Phenylephrine Attenuated Sepsis-Induced Cardiac Inflammation and Mitochondrial Injury Through an Effect on the PI3K/Akt Signaling Pathway.

OBJECTIVE: To investigate whether phenylephrine (PE) inhibits sepsis-induced cardiac dysfunction, cardiac inflammation, and mitochondrial injury through the PI3K/Akt signaling pathway. METHODS: A rat model of sepsis was established by cecal ligation and puncture. PE and/or wortmannin (a PI3K inhibitor) were administered to investigate the role of PI3K/Akt signaling in mediating the effects of PE on inhibiting sepsis-induced cardiac dysfunction, cardiac inflammation, and mitochondrial injury. Hematoxylin-eosin staining, echocardiography, and Langendorff system were used to examine the myocardial injury and function. The concentrations of TNF-α and IL-6 were analyzed by enzyme-linked immunosorbent assay. Intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), myeloperoxidase, mitochondria-related fusion/fission proteins, and PI3K/Akt signaling pathway-associated proteins were analyzed by Western blotting. RESULTS: PE improved the cardiac function and survival in septic rats. PE decreased TNF-α, IL-6, ICAM-1, VCAM-1, and myeloperoxidase contents in the myocardium of septic rats. Meanwhile, PE increased the fusion-related proteins and decreased the fission-related proteins in the myocardial mitochondria of septic rats. On the other hand, PE activated the PI3K/Akt signaling pathway in the cecal ligation and puncture-treated rats, and all the protective effects of PE were abolished by wortmannin. CONCLUSIONS: PE attenuated sepsis-induced cardiac dysfunction, cardiac inflammation, and mitochondrial injury through the PI3K/Akt signaling pathway.

Angiogenesis is repressed by ethanol exposure during chick embryonic development.

It is now known that excess alcohol consumption during pregnancy can cause fetal alcohol syndrome to develop. However, it is not known whether excess ethanol exposure could directly affect angiogenesis in the embryo or angiogenesis being indirectly affected because of ethanol-induced fetal alcohol syndrome. Using the chick yolk sac membrane (YSM) model, we demonstrated that ethanol exposure dramatically inhibited angiogenesis in the YSM of 9-day-old chick embryos, in a dose-dependent manner. Likewise, the anti-angiogenesis effect of ethanol could be seen in the developing vessel plexus (at the same extra-embryonic regions) during earlier stages of embryo development. The anti-angiogenic effect of ethanol was found associated with excess reactive oxygen species (ROS) production; as glutathione peroxidase activity increased while superoxide dismutase 1 and 2 activities decreased in the YSMs. We further validated this observation by exposing chick embryos to 2,2'-azobis-amidinopropane dihydrochloride (a ROS inducer) and obtained a similar anti-angiogenesis effect as ethanol treatment. Semiquantitative reverse transcription-polymerase chain reaction analysis of the experimental YSMs revealed that expression of angiogenesis-related genes, vascular endothelial growth factor and its receptor, fibroblast growth factor 2 and hypoxia-inducible factor, were all repressed following ethanol and 2,2'-azobis-amidinopropane dihydrochloride treatment. In summary, our results suggest that excess ethanol exposure inhibits embryonic angiogenesis through promoting superfluous ROS production during embryo development.

Ginsenoside Rg1 relieves tert-Butyl hydroperoxide-induced cell impairment in mouse microglial BV2 cells.

Microglial activation plays an important role in neurodegenerative diseases associated with oxidative stress. tert-Butyl hydroperoxide (t-BHP), an analog of hydroperoxide, mimics the oxidative damage to microglial cells. It has been reported that ginsenoside Rg1 (G-Rg1), an active ingredient of Panax ginseng, has anti-stress and anti-inflammatory properties. The present study aims to investigate the ability of G-Rg1 to decrease the t-BHP-mediated cell damage of BV2 microglial cells. We performed flow cytometry assays to facilitate the detection of reactive oxygen species as well as Western blotting analyses and immunofluorescence assays using specific antibodies, such as antibodies against phospho-mitogen-activated protein kinases (p-MAPKs), phospho-nuclear factor-κB (p-NF-κB), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), Caspase-3, autophagy marker light chain 3 (LC3), and Becline-1. We found that treatment with 50 µM G-Rg1 protected microglial cells against oxidative damage induced by 10 µM t-BHP.

