion: Hypoxia-induced cardiomyocytes injury was ameliorated by 6 G through suppressing BNIP3 expression with triggering PI3K/AKT/mTOR signalling pathway.

[Effect of Xinling Wan in treatment of stable angina pectoris: a randomized, double-blinded, placebo parallel-controlled, multicenter trial].

To evaluate the effectiveness and safety of Xinling Wan on patients with stable angina pectoris, a randomized, double-blinded, placebo parallel-controlled, multicenter clinical trial was conducted. A total of 232 subjects were enrolled and randomly divided into experiment group and placebo group. The experiment group was treated with Xinling Wan (two pills each time, three times daily) for 4 weeks, and the placebo group was treated with placebo. The effectiveness evaluation showed that Xinling Wan could significantly increase the total duration of treadmill exercise among patients with stable angina pectoris. FAS analysis showed that the difference value of the total exercise duration was between experiment group (72.11±139.32) s and placebo group (31.25±108.32) s. Xinling Wan could remarkably increase the total effective rate of angina pectoris symptom score, and the analysis showed that the total effective rate was 78.95% in experiment group and 42.61% in placebo group. The reduction of nitroglycerin dose was (2.45±2.41) tablets in experiment group and (0.50±2.24) tablets in placebo group on the basis of FAS analysis. The decrease of symptom integral was (4.68±3.49) in experiment group and (3.19±3.31) in placebo group based on FAS analysis. Besides, Xinling Wan could decrease the weekly attack time and the duration of angina pectoris. PPS analysis results were similar to those of FAS analysis. In conclusion, Xinling Wan has an obvious therapeutic effect in treating stable angina pectoris, with a good safety and a low incidence of adverse event and adverse reaction in experiment group.

Astragalus polysaccharide alleviates LPS-induced inflammation injury by regulating miR-127 in H9c2 cardiomyoblasts.

Astragalus polysaccharide (APS) has been widely reported to play an important role in inflammatory response. In this study, we aimed to explore the effects and underlying mechanisms of APS on lipopolysaccharide (LPS)-induced inflammation injury in H9c2 cardiomyoblasts. H9c2 cells were treated with different concentrations of APS, and cell viability was detected by the Cell Counting Kit-8 (CCK-8) assay. Then, the effect of APS on cell viability and apoptosis induced by LPS was determined by CCK-8, flow cytometry, and western blot. The expression and release of inflammatory cytokines were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA). Furthermore, expression of miR-127 in H9c2 cells was analyzed by qRT-PCR, and knocked down by transfection with miR-127 inhibitor. Western blot was used to analyze signaling pathway molecules. APS had no effect on H9c2 cells viability. However, APS could alleviate LPS-induced inflammation injury by increasing cell viability, reducing apoptosis, and inhibiting release of inflammatory cytokines in H9c2 cells ( P < 0.05). Additionally, we found that APS increased toll-like receptor 4 (TLR4) expressions in LPS-treated H9c2 cells. Mechanistically, we found that APS exerted the protective effect by down-regulating LPS-increased expression of miR-127 ( P < 0.05), inhibiting nuclear factor kappa B (NF-κB), JNK and promoting phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathways in LPS-treated H9c2 cells. The results demonstrated that APS could protect H9c2 cells against LPS-induced inflammation injury, which might be partially due to miR-127 down-regulation and regulation of NF-κB, JNK, and PI3K/AKT signaling pathways. These findings indicated that APS might be a potential therapeutic drug for treatment of myocarditis.

Platelet endothelial cell adhesion molecule-1 (PECAM1) plays a critical role in the maintenance of human vascular endothelial barrier function.

Cardiovascular endothelial barrier dysfunction is associated with a number of cardiovascular diseases. This study aims to investigate the role of platelet endothelial cell adhesion molecule-1 (PECAM1) in the maintenance of the vascular endothelial barrier integrate. Human umbilical vein endothelial cells (HUVECs) were cultured into monolayers using as an in vitro model to assess the endothelial barrier function. Knockdown of the gene of PECAM1 markedly reduced the transendothelial resistance and increased the permeability of the HUVEC monolayers. From the wild HUVECs, we detected a complex of PECAM1, claudin1, occluding and endothelial cell selective adhesion molecule (ESAM); such a complex was not detected in the PECAM1-deficient HUVECs. Knockdown of either claudin1, or occludin, or ESAM, did not affect the formation of the tight junction (TJ) complex. Exposure to recombinant interleukin (IL)-13 inhibited the expression of PECAM1 and down-regulated the HUVEC monolayer barrier function. PECAM1 plays an important role in the formation of TJ complex.

Impairment in preattentive processing among patients with hypertension revealed by visual mismatch negativity.

OBJECTIVE: Patients with hypertension show deficits in cognitive function. However, the neural mechanisms underlying the preattentive information processing in hypertensive patients are poorly understood. We seek to investigate whether hypertensive patients have impairments in preattentive information processing. METHODS: We compared visual mismatch negativity (vMMN) between 15 hypertensive patients and 15 age-matched healthy controls, which was elicited by the change of visual duration randomly presented in both peripheral visual fields. In addition, the global cognitive function for all participants was assessed with Mini-Mental State Examination (MMSE). RESULTS: The vMMN in deviant-standard comparison was observed at occipital-temporal regions. Compared with normal healthy controls, the amplitude of vMMN was significantly decreased in hypertensive patients (P < 0.05). Meanwhile, the vMMN peak latency was delayed in the hypertensive group (P < 0.05). However, the MMSE scores of patients with hypertension were not significantly different from those of controls (P > 0.05), and there was no significant correlation between the mean amplitude of vMMN and SBP, DBP, and MMSE in hypertensive individuals, respectively. CONCLUSIONS: These data indicate dysfunction of automatically change detection processing in patients with hypertension. Moreover, the changes of vMMN provide a more objective and reliable assessment for cognitive impairment in hypertensive patients.