Changes in the structure of intestinal mucosal flora in colorectal cancer patients / 南方医科大学学报

OBJECTIVE@#To investigate the changes in bacterial flora in fecal samples, at the tumor loci and in adjacent mucosa in patients with colorectal cancer (CRC).@*METHODS@#We collected fecal samples from 13 patients with CRC and 20 healthy individuals and tumor and adjacent mucosa samples from 6 CRC patients. The differences in bacterial composition between the fecal and mucosa samples were analyzed with 16S rDNA sequencing and bioinformatics methods. We also detected the total number of bacteria in the feces using flow cytometry, isolated and identified the microorganisms in the fecal and mucosa samples using common bacterial culture media. We further tested the effects of 7 isolated bacterial strains on apoptosis of 3 CRC cell lines using lactate dehydrogenase detection kit.@*RESULTS@#The bacterial α-diversity in the feces of healthy individuals and in adjacent mucosa of CRC patients was significantly higher than that in the feces and tumor mucosa in CRC patients (P < 0.05). Lactobacillaceae is a specific bacteria in the feces, while Escherichia, Enterococcus, and Fusobacterium are specific bacteria in tumor mucosa of CRC patients as compared with healthy individuals. Cell experiment with3 CRC cell lines showed that Bacteroides fragilis isolated from the tumor mucosa of CRC patients produced significant inhibitory effects on cell proliferation (P < 0.0001), while the isolated strain Fusobacterium nucleatum obviously promoted the proliferation of the cell lines (P < 0.001).@*CONCLUSION@#The bacterial flora in the feces, tumor mucosa and adjacent mucosa of CRC patients is significantly different from that in the feces of healthy individuals, and the fecal flora of CRC patients can not represent the specific flora of the tumor mucosa. Inhibition of F. nucleatum colonization in the tumor mucosa and promoting B. fragilis colonization may prove beneficial for CRC treatment.

Evaluation of the China Healthcare Improvement Initiative(2018-2020) in tertiary public hospitals in China: results of institutional survey / 中华医院管理杂志

Objective:To evaluate the implementation of the China Healthcare Improvement Initiative(CHII)from 2018 to 2020 in 143 tertiary public hospitals in China.Methods:In March 2019 and from January to March in 2021, 143 tertiary public hospitals in 31 provinces of China were investigated using the unified " medical institution questionnaire Ⅰ" and " medical institution Questionnaire Ⅱ" . The data were collected by means of hospital self-report and expert on-site scoring. Descriptive and inferential statistical analysis were used to analyze the data, and the data of two cross-sectional surveys were compared and analyzed.Results:The average score rate of implementing CHII in 143 sample hospitals in 2020 was 88.9%, which was higher than that in 2018(84.4%). The appointment diagnosis and treatment system, clinical pathway management system, day service, smart hospital and humanistic service were significantly improved. In 2020, the average score rate of logistics service, high quality nursing service and clinical pathway management system was higher than 95%, while the average score rate of day service, telemedicine system and medical social work system was lower than 85%. The total score rate of general hospitals was significantly higher than that of specialized hospitals( P<0.001). In 2020, the proportion of hospitals with full marks in 29 secondary indicators(74.4%)was more than 80%, reaching the standard level. Conclusions:The implementation level of CHII in tertiary public hospitals in China has been improved continuously and made significant progress, but some dimensions and indicators need to be further improved.

Effect and mechanism of aspirin combined with vinorelbine on non-small cell lung cancer / 中国中药杂志