[Effects of antagonist and agonist of nicotinic acetylcholine receptors on injury of rat neurons induced by amyloid ß-protein].

OBJECTIVE: To explore the chronic effects of nicotinic antagonist and agonist on rat neurons injury induced by ß-amyloid protein. METHODS: The rat model of neuron injury was established by the exposure to Aß25-35 and the intervention agent was either methyllycaconitine (MLA) or nicotine (Nic). And the experimental groups were control (distilled water), Aß25-35, MLA (MLA and Aß25-35) and Nic (Nic and Aß25-35). Cellular viability was detected by methyl thiazolyl tetrazolium (MTT) chromatometry while apoptosis and necrosis were detected by flow cytometer. RESULTS: Compared with control, cellular viability decreased while the apoptotic and necrotic rates increased in Aß25-35 group(P = 0.00). The values of cellular viability at (0.75 ± 0.02) and (0.75 ± 0.09) in Aß25-35 and MLA groups respectively were significantly lower than that of Nic group (0.81 ± 0.02, P = 0.01) at Day 3 and 7. No significant differences existed in cellular viability between Aß25-35 and MLA groups. At Day 14, the differences of cellular viability were not obvious in all groups. At Day 21, cell viability of MLA group (0.64 ± 0.10) was significantly higher than those of Aß25-35 (0.57 ± 0.04, P = 0.019) and Nic groups (0.56 ± 0.04, P = 0.008). The apoptotic rate was lower than that of Aß25-35 group (3.70% ± 0.20% vs 4.70% ± 0.46%, P = 0.008) while the necrotic rate lower than that of Aß25-35 group (7.73% ± 0.86% vs 16.30% ± 1.05%, P = 0.00) and Nic group (16.03% ± 1.53%, P = 0.00). However, no significant differences existed in cellular viability or apoptotic and necrotic rate between Aß25-35 and Nic groups. CONCLUSION: With chronic treatment, the protective effect of α7 nicotinic antagonist methyllycaconitine increases whereas that of nicotinic agonist nicotine decreases.

Berberine protects against lipopolysaccharide-induced intestinal injury in mice via alpha 2 adrenoceptor-independent mechanisms.

AIM: To investigate the mechanisms responsible for the protective action of berberine (Ber) against gut damage in endotoxemic mice. METHODS: Male BALB/c mice were administered intragastrically with distilled water (0.1 mL/10 g), Ber (50 mg/kg) alone, yohimbine (2 mg/kg) alone, or Ber (50 mg/kg) in combination with yohimbine (2 mg/kg) for 3 d. On the third day, lipopolysaccharide (LPS, 18 mg/kg) or normal saline was intraperitoneally injected one hour after the intragastric administration. Following the treatment, intestinal injury in the ileum was histopathologically accessed; enterocyte apoptosis was examined using TUNEL method; Toll-like receptor 4 (TLR4) mRNA expression was measured using RT-PCR assay; inhibitor protein-κBα (I-κBα) phosphorylation and myeloperoxidase content were examined using Western blloting. The macrophage inflammatory protein-2 (MIP-2) production was measured using ELISA assay. RESULTS: Mice challenged with LPS caused extensive ileum injury, including a significantly increased injury score, decreased intestinal villus height, reduced gut mucosal weight and increased intestinal permeability. Furthermore, LPS significantly induced enterocyte apoptosis, increased TLR4 mRNA expression, I-κBα phosphorylation, MIP-2 production and myeloperoxidase content in the ileum. Pretreatment with Ber significantly alleviated all the alterations in the ileum in the endotoxemic mice. Pretreatment with the α2-adrenoceptor antagonist yohimbine did not block the protective action of Ber against LPS-induced intestinal injury. In addition, treatment with yohimbine alone did not prevent LPS-induced intestinal injury. CONCLUSION: Pretreatment with Ber provides significant protection against LPS-induced intestinal injury in mice, via reducing enterocyte apoptosis, inhibiting the TLR4-nuclear factor κB-MIP-2 pathway and decreasing neutrophil infiltration that are independent of α2-adrenoceptors.

