草莓白化相关病毒中国分离物全基因组分析

草莓白化相关病毒(strawberrypallidosis-associatedvirus,SPa V)属于长线形病毒科(Closteroviridae)毛形病毒属(Crinivirus),可引起草莓病害,2017年在中国首次报道。采用高通量测序、RACE和RT-PCR技术获得了SPa V中国分离物(FJ)的基因组全长。该病毒含有两条正单链基因组RNA1和RNA2。RNA1全长8 048 nt,5′和3′非编码区序列分别为264和197 nt,含有3个开放阅读框(ORF),分别编码ORF 1a/1b融合蛋白和p9蛋白。RNA2全长7 977 nt,5′和3′非编码区序列分别为248和186 nt,含有8个开放阅读框(ORF),分别编码HSP70h、CPh、CP、CPm、p7、p6、p9和p28等8个蛋白。RNA1和RNA2与美国M1分离物分别具有98.5%和99.0%的核苷酸一致性;系统发育分析结果表明,SPa V中国分离物(FJ)单独处在一个分支。对SPa V来源的小RNA的分析表明,来源于SPa V的小RAN长度以21和22 nt为主。     

草莓病毒1山东分离物全基因组分析

草莓病毒1(strawberry virus 1,StrV-1)属于弹状病毒科(Rhabdoviridae)细胞质弹状病毒属(Cytorhabdovirus),可侵染草莓。利用高通量测序(high-throughput sequencing,HTS)结合RT-PCR和RACE技术从采自山东省烟台市乳山市草莓种植基地的草莓品种‘全明星’上获得了StrV-1山东分离物(StrV-1-CN)的基因组全长(GenBank登录号:MW795715)。该分离物基因组全长为14 051 nt,3’和5’非编码区序列分别为181和767nt,其负义链含有9个开放阅读框(open reading frame,ORF),从3’到5’端分别编码N、P’、P、P3、M、G、P6、P7和L等9个蛋白。序列分析表明,StrV-1-CN与已报道的StrV-1分离物基因组全长核苷酸序列一致性为77%～88%。系统发育分析结果表明,StrV-1-CN与捷克分离物B和1/2017聚在一个分支,亲缘性最近。     

苹果茎沟病毒吉林沙果分离物全基因组序列分析

通过RT-PCR结合RACE技术扩增到苹果茎沟病毒（Apple stem grooving virus,ASGV）吉林沙果分离物（ASGV-JLSG）全长基因组（登录号：FM616381）,共含有6 496个核苷酸（nt）,编码两个彼此重叠的开放阅读框（Open reading frame,ORF）。ORF1（37 ~ 6 354 nt）编码1个241 kD的多聚蛋白,含有甲基转移酶（Methyltransferase）、木瓜蛋白酶（Papain-like-protease）、解旋酶（Nucleotide triphosphate-binding helicase）、RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase）和C端的外壳蛋白（Coat protein,CP）等结构域。ORF2（4 788 ~ 5 750 nt）编码1个36 kD的运动蛋白（Movement protein,MP）。ASGV-JLSG分离物与GenBank公布的29个ASGV分离物全基因核苷酸序列一致性为79.20% ~ 86.60%。系统发育分析结果表明,30个ASGV分离物分成2个组,分离物间没有表现出寄主专一性和地理分布规律。重组分析发现,ASGV-JLSG分离物是苹果茎沟病毒黄花梨分离物（ASGV-HH,JN701424）和柑橘碎叶病毒满头红分离物（CTLV-MTHK,C588948）的重组体。本研究中获得的ASGV-JLSG分离物基因组是来源于中国东北地区的首个ASGV基因组序列。     

