Recombinant duck enteritis virus bearing the hemagglutinin genes of H5 and H7 influenza viruses is an ideal multivalent live vaccine in ducks.

Due to the fact that many avian influenza viruses that kill chickens are not lethal to ducks, farmers are reluctant to use avian influenza inactivated vaccines on ducks. Large numbers of unvaccinated ducks play an important role in the transmission of avian influenza viruses from wild birds to domestic poultry, creating a substantial challenge to vaccination strategies for avian influenza control. To solve this problem, we constructed a recombinant duck enteritis virus (DEV), rDEV-dH5/H7, using a live attenuated DEV vaccine strain (vDEV) as a vector. rDEV-dH5/H7 carries the hemagglutinin gene of two H5 viruses [GZ/S4184/17 (H5N6) (clade 2.3.4.4 h) and LN/SD007/17 (H5N1) (clade 2.3.2.1d)] and an H7 virus [GX/SD098/17 (H7N9)]. These three hemagglutinin genes were stably inherited in rDEV-dH5/H7 and expressed in rDEV-dH5/H7-infected cells. Animal studies revealed that rDEV-dH5/H7 and vDEV induced similar neutralizing antibody responses and protection against lethal DEV challenge. Importantly, rDEV-dH5/H7 induced strong and long-lasting hemagglutinin inhibition antibodies against different H5 and H7 viruses and provided complete protection against challenges with homologous and heterologous highly pathogenic H5 and H7 influenza viruses in ducks. Our study shows that rDEV-dH5/H7 could serve as an ideal live attenuated vaccine to protect ducks against infection with lethal DEV and highly pathogenic avian influenza viruses.

M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes.

Influenza A virus (IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor (M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR, the nuclear accumulation of viral nucleoprotein (NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking, or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin (HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes, thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR-HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

ABTB1 facilitates the replication of influenza A virus by counteracting TRIM4-mediated degradation of viral NP protein.

Influenza A viruses (IAVs) continue to cause tremendous economic losses to the global animal industry and respiratory diseases and deaths among humans. The nuclear import of the vRNP complex, composed of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), nucleoprotein (NP), and viral RNA, is essential for the efficient replication of IAV. Host factors involved in this process can be targeted for the development of countermeasures against IAV infection. Here, we found that Ankyrin Repeat and BTB Domain Containing 1 (ABTB1) promotes the replication of IAV, and positively regulates the nuclear import of the vRNP complex. ABTB1 did not interact directly with NP, indicating that ABTB1 plays an indirect role in facilitating the nuclear import of the vRNP complex. Immunoprecipitation and mass spectrometry revealed that Tripartite Motif Containing 4 (TRIM4) interacts with ABTB1. We found that TRIM4 relies on its E3 ubiquitin ligase activity to inhibit the replication of IAV by targeting and degrading NP within the incoming vRNP complex as well as the newly synthesized NP. ABTB1 interacted with TRIM4, leading to TRIM4 degradation through the proteasome system. Notably, ABTB1-mediated degradation of TRIM4 blocked the effect of TRIM4 on NP stability, and largely counteracted the inhibitory effect of TRIM4 on IAV replication. Our findings define a novel role for ABTB1 in aiding the nuclear import of the vRNP complex of IAV by counteracting the destabilizing effect of TRIM4 on the viral NP protein.

Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α7 is counteracted by TBK1-p62 axis-mediated autophagy.

