Tea and tea drinking: China's outstanding contributions to the mankind.

BACKGROUND: Tea trees originated in southwest China 60 million or 70 million years ago. Written records show that Chinese ancestors had begun drinking tea over 3000 years ago. Nowadays, with the aging of populations worldwide and more people suffering from non-communicable diseases or poor health, tea beverages have become an inexpensive and fine complementary and alternative medicine (CAM) therapy. At present, there are 3 billion people who like to drink tea in the world, but few of them actually understand tea, especially on its development process and the spiritual and cultural connotations. METHODS: We searched PubMed, Google Scholar, Web of Science, CNKI, and other relevant platforms with the key word "tea", and reviewed and analyzed tea-related literatures and pictures in the past 40 years about tea's history, culture, customs, experimental studies, and markets. RESULTS: China is the hometown of tea, tea trees, tea drinking, and tea culture. China has the oldest wild and planted tea trees in the world, fossil of a tea leaf from 35,400,000 years ago, and abundant tea-related literatures and art works. Moreover, tea may be the first Chinese herbal medicine (CHM) used by Chinese people in ancient times. Tea drinking has many benefits to our physical health via its antioxidant, anti-inflammatory, immuno-regulatory, anticancer, cardiovascular-protective, anti-diabetic, and anti-obesity activities. At the moment, COVID-19 is wreaking havoc across the globe and causing severe damages to people's health and lives. Tea has anti-COVID-19 functions via the enhancement of the innate immune response and inhibition of viral growth. Besides, drinking tea can allow people to acquire a peaceful, relaxed, refreshed and cheerful enjoyment, and even longevity. According to the meridian theory of traditional Chinese medicine, different kinds of tea can activate different meridian systems in the human body. At present, black tea (fermented tea) and green tea (non-fermented tea) are the most popular in the world. Black tea accounts for over 90% of all teas sold in western countries. The world's top-grade black teas include Qi Men black in China, Darjeeling and Assam black tea in India, and Uva black tea in Sri Lanka. However, all top ten famous green teas in the world are produced in China, and Xi Hu Long Jing tea is the most famous among all green teas. More than 700 different kinds of components and 27 mineral elements can be found in tea. Tea polyphenols and theaflavin/thearubigins are considered to be the major bioactive components of black tea and green tea, respectively. Overly strong or overheated tea liquid should be avoided when drinking tea. CONCLUSIONS: Today, CAM provides an array of treatment modalities for the health promotion in both developed and developing countries all over the world. Tea drinking, a simple herb-based CAM therapy, has become a popular man-made non-alcoholic beverage widely consumed worldwide, and it can improve the growth of economy as well. Tea can improve our physical and mental health and promote the harmonious development of society through its chemical and cultural elements.

Lipoxin A4 Activates Nrf2 Pathway and Ameliorates Cell Damage in Cultured Cortical Astrocytes Exposed to Oxygen-Glucose Deprivation/Reperfusion Insults.

Lipoxin A4 (LXA4), a potent antioxidant and anti-inflammation mediator, protects brains against cerebral ischemia/reperfusion (I/R) injury in vivo. However, few reports concern its function on astrocytes during cerebral I/R injury. The pathogenesis of cerebral I/R injury involves oxidative stress caused by reactive oxygen species (ROS). Upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) is generally considered to reduce oxidative stress. Nrf2 can induce heme oxygenase-1 (HO-1) expression and glutathione (GSH) release to combat increased oxidative stress. We investigated the effects of LXA4 on astrocytic cell damage, the production of ROS, and Nrf2 pathway, especially on HO-1 expression and GSH release in cultured cortical astrocytes exposed to oxygen-glucose deprivation (OGD)/recovery (OGDR) insults. Primary astrocytes were subjected to a 4-h OGD, followed by 8-h recovery. Cell viability, the production of ROS, and GSH release were measured. Furthermore, Nrf2, HO-1, and p62 expression levels were determined by Western blot. Moreover, Nrf2 location was studied by immunofluorescence staining. Treatment of LXA4 attenuates OGDR-induced cell damage and the production of ROS in a concentration-related manner. LXA4 induced Nrf2 expression and its nuclear translocation, as well as HO-1 expression and GSH release. Moreover, LXA4 induced the excess p62 accumulation. These results indicate that LXA4 can effectively protect against OGDR-induced cell damage in astrocytes, and activation of Nrf2 pathway to reduce oxidative stress may be involved in its protective effects. p62 accumulation induced by LXA4 may be closely related to Nrf2 activation.