Luteolin inhibits microglial inflammation and improves neuron survival against inflammation.

Microglia activation is one of the causative factors for neuroinflammation, which results in brain damage during neurodegenerative disease. Accumulating evidence has shown that the flavonoid luteolin (Lut) possesses potent anti-inflammatory properties; however, its effect on microglia inhibition is currently unknown. Moreover, it is not clear whether Lut also has indirect neuroprotective effects by reducing inflammatory mediators and suppressing microglia activation. In this study, we examined the effects of Lut on lipopolysaccharide (LPS)-induced proinflammatory mediator production and signaling pathways in murine BV2 microglia. In addition, we cocultured microglia and neurons to observe the indirect neuroprotective effects of Lut. Lut inhibited the LPS-stimulated expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) as well as the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Moreover, Lut blocked LPS-induced nuclear factor kappa B (NF-κB) activation. Preincubation of microglia with Lut diminished the neurotoxic effects, owing to the direct anti-inflammatory effects of the compound. Taken together, our findings suggest that Lut may have a potential therapeutic application in the treatment of neuroinflammatory disorders.

Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFalpha production in cardiomyocytes by activating a glycine receptor.

AIM: Previous studies have demonstrated that glycine (GLY) markedly reduces lipopolysaccharide (LPS)-induced myocardial injury.However, the mechanism of this effect is still unclear. The present study investigated the effect of GLY on cytosolic calcium concentration([Ca2+]c) and tumor necrosis factor-alpha (TNFalpha) production in cardiomyocytes exposed to LPS, as well as whether the glycine-gated chloride channel is involved in this process. METHODS: Neonatal rat cardiomyocytes were isolated, and the [Ca2+]c and TNFalpha levels were determined by using Fura-2 and a Quantikine enzyme-linked immunosorbent assay, respectively. The distribution of the GLY receptor and GLY-induced currents in cardiomyocytes were also investigated using immunocytochemistry and the whole-cell patch-clamp technique, respectively. RESULTS: LPS at concentrations ranging from 10 ng/mL to 100 microg/mL significantly stimulated TNFalpha production. GLY did not inhibit TNFalpha production induced by LPS at concentrations below 10 ng/mL but did significantly decrease TNFalpha release stimulated by 100 microg/mL LPS and prevented an LPS-induced increase in [Ca2+]c, which was reversed by strychnine, a glycine receptor antagonist. GLY did not block the isoproterenol-induced increase in [Ca2+]c, but did prevent the potassium chloride-induced increase in [Ca2+]c in cardiomyocytes.Strychnine reversed the inhibition of the KCl-stimulated elevation in [Ca2+]c by GLY. In chloride-free buffer, GLY had no effect on the dipotassium hydrogen phosphate-induced increase in [Ca2+]c. Furthermore, GLY receptor alpha1 and beta subunit-immunoreactive spots were observed in cardiomyocytes, and GLY-evoked currents were blocked by strychnine. CONCLUSION: Cardiomyocytes possess the glycine-gated chloride channel, through which GLY prevents the increase in [Ca2+]c and inhibits the TNFalpha production induced by LPS at high doses in neonatal rat cardiomyocytes.

Therapeutic strategies targeting the LPS signaling and cytokines.

Lipopolysaccharide (LPS) has been recognized as a major player in the pathogenesis of sepsis and neutralization of LPS or inhibition of its signal transduction mechanism is promising new treatment strategy in preclinical experiments. However, these therapeutic approaches have been shown unsuccessful in clinical trials. LPS activates Toll-like receptor 4 (TLR4) and induces pro-inflammatory and anti-inflammatory responses, the altered innate and adaptive immune responses eventually lead to the immunosuppressive state. The future therapeutic efforts in sepsis should focus on the immunosuppressive state. In this article, we will outline the current data on therapeutic strategies targeting LPS, TLR4 and single cytokine in sepsis and discuss the experimental and clinical evaluation of the immunomodulatory action of glycine and berberine. While we have demonstrated berberine in combination with yohimbine can modulate host immune responses in endotoxemia, it seems worthwhile to conduct clinical trials on the safe and efficacy of this new immunomodulatory therapy.