As a major component of the viral ribonucleoprotein (vRNP) complex in influenza A virus (IAV), nucleoprotein (NP) interacts with isoforms of importin α family members, leading to the import of itself  and vRNP complex into the nucleus, a process pivotal in the replication cycle of IAV. In this study, we found that BinCARD1, an isoform of Bcl10-interacting protein with CARD (BinCARD), was leveraged by IAV for efficient viral replication. BinCARD1 promoted the nuclear import of the vRNP complex and newly synthesized NP and thus enhanced vRNP complex activity. Moreover, we found that BinCARD1 interacted with NP to promote NP binding to importin α7, an adaptor in the host nuclear import pathway. However, we also found that BinCARD1 promoted RIG-I-mediated innate immune signaling by mediating Lys63-linked polyubiquitination of TRAF3, and that TBK1 appeared to degrade BinCARD1. We showed that BinCARD1 was polyubiquitinated at residue K103 through a Lys63 linkage, which was recognized by the TBK1-p62 axis for autophagic degradation. Overall, our data demonstrate that IAV leverages BinCARD1 as an important host factor that promotes viral replication, and two mechanisms in the host defense system are triggered-innate immune signaling and autophagic degradation-to mitigate the promoting effect of BinCARD1 on the life cycle of IAV.

A Single Amino Acid Residue R144 of SNX16 Affects Its Ability to Inhibit the Replication of Influenza A Virus.

Influenza A virus (IAV) is an important zoonotic pathogen, posing a severe burden for the health of both animals and humans. Many host factors are involved in the life cycle of IAV to regulate its replication. Herein, we identified sorting nexin-16 (SNX16) as a new host factor that negatively modulates the replication of IAV. When transiently overexpressed in cells, SNX16 appears to be expressed as two obvious bands. Mutagenesis analysis indicated that the amino acid residue R144 of SNX16 was responsible for its two-band expression phenotype. We found that the R144A mutation of SNX16 changed its cellular distribution in A549 cells and partially weakened the inhibitory effect of SNX16 on IAV replication. Further investigation revealed that SNX16 could negatively regulate the early stage of the replication cycle of IAV. Taken together, our results demonstrated that SNX16 is a novel restriction host factor for the replication of IAV by engaging in the early stage of IAV life cycle, and a single amino acid residue at position 144 plays an important role in the cellular distribution and anti-influenza function of SNX16.

LTB4 Promotes Acute Lung Injury via Upregulating the PLCε-1/TLR4/NF-κB Pathway in One-Lung Ventilation.

BACKGROUND: Mechanical ventilation (MV) can provoke acute lung injury (ALI) by increasing inflammation activation and disrupting the barrier in lung tissues even causing death. However, the inflammation-related molecules and pathways in MV-induced ALI remain largely unknown. Hence, the purposes of this study are to examine the role and mechanism of a novel inflammation-related molecule, leukotriene B4 (LTB4), in ALI. METHODS: The functions of LTB4 in one-lung ventilation (OLV) model were detected by the loss-of-function experiments. H&E staining was used to examine the pathologic changes of lung tissues. Functionally, PLCε-1 knockdown and Toll-like receptor 4 (TLR4)/NF-κB pathway inhibitor were used to detect the regulatory effects of LTB4 on the phospholipase Cε (PLCε-1)/TLR4/nuclear factor-kappa B (NF-κB) pathway. The levels of genes and proteins were determined by RT-qPCR and western blotting assay. The levels of inflammation cytokines and chemokines were measured by ELISA. RESULTS: Here, we found LTA4H, leukotriene B (4) receptor 1 (BLT1), LTB4, and PLCε-1 upregulated in OLV rats and associated with inflammatory activation and lung permeability changes of lung tissues. Inhibition of LTB4 alleviated the OLV-induced ALI by inhibiting inflammatory activation and lung permeability changes of lung tissues. For mechanism analyses, LTB4 promoted OLV-induced ALI by activating the PLCε-1/TLR4/NF-κB pathway. CONCLUSION: LTB4 induced ALI in OLV rats by activating the PLCε-1/TLR4/NF-κB pathway. Our findings might supply a new potential therapeutic for OLV-induced ALI.

A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis.