The study aimed to investigate the effect and mechanism of aspirin combined with vinorelbine on the proliferation and apoptosis of non-small cell lung cancer cells. 3-(4-dimethylthiazolyl-2)-2-diphenyltetrazolium bromide(MTT) was used to detect the cytotoxic effect of aspirin and vinorelbine on H460 and A549 cells, and half of inhibitory concentration(IC_(50)) value of drugs as well as synergistic effect were calculated. The results showed that both aspirin and vinorelbine inhibited the cancer cells proliferation by a concentration-dependent manner with IC_(50 )values of 1.553 mmol·L~(-1) and 0.033 μmol·L~(-1) in H460 cells, respectively. The IC_(50 )values of aspirin and vinorelbine were 1.70 mmol·L~(-1)and more than 20 μmol·L~(-1) in A549 cells. The combination index(CI) value was used to evaluate the combined effect of two drugs. Aspirin combined with vinorelbine had synergistic effects at the ratio of 100∶1 on H460 cells and 1∶10 on A549 cells(CI<1). Clone formation and 4',6-diamidino-2-phenylindole(DAPI)/propidium iodide(PI) staining assays were used to verify the effect of the combination of two drugs on proliferation of H460 cells. Compared with the aspirin single group, the combination group had stronger inhibitory effect on the proliferation of H460 cells and the clone formation rate was 49.5%(P<0.05). Furthermore, apoptosis, mitochondrial membrane potential, reactive oxygen species and Western blot experiments were used to explore the synergistic mechanism of aspirin combined with vinorelbine in inhibiting cell proliferation. The results showed that the cancer cell apoptosis rate was 52.8%, the mitochondrial membrane potential was decreased to 33.1%, and the levels of reactive oxygen species was increased to 73.3% in combination group, which were significantly different from those of the single drug treatment groups(P<0.05). Western blot showed that combination group significantly up-regulated the expressions of Bax, p53, cleaved caspase-3 and cytochrome C, while down-regulated the expression of anti-apoptosis proteins such as Bcl-xL and Bcl-2 when compared with single groups. Our results suggested that aspirin combined with vinorelbine could synergistically inhibit the proliferation of H460 cells by inducing the cell apoptosis through the mitochondrial pathway.

Preparation and evaluation of novel purine derivative F7 thermosensitive liposome / 国际药学研究杂志

Objective To prepare F7 thermosensitive liposome and evaluate its physicochemical properties, then investigate its cytotoxicity against tumor cells in vitro. Methods The F7 thermosensitive liposome was prepared by the pH gradient active drug loading method using dipalmitoyl phosphatidylcholine myristoyl lyso-phosphocholine and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy (polyethylene glycol)-2000 as membrane materials. The encapsulation efficiency and drug loading were determined for the F7 thermosensitive liposome by HPLC. The phase transition temperature of F7 thermosensitive liposome was investigated by differential scanning calorimetry;the liposome morphology was observed by atomic force microscopy;the drug release of liposome was examined by dialysis;and the particle size and zeta potential were measured through Malvern particle size analyzer. The cytotoxicity of F7 and F7 thermosensitive liposome was determined by the MTT method, and the freeze-drying process was optimized using the designexpert software. Results The encapsulation efficiency of F7 thermosensitive liposomes was (97.56±0.22) ％, and the drug loading ratio was (1.51±0.01) ％. The phase transition temperature of F7 thermosensitive liposome was 39.9℃, the zeta potential was (-15.10±0.85) mV, the particle size was (86.94±1.21) nm, and the poly disperse coefficient was 0.17±0.01. Compared with the F7 injection, the F7 thermosensitive liposomes showed a stronger, dose-dependent inhibitory effect on the growth of lung cancer H1299 and breast cancer MCF-7 cells. The freeze-dried powder of liposomes dissolved well with the encapsulation efficiency of 95％ and the particle size of approximately 130 nm. Conclusion The F7 thermosensitive liposome prepared by the pH gradient active drug loading method has high encapsulation efficiency and good stability. The preparation method is simple and feasible for further development of the F7 preparation.

EPSAH, an exopolysaccharide from Aphanothece halophytica GR02, improves both cellular and humoral immunity as a novel polysaccharide adjuvant / 中国天然药物

EPSAH is an exopolysaccharide from Aphanothece halophytica GR02. The present study was designed to evaluate its toxicity and adjuvant potential in the specific cellular and humoral immune responses in ovalbumin (OVA) in mice. EPSAH did not cause any mortality and side effects when the mice were administered subcutaneously twice at the dose of 50 mg·kg(-1). Hemolytic activity in vitro indicated that EPSAH was non-hemolytic. Splenocyte proliferation in vitro was assayed with different concentrations of EPSAH. The mice were immunized subcutaneously with OVA 0.1 mg alone or with OVA 0.1 mg dissolved in saline containing Alum (0.2 mg) or EPSAH (0.2, 0.4, or 0.8 mg) on Day 1 and 15. Two weeks later, splenocyte proliferation, natural killer (NK) cell activity, production of cytokines IL-2 from splenocytes, and serum OVA-specific antibody titers were measured. Phagocytic activity, production of pro-inflammatory cytokines IL-1 and IL-12 in mice peritoneal macrophages were also determined. EPSAH showed a dose-dependent stimulating effect on mitogen-induced proliferation. The Con A-, LPS-, and OVA-induced splenocyte proliferation and the serum OVA-specific IgG, IgG1, and IgG2a antibody titers in the immunized mice were significantly enhanced. EPSAH also significantly promoted the production of Th1 cytokine IL-2. Besides, EPSAH remarkably increased the killing activities of NK cells from splenocytes in the immunized mice. In addition, EPSAH enhanced phagocytic activity and the generation of pro-inflammatory cytokines IL-1 and IL-12 in macrophages. These results indicated that EPSAH had a strong potential to increase both cellular and humoral immune responses, particularly promoting the development of Th1 polarization.