Berberine inhibits cytosolic phospholipase A2 and protects against LPS-induced lung injury and lethality independent of the alpha2-adrenergic receptor in mice.

Acute lung injury is still a significant clinical problem having a high mortality rate despite significant advances in antimicrobial therapy and supportive care made in the past few years. Our previous study demonstrated that berberine (Ber) remarkably decreased mortality and attenuated the lung injury in mice challenged with LPS, but the mechanism behind this remains unclear. Here, we report that pretreatment with Ber significantly reduced pulmonary edema, neutrophil infiltration, and histopathological alterations; inhibited protein expression and phosphorylation of cytosolic phospholipase A2; and decreased thromboxane A2 release induced by LPS. Yohimbine, an alpha2-adrenergic receptor antagonist, did not antagonize these actions of Ber. Furthermore, pretreatment with Ber decreased TNF-alpha production and mortality in mice challenged with LPS, which were enhanced by yohimbine, and Ber combined with yohimbine also improved survival rate in mice subjected to cecal ligation and puncture. Taken together, these observations indicate that Ber attenuates LPS-induced lung injury by inhibiting TNF-alpha production and cytosolic phospholipase A2 expression and activation in an alpha2-adrenoceptor-independent manner. Berberine combined with yohimbine might provide an effective therapeutic approach to acute lung injury during sepsis.

Curcumin improves learning and memory ability and its neuroprotective mechanism in mice.

BACKGROUND: Increasing evidence suggests that many neurons may die through apoptosis in Alzheimer's disease (AD). Mitochondrial dysfunction has been implicated in this process of neuronal cell death. One promising approach for preventing AD is based upon anti-apoptosis to decrease death of nerve cells. In this study, we observed the memory improving properties of curcumin in mice and investigated the neuroprotective effect of curcumin in vitro and in vivo. METHODS: The mice were given AlCl(3) orally and injections of D-galactose intraperitoneally for 90 days to establish the AD animal model. From day 45, the curcumin group was treated with curcumin for 45 days. Subsequently, the step-through test, neuropathological changes in the hippocampus and the expression of Bax and Bcl-2 were carried out to evaluate the effect of curcumin on the AD model mice. In cultured PC12 cells, AlCl(3) exposure induced apoptosis. The MTT assay was used to measure cell viabilities; flow cytometric analysis to survey the rate of cell apoptosis; DNA-binding fluorochrome Hoechst 33258 to observe nuclei changes in apoptotic cells and Western blot analysis of Bax, Bcl-2 to investigate the mechanisms by which curcumin protects cells from toxicity. RESULTS: Curcumin significantly improved the memory ability of AD mice in the step-through test, as indicated by the reduced number of step-through errors (P < 0.05) and prolonged step-through latency (P < 0.05). Curcumin also attenuated the neuropathological changes in the hippocampus and inhibited apoptosis accompanied by an increase in Bcl-2 level (P < 0.05), but the activity of Bax did not change (P > 0.05). AlCl(3) significantly reduced the viability of PC12 cells (P < 0.01). Curcumin increased cell viability in the presence of AlCl(3) (P < 0.01). The rate of apoptosis decreased significantly in the curcumin group (P < 0.05) when measured by flow cytometric analysis. Curcumin protected cells by increasing Bcl-2 level (P < 0.05), but the level of Bax did not change (P > 0.05). CONCLUSIONS: This study demonstrates that curcumin improves the memory ability of AD mice and inhibits apoptosis in cultured PC12 cells induced by AlCl(3). Its mechanism may involve enhancing the level of Bcl-2.

[Influence of different treatment patterns on cost-effectiveness in treatment of acute myocardial infarction].

