目的:通过检测毛蕊异黄酮(Calycosin)对流感病毒A/PR/8/34(H1N1)感染的脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells,HUVECs)通透性升高及其肌球蛋白轻链(MLC)磷酸化的影响来探讨其改善内皮细胞通透性的机制.方法:设置正常组、病毒组和毛蕊异黄酮组;将HUVEC接种于96孔板,MTT法检测不同浓度毛蕊异黄酮的细胞毒性;30 MOI PR8病毒感染病毒组与毛蕊异黄酮组HUVECs后,毛蕊异黄酮组加入20μg/mL毛蕊异黄酮溶液,采用内皮细胞跨膜电阻法检测各组在不同时间点的跨膜电阻值;激光共聚焦显微镜观察各组细胞内丝状肌动蛋白(Filamentous actin,F-actin)的表达和分布;Western Blot检测各组MLC、p-MLC蛋白表达水平;将MDCK接种于96孔板,用PR8病毒感染细胞,MTT法检测不同浓度毛蕊异黄酮的抗病毒活性.结果:毛蕊异黄酮对HUVEC的最大无毒浓度为40 μg/mL,细胞存活率约为91.69％;毛蕊异黄酮组细胞通透性与正常组细胞相比无显著差异,但通透性显著低于病毒组(P＜0.05);病毒组的F-actin发生变构、细胞中出现粗大的应力纤维,分布于胞浆中,而毛蕊异黄酮组的F-actin分布于细胞膜、应力纤维减少;与正常组相比,病毒组的p-MLC蛋白表达显著增加,差异有统计学意义(P＜0.05)、与病毒组相比,毛蕊异黄酮组的p-MLC蛋白表达明显下降,差异有统计学意义(P＜0.05);毛蕊异黄酮对流感病毒感染的MDCK细胞的抗病毒作用并不明显.结论:毛蕊异黄酮可通过抑制p-MLC蛋白的表达来抑制流感病毒引起的内皮细胞通透性升高及F-actin变构的作用,从而达到保护内皮细胞的功能.

Mentha haplocalyx has been widely used for its flavoring and medicinal properties and as a traditional Chinese medicine with its anti-inflammation properties. The present study was designed to investigate the anti-inflammatory effects and potential molecular mechanisms of the phenolic fraction of M. haplocalyx (MHP) and its constituent linarin in lipopolysaccharide (LPS)-induced RAW264.7 cells. The high-performance liquid chromatography coupled with linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap MS) was used to analyze the chemical composition of MHP. Using the enzyme-linked immunosorbent assay (ELISA) and quantitative realtime polymerase chain reaction (qRT-PCR), the expression of pro-inflammatory meditators and cytokines was measured at the transcriptional and translational levels. Western blot analysis was used to further investigate changes in the nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and Akt signaling pathways. Fourteen phenolic constituents were identified from MHP based on the data of the mass spectrometry (MS)/MS analysis. MHP and linarin decreased the production of NO, tumor necrosis factor-α (TNF-α), interlenkin-1β (IL-1β), and IL-6. The messenger ribonucleic acid (mRNA) expression levels of inducible NO synthase (iNOS), TNF-α, IL-1β, and IL-6 were also suppressed by MHP and linarin. Further investigation showed that MHP and linarin down-regulated LPS-induced phosphorylation content of NF-κB p65, inhibitor kappa B α (IκBα), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. However, MHP and linarin showed no inhibitory effect on the phosphorylated Akt. These results suggested that MHP and linarin exerted a potent inhibitory effect on pro-inflammatory meditator and cytokines production via the inactivation of NF-κB and MAPKs, and they may serve as potential modulatory agents for the prevention and treatment of inflammatory diseases.

Severe influenza infections are featured by acute lung injury, a syndrome of increased pulmonary microvascular permeability. A growing number of evidences have shown that influenza A virus induces cytoskeletal rearrangement and permeability increase in endothelial cells. Although miRNA's involvement in the regulation of influenza virus infection and endothelial cell (EC) function has been well documented, little is known about the miRNA profiles in influenza-infected endothelial cells. Using human umbilical vein endothelial cells (HUVECs) as cell models, the present study aims to explore the differential miRNAs in influenza virus-infected ECs and analyze their target genes involved in EC permeability regulation. As the results showed, permeability increased and F-actin cytoskeleton reorganized after HUVECs infected with influenza A virus (CA07 or PR8) at 30 MOI. MicroRNA microarray revealed a multitude of miRNAs differentially expressed in HUVECs after influenza virus infection. Through target gene prediction, we found that a series of miRNAs were involved in PKC, Rho/ROCK, HRas/Raf/MEK/ERK, and Ca-2+/CaM pathways associated with permeability regulation, and most of these miRNAs were down-regulated after flu infection. It has been reported that PKC, Rho/ROCK, HRas/Raf/MEK/ERK, and Ca-2+/CaM pathways are activated by flu infection and play important roles in permeability regulation. Therefore, the cumulative effects of these down-regulated miRNAs which synergistically enhanced activation of PKC, Rho/ROCK, Ras/Raf/MEK/ERK, and Ca-2+/CaM pathways, can eventually lead to actin rearrangement and hyperpermeability in flu-infected HUVECs.

