The pulmonary pharmacokinetics and anti-inflammatory effect of anemoside B4 after intratracheal and intravenous administration / 药学学报

Anemoside B4 (B4), a main triterpenoid saponin from a traditional Chinese medicine plant, Pulsatilla chinensis, is a novel anti-inflammatory agent for protection from acute lung injury. We investigated the pulmonary availability and anti-inflammatory efficacy of B4 after intratracheal and intravenous dosing with a view to evaluating the suitability of inhalation delivery. All animal studies were performed under the guidelines approved by the Animal Care and Use Committee of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences (Approval No: SLXD-20181113046). In vitro evaluation of the aerodynamic characteristics and droplet size distribution showed that the aerosols generated by a commercially available nebulizer were well deposited in the respiratory tract. Following intratracheal administration, B4 underwent pulmonary absorption into the bloodstream, rendering an absolute bioavailability of 103%. Compared to intravenous delivery, intratracheal administration dramatically increased the drug availability in lung tissue of rats by more than 1 000-fold, leading to improved and prolonged concentrations of B4 in lung tissue up to 48 h. In addition, the intratracheal administration of B4 resulted in dose-dependent and prolonged anti-inflammatory efficacy in a lipopolysaccharide (LPS)-induced lung injury model in mice. The present results demonstrate that inhalation delivery of B4 is a promising approach to treat pulmonary inflammation with once-daily dosing.

The ethyl acetate extraction of Pileostegia tomentella (ZLTE) exerts anti-cancer effects on H1299 cells via ROS-induced canonical apoptosis / 中国天然药物

Lung cancer is the leading cause of cancer death and the most common malignant tumor, the long-term survival of which has stagnated in the past several decades. Pileostegia tomentella Hand. Mazz is a traditional Chinese medicine called "Zhongliuteng" (ZLT) in the pharmacopeia, which has been proved to possess a potent anti-tumor effect on various cancers. In this study, the effects of ZLT N-butanol extraction (ZLTN) and ZLT ethyl acetate extraction (ZLTE) on the viability of non-small cell lung cancer cell (NSCLC) lines H1299 and A549 were evaluated. Here, we firstly reported that ZLTE significantly inhibited H1299 cells growth without affecting the release of lactate dehydrogenase (LDH). In addition, ZLTE induced caspase-dependent apoptosis in a concentration-dependent manner and increased the expression cleaved-PARP and decreased pro-caspase-3, pro-caspase-7, pro-caspase-8, and pro-caspase-9. Moreover, ZLTE increased the level of cellular reactive oxygen species (ROS) in H1299 cells to lead to apoptosis, which was reversed by N-acetyl-cysteine (NAC). Taken together, our results revealed that ZLTE induced caspase-dependent apoptosis via ROS generation, suggesting that ZLTE is a promising herbal medicine for the treatment of NSCLC.

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment / 中国病理生理杂志

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model.Oil red O staining was used to determine whether the model was established successfully.miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h.The intracellular lipid droplets were observed by Oil red O staining.The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot.The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05).The intracellular cholesterol content was increased gradually (P<0.05) , and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group.Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05).No difference of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.