李属坏死环斑病毒辽宁桃树分离物基因组分析

采用RT-PCR和RACE技术,克隆了李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)辽宁桃树分离物全长基因组。用Vector NTI Advance 11软件进行基因组结构注释。用SDTv.1.0软件分析该分离物与Gen Bank中公布的其他PNRSV分离物间的核酸一致性,并用MEGA5.0软件和RDP3软件对各PNRSV分离物进行系统发育分析和重组分析。结果显示:PNRSV辽宁桃树分离物基因组由3条正义单链RNA组成。RNA1由3 332个核苷酸构成,具有一个开放阅读框(nt 30~3 167),编码一个约117.0k D的复制酶相关蛋白P1。RNA2由2 591个核苷酸构成,具有一个开放阅读框(nt 27~2 426),编码一个约99.0 k D的复制酶相关蛋白P2。RNA3由1 943个核苷酸构成,具有两个互不重叠的开放阅读框分别编码一个运动蛋白(nt 175~1 026)和一个外壳蛋白(nt 1 100~1 774),这两个开放阅读框的间隔区为73个核苷酸。PNRSV辽宁桃树分离物的RNA1、RNA2和RNA3与其他已经公布的PNRSV分离物间,核酸序列一致性分别为91.8%~98.8%,93.3%~99.2%和87.7%~99.0%。基于RNA1、RNA2和RNA3全长序列构建的系统发育进化树,分别将已报道的PNRSV分离物划分为3个、2个和3个组,PNRSV辽宁桃树分离物分别属于RNA1Ⅰ组、RNA2Ⅰ组和PV96组。基于RNA1、RNA2和RNA3全长序列进行的重组分析表明各PNRSV分离物基因组间不存在重组。PNRSV辽宁桃树分离物基因组序列是世界首个来源于桃树的PV96组基因组序列。     

侵染广东辣椒的辣椒脉斑驳病毒的分子特征

辣椒脉斑驳病毒（Chilli veinal mottle virus,ChiVMV）是引起广东辣椒病毒病的主要病原之一,为了明确侵染为害广东辣椒的ChiVMV分子特征,采用分段RT-PCR和RACE扩增方法克隆了ChiVMV广东分离物（ChiVMV-GD）的基因组全序列,结果表明,除poly（A）尾外,ChiVMV-GD基因组大小为9 721 nt,编码一个大小350.44 kD多聚蛋白（位于167 ~ 9 436 nt）,5′–非编码区（5′-UTR）和3′–非编码区（3′-UTR）分别含有166和285 nt,5′–末端结合病毒基因组连接蛋白（VPg）,3′–末端含有poly（A）尾。序列同源性分析结果表明：ChiVMV-GD与ChiVMV其他分离物的基因组序列同源率为79.1% ~ 96.9%,其中与来自中国海南分离物（登录号：GQ981316.1）的同源率最高（96.9%）。系统进化分析结果显示,ChiVMV-GD与海南分离物聚集在一个小分支,说明与海南分离物亲缘关系最近。     

百合斑驳病毒靖宇分离物全基因组序列测定与进化分析

以采自吉林省白山市靖宇县的野生百合感病叶片为试材，采用小RNA深度测序法检测到1株百合斑驳病毒(Lily mottle virus,LMoV),采用分段克隆方法，对LMoV靖宇分离物(LMoV-JY)的全基因组进行测定并分析，以期对吉林省百合病毒病的检测和防控提供参考依据。结果表明：LMoV-JY基因组大小为9 648个核苷酸(nt)序列(GenBank登录号MT795719),该核苷酸序列在第154～9 444位存在1个大的开放阅读框(ORF),编码1个多聚蛋白(分子量351.46 kD)。LMoV-JY与GenBank中登录的其他LMoV分离物的全基因组核苷酸序列比较，其一致性为82.82%～97.85%,在氨基酸水平上的一致性为92.96%～98.64%,其中与百合斑驳病毒大连分离物LMoV-DL(HM222521)的一致性在该2种水平上均为最高。对比LMoV-JY外壳蛋白与其他54个分离物的氨基酸序列，发现可将LMoV分离物分为2个类群。     

侵染白菜的黄瓜花叶病毒分离物基因组的全序列分析

对浙江省杭州地区白菜（Brassica campestris L. ssp. chinensis var. commuis Tsen et Lee.）上获得的CMV分离物（CMV-CTL）进行了全长克隆和基因组序列分析。结果显示：CMV-CTL的RNA1（GenBank序列号：EF213023）全长为3357个核苷酸（nt）,编码993个氨基酸（aa）的1a蛋白;RNA2 （GenBank序列号：EF213024）全长为3047 nt ,编码858 aa的2a蛋白和111 aa的2b蛋白;RNA3 （GenBank序列号：EF213025）全长为2217 nt,编码278 aa的MP蛋白和218 aa的CP蛋白。序列相似性分析表明,CMV-CTL与CMV亚组IB中株系IA相似性最高,RNA 1、RNA 2和RNA3与该株系的相似性分别为91.3%、91.3%和93.6%。CP基因和RNA 3 的5' NTR核酸序列系统发生树分析表明,CMV-CTL与中国大多数CMV分离物一样,属于CMV亚组IB。     

