Network pharmacological analysis and experimental study of Pulsatilla chinensis against inflammatory injury caused by pneumonia in mice infected with influenza virus FM_1 / 中国中药杂志

Network pharmacology and the mouse model of viral pneumonia caused by influenza virus FM_1 were employed to explore the main active components and the mechanism of Pulsatilla chinensis against the inflammatory injury of influenza virus-induced pneumonia. The components and targets of P. chinensis were searched from TCMSP, and the targets associated with influenza virus-induced pneumonia were searched from GeneCards. The common targets between P. chinensis and influenza virus-induced pneumonia were identified with Venn diagram established in Venny 2.1. The herb-component-disease-target(H-C-D-T) network was constructed by Cytoscape 3.7.2. The above data were imported into STRING for PPI network analysis. Gene Ontology(GO) enrichment and KEGG pathway enrichment were performed with DAVID. BALB/cAnN mice were infected with the influenza virus FM_1 by nasal drip to gene-rate the mouse model of pneumonia. Immunohistochemistry was adopted to the expression profiling of inflammatory cytokines in the lung tissues of mice in the blank group, model group, and P. chinensis group 1, 3, 5, and 7 days after infection. The pathological changes of lung and trachea of mice in blank group, model group, and P. chinensis group were observed with light microscope and scanning electron microscope at all the time points. The network pharmacological analysis indicated that 9 compounds of P. chinensis were screened out, with a total of 57 targets, 22 of which were overlapped with those of influenza virus-induced pneumonia. A total of 112 GO terms(P<0.05) were enriched, including 81 terms of biological processes, 11 terms of cell components, and 20 terms of molecular functions. A total of 53 KEGG signaling pathways(P<0.05) were enriched, including TNF signaling pathway, influenza A signaling pathway, NF-κB signaling pathway, MAPK signaling pathway and other signaling pathways related to influenza/inflammation. In the P. chinensis group, the expression of TNF-α and IL-1 in the lung tissue was down-regulated on the 3 rd day after infection, and that of IL-6 in the lung tissue was down-regulated on the 5 th day after infection. Light microscopy and scanning electron microscopy showed that P. chinensis significantly alleviated the pathological damage of lung and trachea compared with the model group. This study reflects the multi-components, multi-targets, and multi-pathways of P. chinensis against influenza virus-induced pneumonia. P. chinensis may reduce the production of proinflammatory cytokines and mediators and block the pro-inflammatory signaling pathways to alleviate viral pneumonia, which provides reference for future research.

Effect of Scutellariae Radix on expression of inflammatory cytokine protein and gene in lung of mice with viral pneumonia caused by influenza virus FM1 infection / 中国中药杂志

Mice models of viral pneumonia were induced by pulmonary adaptive strain FM1 of influenza A virus in Asian mice.RT-PCR and immunohistochemistry were used to dynamically observe the effect of Scutellariae Radix on the protein and gene expression of inflammatory cytokine in the lungs of the model mice infected by influenza virus FM1 at different phases. The partial mechanism of Scutellariae Radix in repairing the immune inflammatory damage of target organs of pneumonia caused by influenza virus was further explored. The results showed that Scutellariae Radix reduced protein and gene expression of proinflammatory cytokines tumor necrosis factor( TNF-α),interleukin IL-1,IL-6 in lung tissues from 3 rd to 5 th day after infection,and increased protein and gene expression of IL-10 and IFN-γ in lung tissues on the 5 th day after infection. Scutellariae Radix may inhibit excessive release of pro-inflammatory cytokines and promote the expression of anti-inflammatory cytokines,thereby inhibiting the systemic inflammatory response syndrome,reducing the immunoinflammatory pathological damage of lung caused by influenza virus FM1 infection,and promoting lung repair of tissue inflammatory lesions.

Early postnatal exposure to lithium in vitro induces changes in AMPAR mEPSCs and vesicular recycling at hippocampal glutamatergic synapses.

