基于转录组测序的金柑类黄酮糖基转移酶基因的初步分析

植物体内类黄酮通常以糖基化的形式存在,其糖基化由UDP–糖基转移酶(UDPglycosyltransferase,UGT)催化完成。以‘浏阳金柑’果实为研究对象,利用UPLCG2-SQTOF技术对其类黄酮种类进行鉴定,共检测出11种类黄酮,包括C–葡萄糖苷类黄酮、C–新橙皮糖苷类黄酮和O–新橙皮糖苷类黄酮。基于转录组测序,筛选出49个金柑果实发育过程中高表达的UGT基因。进化分析显示,13个高表达的FcUGT可能参与了类黄酮的糖基化修饰过程。其中,1个FcUGT编码C–葡萄糖基转移酶,参与了C–葡萄糖苷类黄酮的修饰过程;9个FcUGT编码7–O–葡萄糖基转移酶,可能参与了O–新橙皮糖苷类黄酮的修饰过程;3个FcUGT编码1,2–鼠李糖基转移酶,可能参与了O–或C–新橙皮糖苷类黄酮的修饰过程。     

Therapeutic effect of emodin on constipation induced by loperamide in mice

AIM: To study the therapeutic effect of emodin on loperamide-induced constipation in mice. METHODS: The constipation model of mice was established by lopebutamine treatment, and the effects of emodin on defecation frequency, fecal water content and intestinal transit time of the mice during the observation period were detected. Inflammatory infiltration in colon tissue of the mice was observed by HE staining. Serum nitric oxide (NO) content was detected by commercially available kit. The expression of vasoactive intestinal peptide receptor 1 (VIPR1) and 5-hydroxytryptamine type 4 receptor (5-HT₄ receptor) in mouse colon was determined by immunohistochemistry. The effects of emodin on the expression of transient receptor potential cation channel subfamily V member 1 (TRPV1), glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), nitric oxide synthase (NOS), c-Kit and their ligand stem cell factor (SCF) were detected by Western blot. RESULTS: Emodin significantly increased the number of defecation and fecal water content in the mice during the observation period, and significantly reduced the intestinal transit time and serum NO level in the mice (P<0.05). The results of HE staining showed that emodin significantly reduced the infiltration of colonic inflammation induced by loperamide. Emodin can significantly reduce the increase in VIPR1 induced by loperamine and increased the expression of 5-HT₄ receptor (P<0.01). Emodin increased the expression levels of GDNF and BDNF, reduced the expression levels of TRPV1 and NOS in the colon tissues of loperamine-induced constipation in mice, and significantly increased the expression of c-Kit and SCF (P<0.05 or P<0.01). CONCLUSION: Emodin promotes the defecation behavior of mice with loperamine-induced constipation by increasing intestinal peristalsis and activating a series of smooth muscle contraction-related factors.     

Puerarin protects high glucose-induced injury of vascular relaxation by activation of heme oxygenase-1

AIM: To investigate the effect of puerarin on acute high glucose-induced attenuation of ACh relaxation in isolated rat aortic rings, and to elucidate its underlying mechanism. METHODS: The thoracic aortic rings with endothelium of male Sprague-Dawley rats were mounted on a bath system. Isometric relaxation of aortic rings was measured. RESULTS: ① After incubation with high concentration of glucose (44 mmol/L), the vascular relaxation responses to ACh decreased. ② After coincubation with puerarin (10-10-10-8 mol/L) and high glucose, the high glucose-induced attenuation of ACh relaxation responses of artery was partly inhibited in a dose-dependent manner. ③ After incubation with puerarin for 2 h, the HO-1 activity of thoracic aorta increased. ZnPPIX (an inhibitor of heme oxygenase-1) abrogated the protective effect of puerarin. CONCLUSION: Puerarin prevents the acute high glucose-induced attenuation of endothelium-dependent relaxation in aortic rings. The mechanism might be involved in the activation of heme oxygenase-1.     

