[Optimization of process of sodium aescinate lyophilized powder].

OBJECTIVE: To determine the optimal freeze-drying process of Sodium Aescinate lyophilized powder in order to shorten the lyophilization cycle. METHODS: Using the single factor experiment and L9 (3(4)) orthogonal test to optimize parameters of the herbal liquid volume and concentration, pre-freezing time, pressure, drying time and analytical temperature. RESULTS: The best lyophilization process parameters were as follows: 1.0 mL herbal liquid with concentration of 10 mg/mL, phased cooling style, pre-freezing temperature at - 35 degrees C, 6.5 h; vacuum of 20 Pa;sublimation drying time of 7 h; and desorption-drying temperature of 35 degrees C for 5.5 h. CONCLUSION: Compared with the original process conditions, the product quality is more stable and the freezing-drying cycle time is shorten of 3 h, which can provide technical reference for production process of the freeze-dried powder of sodium aescinate.

Gut-Derived Exosomes Induce Liver Injury After Intestinal Ischemia/Reperfusion by Promoting Hepatic Macrophage Polarization.

Liver injury induced by intestinal ischemia/reperfusion (I/R) is accompanied by the polarization of Kupffer cells, which are specialized macrophages located in the liver. However, the causes of hepatic macrophage polarization after intestinal I/R remain unknown. This study investigated whether gut-derived exosomes contribute to the pathogenesis of liver injury triggered by intestinal I/R in a murine model and explored the underlying mechanisms. Intestinal I/R models were established by temporally clamping the superior mesenteric arteries of mice. Exosomes were isolated from the intestinal tissue of mice that underwent intestinal I/R or sham surgery according to a centrifugation-based protocol. Exosomes were co-cultured with RAW 264.7 macrophages or injected intravenously in mice. Liposomal clodronate was administered intraperitoneally to deplete the macrophages. Macrophage polarization was determined by flow cytometry, immunohistochemistry, and quantitative polymerase chain reaction. Liver injury was assessed by histological morphology and increased serum aspartate aminotransferase and alanine aminotransferase levels. Exosomes from mice intestines subjected to I/R (IR-Exo) promoted macrophage activation in vitro. Intravenous injection of IR-Exo caused hepatic M1 macrophage polarization and led to liver injury in mice. Depleting macrophages ameliorated liver injury caused by intestinal I/R or the injection of IR-Exo. Furthermore, inhibiting exosome release improved intestinal injury, liver function, and survival rates of mice subjected to intestinal I/R. Our study provides evidence that gut-derived exosomes induce liver injury after intestinal I/R by promoting hepatic M1 macrophage polarization. Inhibition of exosome secretion could be a therapeutic target for preventing hepatic impairment after intestinal I/R.

Isolation of extracellular vesicles from intestinal tissue in a mouse model of intestinal ischemia/reperfusion injury.

Extracellular vesicles (EVs) are small membranous particles that contribute to intercellular communications. Separating EVs from tissue is still a technical challenge. Here, we present a rigorous method for extracting EVs from intestinal tissue in a mouse intestinal ischemia/reperfusion (I/R) model, and for analyzing their miRNA content. The isolated EVs show a typical cup shape with a size peak of 120-130 nm in diameter, confirmed by TEM and NTA. They also express EV markers such as CD9, CD63, CD81, Tsg101 and Alix. Real-time qPCR confirmed that these pellets contain miRNAs related to I/R injury. Our study presents a practical way to isolate EVs from intestinal tissue which is suitable for downstream applications such as miRNA analysis, and provides a novel method for investigating the mechanism of intestinal I/R injury.

Mechanism of simvastatin-induced K562 cell apoptosis.

Statins are being widely used for the therapy and prevention of several types of tumors, including human chronic myelogenous leukemia, but the underlying molecular mechanisms still remain unknown. Therefore, inhibition of cell proliferation, apoptosis and involved molecules were investigated in K562 cells after incubation with simvastatin.The results showed that simvastatin diminished K562 cell proliferation and induced apoptosis. At the same time, the level of reactive oxygen species (ROS) and intracellular calcium concentration increased. Furthermore, nitric oxide (NO) content and inducible NO synthase (iNOS) mRNA expression were significantly higher in the simvastatin-treated group than in the corresponding control group. The elevated ROS level and intracellular calcium concentration, enhanced mRNA expression of iNOS and total NO content might be responsible for the apoptotic and anti-proliferative effects of simvastatin in K562 cells.