Establishment of an in vitro blood-brain barrier model by co-culturing rat brain microvascular endothelial cells,pericytes and astrocytes / 中国药理学通报

Aim To establish in vitro blood-brain barrier (BBB) model with characteristics of simulation of in vivo BBB by primi-tive co-culture of brain-microvessel endothelial cells (BMECs) with brain-microvessel pericytes (BMPC)and astrocytes (AS). Methods BMECs,BMPC and AS from SD rats were primitively isolated,purified and cultured,and then primitive culture cells were identified by cellular morphological and immunocytochemi-cal staining methods.Five types of in vitro BBB models were es-tablished by using Millicell culture insert (pore diameter 0.4μm)and their barrier functions were evaluated by detection of transendothelial electrical resistance (TEER),permeability of sodium fluorescent (Na-FLU ),expression of alkaline phospha-tase (AKP)and γ-glutamyl transpeptidase (γ-GT1 ),and simi-larity of permeation amount for positive drugs in vitro and in vivo BBB conditions.Results Primitive culture of BMECs presented typical pebbles-like structure,BMPC presented larger soma with branching property,AS presented slender synapse and shallower cytoplasm.Moreover,immunocytochemical staining results iden-tified primitive cells were targeted cells.TEER value for co-cul-ture of BMECs,BMPC and AS reached (478 ±25 )Ω· cm2 , permeability coefficients (Papp )value of Na-FLU was [(8.23 ± 0.78) ×10 -6 ]cm·s-1 ,expression of AKP and γ-GT1 were (6.90 ±0.27 )King unit · g-1 Pro and (4.39 ±0.32 )μg · g-1 Pro respectively.Moreover,good correlation could be found in Papp for positive controls in vitro and in vivo BBB models (R2=0.92).Conclusion The established in vitro BBB model by using primitive co-culture of BMECs with BMPC and AS posses-ses in vivo BBB properties in cell morphology,structures and barrier functions,and can be used as a powerful tool for studying physiology,pathology of BBB and screening candidate com-pounds.

Effects of recombinant hirudin variant Ⅲ on expression of apoptosis-related genes during galactose-mediated human lens epithelial cells damage / 中华实验眼科杂志

Background Recombinant hirudin variant Ⅲ(rHV3) can effectively prevent galactose-induced human lens epithelial cells LECs injury,but little is known about the molecular mechanism of its action.Objective The present study was to investigate the effects of rHV3 on the expression of apoptosis-related genes in damaged LECs induced by galactose.Methods The rHV3 was extracted by our research group,and the biological activity of rHV3 was identified by titration of thrombase according to Markwardt's method.Human LECs (SRA01/04) were cultured using 125×10-3 mol/L D-galactose+10% FBS+D/F12 medium to establish the damaged human LECs model.rHV3 was added into the medium of the damaged human LECs model.Human LECs were cultured in D/F12 medium containing 10% FBS as normal control.The expression of apoptosis-related genes,such as aldose reductase (AR),bax,bcl2 and p53,in LECs at the mRNA level was detected using RT-PCR.The abundance ratio of target genes was presented with the absorbance (A) of gene mRNA/GAPDH mRNA.Results Compared to the normal control group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were significantly elevated in model group (t=3.90E-06,t=8.44E-04,t=5.15E-08,P＜0.01).However,in the rHV3-treated group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were lower than those of model group (t=5.90E-06,t=1.51E-04,t=3.42E-06,P＜0.01).The bcl2 mRNA/GAPDH mRNA was markedly downregulated in the model group when compared with the normal control group (t=1.86E-05,P＜0.01);while after rHV3 addition,bcl2 mRNA/GAPDH mRNA increased in comparison with the model group (t=8.56E-05,P＜0.01).Conclusion 125×10-3mol/L D-galactose induces the damage and apoptosis of human LECs.rHV3 likely plays a protective function on D-galactose-induced damage of human LECs by inhibiting the polyol pathway and mitochondria-mediated pathway.