Lithium is an effective mood stabilizer but its use is associated with many side effects. Electrophysiological recordings of miniature excitatory postsynaptic currents (mEPSCs) mediated by glutamate receptor AMPA-subtype (AMPARs) in hippocampal pyramidal neurons revealed that CLi (therapeutic concentration of 1 mM lithium, from days in vitro 4–10) decreased the mean amplitude and mean rectification index (RI) of AMPAR mEPSCs. Lowered mean RI indicate that contribution of Ca2+-permeable AMPARs in synaptic events is higher in CLi neurons (supported by experiments sensitive to Ca2+-permeable AMPAR modulation). Co-inhibiting PKA, GSK-3β and glutamate reuptake was necessary to bring about changes in AMPAR mEPSCs similar to that seen in CLi neurons. FM1-43 experiments revealed that recycling pool size was affected in CLi cultures. Results from minimum loading, chlorpromazine treatment and hyperosmotic treatment experiments indicate that endocytosis in CLi is affected while not much difference is seen in modes of exocytosis. CLi cultures did not show the high KCl associated presynaptic potentiation observed in control cultures. This study, by calling attention to long-term lithium-exposure-induced synaptic changes, might have implications in understanding the side effects such as CNS complications occurring in perinatally exposed babies and cognitive dulling seen in patients on lithium treatment.

Establishment of synapses between rat cortical neurons and Neuron-like cells derived from bone marrow stromal cells in vitro / 中华神经医学杂志

Objective To investigate the establishment of synapses between the cortical neurons and the neuron-like cells difierentiated from the marrow stromal cells(BMSCs)in a simulated transplantation system in vitro.Methods The BMSCs from green fluorescent protein(GFP)transgenic mice(GFP-GM-BMSCs) were isolated, cultured and purified in vitro.The third passage of GFP-GM-BMSCs were co-cultured with primary cultured cortical neurons and gliai cells in a simulated transplantation system in serum-free medium conmining 2%B27 supplemented with 20 ng/mL basic fibroblast growth factor(bFGF)and 20 ng/mL epidermal growth factor(EGF).On day 10 of the co-culture,FM1-43,a fluorescent dye specific to active synaptic vesicles,was used to observe synapses formation between the cells under fluorescence microscope. Results The GFP.GM-BMSCsco-cultured with the neural cells in the Serum-free medium containing bFGF and EGF differentiated into neuron-like cells 7 days after the co-culture.On day 10 ofthe co-culture,FM1-43 dye-positive synaptic vesicles were foundin the cell culture,locating mostly in the cell body,processes and terminal sffuctures ofthe neuron-like cells. Conclusions The neuron-like cells derived from GFP-GM-BMSCs can form synapses with the coRical neurons in the simulated cell transplantation system in vitro.

A Promotive Effect of Low-Level Laser on Hair Cell Regeneration Following Gentamicin Induced Ototoxicity in Postnatal Organotypic Culture of Rat Utricles / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: In normal postnatal mammalian inner ear sensory epithelium, regeneration of hair cells is a very rare event, but there is hair cell regeneration with partial restoration of the vestibular sensory epithelium following ototoxic damage. In this study, the effects of low-level laser on hair cell regeneration following gentamicin exposure in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: A long term organotypic culture of 2 to 7 day old rat utricular maculae was established to study aminoglycoside-induced vestibular hair cell renewal. The utricles were exposed to 1 mM of gentamicin for 48 hr and allowed to recover in a culture medium only or in a medium with daily irradiation of low-level laser, whereas the control group was not exposed to gentamicin. Whole-mount utricles were stained with FM1-43, which are known to be an efficient marker, to identify live hair cells in cultured tissues. RESULTS: Loss of hair cells was nearly stopped from 2 days after exposure to gentamicin ; a peak of regeneration was reached after 18 days and sustained for two weeks in the medium with the irradiation of low-level laser. CONCLUSION: These results suggest that low-level laser promotes spontaneous hair cell regeneration following gentamicin damage in utricular explants.

A Promotive Effect of Low Level Laser on Hair Cell Viability in Postnatal Organotypic Culture of Rat Utricles

BACKGROUND AND OBJECTIVES: To culture and maintain mammalian hair cells is still a big challenge. In this study, long-term organotypic culture of rat utricular maculae was established to study vestibular hair cell. The effects of low level laser on hair cell viability in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: Uticular explants were prepared from postnatal 2 to 7 rats and cultured. To improve hair cell survival, the utricles were irradiated daily with low level laser. Whole-mount utricles were stained with FM1-43 which is known to be an efficient marker to identify live hair cells in cultured tissues. Such cells visualized directly through tissue culture dish with cover glass bottom by Confocal laser scanning microscope at specific time points. RESULTS: The explanted utricular hair cells were cultured for up to 31 days in in vitro culture system. In low level laser irradiation group, utricular hair cells were more survived at 24 DIV and 31 DIV. CONCLUSION: These results suggest that low level laser promotes hair cell viability in utricular explants.