OBJECTIVE: To investigate the influences of different treatment patterns on the cost-effectiveness in treating acute myocardial infarction (AMI). METHODS: Data about referral of AMI patients who called for help because of chest pain to the nearby hospitals from October 2003 to December 2005 were collected from the Guangzhou 120 Call Center. All these patients were followed up 6 months after discharge to survey the cost during hospitalization, major treatment, prognosis (death, re-infarction, stroke etc. ), and secondary prevention for coronary heart disease. We used SF-36 scale was used to quantify the health status. RESULTS: 101 AMI patients referred to grade 2 A hospitals (Group A) and 137 patients referred to grade 3 A hospitals (Group B) were successfully followed up. The cost during hospitalization of Group B was (33965 +/- 963) yuan RMB, significantly higher than that of Group A (18943 +/- 893) yuan, P = 0.021). 11 patients of Group B died, and 5 patients suffered from stroke with the mortality and stroke rate both significantly lower than those of Group A (18/101 and 12/101, P = 0.022, P = 0.015). There was no significant difference in the re-infarction rate between the 2 groups. The scores in physical function, general health status, vitality, social function, role-emotional, mental health of Group B were all significantly higher than those of Group A (all P < 0.05) , however, there were not significant differences in body pain and role-physical between these 2 groups. The smoking cessation rate, specialist outpatient department follow-up rate, statins use rate of Group B were significantly higher than those of Group A (P = 0.017, P = 0.016, P = 0.038). CONCLUSION: The 120-grade 3 A hospital CCU pattern is more cost-effective in treatment of AMI.

[Relationship between flash visual evoked potential and severity and prognosis in critically ill patients].

OBJECTIVE: To explore the relationship between flash visual evoked potential (fVEP) and severity and prognosis in critically ill patients in intensive care unit (ICU). METHODS: Sixty-nine critically ill patients were divided into two groups according to survival (35 cases) or death (34 cases) in 28 days. fVEP, Glasgow coma scale (GCS) score, acute physiology and chronic health evaluation II (APACHE II) score and sepsis-related organ failure assessment (SOFA) score of survivors were compared with those of nonsurvivors. Also, according to primary disease, the patients were divided into a group of patients with primary intracranial disease and patients with mental disturbance but without primary intracranial lesion. Above mentioned indexes were compared, and clinical outcome was predicted with their correlation with fVEP in each patient. RESULTS: The latent period of fVEP peak appeared later in nonsurvivors than those in survivors [(228.6+/-41.7) ms vs. (190.5+/-49.2) ms, P<0.01]. APACHE II score (25.9+/-6.4 vs. 22.5+/-6.7) and SOFA score (6.7+/-2.0 vs. 5.4+/-2.5) were higher in nonsurvivors than those in survivors (both P<0.05 ), while the changes in GCS score was in contrary (6.3+/-2.4 vs. 7.0+/-3.0, P<0.05). fVEP peak latency showed a negative correlation with GCS score (r=-0.332, P<0.01). The death rate of the group of patients with primary intracranial lesion was similar to that of the total. fVEP peak latency of the group with no primary intracranial lesion but with mental impairment in nonsurvivors was significantly longer than that of survivors [(226.0+/-46.7) ms vs. (168.8+/-54.1) ms, P<0.05], fVEP peak latency was positively correlated with SOFA score (r=0.526, P<0.05). Area under receiver operator characteristic (ROC) curve of fVEP peak latency was 0.800+/-0.104 (P<0.05) for predicting outcome of patients, while that of SOFA score was 0.650+/-0.131 (P>0.05). The former could be used for predicting death. CONCLUSION: fVEP reflects the prognosis and severity of critically ill patients in ICU. Especially, it maybe used as a tool for predicting death and multiple organ dysfunction syndrome (MODS) in the patients with no primary intracranial lesion but with mental impairment.

Baicalin administration attenuates hyperglycemia-induced malformation of cardiovascular system.

