Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts.

AIMS: Endothelial dysfunction plays a pivotal role in atherosclerosis, but the detailed mechanism remains incomplete understood. Nogo-B is an endoplasmic reticulum (ER)-localized protein mediating ER-mitochondrial morphology. We previously showed endothelial Nogo-B as a key regulator of endothelial function in the setting of hypertension. Here, we aim to further assess the role of Nogo-B in coronary atherosclerosis in ApoE-/- mice with pressure overload. METHODS AND RESULTS: We generated double knockout (DKO) mouse models of systemically or endothelium-specifically excising Nogo-A/B gene on an ApoE-/- background. After 7 weeks of transverse aortic constriction (TAC) surgery, compared to ApoE-/- mice DKO mice were resistant to the development of coronary atherosclerotic lesions and plaque rapture. Sustained elevation of Nogo-B and adhesion molecules (VCAM-1/ICAM-1), early markers of atherosclerosis, was identified in heart tissues and endothelial cells (ECs) isolated from TAC ApoE-/- mice, changes that were significantly repressed by Nogo-B deficiency. In cultured human umbilical vein endothelial cells (HUVECs) exposure to inflammatory cytokines (TNF-α, IL-1ß), Nogo-B was upregulated and activated reactive oxide species (ROS)-p38-p65 signaling axis. Mitofusin 2 (Mfn2) is a key protein tethering ER to mitochondria in ECs, and we showed that Nogo-B expression positively correlated with Mfn2 protein level. And Nogo-B deletion in ECs or in ApoE-/- mice reduced Mfn2 protein content and increased ER-mitochondria distance, reduced ER-mitochondrial Ca2+ transport and mitochondrial ROS generation, and prevented VCAM-1/ICAM-1 upregulation and EC dysfunction, eventually restrained atherosclerotic lesions development. CONCLUSION: Our study revealed that Nogo-B is a critical modulator in promoting endothelial dysfunction and consequent pathogenesis of coronary atherosclerosis in pressure overloaded hearts of ApoE-/- mice. Nogo-B may hold the promise to be a common therapeutic target in the setting of hypertension.

[Inhibition of glutaminolysis alleviates myocardial fibrosis induced by angiotensin II].

The present study was aimed to investigate the role and mechanism of glutaminolysis of cardiac fibroblasts (CFs) in hypertension-induced myocardial fibrosis. C57BL/6J mice were administered with a chronic infusion of angiotensin II (Ang II, 1.6 mg/kg per d) with a micro-osmotic pump to induce myocardial fibrosis. Masson staining was used to evaluate myocardial fibrosis. The mice were intraperitoneally injected with BPTES (12.5 mg/kg), a glutaminase 1 (GLS1)-specific inhibitor, to inhibit glutaminolysis simultaneously. Immunohistochemistry and Western blot were used to detect protein expression levels of GLS1, Collagen I and Collagen III in cardiac tissue. Neonatal Sprague-Dawley (SD) rat CFs were treated with 4 mmol/L glutamine (Gln) or BPTES (5 µmol/L) with or without Ang II (0.4 µmol/L) stimulation. The CFs were also treated with 2 mmol/L α-ketoglutarate (α-KG) under the stimulation of Ang II and BPTES. Wound healing test and CCK-8 were used to detect CFs migration and proliferation respectively. RT-qPCR and Western blot were used to detect mRNA and protein expression levels of GLS1, Collagen I and Collagen III. The results showed that blood pressure, heart weight and myocardial fibrosis were increased in Ang II-treated mice, and GLS1 expression in cardiac tissue was also significantly up-regulated. Gln significantly promoted the proliferation, migration, mRNA and protein expression of GLS1, Collagen I and Collagen III in the CFs with or without Ang II stimulation, whereas BPTES significantly decreased the above indices in the CFs. α-KG supplementation reversed the inhibitory effect of BPTES on the CFs under Ang II stimulation. Furthermore, in vivo intraperitoneal injection of BPTES alleviated cardiac fibrosis of Ang II-treated mice. In conclusion, glutaminolysis plays an important role in the process of cardiac fibrosis induced by Ang II. Targeted inhibition of glutaminolysis may be a new strategy for the treatment of myocardial fibrosis.

Time-restricted feeding alleviates cardiac dysfunction induced by simulated microgravity via restoring cardiac FGF21 signaling.