Design, synthesis and anti-hBV evaluation of adefovir mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs / 药学学报

A series of adefovir mono-L-amino acid esters, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs with more potent anti-HBV activity and lower nephrotoxicity were designed and synthesized. Adefovir bis (L-amino acid) ester was used as lead compound, according to pathological and pharmacological findings that non-steroidal anti-inflammatory drugs can effectively inhibit the organic anion transporter 1 (hOAT1)-mediated adefovir phosphonic acid pairs of anion transport across tubular basement membrane thereby reducing the nephrotoxicity of adefovir. Flatten design principle was used to introducing non-steroidal anti-inflammatory drugs structural fragments to design and synthesize target adefovir mixture ester prodrugs. HepG2 2.2.15 cell line was used as in vitro anti-HBV activity evaluation model. Five compounds exhibited antiviral activity, and compound 18 showed the most potent anti-HBV activity and relatively high selective index (EC50 3.92 micromol L(-1), SI 9.97). HK-2 cell line was used as in vitro model to evaluate nephrotoxicity. Results suggested the target compounds have lower cytotoxicity than the positive control. Moreover, by analyzing the primary structure and activity relationship of these compounds, it could suggest that mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs strategy has significant potential in the acyclic nucleoside phosphonates prodrug design.

Design, synthesis and anti-hBV evaluation of adefovir mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs / 药学学报

A series of adefovir mono-L-amino acid esters, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs with more potent anti-HBV activity and lower nephrotoxicity were designed and synthesized. Adefovir bis (L-amino acid) ester was used as lead compound, according to pathological and pharmacological findings that non-steroidal anti-inflammatory drugs can effectively inhibit the organic anion transporter 1 (hOAT1)-mediated adefovir phosphonic acid pairs of anion transport across tubular basement membrane thereby reducing the nephrotoxicity of adefovir. Flatten design principle was used to introducing non-steroidal anti-inflammatory drugs structural fragments to design and synthesize target adefovir mixture ester prodrugs. HepG2 2.2.15 cell line was used as in vitro anti-HBV activity evaluation model. Five compounds exhibited antiviral activity, and compound 18 showed the most potent anti-HBV activity and relatively high selective index (EC50 3.92 micromol L(-1), SI 9.97). HK-2 cell line was used as in vitro model to evaluate nephrotoxicity. Results suggested the target compounds have lower cytotoxicity than the positive control. Moreover, by analyzing the primary structure and activity relationship of these compounds, it could suggest that mono-L-amino acid ester, mono non-steroidal anti-inflammatory drugs carboxylic ester prodrugs strategy has significant potential in the acyclic nucleoside phosphonates prodrug design.

Expression in Escherichia coli, purification and enzymatic properties of porcine urate oxidase / 生物工程学报

The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase (pUOX), optimize the conditions of the expression of pUOX in recombinant Escherichia coli BL21(DE3), and analyze the in vitro activity and the enzymological properties of pUOX. The pUOX gene was amplified by RT-PCR from the extracted total RNA of porcine liver, and was inserted into the prokaryotic expression vector pET30a(+) to construct a recombinant expression vector pET30a(+)/pUOX. We identified the recombinant vector by endonuclease digestion and sequence analysis. The pUOX gene was amplified and cloned into the vector pET30a(+) successfully. And then the recombinant vector was transformed into E. coli BL21(DE3). The expression of pUOX with a molecular of approximately 41 kD was induced by IPTG. We also optimized the expression conditions of the recombinant protein. The recombinant protein was mostly located in the cytoplasm and it was insoluble. After the inclusion body was solved in 8 mol/L urea and refolding in 2 mol/L urea, the recombinant protein was collected and purified by Ni2+-NTA column. This recombinant protein had a specific activity of 50.61 IU/mg and showed similar properties of optimum temperature and thermal stability, base on the enzymatic assay and analysis of enzymological properties. These results would help to analyze the in vivo activity by testing animal.