羽衣甘蓝Exo70A1基因的分离及表达分析

 以羽衣甘蓝（Brassica oleracea var. acephala）自交不亲和系（S13-b S13-b）为试材,从柱头中分离Exo70A1基因,命名为BoExo70A1,GenBank登录号为JF919716。BoExo70A1全长cDNA序列为2 184 bp,包含56 bp的5′非编码区,211 bp的3′非编码区和一个长度为1 917 bp的开放读码框（ORF）,对应编码一个含有638个氨基酸残基的蛋白质。氨基酸序列比对分析表明,羽衣甘蓝BoExo70A1与油菜BnExo70A1、拟南芥AtExo70A1的一致性分别为99%和94%;BoExo70A1基因结构含12个外显子和11个内含子,内含子5′供体位点和3′受体位点边界序列符合GU-AG规则;Southern杂交结果显示,BoExo70A1在羽衣甘蓝基因组中存在多拷贝;Northern杂交结果显示BoExo70A1在茎、叶、花瓣、花药、柱头、花柱和子房中表达,而且在叶中的表达量最低。     

萝卜中一未知dsRNA全长cDNA克隆及其序列

 采用双链RNA ( double-stranded RNA, dsRNA) 分析法, 发现杭州市近郊的部分萝卜植株及其子代含有约118 kbp的dsRNA; 在聚丙烯酰胺凝胶上可分离为大小接近的dsRNA1和dsRNA2。以dsRNA1为模板, 采用单引物扩增法获得其全长cDNA; 克隆测序确认为1 866 nt; 预测其最大开放阅读框ORF(Open Reading Frame) 为574个氨基酸, 与双分病毒属( Partitivirus) 中部分病毒的RdRp (RNA dependent RNA polymerase) 氨基酸序列具有一定的同源性。推测此dsRNA可能为萝卜黄边病毒( radish yellow edge virus, RYEV) 感染所致。     

陕西杨凌地区TYLCV病毒生物信息学分析研究

采集杨凌五泉、揉谷和李台3个番茄主产区表现矮化、黄化及曲叶症状的植株嫩叶,克隆番茄黄化曲叶病毒(TYLCV)基因全长并测序,依次得到病毒分离物TYLCV-SXYL2、TYLCV-SXYL3和TYLCV-SXYL4。通过多序列比对、系统发育树构建及蛋白质结构和理化性质预测等生物信息学方法进行基因组和蛋白质的特征分析,结果表明,杨凌区3个TYLCV分离物之间全长核苷酸相似度为99.3%~99.4%,是不伴随卫星分子的单组分病毒,属TYLCV-IS株系的不同分离物,全长为2 781 nt;与山东寿光病毒分离物TYLCV-SDSG亲缘关系最近,相似度为99.6%,与陕西泾阳的分离物TYLCV-SX8相似度为99.1%,与以色列株系TYLCV-IS相似度达97.7%~97.8%;编码6个蛋白质,其中CP、Rep、REn为跨膜蛋白,V2、TrAP、C4为胞内蛋白,Rep和REn为稳定蛋白,CP、V2、TrAP、C4为不稳定蛋白。     

Effect of wogonoside on inflammatory response in mice with viral myocarditis induced by Coxsackie virus B3