A Promotive Effect of Low Level Laser on Hair Cell Viability in Postnatal Organotypic Culture of Rat Utricles

BACKGROUND AND OBJECTIVES: To culture and maintain mammalian hair cells is still a big challenge. In this study, long-term organotypic culture of rat utricular maculae was established to study vestibular hair cell. The effects of low level laser on hair cell viability in postnatal organotypic culture of rat utricles were investigated. MATERIALS AND METHOD: Uticular explants were prepared from postnatal 2 to 7 rats and cultured. To improve hair cell survival, the utricles were irradiated daily with low level laser. Whole-mount utricles were stained with FM1-43 which is known to be an efficient marker to identify live hair cells in cultured tissues. Such cells visualized directly through tissue culture dish with cover glass bottom by Confocal laser scanning microscope at specific time points. RESULTS: The explanted utricular hair cells were cultured for up to 31 days in in vitro culture system. In low level laser irradiation group, utricular hair cells were more survived at 24 DIV and 31 DIV. CONCLUSION: These results suggest that low level laser promotes hair cell viability in utricular explants.

Protective Effect of Tanreqing Injection on the Mice Infected with Influenza Virus FM_1 / 中国中医药信息杂志

Objective To investigate the protective effect of Tanreqing injection on the mice infected with influenza virus FM 1 . Methods The experiment includes protection of Tanreqing injection to the mice infected with influenza virus FM1, and effect of Tanreqing injection to the viral titers, pathology and lung index of mice with influenza virus FM 1 . Results Tanreqing injection can reduce the mortality of the mice with influenza virus FM 1 infected. And Tanreqing injection can improve viral titers, pathology, and lung index of the mice infected with influenza virus FM1. Conclusions Tanreqing injection can protect the mice infected with influenza virus FM 1 by resisting the influenza virus and meliorating the lung pathology of the mice infected with influenza virus FM 1 .

Effect of Dureping Injection on Immunological Function of FM1 Infected Mice / 中国中医药信息杂志

Objective To investigate the effect of Dureping injection (DRP) on IL-4, IFN-? mRNA and CD40L protein expression of FM1 infected mice with virus pneumonia, and explore the mechanism of antiviral effect. Methods ICR mice were divided into 7 groups:DRP1, DRP2, DRP3, SHL, Ribavirin, sham and model group. The mice were sacrificed on the 7th after infected, and the lung index was calculated. Then, IFN-?mRNA and IL-10 mRNA in the lung tissue was measured with RT-PCR, CD40L protein in the lung tissue was measured with Western-blot. Results The lung index of mice in model group was significantly higher than sham group (P0.05). Conclusion DRP in vivo could down-regulate the lung index, inhibit the transcription IL-4 mRNA, enhance the transcription of IFN-? and inhibit the expression of CD40L protein in lung of FM1 infected mice.

Antiviral and Antibacterial Effects of the Effective Site of Traditional Chinese Medicine / 中药新药与临床药理

Objective To study the antiviral and antibacterial effects of the effective site of traditional Chinese medicine(TCM-ES).Methods Chicken embryoes were infected with influenza virus A(FM1 strain) and pre-injected with TCM-ES by means of chicken-embryo inoculation technique,and the antiviral effect of TCM-ES on chicken embryo was assayed by detecting the hemagglutination titers in allantoic fluids.Mice were orally pretreated with various dosages of drugs twice daily for 3 days,then were given drugs continuously for another 4 days following FM1 infection.The protective effects of TCM-ES on mice infected with FM1 were assayed by calculating the weight,index of lung,death-protection rate,and life-prolongation rate,etc.Ribavirin was used as the positive control.In addition,the antibacterial effects of TCM-ES were observed by detecting the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) by test-tube dilution method.Results In chicken embryo experiments,TCM-ES showed a potential inhibiting effect on influenza virus with the MIC of 10 mg/mL,which was weaker than ribavirin.The results of animal experiment showed that the body-weight(BW) and pulmonary index of infected model group decreased evidently compared with those of the normal group,TCM-ES groups at the dosages of 750 mg/kg and 1500 mg/kg could reverse the decrease of BW and lung index as compared with the infected models,the difference being insignificant as compared with the normal group.Moreover,TCM-ES also increased the death-protection rate and life-prolongation rate of mice in a dose-dependent manner.TCM-ES at dosage of 10 mg/mL(MIC and MBC) had an antibacterial effect on staphylococcus,while had no effect on gram-negative bacilli.Conclusion TCM-ES has obvious antivirus effect on influenza virus FM1 strain,and also has certain antibacterial effect on staphylococcus,which is worth of further development and research.