Dietary restriction has been well-described to improve health metrics, but whether it could benefit pathophysiological adaptation to extreme environment, for example, microgravity, remains unknown. Here, we investigated the effects of a daily rhythm of fasting and feeding without reducing caloric intake on cardiac function and metabolism against simulated microgravity. Male rats under ad libitum feeding or time-restricted feeding (TRF; food access limited to 8 hours every day) were subjected to hindlimb unloading (HU) to simulate microgravity. HU for 6 weeks led to left ventricular dyssynchrony and declined cardiac function. HU also lowered pyruvate dehydrogenase (PDH) activity and impaired glucose utilization in the heart. All these were largely preserved by TRF. TRF showed no effects on HU-induced loss of cardiac mass, but significantly improved contractile function of cardiomyocytes. Interestingly, TRF raised liver-derived fibroblast growth factor 21 (FGF21) level and enhanced cardiac FGF21 signaling as manifested by upregulation of FGF receptor-1 (FGFR1) expression and its downstream markers in HU rats. In isolated cardiomyocytes, FGF21 treatment improved PDH activity and glucose utilization, consequently enhancing cell contractile function. Finally, both liver-specific knockdown (KD) of FGF21 and cardiac-specific FGFR1 KD abrogated the cardioprotective effects of TRF in HU rats. These data demonstrate that TRF improves cardiac glucose utilization and ameliorates cardiac dysfunction induced by simulated microgravity, at least partially, through restoring cardiac FGF21 signaling, suggesting TRF as a potential countermeasure for cardioprotection in long-term spaceflight.

[Pollution Characteristics and Source Apportionment of n-Alkanes and PAHs in Summertime PM2.5 at Background Site of Yangtze River Delta].

To investigate the pollution characteristics and sources of organic aerosols at a background site of the Yangtze River Delta, day- and night- PM2.5 samples were collected from May 30th to August 15th, 2018 in Chongming Island, China and measured for their normal alkanes (n-alkanes) and polycyclic aromatic hydrocarbons (PAHs) content employing a GC-MS technique. Concentrations of PM2.5, n-alkanes, and PAHs during the entire sampling period were (33±21) µg·m-3, (26±44) ng·m-3, and (0.76±1.0) ng·m-3, respectively. During the entire campaign, 35% of the collected PM2.5 samples were of a particle loading larger than the first grade of the China National Air Quality Standard (35 µg·m-3), suggesting that further mitigation with respect to air pollution in Chongming Island remains imperative. In the period with a PM2.5 concentration higher than 35 µg·m-3, which was classified as the pollution period, concentrations of n-alkanes and PAHs were one order of magnitude higher than those in the period with PM2.5 less than 15 µg·m-3, which was classified as the clean period. During the entire campaign, OC was higher in the daytime than in the nighttime, mainly due to the daytime photooxidation that enhanced the formation of secondary organic aerosols. During the pollution period, concentrations of EC and other pollutants were higher in the nighttime than in daytime, mainly due to the transport of the inland pollutants by the nighttime land breeze. Such a diurnal difference was not observed for the pollutants in clean periods, mainly due to the relatively clean breeze from East China Sea that diluted the air pollution. Diagnostic ratios showed that 67% of n-alkanes in PM2.5 was derived from fossil fuel combustion. PMF analysis further showed that during the pollution period, vehicle exhausts and industrial emissions were the largest sources of PAHs, both accounting for 51% of the total in PM2.5. In contrast, during the clean periods ship emissions were the largest source, contributing about 45% of the total PAHs, exceeding the sum (38%) of vehicle and industrial emissions.

[Diurnal Variations and Source Analysis of Water-soluble Compounds in PM2.5 During the Winter in Liaocheng City].