Influenza virus infection is dependent on host cellular factors, and identification of these factors and their underlying mechanisms can provide important information for the development of strategies to inhibit viral infection. Here, we used a highly pathogenic H5N1 influenza virus to perform a genome-wide CRISPR/Cas9 gene knockout screen in human lung epithelial cells (A549 cells), and found that knockout of transmembrane protein immunoglobulin superfamily DCC subclass member 4 (IGDCC4) significantly reduced the replication of the virus in A549 cells. Further studies showed that IGDCC4 interacted with the viral hemagglutinin protein and facilitated virus internalization into host cells. Animal infection studies showed that replication of H5N1 virus in the nasal turbinates, lungs, and kidneys of IGDCC4-knockout mice was significantly lower than that in the corresponding organs of wild-type mice. Half of the IGDCC4-knockout mice survived a lethal H5N1 virus challenge, whereas all of the wild-type mice died within 11 days of infection. Our study identifies a novel host factor that promotes influenza virus infection by facilitating internalization and provides insights that will support the development of antiviral therapies.

PLCε1 mediates one-lung ventilation injury by regulating the p38/RhoA/NFκB activation loop.

BACKGROUND: Phospholipase C epsilon-1 (PLCε1) might be a novel and potential target in treating inflammatory conditions. In the present study, we aimed to clarify whether PLCε1 is involved in lung injury caused by one-lung ventilation (OLV) and to elucidate the potential molecular mechanism of PLCε1-mediated signaling pathway on OLV induced inflammatory response and injury. METHODS: Male Sprague-Dawley (SD) rats were divided into wide-type (PLCε1-WT) group and PLCε1-KO group, and were treated with OLV for 0.5 h, 1 h, and 2 h respectively. Observation of lung tissue injury in rats was performed by Hematoxylin and eosin (HE) staining and Wet/dry (W/D) radios. In addition, pulmonary microvascular endothelial cells (PMVECs) transfected with PLCε1-si RNA, were stimulated by lipopolysaccharide (LPS). To explore the possible roles of PLCε1 in the OLV induced inflammatory injury and the involved pathway underlying, the lung tissue and bronchoalveolar lavage fluids (BALF) of OLV rats, as well as the PMVECs were prepared for further analysis. Enzyme-linked immunoassay (ELISA) was used to detect the expression of pro-inflammatory factors. The activities of related pathway proteins (NF-κB, phospho-p38, p38, phospho-ERK1/2, ERK1/2, RhoA and ROCK) were also detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. RESULTS: Compared to the PLCε1-WT rats, PLCε1-KOrats exhibited marked alleviation of lung inflammation as shown by great reduction in lung wet/dry weight ratios, decreases in the expressions of pro-inflammatory mediators, and declines in the number of neutrophils and the protein concentration in bronchoalveolar lavage fluid (BALF). Moreover, the increased expressions of RhoA and NF-κB p65 mRNA induced by OLV were significantly inhibited in PLCε1-KO rats. In LPS treated PMVECs, PLCε1-si RNA transfection ones also showed the decrease expression of proinflammatory mediators, reduction in p38 phosphorylation levels and downregulation of RhoA/ROCK signaling activation. Co-cultured with PLCε1-si RNA and BTRB796 (p38 inhibitors) in LPS-stimulated PMVECs resulted in a significant reduction in RhoA and NF-κB activity. In addition, treatment with either ROCK inhibitor (Y-27632) or dominant negative mutant of RhoA (RhoT19 N) significantly reduced the expression of NF-κB in PLCε1-si RNA treated PMVECs. CONCLUSION: The results indicated that PLCε1 played an important role in the inflammatory response induced by OLV. Moreover, through promoting p38/RhoA/ROCK activation loop, PLCε1 promoted NF-κB activation and thereby increased the expressions of inflammatory mediators, which induced the PMVECs inflammation and subsequent injury. The results of this study provide a potential therapeutic target for the reduction of inflammatory response in patients with OLV.

Hierarchical nickel-cobalt selenide nanoparticles/nanosheets as advanced electroactive battery materials for hybrid supercapacitors.