In vitro and in vivo study of cell growth inhibition of simvastatin on chronic myelogenous leukemia cells.

BACKGROUND: Statins, a family of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitors, are being investigated for the therapy and prevention of cancers. Here we aimed to investigate the effects of simvastatin on chronic myelogenous leukemia (CML) cells in vitro and in vivo, and to elucidate the mechanisms. METHODS: Cell proliferation and cell cycle were measured after K562 cells were incubated with simvastatin, and differentially expressed genes were determined by oligonucleotide microarray. Changes of 2 genes obtained by oligonucleotide microarray were validated by real-time RT-PCR, and immunohistochemistry was performed to determine expression of proliferating cell nuclear antigen (PCNA). Finally, a xenograft tumor model was constructed to evaluate the effects of simvastatin in vivo. RESULTS: Simvastatin could inhibit K562 cell proliferation, and the inhibition rate was approximately 30% after treatment with 20 mumol/l simvastatin for 48 h. Cell cycle was arrested in G(1) phase, as shown by flow cytometry results. Fifteen downregulated, 9 upregulated cell cycle-related genes and decreased PCNA protein were observed in the presence of simvastatin. Furthermore, simvastatin exhibited impairment of xenograft tumor growth in nude mice and also blocked cell cycle in G(1) phase. CONCLUSION: Simvastatin can inhibit CML cell proliferation in vitro and in vivo, and its mechanisms might be involved in cell cycle regulation.

[Simvastatin-induced apoptosis of K562 cells is mediated by endoplasmic reticulum stress].

To explore the apoptotic effect of simvastatin on K562 cells through endoplasmic reticulum stress, morphological change of apoptotic cells was observed by Hoechst33258 fluorescent staining under fluorescent microscope. Apoptosis rate of cells was determined with annexinV-FITC/PI double staining by flow cytometry; Intracellular calcium concentration ([Ca2+]i) was measured by laser scanning confocal microscope (LSCM); The expression levels of glucose regulated protein 78 (GRP78) and calpain gene mRNA were determined by RT-PCR; The expression levels of caspase-3, -6, -7, -9, -12, calpain and GRP78 proteins were evaluated by Western blotting. In this study, K562 cells treated with simvastatin for 72 h exhibited typical morphological change of apoptosis cells. After 72 h exposed to 10, 20, 30 micromol x L(-1) simvastatin, the apoptotic rates of K562 cells were 12.41%, 19.08% and 23.41%, respectively. Simvastatin induced the increase of [Ca2+]i in K562 cells, fluorescent intensities were 43, 54, and 64, respectively. The expression levels of GRP78 and calpain gene mRNA were up-regulated. The cleavage and activation of caspase-3, -6, -7, -9, -12 and upregulation of GRP78 expression were determined by Western blotting. These findings suggest that endoplasmic reticulum is an important pathway of apoptosis in cells and participates simvastatin-induced apoptosis in K562 cells. It is implied that simvastatin may be suitable for clinical usage in the treatment of myeloma patients.

[Experimental study of simvastatin induced apoptosis of K562 cells by caspase-12 activation].