Xuebijing Injection () and Resolvin D1 Synergize Regulate Leukocyte Adhesion and Improve Survival Rate in Mice with Sepsis-Induced Lung Injury / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of combined application of Xuebijing Injection ( , XBJ) and resolvin D1 (RvD1) on survival rate and the underlying mechanisms in mice with sepsisinduced lung injury.</p><p><b>METHODS</b>The cecal ligation and puncture (CLP) method was used to develop a mouse sepsis model. Specific pathogen free male C57BL/6 mice were randomly divided into 5 groups (n=20 each): sham, CLP, CLP+XBJ, CLP+RvD1 and CLP+XBJ+RvD1. After surgery, mice in the CLP+XBJ, CLP+RvD1 and CLP+XBJ+RvD1 groups were given XBJ (25 μL/g body weight), RvD1 (10 ng/g body weight), and their combination (the same dose of XBJ and RvD1), respectively. In each group, 12 mice were used to observe 1-week survival rate, while the rest were executed at 12 h. Whole blood was collected for flow cytometric analysis of leukocyte adhesion molecules CD18, lung tissues were harvested for observing pathological changes, and testing the activity of myeloperoxidase (MPO) and the expression of intercellular cell adhesion molecule 1 (ICAM-1).</p><p><b>RESULTS</b>Compared with the CLP group, the histopathological damage of the lung tissues was mitigated, MPO activity was decreased in the CLP+XBJ and CLP+RvD1 groups (P<0.05). In addition, the 1-week survival rate was improved, proportion of CD18-expressing cells in whole blood and ICAM-1 protein expression in lung tissue were decreased in the CLP+XBJ+RvD1 group (P<0.05 or P<0.01).</p><p><b>CONCLUSIONS</b>XBJ together with RvD1 could effectively inhibit leukocyte adhesion, reduce lung injury, and improve the survival rate of mice with sepsis.</p>

NQO1 dependent non-canonical necroptosis mediated by ROS and RIP1/RIP3 in parallel in glioma cancer cells / 中国药理学与毒理学杂志

OBJECTIVE Glioblastomas(GBM)are the most malignant brain tumors in humans and have a very poor prognosis. New therapeutics are urgently needed. Here, we reported 2-methoxy-6-acetyl-7-methyljuglone (MAM)-induced cell death in U87 and U251 glioma cancer cells. METHODS Cells were cultured and treated with MAM, the cell viability was determined by MTT assay and LDH assay. Intracellular reactive oxygen species (ROS) generation was observed by DCF fluorescence. The protein expression was determined by Western blotting. RESULTS MAM induced glioma cancer cell death without caspase activation. The cell death induced by MAM was attenuated by the pharmacological or genetic blockage of necroptosis signaling,including RIP1 inhibitor necrostatin-1s (Nec-1s)and siRNA-mediated gene silencing of RIP1 and RIP3,but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). MAM treated U87 and U251 glioma cancer cells induced RIP1/RIP3 complex formation, ROS level increased, ATP concentration decreased and loss of plasma membrane integrity, further confirmed this process was necroptosis.The essential role of ROS was confirmed by the protective effect of ROS scavenger NAC. Interestingly, MAM induced necroptosis both triggered by RIP1/RIP3 complex and ROS generation. Moreover, MAM induced necroptosis through cytosolic calcium (Ca2 +) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate cell death. Further, we found there exists a feedback loop between RIP1 and JNK activation.Finally,MAM induced necroptosis was inhibited by dicoumarol(a NQO1 inhibitor). Dicoumarol exposed glioma cancer cells were resistant to RIP1/RIP3 complex formation and ROS generation. MAM induced necroptosis was independent of MLKL. CONCLUSION MAM induced non-canonical necroptosis through the NQO1-dependent ROS and RIP1/RIP3 pathway.This study also provided new insights into the molecular regulation of necroptosis in human glioma cancer cells and a promising approach for GBM treatment.

Water-related diseases along the eastern route of the South-to-North Water Diversion Project / 中国人兽共患病学报

The aim of this study is to investigate the epidemic condition of water-related diseases in the eastern route of South-to-North Water Diversion Project (SNWDP).All data were extracted from published literatures in Chinese about water-related diseases in the eastern route of SNWDP.Pooled analysis was used to explore geographical distribution and epidemiology of the disease.A total of 325 articles about water-related diseases were retrieved during 1953 to 2013,and 209 articles were included in this study.Pooling analysis showed that Shandong Province had the largest number of cases for water-related diseases,following by Jiangsu,Hebei,Tianjin,and Anhui.The numbers of cases were relative small before 1960s according to epidemic curve,and the curve peaked in the 1970s,and decreased after the 1980s.A total of 1 383 834 cases of bacillary dysentery was reported,accounting for 84％ of all water-related diseases on these regions of SNWDP,and followed by hepatitis A,hepatitis E,Japanese encephalitis,typhoid and paratyphoid fever and hemorrhagic fever with renal syndrome.Other reported diseases displayed scatter condition and a small numbers of cases.The prevalence of water-related diseases is sporadic and a trend of decline along the regions of the eastern route of SNWDP.