In this study, the effects of Baicalin on the hyperglycemia-induced cardiovascular malformation during embryo development were investigated. Using early chick embryos, an optimal concentration of Baicalin (6 µM) was identified which could prevent hyperglycemia-induced cardiovascular malformation of embryos. Hyperglycemia-enhanced cell apoptosis was reduced in embryos and HUVECs in the presence of Baicalin. Hyperglycemia-induced excessive ROS production was inhibited when Baicalin was administered. Analyses of SOD, GSH-Px, MQAE and GABAA suggested Baicalin plays an antioxidant role in chick embryos possibly through suppression of outwardly rectifying Cl(-) in the high-glucose microenvironment. In addition, hyperglycemia-enhanced autophagy fell in the presence of Baicalin, through affecting the ubiquitin of p62 and accelerating autophagy flux. Both Baicalin and Vitamin C could decrease apoptosis, but CQ did not, suggesting autophagy to be a protective function on the cell survival. In mice, Baicalin reduced the elevated blood glucose level caused by streptozotocin (STZ). Taken together, these data suggest that hyperglycemia-induced embryonic cardiovascular malformation can be attenuated by Baicalin administration through suppressing the excessive production of ROS and autophagy. Baicalin could be a potential candidate drug for women suffering from gestational diabetes mellitus.

Effects of electrical stimulation of ventral septal area on firing rates of pyrogen-treated thermosensitive neurons in preoptic anterior hypothalamus from rabbits.

Although there is considerable evidence supporting that fever evolved as a host defense response, it is important that the rise in body temperature would not be too high. Many endogenous cryogens or antipyretics that limit the rise in body temperature have been identified. Endogenous antipyretics attenuate fever by influencing the thermoregulatory neurons in the preoptic anterior hypothalamus (POAH) and in adjacent septal areas including ventral septal area (VSA). Our previous study showed that intracerebroventricular (I.C.V.) injection of interleukin-1beta (IL-1beta) affected electrophysiological activities of thermosensitive neurons in VSA regions, and electrical stimulation of POAH reversed the effect of IL-1beta. To further investigate the functional electrophysiological connection between POAH and VSA and its mechanisms in thermoregulation, the firing rates of thermosensitive neurons in POAH of forty-seven unit discharge were recorded by using extracellular microelectrode technique in New Zealand white rabbits. Our results show that the firing rates of the warm-sensitive neurons decreased significantly and those of the cold-sensitive neurons increased in POAH when the pyrogen (IL-1beta) was injected I.C.V. The effects of IL-1beta on firing rates in thermosensitive neurons of POAH were reversed by electrical stimulation of VSA. An arginine vasopressin (AVP) V1 antagonist abolished the regulatory effects of VSA on the firing rates in thermosensitive neurons of POAH evoked by IL-1beta. However, an AVP V2 antagonist had no effects. These data indicated that VSA regulates the activities of the thermosensitive neurons of POAH through AVP V1 but not AVP V2 receptor.

Glycine receptors contribute to cytoprotection of glycine in myocardial cells.

BACKGROUND: The classic glycine receptor (GlyR) in the central nervous system is a ligand-gated membrane-spanning ion channel. Recent studies have provided evidence for the existence of GlyR in endothelial cells, renal proximal tubular cells and most leukocytes. In contrast, no evidence for GlyR in myocardial cells has been found so far. Our recent researches have showed that glycine could protect myocardial cells from the damage induced by lipopolysaccharide (LPS). Further studies suggest that myocardial cells could contain GlyR or binding site of glycine. METHODS: In isolated rat heart damaged by LPS, the myocardial monophasic action potential (MAP), the heart rate (HR), the myocardial tension and the activities of lactate dehydrogenase (LDH) from the coronary effluent were determined. The concentration of intracellular free calcium ([Ca(2+)](i)) was measured in cardiomyocytes injured by LPS and by hypoxia/reoxygenation (H/R), which excludes the possibility that reduced calcium influx because of LPS neutralized by glycine. Immunohistochemistry was used to detect the GlyR in myocardial tissue. GlyR and its subunit in the purified cultured cardiomyocytes were identified by Western blotting. RESULTS: Although significant improvement in the MAP/MAPD(20), HR, and reduction in LDH release were observed in glycine + LPS hearts, myocardial tension did not recover. Further studies demonstrated that glycine could prevent rat mycordial cells from LPS and hypoxia/reoxygenation injury (no endotoxin) by attenuating calcium influx. Immunohistochemistry exhibited a positive green-fluorescence signaling along the cardiac muscle fibers. Western blotting shows that the purified cultured cardiomyocytes express GlyR beta subunit, but GlyR alpha1 subunit could not be detected. CONCLUSIONS: The results suggest that glycine receptor is expressed in cardiomyocytes and participates in cytoprotection from LPS and hypoxia/reoxygenation injury. Glycine could directly activate GlyR on the cardiomyocytes and prevent calcium influx into the cardiomyocytes.