Effect of ginkgolide B on the platelet-activating factor induced changes of chemotaxis and cytoskeleton of macrophages / 药学学报

<p><b>AIM</b>To study the inhibitory effect of ginkgolide B (BN52021) on the PAF induced changes of chemotaxis of murine peritoneal macrophages and the related polymerization of F-actin.</p><p><b>METHODS</b>Chemotaxis assays were performed using a modified 48-well Boyden chamber. Actin polymerization of murine peritoneal macrophages was analyzed by flow cytometry using a specific fluorescent stain.</p><p><b>RESULTS</b>Peritoneal macrophages significantly migrated toward platelet-activating factor (PAF) through a micropore filter; however, in the presence of PAF receptor antagonist BN52021 (0.01 nmol x L(-1) -0.1 micromol x L(-1)), the migration was significantly inhibited. Moreover, BN52021 inhibited the actin polymerization of murine peritoneal macrophages induced by PAF in the presence of Ca2+, but not in Ca2+ -free medium.</p><p><b>CONCLUSION</b>The results suggested that preventing polymerization of F-actin may be a pathway by BN52021 to inhibit the chemotaxis of macrophages, and this effect seems to be Ca2+ dependent. The data further indicated that inhibition of PAF induced macrophage chemotaxis is an important mechanism underlying the anti-inflammatory action of BN52021.</p>

Inhibitory effect of ginkgolide B on angiogenesis in chronic inflammation / 药学学报

<p><b>AIM</b>To investigate the inhibitory effect of ginkgolide B on angiogenesis in chronic inflammation and the possible mechanisms.</p><p><b>METHODS</b>The murine chronic granulomatous air pouch model was used to observe the anti-angiogenesis effect of ginkgolide B. The vascular index was determined by colorimetry of carminic acid, and angiogenesis was observed by histology method. The interleukin-1beta (IL-1beta) levels in mice serum and in supernatants of U937 cell culture stimulated by phorbol 12-myristate 13-acetate (PMA) were detected by radioimmunoassay (RIA). The tumor necrosis factor-alpha (TNF-alpha) levels in mice serum and in supernatant of U937 cell culture were measured by cytotoxicity bioassay. The mRNA expression of IL-1beta and TNF-alpha of U937 cell culture was investigated by RT-PCR.</p><p><b>RESULTS</b>Oral administration of ginkgolide B 25 and 100 mg x kg(-1) was shown to significantly inhibit the vascular index of murine chronic granulomatous air pouch model with the inhibitory rate of 22.52% and 25.29%, respectively. This result was supported by histological observation. Concomitantly, the IL-1beta levels in mice serums were also significantly decreased with the inhibitory rate of 50.61% and 58.66%; so were the TNF-alpha levels with the inhibitory rate of 28.91% and 52.41%. Ginkgolide B at concentration of 1 x 10(-5) to 1 x 10(-8) mol x L(-1) could also reduce both the IL-1beta and TNF-alpha contents in the supernatants of U937 cell culture stimulated by PMA, but the scopes of changes were much different. For IL-1beta the IC50 was 1.93 x 10(-8) mol x L(-1), while ginkgolide B at concentration of 1 x 10(-5) mol x L(-1) only decreased the release of TNF-alpha by 25.99%. Furthermore, ginkgolide B at concentrations of 1 x 10(-5) to 1 x 10(-7) mol x L(-1) was shown to significantly inhibit TNF-alpha mRNA expression of U937 cells; and at concentrations of 1 x 10(-5) and 1 x 10(-6) mol x L(-1) could inhibit IL-1beta mRNA expression.</p><p><b>CONCLUSION</b>Ginkgolide B was shown to significantly inhibit angiogenesis of the murine chronic granulomatous air pouch model, reduce the IL-1beta and TNF-alpha levels in mice serums, and significantly inhibit IL-1beta and TNF-alpha mRNA expression and protein secretion in supernatants of U937 cell culture. It was suggested that reduction of proangiogenic cytokines IL-1beta and TNF-alpha secretion may contribute to the anti-angiogenesis effect of ginkgolide B in the murine chronic granulomatous air pouch model.</p>