Lipoxin A4 ameliorates cerebral ischaemia/reperfusion injury through upregulation of nuclear factor erythroid 2-related factor 2.

OBJECTIVES: Lipoxin A4 (LXA4) is a potent anti-inflammatory mediator that exerts a neuroprotective effect following cerebral ischaemia/reperfusion (I/R) injury. However, little is known about the underlying mechanisms. Upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) is generally considered to reduce cerebral I/R injury. Nuclear factor erythroid 2-related factor 2 can induce haeme oxygenase-1 (HO-1) and glutathione (GSH) expression to combat increased oxidative stress. The present study aimed to investigate the effects of Nrf2 signalling on LXA4-mediated neuroprotection. METHODS: Adult male Sprague Dawley rats were subjected to 2-hour middle cerebral artery occlusion followed by 24-hour reperfusion. Rats were randomly divided into four groups: Sham, I/R, LXA4, and LXA4+butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2) (all n = 24). Brain infarction was detected by 2,3,5-triphenyltetrazolium chloride staining. After 24 hours of reperfusion, Nrf2, HO-1, and p62 expression levels were determined by western blot, and GSH synthesis was assessed. RESULTS: Lipoxin A4 effectively reduced infarct volumes and improved neurological scores. These effects were partially blocked by Boc2, a specific antagonist of the LXA4 receptor (ALXR). Lipoxin A4 induced Nrf2 expression and its nuclear translocation, as well as HO-1 expression and GSH synthesis; Boc2 did not block these effects. The excess p62 accumulation induced by LXA4 might be closely related to Nrf2 activation. DISCUSSION: Overall, our data suggest that Nrf2 upregulation is involved in the neuroprotective effects of LXA4 and may be ALXR independent.

Lipoxin A4 inhibits 5-lipoxygenase translocation and leukotrienes biosynthesis to exert a neuroprotective effect in cerebral ischemia/reperfusion injury.

Lipoxin A(4) (LXA(4)), a biologically active eicosanoid with anti-inflammatory and pro-resolution properties, was recently found to have neuroprotective effects in brain ischemia. As 5-lipoxygenase (5-LOX) and leukotrienes are generally considered to aggravate cerebral ischemia/reperfusion (I/R) injury, we investigated their effects on LXA(4)-mediated neuroprotection by studying middle cerebral artery occlusion (MCAO)/reperfusion in rats and oxygen-glucose deprivation (OGD)/recovery in neonatal rat astrocyte primary cultures. LXA(4) effectively reduced infarct volumes and brain edema, and improved neurological scores in the MCAO/reperfusion experiments; this effect was partially blocked by butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2), a specific antagonist of the LXA(4) receptor (ALXR). Total 5-LOX expression did not change, regardless of treatment, but LXA(4) could inhibit nuclear translocation induced by MCAO or OGD. We also found that LXA(4) inhibits the upregulation of both leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) and the phosphorylation of extracellular signal-regulated kinase (ERK) induced by MCAO or OGD. The phosphorylation of the 38-kDa protein kinase (p38) and c-Jun N-terminal kinase (JNK) was not altered throughout the experiment. These results suggest that the neuroprotective effects of LXA(4) are probably achieved by anti-inflammatory mechanisms that are partly mediated by ALXR and through an ERK signal transduction pathway.

[Effect of lipoxin A(4) on lipopolysaccharide-induced endothelial hyperpermeability in human umbilical vein endothelial cell].