AIM: To investigate the effect of wogonoside on the inflammatory response of mice with Coxsackie virus B3 (CVB3)-induced myocarditis and its possible regulatory mechanism. METHODS: A mouse model of viral myocarditis was constructed by infecting BALB/c mice with CVB3. BALB/c mice (n=40) were randomized into 4 groups: normal group, CVB3-induced viral myocarditis group, CVB3-induced viral myocarditis combined with wogonoside treatment group and CVB3-induced viral myocarditis combined with wogonoside plus AKT agonist treatment group. All the mice were sacrificed 7 days after treatment. In the first 3 groups, HE staining was applied to detect the infiltration of inflammatory cells in the myocardium, ELISA was applied to detect the serum levels of interleukin-1β (IL-1β) and IL-6, while Western blot was applied to detect the protein expression of inflammatory factors and the activation of AKT/NF-κB pathway. Inaddition, the activation of AKT/NF-κB pathway in the 4 groups was detected by Western blot analysis. RESULTS: HE staining showed that there was a large amount of inflammatory cell infiltration in the myocardium of CVB3-induced viral myocarditis mice, as compared with the normal group, which was significantly reduced by wogonoside treatment (P<0.05). The serum levels of IL-1β and IL-6 in the mice after CVB3 infection were significantly higher than those in normal group (P<0.05), which was also significantly reduced by wogonoside treatment (P<0.05). Western blot analysis indicated that wogonoside treatment significantly reduced the expression of inflammatory factors IL-1β and IL-6, and the phosphorylation of AKT/NF-κB pathway-related proteins in the myocardial tissue (P<0.05). After administration of AKT agonist, the inhibitory effect of wogonoside on NF-κB phosphorylation and inflammatory factors expression was significantly eliminated (P<0.05). CONCLUSION: Wogonoside attenuates the inflammatory response of mice with viral myocarditis by inhibiting the AKT/NF-κB pathway.     

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

AIM To investigate the mechanism of long noncoding RNA （lncRNA） FEZF1-AS1 regulating microRNA-363-3p （miR-363-3p） on the viability and apoptosis of lipopolysaocharide （LPS）-induced vascular endothelial cells. METHODS Human umbilical vein endothelial cells （HUVECs） were cultured in vitro. pcDNA-NC, pcDNA-FEZF1-AS1, anti-miR-NC, anti-miR-363-3p, miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h. RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p. The cell viability was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p. Western blot was used to determined the expression of cyclin D1, Ki67 and cleaved caspase-3. RESULTS Compared with control group, the expression level of FEZF1-AS1 in LPS group was significantly reduced （P<0.05）, and the expression level of miR-363-3p was significantly increased （P<0.05）. Compared with pcDNA-NC+LPS group, the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Compared with anti-miR-NC+LPS group, the cell viability in anti-miR-363-3p+LPS group was significantly increased （P<0.05）, the apoptotic rate was significantly reduced （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly increased （P<0.05）, and the protein level of cleaved caspase-3 was significantly reduced （P<0.05）. Dual-luciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p. Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group, the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced （P<0.05）, the apoptotic rate was significantly increased （P<0.05）, the protein levels of cyclin D1 and Ki67 were significantly reduced （P<0.05）, and the protein level of cleaved caspase-3 was significantly increased （P<0.05）. CONCLUSION Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.     

Effects of naringenin on PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways in rats with myocardial ischemia/reperfusion injury

AIM To observe the effect of naringenin on cardiac injury in ischemia/reperfusion （I/R） rats, and to explore whether the role of naringenin is involved in PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways. METHODS SD rats （n=48） were randomly divided into sham operation （sham） group, model （I/R） group, naringenin treatment （NAR） group and naringenin+LY294002 （NL） group. Myocardial I/R injury model was prepared by ligation of left anterior descending coronary artery of rats for 30 min followed by reperfusion for 120 min. After reperfusion, the serum levels of cardiac troponin I （cTnI） was measured by ELISA. HE staining, TTC staining and TUNEL staining were used to detect the myocardial histopathological changes, myocardial infarction area and myocardial cell apoptotic rate. The mRNA levels of endoplasmic reticulum stress-related indicators glucose-regulated protein 78 （GRP78）, C/EBP homologous protein （CHOP） and caspase-12 were detected by RT-qPCR. The protein levels of cleaved caspase-3, GRP78, CHOP, caspase-12, p-PI3K and p-AKT were determined by Western blot. RESULTS Compared with I/R group, the serum content of cTnI, myocardial pathological damage, myocardial infarction area and myocardial cell apoptotic rate were significantly reduced （P<0.05）, the protein levels of cleaved caspase-3, GRP78, CHOP and caspase-12 were decreased （P<0.05）, and the protein levels of p-PI3K and p-AKT were increased in NAR group （P<0.05）. LY294002 attenuated the protective effect of naringenin to some extent. CONCLUSION Naringenin reduces myocardial I/R injury in rats possibly by activating PI3K/AKT signaling pathway and subsequently regulating endoplasmic reticulum stress and its related apoptotic pathways.     