OBJECTIVE: To explore the apoptotic effect of simvastatin on K562 cells through Caspase-12 activation. METHODS: Morphological changes of apoptotic cells were observed by Hoechst33258 fluorescent staining under fluorescent microscope; Apoptosis rate of cells was determined with annexin V-FITC/PI double staining by flow cytometry; Intracellular calcium concentration ([ca2+]i) was measured by Laser Scanning Confocal Microscope(LSCM); The expression levels of GRP78 and Calpain gene mRNA were determined by RT-PCR; The expression levels of Caspase-3,-6,-7,-9,-12 and GRP78 proteins were evaluated by Western blot. RESULTS: Typical morphological changes of K562 apoptosis cells were observed post 72 hours treated with 10, 20, 30 micromol/L simvastatin. The apoptotic rates of K562 cells were (12.41 +/- 0.32)%, (19.08 +/- 0.26)% and (23.41 +/- 0.36)%, respectively. The fluorescent intensities were 43 +/- 2.9, 54 +/- 2.7 and 64 +/- 2.6, respectively in K562 cells treated with 10, 20, 30 micromol/L simvastatin, which represented the increase of [ca2+]i The expression levels of GRP78 and Calpain gene mRNA were up-regulated. And the cleavage and activation of Caspase-3,-6,-7,-9,-12 and upregulation of GRP78 expression were demonstrated by Western blot detection for the treated cells. CONCLUSION: Caspase-12 is a important pathway of apoptosis in cells and participates simvastatin-induced apoptosis in K562 cells.

[Detection of platelet Ca2+(i), CD62P, CD63 and plasma CD62P in cirrhosis patients].

OBJECTIVES: To study on the relationship between platelet Ca2+(i), CD62P, CD63, serum CD62P (SCD62P) and cirrhosis patients. METHODS: Platelet CD62P, CD63 were determined with flow cytometry, SCD63P with ELISA, and Ca2+(i) in platelet was determined with fluorophotometry. RESULTS: Platelet Ca2+(i), CD62P, CD63, and SCD62P levels in cirrhosis patients were (103.1+/-22.2)nmol/L, (47.6+/-20.0)%, (47.1+/-24.6)%, and (67.6+/-37.6)microg/L, and in controls were (57.6+/-13.1)nmol/L, (3.1+/-0.7)%, (2.5+/-0.7)%, and (24.0+/-6.5)microg/L, respectively. The levels in the former were higher than those in the latter (t > or = 6.148, P<0.05). The above levels in upper gastrointestinal haemorrhage group were much higher than those in the non-haemorrhage group (120.3nmol/L+/-18.8nmol/L vs 91.1nmol/L+/-14.3nmol/L, 64.9%+/-14.7% vs 34.6%+/- 11.9%, 70.9%+/-14.5% vs 30.2%+/-14.4%, and 103.6microg/L+/-14.9microg/L vs 40.8microg/L+/-24.0microg/L, respectively, t > or = 5.380, P<0.05). But the numbers of platelet between the two groups were no obvious difference. CONCLUSIONS: Platelet in the cirrhosis patients is greatly active, and the detection of platelet CD62P, CD63, SCD62P has a certain value in judging the degree of cirrhosis.

Reduction of preanalytical errors in clinical laboratory through multiple aspects and whole course intervention measures.

OBJECTIVE: Errors in preanalytical phase decrease the accuracy of reports from clinical laboratory department. Considering the disqualified rate of preanalytical sample in our hospital, we performed several intervention measures to improve the situation. METHODS: The disqualified sample types and major causes of errors in the preanalytical phase were investigated in clinical laboratory department from September 2008 to August 2009. In the following year, we utilized multiple measures to properly intervene the key points of whole sample collection process, and the preanalytical errors were reanalyzed trimonthly, then the disqualification rate of total, major disqualified sample types and different test groups were calculated to evaluate the effects of the intervention measures. RESULTS: The total disqualification rate in the preanalytical phase obtained from September 2008 to August 2009 was 1.36%, and the major types of disqualified samples were coagulation of anticoagulant sample, sample inadequacy, sample container error, sample information error and sample type error. After one year intervention through key points of whole preanalytical sample collection process, the total disqualification rate dropped to 0.94%, and the disqualification rate of coagulation of anticoagulant sample, sample inadequacy, sample container error, sample information error, and sample type error decreased by 20.45%, 28.00%, 25.00%, 76.92%, and 66.66%, respectively. As for test groups, the decreasing amplitude of biochemical, routine, immunological, microbiological and emergency test group was 47.36%, 33.33%, 20.00%, 50.00%, and 21.43%, respectively. CONCLUSIONS: The overall effect of the interventions is very good, and the disqualification rate of the main causes decreases to various degrees.