Transition metal selenides are very ideal as electroactive battery material for hybrid supercapacitors owing to their high electrochemical activity and metallic electrical conductivity, but their environmental friendly fabrication and complex structure construction is still a big challenge. Here, a simple low-temperature selenization method has been developed to green synthesize a series of Ni-Co selenide samples with a hierarchical nanoparticle/nanosheet structure. The hierarchical structure has been constructed by the recrystallization of sample during the selenization process, which can be greatly influenced by the Co composition. The specific hierarchical structure provides more electroactive sites for charge storage, and the optimized synergy between the Ni and Co compositions further greatly tunes the charge storage performance, as a result, the Ni0.67Co0.33Se2 shows the best performance with a specific capacity of 447 C g-1 at 1 A g-1, which is much higher than the Ni-Co selenides with the other Ni to Co ratios and the Ni-Co oxides. The Ni0.67Co0.33Se2 retains 97% of the specific capacity after 2000 cycles, demonstrating its excellent cycling stability. A hybrid supercapacitor has been assembled using Ni0.67Co0.33Se2 as the positive electrode, which shows both high specific energy and specific power performances.

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry.

Influenza A virus (IAV) coopts numerous host factors to complete its replication cycle. Here, we identify free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with small interfering RNA (siRNA) targeting FFAR2 or the FFAR2 pathway agonists 2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide (4-CMTB) and compound 58 (Cmp58) [(S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide] dramatically inhibited the nuclear accumulation of viral nucleoprotein (NP) at early time points postinfection, indicating that FFAR2 functions in the early stage of the IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (vRNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2-knocked-down or 4-CMTB- or Cmp58-treated A549 cells. Further studies showed that FFAR2 associated with ß-arrestin1 and that ß-arrestin1 interacted with the ß2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either ß-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the ß-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three G protein-coupled receptor (GPCR) kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells.IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low-pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2-ß-arrestin1-AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

Protective efficacy in farmed ducks of a duck enteritis virus-vectored vaccine against H5N1, H5N6, and H5N8 avian influenza viruses.

Ducks play a key role in the maintenance and spread of avian influenza viruses (AIVs) in nature, and control of AIVs in ducks has important implications for AIV eradication from poultry. We previously constructed a recombinant duck enteritis virus (DEV), rDEVus78HA, that expresses the HA gene of an H5N1 AIV and showed that rDEVus78HA immunization provides complete protection against both DEV and H5N1 AIV challenge in specific-pathogen-free ducks. In this study, we performed a 60-week clinical trial and found that this rDEVus78HA vaccine can function as a bivalent vaccine in farmed ducks against lethal challenge with DEV and H5N1 virus. Moreover, we found that rDEVus78HA-vaccinated ducks were efficiently protected against challenges with recently isolated heterologous H5N6 and H5N8 viruses. Our results demonstrate that rDEVus78HA could be extremely valuable for the control of DEV and H5 AIVs in ducks.

Recombinant duck enteritis viruses expressing the Newcastle disease virus (NDV) F gene protects chickens from lethal NDV challenge.

Newcastle disease virus (NDV) is a major threat to poultry worldwide. Virulent Newcastle disease virus infection can cause 100% morbidity and mortality in chickens. Vaccination is the most effective way to prevent and control NDV outbreaks in poultry. Previously, we demonstrated that a duck enteritis virus (DEV) vaccine strain is a promising vector to generate recombinant vaccines in chickens. Here, we constructed two recombinant DEVs expressing the F protein (rDEV-F) or HN protein (rDEV-HN) of NDV. We then evaluated the protective efficacy of these recombinant DEVs in specific-pathogen-free chickens. rDEV-F induced 100% protection of chickens from lethal NDV challenge after a single dose of 104 TCID50, whereas rDEV-HN did not induce effective protection. rDEV-F may therefore serve as a promising vaccine candidate for chickens. This is the first report of a DEV-vectored vaccine providing robust protection against lethal NDV infection in chickens.