Cyanidin attenuates pressure overload-induced cardiac remodeling in mice via mitochondrial fusion enhancement

AIM To investigate the effect of cyanidin （Cyn） on pressure overload-induced cardiac remodeling and the underlying mechanism. METHODS Six-week-old male C57BL/6 mice （n=120） were divided into 4 groups： sham group （n=20）, sham+Cyn group （n=20）, transverse aortic constriction （TAC） group （n=40） and TAC+Cyn group （n=40）. The model of cardiac chronic pressure overload was induced by TAC, and the first day of TAC was defined as day 0. The animals in sham+Cyn group and TAC+Cyn group were treated with Cyn dissolved in DMSO and normal saline （5 mg·kg^-1·d^-1） for 5 d before TAC, while the animals in sham group and TAC group were treated with the same amount of DMSO and normal saline. Four weeks after TAC, the survival rate of the animals in each group was analyzed, the heart function of the mice was measured by ultrasound echocardiography, and the heart weight/body weight and lung weight/body weight were calculated. The cross-sectional area of the cardiomyocytes was measured by wheat germ agglutinin staining and hematoxylin-eosin staining. The degree of cardiac oxidative stress was evaluated by dihydroethidium staining and measurement of superoxide dismutase （SOD） and malondialdehyde （MDA） levels. The cardiomyocyte apoptosis was detected by TUNEL method. The mRNA expression levels of atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP） and β-myosin heavy chain （β-MHC） were detected by RT-qPCR, and the protein expression levels of Bax, Bcl-2, optic atrophy protein 1 （OPA1） and dynamin-related protein 1 （Drp1） were determined by Western blot. The mitochondrial morphological changes were observed by transmission electron microscopy. RESULTS Compared with TAC group, the survival rate of the mice in TAC+Cyn group was significantly increased （P<0.05）, the myocardial apoptosis, the cross-sectional area of myocardial cells, the heart weight/body weight, the lung weight/body weight, the level of reactive oxygen species and the MDA content were decreased （P<0.05）, and the SOD was activated （P<0.05）. M-mode ultrasound tests showed that Cyn treatment significantly increased left ventricular ejection fraction and left ventricular fractional shortening in the mice after TAC （P<0.05）, while left ventricular end-diastolic diameter and left ventricular posterior wall thickness in diastole were reduced （P<0.05）. Transmission electron microscopic observation showed that the number of myocardial mitochondria was increased and the mitochondrial area was decreased after TAC （P<0.05）, while treatment with Cyn increased the area of myocardial mitochondria and decreased the mitochondrial number （P<0.05）. Compared with sham group, the protein level of OPA1 in TAC group was significantly reduced （P<0.05）, while treatment with Cyn significantly increased the protein level of OPA1. CONCLUSION Cyanidin significantly increases the survival rate, improves the cardiac function and attenuates the cardiac remodeling of the mice after TAC. The mechanism may be related to the inhibition of myocardial mitochondrial OPA1 cleavage and the promotion of mitochondrial fusion.     

Effect of intermittent hypoxia on bladder detrusor cell apoptosis and regulatory mechanism of Alpiniae oxyphyllae Fructus