Emodin inhibits proliferation and blocks cell cycle of human breast cancer MCF-7 cells

AIM：To investigate the effects of emodin on the proliferation of human breast cancer MCF-7 cells and its mechanisms. METHODS：MTT assay was used to observe the viability of MCF-7 cells. The cell cycle distribution and apoptosis of MCF-7 cells was analyzed by flow cytometry. The membrane surface morphology and three-dimensional ultrastructure of MCF-7 cells were observed under atomic force microscope (AFM). RESULTS：MTT assay showed that emodin could inhibit MCF-7 cell proliferation in a dose-dependent manner. Flow cytometric analysis demonstrated that emodin induced cell cycle arrest at G0/G1 phase. Annexin V/PI double staining confirmed that emodin had no effect on cell apoptosis. AFM images revealed that the cell nuclear area was full and the surface of cell membrane was flat and smooth in control group. Compared with control group, the cell nuclear area collapsed and shrank in emodin group at 48 h. The cell membrane ultrastructure showed that the particles in emo-din group had an intensive distribution. The height of cell nuclear area was decreased, and the surface average roughness (Ra) and root mean square roughness (Rq) were elevated in emo-din group compared with control group. CONCLUSION: Emodin has cytotoxicity on MCF-7 cells via cell cycle arrest at G0/G1 phase and ultrastructural changes.     

Protective effect of emodin on lung injury induced by hepatic fibrosis in rats

AIM:To explore the protective effect of emodin on lung injury induced by hepatic fibrosis in rats. METHODS:The hepatic fibrosis rat model was established with multiple pathogenic factors (CCl 4, ethanol, high fat, high cholesterol and low choline) and treated with different doses (20 mg/kg and 40 mg/kg) of emodin for 4 weeks. The hepatic index was measured. The biochemical indexes, endotoxin, homocysteine, albumin, aspartate aminotransferase,alanine aminotransferase, total bilirubin, total cholesterol and triglyceride, and hepatic fibrosis indexes, hyaluronic acid, laminin, collagen IV and procollagen Ⅲ, were detected. The histopathological changes of the liver were observed. The pulmonary index was determined. The histopathological changes of the lungs were observed. The levels of tumor necrosis factor α (TNF-α), malondialdehyde (MDA), nitric oxide (NO) and peroxynitrite (ONOO-) in the lung tissues were analyzed. RESULTS:The rat hepatic fibrosis model was successfully established. In model group, lung edema and inflammation occurred, and the pulmonary index and the levels of TNF-α, MDA, NO and ONOO- in the lung tissues were increased significantly. In emodin treatment groups, the pulmonary indexes were lower than that in model group. The pathological injury of the lung tissues was alleviated. The levels of TNF-α, MDA, NO and ONOO- in the lung tissues were decreased. CONCLUSION:Emodin has a protective effect on lung injury induced by hepatic fibrosis in rats.     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.     

Vasodilatation induced by Jumi extraction and its mechanisms

AIM: The objectives of the present study were to examine the effect of Jumi (JM) extraction on relaxation of isolated rat aortic rings, and to elucidate its mechanisms. METHODS: The thoracic aortic rings with and without endothelium of male Sprague-Dawley rats were mounted on a bath system. Vasodilatation of aortic rings preconstricted with 10-6 mol/L of phenylephrine (PE) was measured. RESULTS: JM extraction (0.5-8 g/L) caused a concentration-dependent relaxation in aortic rings. The extent of relaxation was larger in endothelium-intact aortic rings than that in endothelium-denuded aortic rings. Both L-NAME［a nitric oxide synthase (NOS) inhibitor］ and high potassium (20 mmol/L KCl) partly abolished the relaxation action of JM extraction in endothelium-intact aortic rings. Pretreatment with L-NAME also inhibited the relaxation response to JM extraction in endothelium-denuded aortic rings. After incubation with JM extraction, NOS activities enhanced both in endothelium-intact and endothelium-denuded aortic rings. CONCLUSION: JM extraction causes relaxation of aortic rings through endothelium-dependent and independent pathways. The mechanisms might be involved in NOS and endothelium-derived hyperpolarizing factor.     