Objective:To investigate the effect of berberine on the cytoskeleton and permeability of human umbilical vein endothelial cells (HUVECs) infected with influenza virus A/PR/8/34 (H 1N 1).Methods:Influenza virus A/PR/8/34 were passaged in embryonated chicken eggs,and samples of allantoic fluid were stored at-80 ℃.Virus titer was measured by plaque assay.HUVEC were seeded in 96-well plates.MTT assay was used to detect the toxicity of berberine to HUVEC.The HUVECs in the virus and berberine group of HUVEC were stimulated with 30 MOI A/PR/8/34,and the cells in the berberine group were then added with 5 μ g/mL berberine solution.The resistance values were measured at 0,2,6,12,24 and 36 h,respectively.The cytoskeleton morphology was observed by laser confocal microscopy.Results:The virus titer of influenza virus A/PR/8/34 is (2-3) × 108 PFU/mL.The maximum non-toxic concentration of berberine to HUVEC was 10 μ g/mL,and the cell survival rate was about 94.05％.The permeability of the berberine group was significantly lower than that of the virus group (P＜0.05),and the difference was not significant with the non-infected group (P＞0.05).Compared with the virus group,the filamentous actin (F-actin) in HUVECs of the berberine group was distributed in the cell membrane,with the stress fibers decreased,and the edge of the cells recovered to flat Irregular polygons,consistent with the performance of non-infected cells.Conclusion:Berberine has the effect of improving skeleton remodeling,cell shrinkage and cell morphological changes of endothelial cells infected with influenza virus,which can protect the endothelial cells from permeability increase induced by influenza virus infection.

Objective To observe the impacts of xijiao dihuang tang and yinqiao san( combined medicine)on MLC phosphrylation signaling pathways in lung tissue of the mouse model of influenza viral in-fection and determine the molecular mechanism of the combined medicine on its anti -viral effect. Meth-ods Fifty male Balb/c mice were randomized into five groups according to the weight,10 mice in each one, i. e. normal group,viral group,the low-dose,middle-dose and high-dose medication groups. Except the mice in the normal group,the mice in the rest groups were infected with A/FM/1/34(H1N1)via nasal drop. One hour later,in the viral group,the lavage with distilled water was used,twice a day. In the rest three groups,the lavage with the combined medicine was used at the low,middle and high doses separately,twice a day. After infection,on the 4th day,the eyeballs were extracted and the mice were sacrificed. RT-PCR was used to determine viral RNA level in lung tissue. Western bolt was used to determine the protein expressions of MLC,p-MLC,PKC,p-PKC,ROCK-1,ERM and p-ERM. Results Compared with the viral group, RNA level in the middle-dose medication group was reduced significantly,indicating the significant differ-ence(P﹤0. 01),that in the high-dose medication and low-dose medication group did not change appar-ently. In the middle-dose medication group,the expressions of Rho/ROCK and PKC and the protein expres-sions of p-MLC and p-ERM were reduced significantly,indicating the significant differences(P﹤0. 05). Compared with the normal group,in the viral group,the expressions of ROCK1 and PKC,and the protein ex-pressions of p-MLC and p-ERM were increased significantly,indicating the significant differences( P﹤0. 05). Conclusion The combined medication acts on antiviral effect through the suppression of the activation of Rho/ROCK and PKC signal pathways,the inhibition of p-MLC protein expression and the reducing of viral duplication.

目的:建立小鼠流感性肺炎模型,明确呼吸道流感病毒感染过程中是否存在特异性的肠道病理损伤,并探讨了其在“肺与大肠相表里”研究中的应用.方法:BALB/c小鼠48只随机分为正常组、病毒组.病毒组给予50 μL、50LD5o病毒液滴鼻建立流感病毒感染的小鼠肺炎模型,感染后第3、5、6天分别取材,HE染色检测肺脏、大肠、小肠、心脏、肝脏、肾脏组织病理结果,血生化检测血清谷草转氨酶(AST)、谷丙转氨酶(ALT)和尿素氮(BUN)水平.结果:流感病毒感染后,病毒组小鼠肺组织出现明显的炎性病变外,随时间延长肺组织充血水肿出血加重;小肠黏膜腺体排列紊乱,绒毛粗大、部分脱落,黏膜固有层炎性细胞浸润:大肠黏膜固有层、肌层大量炎性细胞浸润,腺体结构消失、脱落;而心脏、肝脏、肾脏无明显病理改变;病毒组小鼠血清AST、ALT、BUN较正常组相比,差异无统计学意义.结论:小鼠流感病毒性肺炎模型中存在小肠、大肠特异性免疫病理损伤,是研究中医“肺与大肠相表里”理论及相关临床治法的理想模型.