To investigate the diurnal variations and sources of water-soluble compounds in Liaocheng City, PM2.5 samples were collected between January and February 2017. The PM2.5 samples were analyzed for the compositions, concentrations, and sources of water-soluble inorganic ions, oxalic acid, and levoglucosan. The sources of these chemical compound were investigated using principal component analysis (PCA) and multiple linear regression (MLR) modeling. The results showed that the mass concentrations of PM2.5during the nighttime were higher than those during the daytime, and the average concentrations exceeded the National Ambient Air Quality Standard (GB 3095-2012) by more than 1.8 times. Moreover, atmospheric pollution was worse during the day than during the night. SNA (SO42-, NO3-, and NH4+) were the dominant species among the inorganic ions, the relative abundance of which with respect to the total concentrations of inorganic ions was 73.4% and 77.1% during the daytime and nighttime, respectively. The ratios of anion to cation equivalents (AE/CE) were less than one, suggesting that the PM2.5 was slightly alkaline, and the degree of acidity at night was stronger than during the day. The results of the correlation analyses suggested that aqueous-phase oxidation was the major formation pathway of oxalic acid, which is driven by acid-catalyzed oxidation. The oxalic acid was mainly influenced by biomass burning during the winter in Liaocheng City. The results of the PCA-MLR model suggested that water-soluble compounds in Liaocheng City were mostly from vehicular emissions and secondary oxidation, biomass burning, while the impacts of mineral dust and coal burning were relatively minor.

Anti-nociceptive effect of patchouli alcohol: Involving attenuation of cyclooxygenase 2 and modulation of mu-opioid receptor.

OBJECTIVE: To explore the anti-nociceptive effect of patchouli alcohol (PA), the essential oil isolated from Pogostemon cablin (Blanco) Bent, and determine the mechanism in molecular levels. METHODS: The acetic acid-induced writhing test and formalin-induced plantar injection test in mice were employed to confirm the effect in vivo. Intracellular calcium ion was imaged to verify PA on mu-opioid receptor (MOR). Cyclooxygenase 2 (COX2) and MOR of mouse brain were expressed for determination of PA's target. Cellular experiments were carried out to find out COX2 and MOR expression induced by PA. RESULTS: PA significantly reduced latency period of visceral pain and writhing induced by acetic acid saline solution (P<0.01) and allodynia after intra-plantar formalin (P<0.01) in mice. PA also up-regulated COX2 mRNA and protein (P<0.05) with a down-regulation of MOR (P<0.05) both in in vivo and in vitro experiments, which devote to the analgesic effect of PA. A decrease in the intracellular calcium level (P<0.05) induced by PA may play an important role in its anti-nociceptive effect. PA showed the characters of enhancing the MOR expression and reducing the intracellular calcium ion similar to opioid effect. CONCLUSIONS: Both COX2 and MOR are involved in the mechanism of PA's anti-nociceptive effect, and the up-regulation of the receptor expression and the inhibition of intracellular calcium are a new perspective to PA's effect on MOR.

Pomegranate leaf attenuates lipid absorption in the small intestine in hyperlipidemic mice by inhibiting lipase activity.

Pomegranate leaf (PGL) has a definite role in regulating lipid metabolism. However, pharmacokinetic results show the main active ingredient, ellagic acid, in PGL has lower oral bioavailability, suggesting that the lipid-lowering effect of PGL may act through inhibiting lipid absorption in the small intestine. Our results demonstrated that pomegranate leaf and its main active ingredients (i.e., ellagic acid, gallic acid, pyrogallic acid and tannic acid) were capable of inhibiting pancreatic lipase activity in vitro. In computational molecular docking, the four ingredients had good affinity for pancreatic lipase. Acute lipid overload experiments showed that a large dosage of PGL significantly reduced serum total cholesterol (TG) and triglycerides (TC) levels in addition to inhibiting intestinal lipase activity, which demonstrated that PGL could inhibit lipase activity and reduce the absorption of lipids. We also found that PGL could reverse the reduced tight-junction protein expression due to intestinal lipid overload, promote Occludin and Claudin4 expression in the small intestine, and enhance the intestinal mucosal barrier. In conclusion, we demonstrated that PGL can inhibit lipid absorption and reduce blood TG and TC by targeting pancreatic lipase, promoting tight-junction protein expression and thereby preventing intestinal mucosa damage from an overload of lipids in the intestine.

Mechanism underlying berberine's effects on HSP70/TNFα under heat stress: Correlation with the TATA boxes.

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.

TRPM8 in the negative regulation of TNFα expression during cold stress.