Expression of neuronal nuclear antigen and neuron-specific enolase in developing human embryonic small intestines

AIM：To investigate the neuronal nuclear antigen (NeuN) and neuron-specific enolase (NSE) distribution in the developing stage of human embryonic small intestines and their clinical significance. METHODS：Sixteen cases of human embryos at 2~4 months of gestational age were used in the present study. The technique of immunohistochemical staining was used to detect the expression and distribution of NeuN and NSE in small intestinal walls. RESULTS：At 2~4 months of gestational age, NSE was strongly expressed in neurons and nerve fibers of the small intestinal myenteric nerve plexuses in human embryos. The numbers of NSE-positive cells and fibers gradually increased in the small intestinal submucosa with the increase in gestational age, and a few NSE-positive cells located in the small intestinal glands of human embryos. NeuN-positive cells scattered in the epithelium and glands of the small intestinal mucosa, and the number of NeuN-positive cells gradually increased with the increase in gestational age. But there were no NeuN-positive cells in the small intestinal submucosa. CONCLUSION: The expression and distribution of NeuN are not consistent with those of NSE during the development of human embryonic small intestines, and both of them may be involved in the development of neurons and neuroendocrine cells in small intestinal walls.     

Relationship between UGT1A1 gene polymorphisms and toxicity/efficacy of irinotecan-based chemotherapy in metastatic colorectal cancer

AIM: To investigate the correlation of UGT1A1 ^*28 and UGT1A1 ^*6 gene polymorphisms with irinotecan-associated adverse events and efficacy in the patients with metastatic colorectal cancer (mCRC) treated with irinotecan-based chemotherapy. METHODS: Analysis of UGT1A1 ^*28 and UGT1A1 ^*6 gene polymorphisms was performed in 207 gastrointestinal cancer patients admitted to our hospital from April 2010 to March 2012 by amplifying the gene fragments using PCR and direct sequencing. Fifty six cases with mCRC treated with irinotecan were chosen to observe the adverse events and efficacy during chemotherapy, and the time to progression (TTP) was also recorded. The incidence of different genotypes was compared. RESULTS: The distribution of the genotypes in 207 gastrointestinal cancer patients was as follows: UGT1A1 ^*28 wild-type (WT) genotype TA6/6 (164, 79.2%), heterozygous genotype TA6/7 (41, 19.8%), and homozygous genotype TA7/7 (2, 1.0%); UGT1A1 ^*6 WT genotype G/G (154, 74.4%), heterozygous genotype G/A (51, 24.6%), and homozygous genotype A/A (2, 1.0%). In the 56 mCRC cases, the incidence of grade 3 and 4 delayed diarrhea and neutropenia in the patients carrying UGT1A1 ^*6 (G/A and A/A) was higher than that in the WT genotype (6/6) (38.9% vs 7.9%,61.1% vs 29.0%, both P<0.05). The incidence of grade 3 and 4 thrombocytopenia in the patients carrying UGT1A1 ^*28 (TA6/7 and TA7/7) was higher than that in the WT genotype (TA6/6) (33.3% vs 2.1%, P<0.05). No significant difference of TTP and chemotherapeutic effect was observed between different genotypes. CONCLUSION: The UGT1A1 ^*6 (G/A and A/A) genotypes increase the risk of grade 3 and 4 delayed diarrhea and neutropenia, and the UGT1A1 ^*28 (TA6/7 and TA7/7) genotypes increase the risk of grade 3 and 4 thrombocytopenia in mCRC patients treated with irinotecan-based chemotherapy.     