Inhalation treatment of idiopathic pulmonary fibrosis with curcumin large porous microparticles.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with high mortality and poor prognosis. Curcumin shows anti-inflammatory effect by suppressing pro-inflammatory cytokines and inhibiting NF-κB mediated inflammation. Here, we developed inhalable curcumin-loaded poly(lactic-co-glycolic)acid (PLGA) large porous microparticles (LPMPs) for the treatment of IPF. Curcumin LPMPs were rough and loose particles with many pores on the surfaces and channels in the inner spaces. The mean geometric diameter of them was larger than 10 µm while the aerodynamic diameter was only 3.12 µm due to their porous structures. They showed a fine particle fraction (FPF) <4.46 µm of 13.41%, 71% cumulative release after 9 h, and more importantly, they avoided uptake by alveolar macrophages. Therefore, most of released curcumin had opportunities to enter lung tissues. Rat pulmonary fibrosis models were established via once intratracheal administration of bleomycin. Curcumin powders and curcumin LPMPs were administered on Days 2, 7, 14, and 21. Curcumin LPMPs remarkably attenuated lung injuries, decreased hydroxyproline contents, reduced the synthesis of collagen I, and inhibited the expressions of TNF-α, TGF-ß1, NF-κB p65 and MMP9. Moreover, curcumin LPMPs showed higher antifibrotic activity than curcumin powders. Curcumin LPMPs are a promising inhalable medication for the treatment of IPF.

Inhalation treatment of primary lung cancer using liposomal curcumin dry powder inhalers.

Lung cancer is the leading cause of cancer-related deaths. Traditional chemotherapy causes serious toxicity due to the wide bodily distribution of these drugs. Curcumin is a potential anticancer agent but its low water solubility, poor bioavailability and rapid metabolism significantly limits clinical applications. Here we developed a liposomal curcumin dry powder inhaler (LCD) for inhalation treatment of primary lung cancer. LCDs were obtained from curcumin liposomes after freeze-drying. The LCDs had a mass mean aerodynamic diameter of 5.81 µm and a fine particle fraction of 46.71%, suitable for pulmonary delivery. The uptake of curcumin liposomes by human lung cancer A549 cells was markedly greater and faster than that of free curcumin. The high cytotoxicity on A549 cells and the low cytotoxicity of curcumin liposomes on normal human bronchial BEAS-2B epithelial cells yielded a high selection index partly due to increased cell apoptosis. Curcumin powders, LCDs and gemcitabine were directly sprayed into the lungs of rats with lung cancer through the trachea. LCDs showed higher anticancer effects than the other two medications with regard to pathology and the expression of many cancer-related markers including VEGF, malondialdehyde, TNF-α, caspase-3 and BCL-2. LCDs are a promising medication for inhalation treatment of lung cancer with high therapeutic efficiency.

H7N9 virulent mutants detected in chickens in China pose an increased threat to humans.

Certain low pathogenic avian influenza viruses can mutate to highly pathogenic viruses when they circulate in domestic poultry, at which point they can cause devastating poultry diseases and severe economic damage. The H7N9 influenza viruses that emerged in 2013 in China had caused severe human infections and deaths. However, these viruses were nonlethal in poultry. It is unknown whether the H7N9 viruses can acquire additional mutations during their circulation in nature and become lethal to poultry and more dangerous for humans. Here, we evaluated the evolution of H7N9 viruses isolated from avian species between 2013 and 2017 in China and found 23 different genotypes, 7 of which were detected only in ducks and were genetically distinct from the other 16 genotypes that evolved from the 2013 H7N9 viruses. Importantly, some H7N9 viruses obtained an insertion of four amino acids in their hemagglutinin (HA) cleavage site and were lethal in chickens. The index strain was not lethal in mice or ferrets, but readily obtained the 627K or 701N mutation in its PB2 segment upon replication in ferrets, causing it to become highly lethal in mice and ferrets and to be transmitted efficiently in ferrets by respiratory droplet. H7N9 viruses bearing the HA insertion and PB2 627K mutation have been detected in humans in China. Our study indicates that the new H7N9 mutants are lethal to chickens and pose an increased threat to human health, and thus highlights the need to control and eradicate the H7N9 viruses to prevent a possible pandemic.