AIM To investigate the effect of intermittent hypoxia （IH） on bladder detrusor cells apoptosis and calcium channel, and to discuss the regulatory mechanism of Alpiniae oxyphyllae Fructus （AOF）. METHODS IH model of bladder detrusor cells was established by treating the cells with 6 cycles of 5% O₂ for 60 min and 20% O₂ for 30 min. Human bladder detrusor cells were cultured in vitro, randomly divided into 6 groups, each group had 8 holes. P₂X₃ receptor antagonist + IH （A） group, M₃ receptor antagonist + IH （B） group, β₃ receptor antagonist + IH （C） group, AOF + IH （D） group, saline + IH control （NC） group and air simulation control （AC） group were set up. The cells density and morphology were identified by the methods of counting chamber and immunofluorescence light microscopy （LM） after interventions. The apoptosis was analyzed by flow cytometry. Calcium channel expression was detected by patch clamp. RESULTS （1） Compared with the cells in AC group, the cells density and activity were significantly increased in NC group （P<0.05）; some cells appeared protrusions, turned round and blur in cell borders. （2） The results of immunofluorescence for detecting α-SMA protein expression showed that, compared with the cells in group AC, the mean absorbance （MA） in group NC was significantly increased （F=3.25, P<0.05）; compared with the cells in group NC, that in group A and group D was both decreased significantly （P<0.05）. （3） Compared with the cells in group AC, the apoptotic rate was significantly decreased in group NC （P<0.05）; Compared with the cells in group NC, the apoptotic rates in group A and group D were both significantly increased （P>0.05）. （4） Compared with the cells in group AC, calcium ion channel expression was significantly decreased （P<0.05）. Compared with the cells in group NC, calcium ion channel expression in AOF （100 mg/L） and AOF （50 mg/L） group was significantly increased （P<0.05）. CONCLUSION IH regulates bladder detrusor cells proliferation and apoptosis through P₂X₃ bladder nerve receptors, high or moderate dose of AOF may change calcium channel and play a protective role in IH induced cell damage.     

Geniposide attenuates cognitive dysfunction in sleep-deprived rats by inhibiting TLR4/NF-κB signaling pathway

AIM To investigate the effects of geniposide （Gen） on Toll like receptor 4/nuclear factor-κB （TLR4/NF-κB） signaling pathway and cognitive dysfunction in sleep deprived rats. METHODS Wistar rats （n=120） were randomly divided into normal control （NC） group, model （M） group, low-dose （5 g·kg^-1·d^-1） Gen （Gen-L） group, medium-dose （10 g·kg^-1·d^-1） Gen （Gen-M） group, high-dose （20 g·kg^-1·d^-1） Gen （Gen-H） group and Gen-H+LPS （0.4 mg·kg^-1·d^-1, tail vein injection） group. After 7 days of intervention, the sleep deprivation model of rats in M group, Gen-L, Gen-M, Gen-H and Gen-H+LPS group was established by improved small platform water environment. The escape latency of Morris water maze experiment and the behavior correct rate of Y maze experiment were measured. The serum levels of S100B and neuron-specific enolase （NSE）, and the levels of interleukin-1β （IL-1β）, IL-6 and tumor necrosis factor-α （TNF-α） in hippocampus were detected by ELISA. The mRNA levels of TLR4 and NF-κB p65 were detected by RT-qPCR, and the protein levels of TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with NC group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the mRNA and protein expression of TLR4 and NF-κB p65 were increased significantly in M group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. Compared with M group, the escape latency, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were decreased significantly in Gen-L, Gen-M and Gen-H groups （P<0.01）, and the behavior correct rate was increased in turn （P<0.01）. Compared with Gen-H group, the escape latency, the serum levels of S100B and NSE, the hippocampal levels of IL-1β, IL-6 and TNF-α, and the expression of TLR4 and NF-κB p65 at mRNA and protein levels were increased significantly in Gen-H+LPS group （P<0.01）, and the behavior correct rate was decreased significantly （P<0.01）. CONCLUSION Geniposide may inhibit the TLR4/NF-κB p65 signaling pathway to effectively improve cognitive function in sleep-deprived rats and reduce hippocampus inflammation.     

Role and potential mechanism of fecal microbiota transplantation in treatment of chronic hepatitis B