Objective: It has been documented that ezrin/radixin/moesin（ERM） phosphorylation by the p38 mitogen-activated protein kinase（MAPK）, Rho/ROCK, and protein kinase C（PKC） pathways leads to filamentous actin（F-actin） reorganization and microvascular endothelial cell hyperpermeability. In this study, we investigated the effects of Xijiao Dihuang Decoction combined with Yinqiao Powder（XDY） on influenza virus（IV）-induced F-actin restructuring and ERM phosphorylation regulated by the Rho/Rho kinase 1（ROCK）, p38 MAPK, and PKC signaling pathways in pulmonary microvascular endothelial cells（PMVECs）.Methods: Serum containing XDY（XDY-CS; 13.8 g/kg） was acquired using standard protocols for serum pharmacology. Primary PMVECs were obtained from male Wistar rats and cultured. After adsorption of IV A（multiplicity of infection, 0.01） for 1 h, medium with 20% XDY-CS was added to the PMVECs. The distributions of F-actin and phosphorylated ERM were determined by confocal microscopy, and F-actin expression was measured by flow cytometry. The expression levels of ROCK1, phosphorylated myosin phosphatase target-subunit（p-MYPT）, phosphorylated MAPK kinase, phosphorylated p38（p-p38）, phosphorylated PKC（p-PKC）, and phosphorylated ERM（p-ERM） were determined by western blotting.Results: F-actin reorganization in IV-infected PMVECs was reversed by XDY-CS treatment,which was accompanied by reduced p-ERM production. The p-ERM protein accumulated at plasma membrane of PMVECs infected with IV, which was also inhibited by XDY-CS treatment.In addition, XDY-CS treatment drastically reduced the levels of p-p38, ROCK1, p-MYPT, and pPKC induced by IV infection in PMVECs.Conclusion: These results show that XDY-CS inhibited influenza-induced F-actin reorganization in PMVECs by down-regulating p-ERM expression via inhibition of the Rho/ROCK, p38 MAPK, and PKC pathways. In conclusion, XDY could reduce the damage to endothelial cytoskeleton induced by IV infection, thus protecting the barriers of PMVECs.

近年来,中医药治疗甲型H1N1流感取得了较好的临床疗效.尽管目前临床对甲型H1N1流感的治疗仍以西药为主,但西药抗病毒药存在副作用大,易引起耐药性等缺点,限制了治疗的效果.中医治疗甲型H1N1流感则具有独特的思路,根据甲型H1N1流感不同进展阶段的不同证型,选择不同的中药进行”辨证论治”,不仅能减轻西药的不良反应,而且在疾病的治疗上也有独特的功效.我们总结了近来学者们对中医成药治疗甲型H1N1流感的临床及基础研究,将甲型H1N1流感分为轻、中、重三种,并分别根据症状与中医证型相匹配,梳理了临床用于治疗甲型H1N1流感的中医成药的适应证型与作用机制.因此,在西药进行抗病毒治疗的同时,根据疾病进展不同阶段的中医的证型,选用不同的中医方药,可有效减少西药的不良反应,取得更好的疗效.

OBJECTIVETo construct lentivirus siRNA vectors targeting ezrin (E), radixin (R) and moesin (M), transfect rat primary pulmonary microvascular endothelial cells (PMVECs) with the vectors and observe the effects of the transfection on E, R, M gene expressions and influenza virus replication.METHODSAccording to the principles of siRNA synthesis, siRNA sequences targeting E, R and M genes were synthesized and cloned into lentiviral plasmid GV248 to construct lentiviral siRNA vectors Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. Random Lenti-control-siRNA was also constructed targeting nonsense sequences. Rat PMVECs were cultured by peripheral lung tissue-sticking method. Rat PMVECs were transfected with Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA, respectively. The levels of ezrin, radixin and moesin mRNA and proteins were detected by real-time quantitative PCR and Western blotting, the distributions of ERM proteins were detected using confocal laser scanning microscope; After lentivirus-transfected PMVECs were infected with influenza virus, the effects of lentiviral vectors on influenza virus replication were measured by real-time quantitative PCR.RESULTSLenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA and Lenti-control-siRNA were successfully constructed according to sequencing identification of recombinant plasmids. The infection efficacy of lentiviral siRNA vectors in rat PMVECs was above 80% at a multiplicity of infection (MOI) of 10. The transcription and expressions of ezrin, radixin and moesin in rat PMVECs were significantly inhibited by Lenti-E-siRNA, Lenti-R-siRNA, Lenti-M-siRNA respectively. Lentivirus control (containing no target genes) and Lenti-control-siRNA inhibited influenza virus replication in influenza virus-infected PMVECs, while Lenti-E-siRNA, Lenti-R-siRNA and Lenti-M-siRNA increased the levels of influenza virus mRNA in the infected PMVECs.CONCLUSIONLentiviral siRNA vectors targeting E, R and M genes were constructed successfully and could inhibit E, R and M gene expressions specifically and effectively. Lentiviral vectors could inhibit influenza virus replication whereas lentiviral siRNA vectors targeting E, R and M facilitated influenza virus replication.