Reduction of preanalytical errors in laboratory by establishment and application of training system.

OBJECTIVES: Errors in preanalytical phase occupied for almost half of total errors in clinical laboratory, and the causes are related to medical staff's quality awareness and behaviors. In order to reduce the preanalytical errors in our hospital, we established and applied a training system to improve the situation. METHODS: The disqualified sample types and major causes of errors in the preanalytical phase were investigated in clinical laboratory department from September 2008 to August 2009. In the following year, we established and applied a training system to affect the quality awareness and behaviors of medical staff. Questionnaire investigation was analyzed to illustrate the changes of respondents' quality awareness and behavior, and the preanalytical errors were reanalyzed according to different departments to evaluate the effects of the intervention measures. RESULTS: The total disqualification rate in the preanalytical phase obtained from September 2008 to August 2009 was 1.36%, and the major types of disqualified samples were coagulation of anticoagulant sample, sample inadequacy, sample container error, sample information error, and sample type error. After application of established training system, respondents' quality awareness on preanalytical samples changed dominantly, and respondents' own behavior and behavior to others also changed notably. The total disqualification rate in preanalytical phase dropped to 0.94%, among 33 clinical departments, the preanalytical errors in 25 departments decreased to various degrees, and 10 departments had overall decreasing amplitude over 50%. CONCLUSIONS: The overall effect of the application of established training system is very good, and the disqualification rate of the major departments decrease to various degrees.

Study on the resistant genes to carbapenems and epidemiological characterization of multidrug-resistant Acinetobacter baumannii isolates.

The purpose of this study was to examine the carbapenemase-encoding resistance genes and analyze homologous of multidrug-resistant Acinetobacter baumannii (MRAB) isolates from nosocomial infections. Seventy-six A. baumannii strains were collected from inpatients and object surface of devices in intensive care units from May 2008 to February 2011. Antibiotic susceptibility testing of 18 antimicrobial agents was performed. The presence of carbapenemase-encoding resistance genes was investigated by polymerase chain reaction. Genotyping and dendrogram analysis of A. baumannii strains from nosocomial infections were performed using the DiversiLab System. All of the 76 clinical A. baumannii isolates were shown multidrug resistance. The bla(OXA-23) gene was identified in the 76 MRAB strains, while bla(OXA-24), bla(OXA-58), VIM, IMP-1, IMP-4, SIM, and blaNDM-1 were absent in all. Twenty-four A. baumannii strains from the samples with nosocomial infections were classified into four unrelated groups and nine patterns. In conclusion, production of bla(OXA-23) in MRAB is one of the molecular mechanisms responsible for carbapenem resistance. The MRAB strains from unrelated groups show different drug resistance, but the homologous strains also have different drug resistance. Homologous analysis can provide scientific evidence for evaluation of epidemic status of nosocomial infection caused by MRAB.

[Establishment of protein fingerprint database of Salmonella paratyphi A using SELDI-TOF-MS].

AIM: To establish a protein fingerprint database of Salmonella paratyphi A by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). METHODS: Thirty-six clinical bacterial isolates and 96 control bacteria isolates were collected and identified using 16S rDNA sequencing. Bacterial proteins were detected by SELDI-TOF-MS, and all protein fingerprints were analyzed by ProteinChip and Biomarker Wizard software. The analysis results were used to set up a classification tree model by means of BioMarker Patterns software. At the same time, the data were tested by a blinded validation. RESULTS: In the range of M(r); 3 000-20 000, we obtained 104 protein peaks, of which 90 were of statistical significance (P<0.01). A protein peak with mass-to-charge ratio(M/Z) 10 061.7 was chosen to establish the classification tree model of Salmonella paratyphi A, and the sensitivity and specificity of Salmonella paratyphi A diagnosis was 100% as shown by the blinded validation. CONCLUSION: The classification tree model of Salmonella paratyphi A can be not only established using SELDI-TOF-MS technology, but also used for the rapid identification of Salmonella paratyphi A.

Column chromatographic extraction and preparation of cordycepin from Cordyceps militaris waster medium.