AIM To investigate the effect of fecal microbiota transplantation （FMT） on the treatment of chronic hepatitis B （CHB） and the potential mechanism. METHODS Fifty C57BL/6J mice （6~8 weeks old） were divided into 5 groups： control group, CHB group, entecavir （ETV） group, comprehensive treatment （ETV+FMT, EFMT） group, and blocker （TAK-242+ETV+FMT, EFMT-TAK） group. The mice in each group were given corresponding treatment. The general condition of the mice was observed daily, and fecal specimens were kept every 10 d. The mice were sacrificed after 12 weeks, and the liver tissues and blood samples were collected. HE staining was used for histological scoring. Serum hepatitis B surface antigen （HBsAg） and interleukin-18 （IL-18） levels were measured by ELISA. Toll-like receptor 4 （TLR4） expression was detected by flow cytometry. Intestinal flora diversity was analyzed by high-throughput sequencing. RESULTS （1） Compared with control group, the body weight of the mice in CHB group was significantly reduced （P<0.05）. The body weight loss of the mice in ETV group, EFMT group and EFMT-TAK group was reversed to some extent as compared with CHB group （P<0.05）. （2） The histological score of the mice in CHB group was significantly higher than that in control group （P<0.05）. The score in ETV group was lower than that in CHB group （P<0.05）. The scores in EFMT group and EFMT-TAK group were lower than that in ETV group （P<0.05）, and that in EFMT-TAK group had a further downward trend compared with EFMT group （P<0.05）. （3） Compared with control group, the serum level of HBsAg in the CHB mice was significantly increased （P<0.05） and decreased after ETV treatment （P<0.05）. The HBsAg level in both EFMT group and EFMT-TAK group was significantly lower than that in ETV group （P<0.05）. （4） The IL-18 level in CHB group was significantly higher than that in control group （P<0.05）. After ETV treatment, the IL-18 level was decreased （P<0.05）, and that in both EFMT group and EFMT-TAK group was decreased more than that in ETV group （P<0.05）. （5） TLR4 expression in CHB group was higher than that in control group （P<0.05）, that in ETV group was lower than CHB group （P<0.05）, and that in EFMT group was further decreased （P<0.05）. （6） The heat map analysis at the class level showed that the abundances of Gammaproteobacteria, Deltaproteobacteria and Negativicutes in CHB group were significantly higher than those in control group, and those of Deltaproteobacteria and Negativicutes in EFMT group were close to those in control group. The heat map analysis at the family level indicated that the abundances of Burkholderiaceae, Desulfovibrionaceae and Veillonellaceae in CHB group were significantly higher than those in control group, while those in ETV group and EFMT group gradually approached normal levels. The α diversity index in CHB group was significantly decreased, while the diversity in ETV group was increased, that in EFMT group was further increased, and that in EFMT-TAK group was the highest. CONCLUSION FMT plays an active role in the treatment of CHB. The mechanism may be related to reducing the level of IL-18 and improving the structure and diversity of intestinal flora. The TLR4 signaling pathway is involved.     

Effect of compound of Epimedium, Astragalus and Radix Puerariae on expression of ADAM10 in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin expression knock-down

AIM To explore the effect of compound of Epimedium, Astragalus and Radix Puerariae on the expression of a disintegrin and metalloproteinase 10 （ADAM10） in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin （HAMP） expression knock-down for analyzing the pathogenesis of Alzheimer disease （AD） at cell level. METHODS Hippocampal neuron HT22 cells were cultured in vitro and randomly divided into 7 groups： control group, Aβ group （Aβ_25-35-induced HT22 cells）, RNAi group （HAMP gene was silenced in HT22 cells）, Aβ+RNAi group （HAMP gene expression in Aβ_25-35-induced HT22 cells was silenced）, Aβ+TCM group （Aβ_25-35-induced HT22 cells were treated with Epimedium, Astragalus root and Radix Puerariae effective components）, RNAi+TCM group （HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components） and Aβ+RNAi+TCM group （Aβ_25-35-induced HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components）. The silence efficiency of HAMP siRNA was detected by qPCR and Western blot. The ADAM10 expression in each group was determined by immunofluorescence, qPCR and Western blot. RESULTS The HAMP siRNA-3 sequence had the highest interference efficiency. Compared with control group, the expression levels of ADAM10 in Aβ group, RNAi group and Aβ+RNAi group were decreased （P<0.05）. Compared with Aβ group,the expression levels of ADAM10 in Aβ+RNAi group was also decreased （P<0.05）, and the expression levels of ADAM10 in Aβ+TCM group was increased （P<0.05）. Compared with RNAi group, the expression levels of ADAM10 in Aβ+RNAi group was decreased （P<0.05）, while the expression levels of ADAM10 in RNAi+TCM group was increased （P<0.05）. Compared with Aβ+RNAi group, the expression levels of ADAM10 in Aβ+RNAi+TCM group was increased （P<0.05）. CONCLUSION The effective components of Epimedium, Astragalus and Radix Puerariae compound promotes the expression of ADAM10 in Aβ_25-35-induced HT22 cells, which mechanism may be related to the expression of HAMP.