Ginsenosides from stems and leaves of ginseng prevent ethanol-induced lipid accumulation in human L02 hepatocytes.

OBJECTIVE: To investigate the effect of ginsenosides from stems and leaves of ginseng on ethanol-induced lipid deposition in human L02 hepatocytes. METHODS: L02 cells were exposed to ethanol for 36 h and treated with or without ginsenosides. The viability of L02 cells was evaluated by methylthiazolyldiphenyl-tetrazolium bromide assay and the triglyceride (TG) content was detected. Lipid droplets were determined by oil red O staining. Intracellular reactive oxygen species (ROS) production and the mitochondrial membrane potential were tested by flow cytometry. The ATP level was measured by reverse phase high performance liquid chromatography. The expression of cytochrome p450 2E1 (CYP2E1) and peroxisome proliferator-activated receptor α (PPARα) was detected by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. RESULTS: Ethanol exposure resulted in the increase of TG level, lipid accumulation and ROS generation, and the decrease of mitochondrial membrane potential and ATP production in the cells. However, ginsenosides significantly reduced TG content (9.69±0.22 µg/mg protein vs. 4.93±0.49 µg/mg protein, P<0.01), and ROS formation (7254.8±385.7 vs. 5825.2±375.9, P<0.01). Meanwhile, improvements in mitochondrial membrane potential (10655.33±331.34 vs. 11129.52±262.35, P<0.05) and ATP level (1.20±0.18 nmol/mg protein vs. 2.53±0.25 nmol/mg protein, P<0.01) were observed by treatment with ginsenosides. Furthermore, ginsenosides could down-regulate CYP2E1 expression (P<0.01) and upregulate PPARα expression (P<0.01) in ethanol-treated cells. CONCLUSIONS: Ginsenosides could prevent ethanol-induced hepatocyte steatosis in vitro related to the inhibition of oxidative stress and the improvement of mitochondrial function. In addition, the modulation of CYP2E1 and PPARα expression may also play an important role in the protective effect of ginsenosides against lipid accumulation.

From the Cover: Exposing Imidacloprid Interferes With Neurogenesis Through Impacting on Chick Neural Tube Cell Survival.

As a neonicotinoid pesticide, imidacloprid is widely used to control insects in agriculture and fleas on domestic animals. However, it is not known whether imidacloprid exposure negatively affects neurogenesis during embryonic development. In this study, using a chick embryo model, we investigated the effects of imidacloprid exposure on neurogenesis at the earliest stage and during late-stage embryo development. Exposing HH0 chick embryos to imidacloprid in EC culture caused neural tube defects (NTDs) and neuronal differentiation dysplasia as determined by NF/Tuj1 labeling. Furthermore, we found that F-actin accumulation on the apical side of the neural tube was suppressed by exposure to imidacloprid, and the expression of BMP4 and Shh on the dorsal and ventral sides of the neural tubes, respectively, were also reduced, which in turn affects the dorsolateral hinge points during bending of the neural plate. In addition, exposure to imidacloprid reduced cell proliferation and increased cell apoptosis, as determined by pHIS3 labeling and TUNEL staining, respectively, also contributing to the malformation. We obtained similar results in late-stage embryos exposed to imidacloprid. Finally, a bioinformatics analysis was employed to determine which genes identified in this study were involved in NTDs. The experimental evidence and bioinformatics analysis suggested that imidacloprid exposure during chick embryo development could increase the risk of NTDs and neural dysplasia.

Imidacloprid Exposure Suppresses Neural Crest Cells Generation during Early Chick Embryo Development.