OBJECTIVE: To explore whether lipoxin A(4) (LXA(4))could prevent lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVEC) monolayer hyperpermeability and its possible mechanism. METHODS: Human umbilical cords were obtained from women with normal pregnancy immediately after delivery from Tongji Hospital Affiliated of Tongji Medical College. Primary HUVEC were isolated from umbilical veins and subcultured, then, HUVEC were divided into four groups:control group; LPS group (10 mg/L of LPS); LPS + LXA(4) group(10 mg/L of LPS and 100 nmol/L of LXA(4)); LPS + LXA(4) + BOC-2 group [10 µmol/L of BOC-2, an effective antagonist of formyl peptide receptor like 1 (FPRL-1)]. All expriments were performed after cells were treated for 24 hours. Endothelial permeability was measured by fluorescein isothiocyan-ate labelled bovine serum albumin (FITC-BSA) clearance across the monolayer; tumor necrosis factor α (TNF-α) mRNA and secretion were detected by reverse transcriplase (RT)-PCR and ELISA assay respectively, and nuclear factor κB (NF-κB) protein change was determined by western blot. RESULTS: (1) LPS induced a significant increase in the permeability [Pa value of LPS group was (183.1 ± 1.7)%], while co-administrating with LXA(4) obviously attenuated this LPS-induced hyperpermeability, Pa value of LPS + LXA(4) group was (103.1 ± 2.2)%, LPS + LXA(4) + BOC-2 group was (162.2 ± 2.8)%, control group was 100%, the permeability of HUVEC monolayer was significantly increased by LPS which was (83.1 ± 1.7)% of control (P < 0.01), however, it was notably inhibited by LXA(4) (P < 0.05); the blockade of FPRL-1 could attenuate the effect of LXA(4), that is, there was no difference between the LPS + LXA(4) + BOC-2 group and the LPS group. (2) After treatment with different concentration of LPS(0, 0.1, 1, 10 mg/L), the mRNA expressions of TNF-α were increased (1.11 ± 0.11, 1.27 ± 0.03, 1.60 ± 0.06, 1.82 ± 0.04, respectively), compared with the control group, at the concentration of 1, 10 mg/L LPS, the difference was statistically significant (P < 0.05). (3) The increased levels of NF-κB and inflammatory mediator TNF-α in the LPS group were both inhibited by LXA(4). Levels of NF-κB protein and TNF-α mRNA secretion in LPS treated group (0.53 ± 0.06 and 0.81 ± 0.09, respectively) were both inhibited by LXA(4)(0.19 ± 0.05 and 0.41 ± 0.07, respectively, and both had significant difference, P < 0.05). (4) Levels of TNF-α in HUVEC culture medium of LPS group [(31.94 ± 0.01) ng/L] was significantly higher than the control group [(18.17 ± 0.03) ng/L, P < 0.05], LPS + LXA(4) group [(15.72 ± 0.07) ng/L] was significantly lower than the LPS group (P < 0.05). CONCLUSION: Our findings demonstrated that LXA(4) could prevent the endothelial cell hyperpermeability induced by LPS in HUVEC under which the possible mechanism was through inhibiting the expression of NF-κB and its related cytokines through receptor-dependent.

[Effect of lipoxin A(4) on lipopolysaccharide-induced oxidant stress in human umbilical vein endothelial cells].