Curcumin ameliorates high glucose-induced dysfunction of vasoconstriction via heme oxygenase-1 and GC pathway

AIM: To explore the protective effect of curcumin on high glucose-induced decrease in contraction of isolated rat aortic rings, and to elucidate its underlying mechanism. METHODS: The thoracic aortic rings with endothelium of male Sprague-Dawley rats were mounted on a bath system. Isometric contractions of aortic rings were measured. HO activity was also evaluated. RESULTS: (1)Four hours after incubated with 44 mmol/L of glucose (high glucose), the vascular contraction responses to phenylephrine (PE) decreased compared to control group (containing 11 mmol/L of glucose). (2)Coincubation with curcumin (3×10-11-3×10-10 mol/L) and high glucose, the high glucose-induced decrease in contraction responses to PE of arteries was partly inhibited. (3)Four hours after incubation with curcumin, the HO activity in thoracic aorta increased. ZnPP, an inhibitor of HO-1, completely abrogated the protection effect of curcumin. (4)Methylene blue, an inhibitor of guanylate cyclase (GC), partly abolished the protective effect of curcumin. CONCLUSION: Curcumin prevents the high glucose-induced decrease in contraction responses to PE in intact aortic rings. The mechanism might be mainly involved in the activation of HO-1 and GC.     

Effect of peroxisome proliferator-activated receptor γ agonist rosiglitazone on intestinal injury in rats undergoing orthotopic autologous liver transplantation by inhibiting inflammatory response

AIM: To investigate the effect of rosiglitazone, a peroxisome proliferators-activated receptor γ(PPARγ) agonist, on the expression of PPARγ, the activation of NF-κB and intestine injury in the rats undergoing orthotopic autologous liver transplantation(OALT).METHODS: Sprague-Dawley male rats were randomly divided into 4 groups:control group, sham group, OALT group and rosiglitazone(0.3 mg/kg, iv) pretreatment(ROS+OALT) group. The OALT model was established, and the intestinal tissues were collected 8 h after the liver reperfusion. The intestinal tissue sections were stained to visualize the damage. The expression of PPARγ and NF-κB in the tissues, the concentrations of diamine oxidase(DAO) and fatty acid-binding protein 2(FABP2) in the serum and the concentration of TNF-α and IL-6 in the tissues were measured.RESULTS: Compared with sham group, the intestinal mucosa of the rats showed obvious pathological injury after liver reperfusion in OALT group and ROS group, the Chiu,s scores of intestinal mucosa was significantly higher, and the serum concentrations of DAO and FABP2 increased(P<0.05). After rosiglitazone pretreatment, the injury of intestinal mucosa of the rats was alleviated, the Chiu,s scores was lower and the serum concentrations of DAO and FABP2 decreased(P<0.05), the PPARγ expression was obviously up-regulated in the intestinal tissues, the nuclear translocation of NF-κB was reduced and the concentrations of IL-6 and TNF-α were decreased.CONCLUSION: During perioperative period of OALT in rats, the inflammatory responses are obvious. Furthermore, obvious intestinal injury occurs. PPARγ agonist rosiglitazone obviously up-regulates PPARγ expression and inhibits the inflammation in the intestines, thus protecting against intestinal injury in rats undergoing OALT.     

鱼腥草糖基转移酶基因UGT75C1 的克隆及原核#br# 表达

 根据已经获得的鱼腥草UGT75C1 转录本序列设计1 对引物,采用RT-PCR 方法获得UGT75C1 基因cDNA 序列,并对UGT75C1 蛋白进行理化性质分析,并预测该蛋白功能;利用实时荧光定量PCR 方法检测了UGT75C1 基因在鱼腥草的地下茎、地上茎、叶、花中的表达情况,将克隆得到的UGT75C1 基因完整开放阅读框连接到原核表达载体pGEX4T-1 上,转化大肠杆菌E. coli BL21（DE3）,通过IPTG 诱导表达,SDS-PAGE 检测表达产物。克隆获得的UGT75C1 基因长为1 787 bp,开放阅读框1 461 bp, 编码486 个氨基酸。生物信息学预测UGT75C1 蛋白含跨膜区,不含信号肽,具有糖基转移酶的PSPG motif。 UGT75C1 在鱼腥草的叶片中表达丰度最高,其他器官中表达量相对较低,花中表达量最低;该基因原核 表达产物与预期大小一致,显示原核表达成功,为下一步研究其功能奠定了基础。     