Classical formula, well-known for prominent clinical efficacy has complicated mechanism of action and unknown active substances due to varieties of compositions, which has constrained its in modern clinical medicine at a global platform.High per-formance liquid chromatography ( HPLC) and high liquid chromatography-mass spectrometry ( HPLC-MS) techniques, widely used in pharmaceutical analysis and quality control, has a multiple usage in active substance identification and content assessment of classical formula, ranging from sample pre-assessment handling, quantitative and qualitative analysis, pharmacokinetic research. Co-immunoprecipitation can be used to detect the effects of classical formula on the interactions between proteins, and confocal la-ser techniques can be used to observe the effects of classical formula on the distribution and transfer of proteins and ion.Therefore, the paper also briefed the use of those two techniques in the research of classical formula.

目的：建立小鼠流感性肺炎模型，明确呼吸道流感病毒感染过程中是否存在特异性的肠道病理损伤，并探讨了其在“肺与大肠相表里”研究中的应用。方法：BALB／c小鼠48只随机分为正常组、模型组。模型组给予25μL 50LD50病毒液滴鼻建立流感病毒感染的小鼠肺炎模型，感染后0、3、5、6 d分别取材，HE染色检测肺脏和大肠、小肠、心脏、肾脏和肝脏组织病理结果，血生化检测血清尿素氮（ BUN）、谷丙转氨酶（ ALT）和谷草转氨酶（ AST）水平。结果：流感病毒感染后，模型组小鼠肺组织出现明显的炎性病变外，随时间延长肺组织充血水肿出血加重；小肠黏膜腺体排列紊乱，绒毛粗大、部分脱落，黏膜固有层淋巴细胞大量浸润；大肠黏膜固有层、肌层大量淋巴细胞浸润，腺体结构消失、脱落；而心脏、肝脏和肾脏无明显病变；模型组小鼠血清BUN、ALT和AST较正常组相比，无显著差异。结论：小鼠流感病毒性肺炎模型中存在小肠、大肠特异性免疫病理损伤，是研究中医“肺与大肠相表里”理论及相关临床治法的理想模型。

Severe influenza infections are featured by acute lung injury, a syndrome of pulmonary microvascular leak. A growing number of evidences have shown that the pulmonary microvascular endothelial cells (PMVEC) are critical target of influenza virus, promoting microvascular leak. It is reported that there are multiple mechanisms by which influenza virus could elicit increased pulmonary endothelial permeability, in both direct and indirect manners. Ezrin/radixin/moesin family proteins, the linkers between plasma membrane and actin cytoskeleton, have been reported to be involved in cell adhesion, motility and may modulate endothelial permeability. Studies have also shown that ERM is phosphorylated in response to various stimuli via p38MAPK, Rho/ROCK or PKC pathways. However, it is unclear that whether influenza infection could induce ERM phosphorylation and its relocalization. In the present study, we have found that there are cytoskeletal reorganization and permeability increases in the course of influenza virus infection, accompanied by upregulated levels of p-ERM. p-ERM's aggregation along the periphery of PMVEC upon influenza virus infection was detected via confocal microscopy. Furthermore, we sought to determine the role of p38MAPK, Rho/ROCK and PKC pathways in ERM phosphorylation as well as their involvement in influenza virus-induced endothelial malfunction. The activation of p38MAPK, Rho/ROCK and PKC pathways upon influenza virus stimulation were observed, as evidenced by the evaluation of phosphorylated p38 (p-p38), phosphorylated MKK (p-MKK) in p38MAPK pathway, ROCK1 in Rho/ROCK pathway and phosphorylated PKC (p-PKC) in PKC pathway. We also showed that virus-induced ERM phosphorylation was reduced by using p38MAPK inhibitor, SB203580 (20 μM), Rho/ROCK inhibitor, Y27632 (20 μM), PKC inhibitor, LY317615 (10 μM). Additionally, influenza virus-induced F-actin reorganization and hyperpermeability were attenuated by pretreatment with SB203580, Y27632 and LY317615. Taken together, we provide the first evidence that p38MAPK, Rho/ROCK and PKC are involved in influenza-induced cytoskeletal changes and permeability increases in PMVEC via phosphorylating ERM.