OBJECTIVE: To explore the effects of lipoxin A(4) (LXA(4)) on lipopolysaccharide (LPS)-induced oxidative stress in human umbilical veins endothelial cells (HUVEC) and the possible mechanism. METHODS: Neonatal umbilical cords were obtained from normal term pregnant women with cesarean section within 4 hours and then were used to isolate HUVEC for subculture. HUVEC were divided into four groups:control group; LPS group (10 µg/ml of LPS); LPS + LXA(4) group (10 µg/ml of LPS and 100 nmol/L of LXA(4)); LXA(4) group (100 nmol/L of LXA(4)). All expriments were performed after cells treated for 12 and 24 hours respectively. Immunofluorescence was used to detect the expression of VIII foctor and nuclear translocation of nuclear factor-erythroid-2-related factor 2 (Nrf2); the mRNA expression of Nrf2, heme oxygenase 1 (HO-1) and reduced form of nicotinamide-adenine dinucleotide quinone oxidoreductase-1 (NQO1) were evaluated by reverse transcription-PCR. RESULTS: (1) The flavovirens fluorescence was observed in the cytoplasm under fluorescence microscope, which confirmed the existence of VIII factor which specifically expressed in endothelial cells, especially in HUVEC. (2) Immunofluorescent results showed that in control group, Nrf2 protein expressed in the cytosol rather than in the nucleus. In LPS group, the expression of Nrf2 protein obviously increased in the nucleus while decreased in the cytosol after 12 hours. However, after LPS treatment for 24 hours, Nrf2 expression reduced in the cytosol and nucleus. In co-treatment with LPS and LXA(4) group, the expression of Nrf2 protein was much higher than that in LPS group after 12 hours or 24 hours. Furthermore, Nrf2 protein also mostly expressed in the cytosol in LXA(4) group. (3) After stimulation for 12 hours, compared with control group, the gene expression of Nrf2 and HO-1 were significantly enhanced in LPS group (0.581 ± 0.019 and 0.081 ± 0.009, P < 0.05) and in LPS + LXA(4) group (0.692 ± 0.048 and 0.136 ± 0.018, P < 0.05), the level of NQO1 mRNA in LPS group and LPS + LXA(4) group were 0.381 ± 0.009 (P > 0.05) and 0.574 ± 0.034 (P < 0.05). After treatment for 24 hours, compared with control goup, the gene expressions of Nrf2 and NQO1 were down-regulated in LPS group (0.180 ± 0.017 and 0.472 ± 0.064, P < 0.05). But in LPS + LXA(4) group the expression of Nrf2 and NQO1 were upregulated (0.532 ± 0.051 and 0.830 ± 0.068, P < 0.05, compared with treatment for LPS group). The mRNA expressions of Nrf2, HO-1 and NQO1 were increased in LPS + LXA(4) group compared with LPS group (P < 0.05). In addition, there was no markedly difference in the expressions of Nrf2, HO-1 and NQO1 between control and LXA(4) group after 12 hours and 24 hours (P > 0.05). CONCLUSION: Through activating nuclear translocation of Nrf2 protein from cytoplasm, LXA(4) upregulates the Nrf2 downstream enzymes, such as NQO1 and HO-1 to protect HUVEC against the oxidative stress induced by LPS.

Effects of exogenous annexin-1 on lipopolysaccharide-induced proliferation and reactive oxygen species production partially through modulation of CRAC channels but independent of NF-kappaB pathway.

OBJECTIVE: To investigate the effects of exogenous annexin-1 (ANXA1) on lipopolysaccharide(LPS)-induced proliferation, reactive oxygen species (ROS) production, and calcium signal transduction in RAW264.7 macrophages. METHODS: RAW264.7 macrophages were treated with or without LPS in the absence or presence of ANXA1. The proliferation effects were detected by Cell Counting Kit-8 assay. ROS were quantified by flow cytometry and fluorescence microscopy. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was analyzed by laser confocal scanning microscopy. IkappaBalpha degradation and NF-kappaB translocation were tested by Western blot. RESULTS: Exogenous ANXA1 inhibited LPS-induced proliferation and ROS production in a dose-dependent manner. LPS evoked [Ca(2+)](i) increase through CRAC channels, and ANXA1 suppressed LPS-induced [Ca(2+)](i) increase in a dose-dependent manner. The CRAC channels were associated with LPS-induced proliferation and ROS production. Exogenous ANXA1 had no effect on LPS-induced IkappaB degradation and NF-kappaB translocation. CONCLUSIONS: ANXA1 inhibited LPS-induced proliferation and ROS production in RAW264.7 macrophages partially through modulation of CRAC channels but independent of the NF-kappaB pathway.

[Protective effects and its mechanism of lipoxins on human umbilical vein endothelial cells under hypoxia].