The relations among PAMP,ADM and AngⅡ on vascular tissue

AIM: To elucidate the relations among proadrenomedullin N terminal 20 peptide(PAMP), adrenomedullin(ADM)and angiotensin(Ang).METHODS: Tissue slices of rat aorta were incubated as follows:(I) increasing concentrations of AngⅡ(10^-9, 10^-8, 10^-7 mol/L); increasing concentrations of PAMP(10^-9,10^-8,10^-7mol/L).The tissue and incubation concentrations of PAMP,ADM and AngⅡ were measured by the radioimmunoassay (RIA).RESULTS: The tissue and incubation concentrations of PAMP and ADM were concentration-dependently increased by AngⅡ,but the tissue and incubation concentrations of AngⅡ can not effected by PAMP.CONCLUSION: AngⅡ markedly stimulate the release of ADM and PAMP.It may be one of the factors which regulate the synthesis and release of ADM and PAMP.The regulation may play an important role in homeostasis regulation of cardiovascular system.     

Effects of emodin on activity of sphingomylinase and content of ceramide in rabbit aorta of experimental atherosclerosis

AIM: To study the changes of the sphingomylinase activity and ceramide content in rabbit aorta of experimental atherosclerosis and investigate the effects of emodin on them. METHODS: The qualified rabbits were fed with food containing 1% cholesterol and 5% lard for 10 weeks to establish the animal models. The concentration of cholesterol (TC) was assayed by a enzyme method. Trace-fast-test method was used to test the activity of superoxide dismutase (SOD) and motified- BAMuGuoFu methods was employed to assay the content of myocardial malondialdehyde (MDA). Radiolabeled -enzyme-tracing was used to detect the activity of the sphingomyelinase,and thin-layered scanning was conducted to analyze the content of the ceramide in aorta. RESULTS: The ceramide content in aorta and the sphingomyelinase activity were markedly increased in the rabbits with experimental atherosclerosis. The increase was positively correlated with the content of TC and MDA and negatively correlated with the activity of SOD in blood. Compared to the model animals, emodin at concentration of 5 mg·kg^-1, 10 mg·kg^-1 and 20 mg·kg^-1 respectively reduced the area of plague on endothelium in rabbit's aortic artery and elevated the activity of SOD (P<0.05). The activity of sphingomylinase and the content of ceramide were decreased at the same time (P<0.05). 10 mg·kg^-1 emodin proved to be more effective than 5 mg·kg^-1 and 20 mg·kg^-1 emodin (P<0.01). CONCLUSIONS: Our data suggest that atherosclerosis is related to ceramide signal transduction initiated by factors such as oxidative stress in hypercholesterolemia. The emodin prevents the development of atherosclerosis probably by interfering with the above pathway.     

Effects of cholecystokinin octapeptide on changes in rabbit thoracic aortic reactivities induced by lipopolysaccharides in vitro

AIM and METHODS: To elucidate the mechanism of anti-endotoxic shock of cholecystokinin octapeptide(CCK-8), the effects of CCK-8 on changes in rabbit thoracic aortic reactivities induced by lipopolysaccharides(LPS) in vitro were studied, and the ultrastructure of the endothelial cells was observed under scanning electron microscope. RESULTS: Incubation of thoracic aortic rings(TARs) with LPS(100 mg/L) resulted in an time-dependent impairment of the endothelium-dependent relaxations to acetylcholine(incubation for 3, 7, 14 h), a reduction of contractive response to phenylphrine(incubation for 14 h) and ultrastructural injury in endothelial cells(incubation for 7 h), all of which were alleviated by concomitant incubation with CCK-8(1 mg/L). In contrast, neither the vascular contractions nor the relaxations were affected by CCK-8 (1 mg/L) alone. CONCLUSION: CCK-8 improved the vascular reactivities in the presence of LPS, which may be one of the anti-endotoxic shock mechanisms of CCK.     