Imidacloprid is a neonicotinoid pesticide that is widely used in the control pests found on crops and fleas on pets. However, it is still unclear whether imidacloprid exposure could affect early embryo development-despite some studies having been conducted on the gametes. In this study, we demonstrated that imidacloprid exposure could lead to abnormal craniofacial osteogenesis in the developing chick embryo. Cranial neural crest cells (NCCs) are the progenitor cells of the chick cranial skull. We found that the imidacloprid exposure retards the development of gastrulating chick embryos. HNK-1, PAX7, and Ap-2α immunohistological stainings indicated that cranial NCCs generation was inhibited after imidacloprid exposure. Double immunofluorescent staining (Ap-2α and PHIS3 or PAX7 and c-Caspase3) revealed that imidacloprid exposure inhibited both NCC proliferation and apoptosis. In addition, it inhibited NCCs production by repressing Msx1 and BMP4 expression in the developing neural tube and by altering expression of EMT-related adhesion molecules (Cad6B, E-Cadherin, and N-cadherin) in the developing neural crests. We also determined that imidacloprid exposure suppressed cranial NCCs migration and their ability to differentiate. In sum, we have provided experimental evidence that imidacloprid exposure during embryogenesis disrupts NCCs development, which in turn causes defective cranial bone development.

Effects of Senegenin against hypoxia/reoxygenation-induced injury in PC12 cells.

OBJECTIVE: To investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia/reoxygenation (H/R) in highly differentiated PC12 cells. METHODS: The cultured PC12 cells were treated with H/R in the presence or absence of Sen (60 µmol/L). Four groups were included in the experiment: control group, H/R group, H/R+Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase (LDH) in culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) and intracellular free calcium ([Ca(2+)]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and activity of NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively. RESULTS: Sen significantly elevated cell viability (P<0.05), decreased the leakage of LDH (P<0.05) and apoptosis rate (P<0.05) in H/R-injured PC12 cells. Sen maintained the value of △Ψm (P<0.05) and suppressed the activity of caspase-3 (P<0.05). Moreover, Sen reduced ROS accumulation P<0.05) and [Ca(2+)]i increment (P<0.05) by inhibiting the activity of NOX (P<0.05). CONCLUSION: Sen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and [Ca(2+)]i, thereby suppressing the mitochondrial pathway of cellular apoptosis.

A clinical study of Yigu capsule in treating postmenopausal osteoporosis.

OBJECTIVE: To observe the efficacy and safety of Yigu capsule (YGC), a Chinese herbal compound preparation, in treating postmenopausal osteoporosis (PMO) and to explore its possible mechanism. METHODS: The clinical study was conducted in a prospective, randomized, double blinded method lasting for 6 months with placebo and positive control. Two hundred and ten PMO patients with confirmed diagnosis were assigned into the YGC group, the calciferol group and the placebo group. Besides being administered element calcium, they were treated with YGC, calciferol capsule and placebo capsule respectively. And such symptoms as newly found fracture and ostealgia, bone mineral density (BMD) of the 2nd-4th lumbar vertebrae (L(2-4)) and upper femur, blood and urinary indexes for bone metabolism, sex hormone level and adverse reaction that occurred in patients were observed. RESULTS: In the YGC group, the total effective rate was 95.50%, with no new occurrence of fractures, which was significantly better than that in the other two groups (P < 0.05). Moreover, in the YGC group, the increase rate of BMD was 9.83% in L(2-4), 4.09% in femoral neck, 4.60% in Wards triangle, 3.00% in greater trochanter, which was also better than that in the placebo group (P < 0.05, P < 0.01). As compared with the placebo group, levels in the YGC group of urinary oxyproline hydroxyproline/creatinine, urinary calcium/creatinine were significantly lower, serum and bone alkaline phosphatase, osteocalcin, estradiol and estradiol/testosterone were significantly higher, but no difference was shown in the comparison of testosterone level. In the observation period, no abnormality in blood or urine routine, liver or renal function was found. Only mild, transient gastro-intestinal response occurred in individual patients, but it did not affect the treatment. CONCLUSION: YGC could treat PMO effectively, as it could obviously increase the BMD of lumbar vertebrae and coxafemoral bone, elevate the alleviating rate of ostealgia and incessant motion time, yet causing no newly found compressive fracture of vertebrae, or and any related adverse reaction. YGC could not only promote the formation, but also inhibit the absorption of bone as well as increase the sex hormone level. Therefore, it is a pure Chinese herbal compound preparation worthy of further research and development.