OBJECTIVE: To investigate protective effects and mechanisms of lipoxinA(4) (LXA(4)) on human umbilical vein endothelial cells (HUVEC) under hypoxia in vitro. METHODS: The HUVEC culture were divided into groups as followed: added M199 culture medium as normal control groups, added CoCl2 to mimic hypoxia in vitro as hypoxia group and added different concentrations of LXA(4) (1, 10, 100 nmol/L) were added to the induced hypoxial HUVEC as agents intervention group. Morphological changes of HUVEC were observed by using inverted phase contrast microscope. The influence of LXA(4) on cell survival was investigated by methyl thiazolyl tetrazolium (MTT) assaying method after the treatment with different concentrations of LXA(4) and 100 nmol/L lipoxinA(4) according to different time (4, 8, 12 and 24 hours). The expression of von-willebrand factor (vWF) was detected by immunocytochemistry method. The changes of cytosolic Ca(2+) were measured by laser scanning confocal microscope. RESULTS: (1) Morphological changes:the cells under hypoxia lost its normal shapes and showed necrosis, while the cells cocultured with 100 nmol/L LXA(4) were normal appropriately. (2) Survival rate: the survival rates of HUVEC under hypoxia was (40.1 +/- 3.9)% and increased to (52.9 +/- 1.4)%, (64.1 +/- 3.3)%, (76.6 +/- 1.6)% respectively when added with LXA(4) with concentration of 1, 10, 100 nmol/L into culture medium. There was significant different survival rate when compared with that of hypoxia group. (3) The level of vWF: The expression of vWF was decreased with the increasing concentrations of LXA(4) added into culture medium, the gray values were 203.9 +/- 0.7 in 1 nmol/L, 204.6 +/- 0.9 in 10 nmol/L, 191.8 +/- 0.5 in 100 nmol/L respectively, which reached statistical difference in comparison with that of hypoxia groups (P < 0.05). (4) Confocal analysis: the intracellular free Ca(2+) concentrations of HUVEC were intensified with LXA(4) treatment. CONCLUSIONS: LXA(4) plays an important role in keeping the normal shape of HUVEC under hypoxia, can enhance survival of hypoxial HUVEC and decrease the level of vWF in cytoplasm. The protective mechanism might be via decreasing mitochondria Ca(2+) overload and increasing cytoplasm Ca(2+) by nucleus Ca(2+) transference.

Lipoxin A(4) inhibited hepatocyte growth factor-induced invasion of human hepatoma cells.

AIM: Inflammation is a critical component of tumor progression. Lipoxin A(4) (LXA(4)) has been approved for potent anti-inflammatory properties. Recently, it was reported that LXA(4) repressed the expression and activity of cyclooxygenase-2 (COX-2), which is essential for invasion. However, there are few reports dealing with its effects on cancer. To explore whether LXA(4) regulate invasion, the effects of LXA(4) and its receptor agonist BML-111 on hepatocyte growth factor (HGF)-induced invasion of hepatoma cells and the possible mechanisms were researched. METHODS: Lipoxin A(4) receptor (ALX) expression in HepG2 cells were measured through reverse transcription polymerase chain reaction and western blot. Cytotoxicity of LXA(4) and BML-111 to HepG2 cells was detected by MTT and ((3)H)-TdR incorporation assay. Cell migration and invasion assays were performed using a Boyden chemotaxis chamber. COX-2 expression was detected by real-time polymerase chain reaction and western blot, respectively. Moreover, the expressions of matrix metalloproteinases (MMP)-2, MMP-9, IkappaBalpha and nuclear factor-kappaB (NF-kappaB) p65 were observed via western blot, and NF-kappaB transcriptional activity was tested by transfections and luciferase activities assay. RESULTS: ALX expression was detected in HepG2 cells, and suitable concentrations of LXA(4) and BML-111 had no cytotoxicity to cells. LXA(4) and BML-111 inhibited HGF-induced migration and invasion; downregulated COX-2, MMP-2 and -9; restrained HGF-induced IkappaBalpha degradation, NF-kappaB translocation and the transcriptional activity of NF-kappaB in HepG2 cells. Furthermore, exogenous PGE2 could reverse the inhibitory effects of LXA(4) also BML-111 on HGF-induced invasion and migration partially. CONCLUSION: LXA(4) inhibited HGF-induced invasion of HepG2 cells through NF-kappaB/COX-2 signaling pathway partially.

Formyl-peptide receptor like 1: a potent mediator of the Ca2+ release-activated Ca2+ current ICRAC.

In electrically non-excitable cells, one major source of Ca(2+) influx is through the store-operated (or Ca(2+) release-activated Ca(2+)) channel by which the process of emptying the intracellular Ca(2+) stores results in the activation of Ca(2+) channels in the plasma membrane. Using both whole-cell patch-clamp and Ca(2+) imaging technique, we describe the electrophysiology mechanism underlying formyl-peptide receptor like 1 (FPRL1) linked to intracellular Ca(2+) mobilization. The FPRL1 agonists induced Ca(2+) release from the endoplasmic reticulum and subsequently evoked I(CRAC)-like currents displaying fast inactivation in K562 erythroleukemia cells which expresses FPRL1, but had almost no effect in K562 cells treated with FPRL1 RNA-interference and HEK293 cells which showed no FPRL1 expression. The currents were impaired after either complete store depletion by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin, or after inhibition of PLC by U73122. Our results present the first evidence that FPRL1 is a potent mediator in the activation of CRAC channels.