Large amounts of solid medium containing cordycepin, used in the industrial production of Cordyceps militaris through solid fermentation, are discarded as waste and contaminate the environment. We have developed a new column chromatographic extraction (CCE) method for the extraction of cordycepin from this waste and a preparation method for further separation and purification. Dried waste material was imbibed in four times its volume of water for 6 h, transferred to columns and eluted with water. Eluates were directly separated with macroporous resin DM130 columns followed by purification steps, including precipitation, crystallization, and polyamide column chromatography. Extraction rates of more than 97% were obtained with 12 volumes of water for a single column and 4 volumes of water for eluates circulated through 3 different columns designed to concentrate cordycepin. Cordycepin (98% pure) was obtained following the separation and purification processes, with an overall recovery rate of more than 90%. The CCE method has high extraction efficiency, uses a minimum volume of solvent and can be used for both quantitative analysis and large preparations of cordycepin from waste. The preparation method is simple, highly efficient, energy-saving, environmentally friendly, and has been demonstrated to be effective for large preparations of cordycepin from waste with low equipment and operating costs.

Rapid identification of Staphylococcus aureus by surface enhanced laser desorption and ionization time of flight mass spectrometry.

Staphylococcus aureus (S. aureus), a vital nosocomial pathogen, is responsible for several diseases. With the increasing isolation rate in clinical specimens, rapid identification of this bacterial species is required. But present identification via conventional methods is time-consuming and lacks accuracy. The purpose of the current study was to evaluate the use of surface enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) for rapid identification of S. aureus. A total of 120 clinical isolates of S. aureus and 153 non-S. aureus species were identified by conventional methods, and the species nature of all staphylococci was further confirmed by 16S rDNA sequencing. All strains observed were analyzed by SELDI-TOF MS. An identification model for S. aureus was developed and validated by an artificial neural network. The model based on 6 protein peaks exhibited a sensitivity of 98.4% and specificity of 98.6%. This strategy has the potential for rapid identification of S. aureus.

[Experimental research of targeting hTERT gene inhibited in hepatocellular carcinoma therapy by RNA interference].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) is a new gene blocking technology that silences target gene at post-transcription level induced by the small interference RNA (siRNA). RNAi has been demonstrated great prospect in gene functional research and gene therapy areas. Nowadays, RNAi has been reported to be used to inhibit the expression of endogenous genes including cyclophilin, GAPDH, p53, and c-myc; and there were some progresses in the therapy of the diseases caused by AIDS and hepatitis viruses with RNAi. However, hTERT gene, which was highly expressed in hepatocellular carcinoma and other malignant neoplasm, has not been researched by RNAi. In present research, we utilized RNAi to inhibit hTERT gene expression in vitro and in vivo, investigated the feasibility and specificity of gene therapy for hepatocellular carcinoma. METHODS: Small interference RNAs homologous to hTERT gene were designed,pTZU6+1-shRNA-hTERT vector was constructed and transfected into hepatocellular carcinoma SMMC-7721 cells and transplanted SMMC-7721 tumor in nude mice to induce RNAi. The changes of hTERT gene expression and tumor cell proliferation in both siRNA treatment groups and control group were determined by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunochemistry in vitro and in vivo. RESULTS: The expression of hTERT had been obviously inhibited by RNAi in vitro. The inhibition rate of cell growth was 37.5% after pTZU6+1-shRNA-hTERT vector was transfected to hepatocellular carcinoma SMMC-7721 cells; the phase of cell cycle indicated the reduction of S phase, while G(1)/G(0) phase increased. The mRNA level of hTERT decreased from 99.4% to 53.1%, its protein expression reduced from 86.3% to 46.6%. The tumor size reduced after treated with pTZU6+1-shRNA-hTERT vector in vivo; hTERT mRNA level decreased from 99.1% to 76.2%, and its protein expression decreased from 87.2% to 61.8% in siRNA treatment group. In contrast, there were no changes in control groups in vitro and in vivo. CONCLUSION: RNAi inhibits the hTERT gene expression and proliferation of hepatocellular carcinoma SMMC-7721 cells with specificity, and is a possible new approach for neoplasm gene therapy.