Transient Receptor Potential Melastatin-8 (TRPM8) reportedly plays a fundamental role in a variety of processes including cold sensation, thermoregulation, pain transduction and tumorigenesis. However, the role of TRPM8 in inflammation under cold conditions is not well known. Since cooling allows the convergence of primary injury and injury-induced inflammation, we hypothesized that the mechanism of the protective effects of cooling might be related to TRPM8. We therefore investigated the involvement of TRPM8 activation in the regulation of inflammatory cytokines. The results showed that TRPM8 expression in the mouse hypothalamus was upregulated when the ambient temperature decreased; simultaneously, tumor necrosis factor-alpha (TNFα) was downregulated. The inhibitory effect of TRPM8 on TNFα was mediated by nuclear factor kappa B (NFκB). Specifically, cold stress stimulated the expression of TRPM8, which promoted the interaction of TRPM8 and NFκB, thereby suppressing NFκB nuclear localization. This suppression consequently led to the inhibition of TNFα gene transcription. The present data suggest a possible theoretical foundation for the anti-inflammatory role of TRPM8 activation, providing an experimental basis that could contribute to the advancement of cooling therapy for trauma patients.

Mitochondria play an important role in the cell proliferation suppressing activity of berberine.

After being studied for approximately a century, berberine (BBR) has been found to act on various targets and pathways. A great challenge in the pharmacological analysis of BBR at present is to identify which target(s) plays a decisive role. In the study described herein, a rescue experiment was designed to show the important role of mitochondria in BBR activity. A toxic dose of BBR was applied to inhibit cell proliferation and mitochondrial activity, then α-ketobutyrate (AKB), an analogue of pyruvate that serves only as an electron receptor of NADH, was proven to partially restore cell proliferation. However, mitochondrial morphology damage and TCA cycle suppression were not recovered by AKB. As the AKB just help to regenerate NAD+, which is make up for part function of mitochondrial, the recovered cell proliferation stands for the contribution of mitochondria to the activity of BBR. Our results also indicate that BBR suppresses tumour growth and reduces energy charge and mitochondrial DNA (mtDNA) copy number in a HepG2 xenograft model. In summary, our study suggests that mitochondria play an important role in BBR activity regarding tumour cell proliferation and metabolism.

Brazilein inhibits neuronal inflammation induced by cerebral ischemia and oxygen-glucose deprivation through targeting NOD2 expression.

Brazilein is reported to have immunosuppressive effect on cardiovascular and cerebral-vascular diseases. The essential roles of innate immunity in cerebral ischemia are increasingly identified, but no studies concerning the influence of brazilein on the innate immunity receptors have been reported. The present study was designed to investigate the regulation of NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) by brazilein for its protection of neuron in cerebral ischemia in vivo and oxygen-glucose deprivation in vitro. The results showed that brazilein could reverse the elevated expression of NOD2 and TNFα (tumor necrosis factor alpha) elicited by cerebral ischemia and reperfusion. This reduction could also be detected in normal mice and C17.2 cells, indicating that this suppressive effect of brazilein was correlated with NOD2. The results from GFP reporter plasmid assay suggested brazilein inhibited NOD2 gene transcription. In conclusion, brazilein could attenuate NOD2 and TNFα expression in cerebral ischemia and NOD2 may be one possible target of brazilein for its immune suppressive effect in neuro-inflammation.

TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine.

Berberine (BBR) is a natural compound with variable pharmacological effects and a broad panel of target genes. We investigated berberine's pharmacological activities from the perspective of its nucleotide-binding ability and discovered that BBR directly regulates gene expression by targeting TATA boxes in transcriptional regulatory regions as well as the poly adenine (poly (A)) tail at the mRNA terminus. BBR inhibits gene transcription by binding the TATA boxes in the transcriptional regulatory region, but it promotes higher levels of expression by targeting the poly (A) tails of mRNAs. The present study demonstrates that TATA boxes and poly (A) tails are the first and second primary targets by which BBR regulates gene expression. The final outcome of gene regulation by BBR depends on the structure of the individual gene. This is the first study to reveal that TATA boxes and poly (A) tails are direct targets for BBR in its regulation of gene expression. Our findings provide a novel explanation for the complex activities of a small molecule compound in a biological system and a novel horizon for small molecule-compound pharmacological studies.

Protection of Gastrointestinal Mucosa from Acute Heavy Alcohol Consumption: The Effect of Berberine and Its Correlation with TLR2, 4/IL1ß-TNFα Signaling.