Differential response of human fetal smooth muscle cells from arterial duct to retinoid acid.

AIM: The aim of the present study was to understand the role of retinoic acid (RA) in the development of isolated patent ductus arteriosus and the features of arterial duct-derived vascular smooth muscle cells (VSMC). METHODS: The VSMC were isolated, and the biological characteristics and the response to RA were investigated in the arterial duct, aorta, and pulmonary artery VSMC from 6 human embryonic samples. Western blotting, immunostaining, and cell-based ELISA were employed to analyze the proliferation regulation of VSMC. RESULTS: The VSMC from the arterial duct expressed proliferating cell nuclear antigen (PCNA) at a significantly lower rate than those from the aorta and pulmonary artery, but expressed a higher level of Bax and Bcl-2. The expression level of PCNA or Bcl-2 was associated with the embryonic age. The effects of RA on the VSMC from the arterial duct were quite different from those from the aorta and pulmonary artery. In arterial duct VSMC, RA stimulated PCNA expression, but such stimulation could be suppressed by CD2366, an antagonist of nuclear retinoid receptor activation. In aorta or pulmonary artery VSMC, the expression response of PCNA to RA was insignificant. The ratio of Bax/Bcl-2 decreased in arterial duct VSMC after RA treatment due to the significant inhibition of Bax expression. CONCLUSION: The VSMC from the arterial duct possessed distinct biological behaviors. RA might be important in the development of ductus arteriosus VSMC.

Lipoxin A4 negatively regulates lipopolysaccharide-induced differentiation of RAW264.7 murine macrophages into dendritic-like cells.

BACKGROUND: Lipoxins (LXs), endogenous anti-inflammatory and pro-resolving eicosanoids generated during various inflammatory conditions, have novel immunomodulatory properties. Because dendritic cells (DCs) play crucial roles in the initiation and maintenance of immune response, we determined whether LXs could modulate the maturation process of DCs and investigated the effects of lipoxin A(4) (LXA(4)) on lipopolysaccharide (LPS)-induced differentiation of RAW264.7 cells into dendritic-like cells. METHODS: RAW264.7 cells were cultured in vitro with 1 microg/ml LPS in the absence or presence of LXA(4) for 24 hours, and cellular surface markers (MHC-II, CD80 (B7-1), CD86 (B7-2)) were measured by flow cytometry (FCM). Mixed lymphocyte reaction was performed to evaluate the allostimulatory activity. Cytoplastic IkappaB degradation and nuclear factor kappa B (NF-kappaB) translocation were detected by Western blotting. Luciferase reporter plasmid was transiently transfected into RAW264.7 cells, and luciferase activity was determined to measure the transcriptional activity of NF-kappaB. RESULTS: LXA(4) reduced the ratio of LPS-treated RAW264.7 cells to DCs with morphological characteristics and inhibited the expression of MHC II. LPS-induced up-regulation of CD86 was moderately suppressed by LXA(4) but no obvious change of CD80 was observed. Moreover, LXA(4) weakened the allostimulatory activity of LPS-treated RAW264.7 cells. These alterations of LPS+LXA(4)-treated cells were associated with a marked inhibition of IkappaB degradation, NF-kappaB translocation and then the transcriptional activity of NF-kappaB. CONCLUSIONS: LXA(4) negatively regulates LPS-induced differentiation of RAW264.7 cells into dendritic-like cells. This activity reveals an undescribed mechanism of LXA(4) to prevent excessive and sustained immune reaction by regulating maturation of DCs.

Posttreatment with aspirin-triggered lipoxin A4 analog attenuates lipopolysaccharide-induced acute lung injury in mice: the role of heme oxygenase-1.