[Application of random forest algorithm in fingerprint of Chinese medicine: different brands of Xiasangju granules as example].

To establish a random forest algorithm for identifying and classifying different brands of Xiasangju granules, and provide effective reference for identifying multi-index complex fingerprint. HPLC method was used to collect the fingerprint of 83 batches of Xiasangju granules from different manufacturers. The classification of Xiasangju granules samples based on chromatographic fingerprints was identified by chemometric methods including principal component analysis (PCA), partial least squares discriminate analysis (PLS-DA) and random forest analysis (RF). The superiority of the above three chemometric methods was compared. The results showed that the fingerprints of 83 batches of Xiasangju granules were established in this study. PCA could only explicate 56.52% variance contribution rate and could not completely classify the samples; PLS-DA analysis was superior to PCA, explicating 63.43% variance contribution rate and could obtain certain separation; RF could well classify the samples into 3 types, and the predication accuracy of the proposed method was 96.5%. Therefore, The results indicate that RF combined with HPLC fingerprint could effectively construct traditional Chinese medicine quality control and analysis system.

Inhalable Andrographolide-ß-cyclodextrin Inclusion Complexes for Treatment of Staphylococcus aureus Pneumonia by Regulating Immune Responses.

Bacterial pneumonia is a serious disease with high mortality if no appropriate and immediate therapy is available. Andrographolide (AG) is an anti-inflammatory agent extracted from a traditional Chinese herb andrographis paniculata. Oral AG tablets and pills are clinically applied for treatment of upper respiratory tract infections. However, the low solubility and bioavailability of AG lead to high doses and long-term therapy. Here we developed an andrographolide-ß-cyclodextrin inclusion complex (AG-ß-CD) for inhalation therapy of Staphylococcus aureus pneumonia. AG-ß-CD was identified with X-ray diffraction and FT-IR. Surprisingly, both AG-ß-CD and AG showed little in vitro anti-S. aureus activity. However, pulmonary delivery of AG, AG-ß-CD, or penicillin had significant anti-S. aureus pneumonia effects. Leukocytes, neutrophils, white blood cells, total proteins, TNF-α, IL-6, NF-κB p65 expression, and bacterial colonies in the bronchoalveolar lavage fluids were detected. Pulmonary delivery of AG and AG-ß-CD led to bacterial inhibition and inflammation alleviation by regulating immune responses, while penicillin only killed bacteria without significant immune regulation. Moreover, the antipneumonia activity of AG-ß-CD was much higher than that of AG, probably resulting from locally accelerated AG dissolution due to ß-CD inclusion. The aerodynamic diameter of AG-ß-CD powders was 2.03 µm, suitable for pulmonary delivery. Inhalable AG-ß-CD is a promising antibacterial and anti-inflammatory medicine for the treatment of S. aureus pneumonia by regulating immune responses, and the effect is enhanced by ß-CD inclusion. AG and its formulations might be potent weapons against the resistant bacterial pneumonia due to their specific mechanism in the future.

Effect of Sophora flavescens alkaloid on aerobic vaginitis in gel form for local treatment.

OBJECTIVE: To investigate the effect of Sophora flavescens alkaloid (SFA) in gel form on aerobic vaginitis (AV) and the possible mechanism underlying the effects. METHODS: AV rat models were prepared by intravaginal inoculation of Escherichia coli and Staphylococcus aureus. SFA gel and placebo gel were intravaginally administered. In vivo antibacterial effects, vaginal microenvironment, vaginal smears, pathological tissues of vaginas, and retention of gel in the vaginal cavity were investigated. RESULTS: SFA gel had much higher antibacterial effect than placebo gel. SFA gel protected the vaginal mucosa from erosion of bacteria. At the same time, they inhibited the inflammatory responses, exhibiting little leukocytes and parabasal cells. Furthermore, the number of vaginal Lactobacilli remarkably increased following administration of SFA gel. However, the vaginal pH did not recover to the healthy acidic levels after treatment due to the buffering effect of gel. The gel of a fluorescent agent, Cyanine 7, showed very long retention time in the vaginal cavity, up to more than 24 h, much longer than the solutions. CONCLUSION: The SFA gel is a promising medicine for local treatment of AV with the advantages of anti-bacteria, protection of vaginal mucosa, increase of Lactobacilli, and long retention time in the vaginal cavity.