The purpose of the present study is to confirm the protective effect of berberine (BBR) on gastrointestinal injury caused by acute heavy alcohol exposure, an effect that has not been reported previously. Our research details how BBR protects against gastrointestinal injuries from acute alcohol exposure using both in vivo and in vitro experiments. Acute high alcohol concentrations lead to obvious damage to the gastrointestinal mucosa, resulting in necrosis of the intestinal mucosa. Oral administration of BBR was able to significantly reduce this alcohol-induced damage, inhibit increases of alcohol-induced TNFα and IL-1ß expression in gastrointestinal mucosa as well as their upstream signals TLR2 and TLR4, and regulate cytokines that modulate tight junctions. Alcohol consumption is a popular human social behavior worldwide, and the present study reports a comprehensive mechanism by which BBR protects against gastrointestinal injuries from alcohol stress, providing people with a novel application of BBR.

[Transmembrane transport behavior of in vitro HepG2 cells of ananas and its effect on lipids and glucose distribution].

Pineapple (Ananas comosus) leaves contain mainly phenolic components with antioxidant and hypolipidemic effects. One of the principle components is p-coumaric acid. In this study, the transport behavior of p-coumaric acid, was observed after the administration of pineapple leaf phenols in vitro. Simultaneously, the effect of the phenols on glucose, total cholesterol and triglycerides transportation and metabolism in HepG2 cells was also observed. The results showed that the phenols had good transport characteristics. 5 min after the administration, p-coumaric acid of the phenols could be detected, and the content of p-coumaric acid reached the peak concentration after 60 min of the administration. p-coumaric acid of phenols have time-and dose-dependent manner. While promoting glucose transporter (GLUT4) and low density lipoprotein receptor (LDLR) expression, the phenols decreased intracellular lipid content. This reduction of intracellular lipid content was highly correlated with the promotion of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) expression, while the reduction of intracellular glucose levels was correlated with glycogen synthesis in the cells.

Transport and metabolism behavior of brazilein during its entrance into neural cells.

Brazilein, a natural small molecule, shows a variety of pharmacological activities, especially on nervous system and immune system. As a potential multifunctional drug, we studied the distribution and the transport behavior and metabolic behavior of brazilein in vivo and in vitro. Brazilein was found to be able to distribute in the mouse brain and transport into neural cells. A metabolite was found in the brain and in the cells. Positive and negative mode-MS/MS and Q-TOF were used to identify the metabolite. MS/MS fragmentation mechanisms showed the methylation occurred at the 10-hydroxyl of brazilein (10-O-methylbrazilein). Further, catechol-O- methyltransferase (COMT) was confirmed as a crucial enzyme correlated with the methylated metabolite generation by molecular docking and pharmacological experiment.

Effect of berberine on cell cycle arrest and cell survival during cerebral ischemia and reperfusion and correlations with p53/cyclin D1 and PI3K/Akt.

Berberine acted as a natural medicine with multiple pharmacological activities. In the present study, we examined the effect of berberine against cerebral ischemia damage from cell cycle arrest and cell survival. Oxygen-glucose deprivation of PC12 cells and primary neurons, and carotid artery ligation in mice were used as in vitro and in vivo cerebral ischemia models. We found that the effect of berberine on cell cycle arrest during ischemia was mediated by decreased p53 and cyclin D1, increased phosphorylation of Bad (higher expression of p-Bad and higher ratio of p-Bad to Bad) and decreased cleavage of caspase 3. Meanwhile, berberine activated the PI3K/Akt pathway during the reperfusion, especially the phosphor-activation of Akt, to promote the cell survival. The neural protective effect of berberine was remained in the presence of inhibitor of mitogen-activated protein/extracellular signal-regulated kinase (MEK), but was suppressed by the inhibitors of PI3K and Akt. We demonstrated that berberine induced cell cycle arrest and cell survival to resist cerebral ischemia injury.

Novel effect of berberine on thermoregulation in mice model induced by hot and cold environmental stimulation.

The purpose of this study was to assess the effects of berberine (BBR) on thermoregulation in mice exposed to hot (40°C) and cold (4°C) environmental conditions. Four groups of mice were assembled with three different dosages of BBR (0.2, 0.4, and 0.8 mg/kg) and normal saline (control). In room temperature, our largest dosage of BBR (0.8 mg/kg) can reduce rectal temperatures (Tc) of normal mice. In hot conditions, BBR can antagonize the increasing core body temperature and inhibit the expression of HSP70 and TNFα in mice; conversely, in cold conditions, BBR can antagonize the decreasing core body temperature and enhance the expression of TRPM8. This study demonstrates the dual ability of BBR in maintaining thermal balance, which is of great relevance to the regulation of HSP70, TNFα and TRPM8.