BACKGROUND: We hypothesized that posttreatment with 15-epi-16-parafluoro-phenoxy lipoxin A4 (ATL) could attenuate lipopolysaccharide (LPS)-induced acute lung injury in mice. METHODS: All the animals were randomly assigned to one of six groups (n = 6 per group). In the sham-vehicle group, mice were treated with 0.9% saline 60 min after they were challenged with saline. The sham-ATL group was identical to the sham-vehicle group except that ATL (0.7 mg/kg, IV) was administered, and the sham-ZnPP group was identical to the sham-vehicle group except that Zinc protoporphyrin IX (ZnPP, 25 mg/kg IV) was administered. In the LPS-vehicle group, mice were treated with vehicle 60 min after they were challenged with LPS. The LPS-ATL group was identical to the LPS-vehicle group but received ATL. The ZnPP-ATL-LPS group was identical to the LPS-ATL group, but ZnPP was administered 30 min before ATL. RESULTS: Inhalation of LPS increased inflammatory cell counts, tumor necrosis factor-alpha, and protein concentration in bronchoalveolar lavage fluid and also induced lung histological injury and edema. Posttreatment with ATL inhibited tumor necrosis factor-alpha, nitric oxide, and malondialdehyde production, with the outcome of decreased pulmonary edema, lipid peroxidation, and the infiltration of neutrophils in lung tissues. In addition, ATL promoted the formation of heme oxygenase-1 in the lung tissues. Heme oxygenase-1 activity was also increased in the lung tissues after ATL stimulation. The beneficial effects of ATL were abolished by ZnPP. CONCLUSIONS: This study demonstrates that posttreatment with ATL significantly reduces LPS-induced acute lung injury in mice.

Experimental study on the action of allitridin against human cytomegalovirus in vitro: Inhibitory effects on immediate-early genes.

Garlic (Allium sativum) extraction has been reported having anti-HCMV efficacy. This study was aimed to investigate the effect of allitridin (diallyl trisulfide, a compound from A. sativum extraction) on the replication of HCMV and the expression of viral immediate-early genes. In HCMV plaque-reduction assay, allitridin appeared a dose-dependent inhibitory ability with EC(50) value of 4.2 microg/ml (selective index, SI=16.7). Time-of-addition and time-of-removal studies showed that allitridin inhibited HCMV replication in earlier period of viral cycle before viral DNA synthesis. Both immediate early gene (ie1) transcription and IEA (IE(1)72 and IE(2)86) expression was suppressed by allitridin, but not by GCV in a single HCMV cycle format. In addition, allitridin appeared stronger inhibition on IE(2)86 than on IE(1)72. Decrease of viral DNA load in infected cells was also detected under allitridin treatment, probably due to an indirect consequence of the reduction in ie gene transcription. In summary, this study indicated that allitridin has anti-HCMV activity and the mechanism is associated with suppression of ie gene transcription.

Close functional coupling between Ca2+ release-activated Ca2+ channels and reactive oxygen species production in murine macrophages.

AIM: To investigate the role of Ca(2+) release-activated Ca(2+) (CRAC) channels in the ROS production in macrophages. METHODS: The intracellular [Ca(2+)](i) was analyzed by confocal laser microscopy. The production of ROS was assayed by flow cytometry. RESULTS: Both LPS and thapsigargin induced an increase in intracellular [Ca(2+)](i), either in the presence or absence of extracellular Ca(2+) in murine macrophages. The Ca(2+) signal was sustained in the presence of external Ca(2+) and only initiated a mild and transient rise in the absence of external Ca(2+). CRAC channel inhibitor 2-APB completely suppressed the Ca(2+) entry signal evoked by thapsigargin, and suppressed approximately 93% of the Ca(2+) entry signal evoked by LPS. The increase in intracellular [Ca(2+)](i) was associated with increased ROS production, which was completely abolished in the absence of extracellular Ca(2+) or in the presence of CRAC channel inhibitors 2-APB and Gd(3+). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in ROS production and completely inhibited thapsigargin and LPS-evoked responses. Conclusions. These findings indicate that the LPS-induced intracellular [Ca(2+)](i) increase depends on the Ca(2+) entry through CRAC channels, and close functional coupling between CRAC and ROS production in murine macrophages.

An experimental study on astrocytes promoting production of neural stem cells derived from mouse embryonic stem cells.