Role of cytokeratin 8 on changes of intestinal epithelial permeability induced by corticotropin-releasing factor

AIM: To investigate the role of cytokeratin 8 (CK8) on the change of intercellular permeability of intestinal epithelial cells induced by corticotropin-releasing factor (CRF). METHODS: The expression levels of CRF receptor 1 (CRFR1) and CRFR2 on human colon adenocarcinoma HT29 cell surface were determined by immunofluorescence staining. After treatment with 100 nmol/L CRF for 72 h, the translocation of FITC-labelled dextran was measured in a Transwell chamber. The structural changes of tight junctions were observed under transmission electron microscope. The expression levels of CK8, and tight junction proteins ZO-1 and occludin were determined by Western blot. The activity of protein kinase C (PKC) was detected by ELISA. Furthermore, the effects of CRF on intestinal epithelial permeability were examined in CK8-silencing HT29 cells, which were constructed by infection with sh-CK8 lentivirus. RESULTS: CRF treatment increased the permeability of FITC-labelled dextran (P<0.05), caused the opening of tight junctions, and induced increased fluorescence intensity of CK8. The expression levels of occludin and ZO-1 were down-regulated (P<0.05). PKC activity was decreased at 1 h after CRF treatment (P<0.05). CRF-induced increase in the permeability and down-regulation of occludin were not blocked by CK8 silencing. Nevertheless,CK8 silencing blocked the effects of CRF regarding the decrease in the expression levels of ZO-1 and the increase in PKC activity (P<0.05). CONCLUSION: CK8 may be involved in CRF-induced increase in intestinal epithelial permeability by inhibiting the activity of PKC, and there may be other signaling pathways involved.     

HCMV-infected human THP-1 cells induce expression of HLA-G and its receptors

AIM: To investigate the differential expression of human leukocyte antigen-G (HLA-G) isoforms and its receptors in human monocyte line THP-1 after human cytomegalovirus (HCMV) infection for exploring the role of HLA-G in HCMV escaping the immune response of the organism.METHODS: THP-1 cells were infected with HCMV Towne strain. The expression of HLA-G isoforms at mRNA and protein levels was determined by RT-PCR and Western blot, respectively. The surface expression of HLA-G and its receptors ILT2/ILT4 and the cell viability were analyzed by flow cytometry. The levels of soluble HLA-G (sHLA-G) and IL-10 were measured by ELISA.RESULTS: After infection of the THP-1 cells with HCMV, no obvious apoptosis in the cells was observed, and the viability of the cells was high. A significant up-regulation of HLA-G1, -G3, -G4 and -G5 at mRNA expression level 1 d after infection was found, while the protein expression of HLA-G1 and HLA-G5 isoforms was mainly detected. The expression of HLA-G/ILT2/ILT4 was evidently up-regulated 1 d after infection. The level of sHLA-G was significantly increased 1 d after infection as compared with control group (P<0.01). The expression of IL-10 was obviously up-regulated 1 d post-infection as compared with control group.CONCLUSION: The differential expression of HLA-G isoforms and secretion of the receptors ILT2/ILT4 and IL-10 in the THP-1 cells are induced after HCMV infection. This study provides experimental evidence for evaluating the immune mechanism of HCMV infection.     

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《园艺学报》是中国园艺学会和中国农业科学院蔬菜花卉研究所主办的学术期刊,创刊于1962年,刊载有关果树、蔬菜、观赏植物、茶及药用植物等方面的学术论文、研究报告、专题文献综述、问题与讨论、新技术新品种以