Alanine solution as enzyme reaction buffer used in A to O blood group conversion / 中国实验血液学杂志

The aim of this study was to investigate the effect of alanine solution as α-N-acetylgalactosaminidase enzyme reaction buffer on the enzymatic activity of A antigen. The binding ability of α-N-acetylgalactosaminidase with RBC in different reaction buffer such as alanine solution, glycine solution, normal saline (0.9% NaCl), PBS, PCS was detected by Western blot. The results showed that the efficiency of A to O conversion in alanine solution was similar to that in glycine solution, and Western blot confirmed that most of enzymes blinded with RBC in glycine or alanine solution, but few enzymes blinded with RBC in PBS, PCS or normal saline. The evidences indicated that binding of enzyme with RBC was a key element for A to O blood group conversion, while the binding ability of α-N-acetylgalactosaminidase with RBC in alanine or glycine solution was similar. It is concluded that alanine solution can be used as enzyme reaction buffer in A to O blood group conversion. In this buffer, the α-N-acetylgalactosaminidase is closely blinded with RBC and α-N-acetylgalactosaminidase plays efficient enzymatic activity of A antigen.

Holstein-Friesian RBC as human blood substitute / 中国实验血液学杂志

α-Gal, the main xenotransplantation antigen, can lead to hyperacute rejection (HAR) in xenotransplantation. This study was purposed to investigate the effect of recombinant α-galactosidase (α-Gal antigen) on the Holstein-Friesian(H-F) red blood cells (RBC). The enzymelysis method was used to digest the α-Gal antigen on H-F RBC; the saline and anti-human globulin methods were used to perform the agglutination test of H-F RBC and human plasma; the flow cytometry was used to detect the α-Gal antigen on surface of H-F RBC, fluorescence intensity of FITC-IB4 and FITC-IgG labeled RBC. The results indicated that the saline and anti-human globulin method showed α-galactosidase-treated H-F RBC fail to agglutinate with human pooled plasma; the flow cytometry showed the fluorescence intensity of FITC-IB4 and FITC-IgG labeled RBC decrease 99.0% and 87.8%, respectively. It is concluded that the novel α-galactosidase can be used to cleared the α-Gal antigen on the surface of H-F RBC and α-galactosidase-treated H-F RBC may be considered as human blood substitute.

Removal of αGal xenotransplantation antigen by a novel α-galactosidase / 中国实验血液学杂志

αGal, a xenotransplantations antigen (XTA), can lead to hyper acute reaction (HAR) in xenotransplantation. α-Galactosidase from B. fragilis is a novel galactosidase belong to CAZy GH110 which can clear the terminal αGal from branched and linear oligosaccharides. This study was purposed to investigate the removal effect of a novel α-galactosidase on α-Gal XTA on surface of red blood cells. The αGal XTA from the red blood cells of cattle, pig, dog and rabbit was digested by using recombinant α-galactosidase; the α-Gal antigens on surface of cells was detected by flow cytometry. The results showed that the XTA was disappeared completely or mainly. It is concluded that the novel α-galactosidase is a potential enzyme to remove the XTA on the surface of xenotransplants and can be used to overcome the HAR in xenotransplantation.

A reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human blood type B→O conversion / 中国实验血液学杂志

This study was aimed to prepare a reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human B to O blood group conversion. Based on the construction of recombinant E. Coli (DE3) which can express α-galactosidase, the inducing time and inducer concentration were optimized for high expression of α-galactosidase. Then, the expression products in supernatant were purified by cation and anion exchange column chromatography. The purified α-galactosidase was used to treat B group red blood cells in phosphate buffer (pH 6.8) for 2 hours to prepare O group red blood cells. The results showed that the optimal inducing conditions for α-galactosidase expression were IPTG 0.1 mmol/L, 37°C and 2 hours. The specific enzyme activity of purified protein increased from 0.42 U/mg to 2.1 U/mg as compared with pre-purification. And, the conditions of B to O blood group conversion were 26°C, pH 6.8 (neutral pH condition) and 2 hours. Moreover, 225 µg of the enzyme could converse 1 ml B red blood cells to O completely. It is concluded that the technology of expression and purification of recombinant α-galactosidase has been established, and the purified protein can converse B red blood cells to O completely, which means that an effective enzyme conversing B red blood cells to O has been obtained.

B to O erythrocyte conversion by the recombinant alpha-galactosidase / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human group O red blood cells have great benefit in specialized transfusion areas such as armed conflict and natural calamity. The group B antigen differs structurally from group O antigen only by the addition of one terminal alpha-linked galactose residue. In this study we aimed to remove the terminal galactose from group B red blood cell to get group O red blood cell.</p><p><b>METHODS</b>alpha-galactosidase cDNA was cloned by RT-PCR from Catimor coffee beans grown on Hainan Island of China. The vector for alpha-galactosidase cDNA expression was constructed and transferred into Pichia pastoris cells by electroporation. The transgenic cells were cloned by fermentation and the recombinant alpha-galactosidase was purified by ion exchange chromatography. After studying the biochemical characters of alpha-galactosidase, we have used it in converting human erythrocytes from group B to group O.</p><p><b>RESULTS</b>The purity of recombinant alpha-galactosidase was higher than 96%, which was thought to be suitable for the use of blood conversion. Enzymatically converted human group O red blood cells (ECHORBC) exhibited membrane integrity, metabolic integrity, normal cell deformation and morphology. There were no coagulation between ECHORBC and any group of human blood. The ECHORBC will keep normal structure and function for a period of 21 days at 4 degrees C in monoammoniumphosphate nutrient solution. Experiments with Rhesus monkeys and gibbons showed that transfusion of enzymatically converted erythrocytes was safe.</p><p><b>CONCLUSION</b>ECHORBC can be easily obtained from group B red blood cell by alpha-galactosidase digestion. This study suggests that ECHORBC could be transfused to patients safely and efficiently.</p>

Preparation of transfusable human universal red blood cell with recombinant alpha-galactosidase / 中国实验血液学杂志

In order to meet the demand for safe transfusion in special conditions and to utilize the donated blood supply efficiently, technology has been developed to convert erythrocytes from type A, B, or AB to "universal donor" blood. Conversion of blood type B to O was performed by means of recombinant alpha-galactosidase digestion. The results showed that blood type B to O was converted successfully, 1 transfusion unit of red cells of group B (100 ml totally) could converted to universal blood cells in the optimal conditions including pH 5.6, 26 degrees C, 2 hours, obturation and sterilization. It is concluded that the universal red blood cells converted from group B to group O are conformed to demand of identification rules of biological products, no harmful effects of alpha-galactosidase on cell structure and function are observed. The converted red cells can stored in 4 degrees C for 21 days.

Establishment of Loop-mediated Isothermal Amplification (LAMP) Method for Campylobacter jejuni Detection / 微生物学通报

A rapid LAMP detection method with primers designed on genus-specific region identified in the gyrA gene was established in this assay. All four Campylobacter jejuni from different sources were detected positive and fourteen non Campylobacter bacteria were negative, which shows excellent specificity of the primers. Compared with plate count and PCR method, the LAMP method and the PCR method had equal sensitivity, which were three orders of magnitude higher than plate count. In this assay, we also found out that the treatment of DNase could reduce the dead bacteria DNA effectively. The LAMP detection on chicken indicated relatively good result on detection of Campylobacter jejuni combining with treatment of DNase.