BACKGROUND: The production of neural stem cells (NSCs) derived from embryonic stem (ES) cells was usually very low according to previous studies, which was a major obstacle for meeting the needs of clinical application. This study aimed at investigating whether astrocytes could promote production of NSCs derived from ES cells in vitro. METHODS: Mouse ES cells line-D3 was used to differentiate into NSCs with astrocytes as inducing stromal cells by means of three-stage differentiation procedure. Another group without astrocytes served as control. The totipotency of ES cells was identified by observation of cells' morphology and formation of teratoma in severe combined immunodeficiency disease (SCID) mice. The quantity and purity of NSCs derived from ES cells were analyzed using clonogenic assay, immunohistochemical staining and flow cytometry assay. The plasticity of NSCs was detected by differentiating test. Octamer-binding transcription factor 4 (Oct-4) and nestin, the specific marker genes of ES cells and NSCs respectively, were detected continuously using reverse transcription-polymerase chain reaction (RT-PCR) method to monitor the process of cell differentiation. RESULTS: The ES cells of D3 line could maintain the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. At the end of two-stage of inducing process, 23.2 +/- 3.5 neurospheres per plate formed in astrocyte-induced group and only 0.8 +/- 0.3 per plate in the control group (clonogenic assay, P < 0.01), and the ratio of nestin positive cells was (50.2 +/- 2.8)% in astrocyte-induced group and only (1.4 +/- 0.5)% in the control group (flow cytometry, P < 0.01). With the induction undergoing, the expression of Oct-4 gradually decreased and then disappeared, while the expression of nestin was increased step by step, and the ratio of nestin positive cells was up to 91.4% by the three-stage differentiation. The nestin positive cells could be further induced into neurons, astrocytes, and oligodendrocytes in differentiating medium supplemented with fetal calf serum. The results of differentiating test showed that the ratio of NF-200 and NSE positive cells was (42.7 +/- 2.6)% in astrocyte-induced group and only (11.2 +/- 1.8)% in the control group (P < 0.01). CONCLUSIONS: Astrocytes can not only increase the production of NSCs derived from ES cells but also promote the differentiation of NSCs toward neuronal lineage.

[Effect of tetramethylpyrazine on lipopolysaccharides induced macrophage cyclo-oxidase-2 expression and apoptosis of cardiac myocytes].

OBJECTIVE: To observe the effect of tetramethylpyrazine (TMP) on lipopolysaccharides (LPS) induced macrophage cyclo-oxidase-2 (COX-2) gene expression and activity in RAW264.7 mice, and to further investigate the effect and mechanism of TMP on LPS induced apoptosis of cardiac myocytes in suckling mice. METHODS: RT-PCR and Western Blot (WB) were used to investigate the macrophage COX-2 gene expression, ELISA was used to measure its activity, fluorescence microscopy was used to determine the apoptosis of murine neonatal cardiac myocyte, and fluorescence spectrophotometry was used to detect the concentration of intracellular calcium ion (Ca2+). RESULTS: TMP of 10(-6) mol/L could significantly reduce the COX-2 mRNA and protein expression (P < 0.05), in 10(-5) mol/L and 10(-4) mol/L could significantly decrease the COX-2 expression (P < 0.01) stimulated by LPS, but couldn't influence the activity of COX-2 by different TMP concentration. TMP in 10(-5) mol/L could significantly lower the concentration of intracellular Ca2+ in cardiac myocyte, and antagonize the LPS induced apoptosis of cardiac myocyte in suckling mice (P < 0.05). CONCLUSION: TMP has the pharmacological effect in inhibiting LPS induced macrophage COX-2 expression and apoptosis of cardiac myocyte in suckling mice.

[Expression of cyclooxygenase-2 mRNA and identification of its spliceriant in human myometrium obtained from women in labor].

OBJECTIVE: To investigate the expression of cyclooxygenase-2 (COX-2) in human lower segments of myometrium obtained from women in labor and those not in labor and identify the splice variant of COX-2. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for investigating the expression of COX-2. According to the sequence of rat COX-2 splice variant, the primers were designed and synthesized, then the splice variant of COX-2 in human myometrium from woman in labor was identified, cloned into vector and sequenced. RESULTS: The results showed that (1) The Expression of COX-2 mRNA was lower in human myometrium obtained from women who were not in labor than those in labor. (2) A new band of COX-2 was obtained in myometrium from a woman in labor. The fragment includes an unspliced intron, which locates between exons 7 and 8. CONCLUSION: COX-2 gene is not only expressed highly in human myometrium from women in labor, but also produced splicing variant by alternative splicing.