[Preparation of liposomal artesunate dry powder inhalers and the effect on the acute lung injury of rats].

Artesunate is one of artemisinin derivatives with anti-malarial and anti-inflammatory activities though its water solubility and bioavailability are low. Acute lung injury (ALI) is a seriously dispersive lung disease with a high mortality. In this study, artesunate liposomes were prepared with the film dispersion method, and then lyophilized to obtain the liposomal artesunate dry powder inhalers(LADPIs). The LADPIs were pulmonary-delivered into the lung to treat ALI in rats. The artesunate liposomes had the capsulation efficiency of 71.4%, the particle size of 47.3 nm, and the zeta potential of -13.7 m V. The LADPIs had the aerodynamic particle size of 4.2 µm and the fine particle fraction (FPF) of 34.5%. ALI was established in rats by instilling lipopolysaccharide (LPS) into the lungs. The rats quickly showed a reduction in movement and acceleration in breath followed by diarrhea and so on. The LADPIs were directly administrated into the lungs of ALI rats through airways after 1 h of LPS challenge. The treatment induced a reduction in ALI syndromes. Two inflammatory factors, including TNF-α and IL-6, were significantly reduced by the artesunate powder in the LADPI group similarly to the reduction in the positive drug dexamethasone group (P < 0.05). Therefore, the anti-inflammatory effect of LADPIs contributed to the anti-ALI activity. Furthermore, the liposomal formulation improved drug bioavailability in the lung and increased therapeutic efficiency. The LADPIs are promising medicines for therapy of ALI through local drug administration.

Effect of Rab23 on the proliferation and apoptosis in breast cancer.

Rab23, as a negative regulatory molecule of the Hedgehog (Hh) signaling pathway, may be a new target for treating carcinoma. In the present study, we aimed to determine whether Rab23 is expressed in breast cancer cells and whether Rab23 affects the viability and proliferation of breast cancer cells. We evaluated Rab23 expression in several breast cancer cell lines including MDA-MB-231, Bcap37 and MCF-7 by reverse transcription-PCR (RT-PCR), western blotting and immunofluorescence in vitro. We assessed cell growth and proliferation by 3-(4,5-dimethylthiazol­2-y1)­3,5-diphenyltetrazolium bromide (MTT), colony formation and bromodeoxyuridine (BrdU) incorporation assays. The distribution of the cell cycle and the rate of apoptosis were assessed using flow cytometry (FCM). In addition, we determined the mechanisms by which Rab23 regulates the Hh pathway by detecting the level of Gli molecules by RT-PCR. We found that Rab23 mRNA and protein levels were expressed in breast cancer cells, and the expression of Rab23 in MDA-MB-231 cells was higher than that in the MCF-7 cells. Rab23 protein was primarily expressed and localized in the cytoplasm surrounding the nucleus. The MTT assay showed that the absorbance value at A(490 nm) of the Rab23­transfected group was reduced in comparison with the control group. The number of colonies formed in the breast cancer cells was significantly reduced and BrdU labeling was weakened in the group transfected with Rab23. The results of FCM showed that overexpression of Rab23 protein caused cell cycle arrest in the G1 phase and a decrease in the S phase population as well as induction of apoptosis. Furthermore, Rab23 decreased Gli1 and Gli2 mRNA levels when compared with the control group. Our results indicate that Rab23 is expressed in breast cancer cells, and ectopic expression of Rab23 inhibits the growth and proliferation as well as induces cell apoptosis in breast cancer cells. These effects may be due to the inhibition by Rab23 of Gli1 and Gli2 mRNA expression. These results suggest that Rab23 is a potential target for the treatment of breast cancer.