Inhibitory effect of astragalus membranaceus injection on apoptosis and autophagy induced by enterovirus 71 / 中国生物制品学杂志

@#Objective To investigate the effect of astragalus membranaceus（AM）injection on apoptosis and autophagy of human gastric epithelial cell line（GES⁃1）induced by enterovirus 71（EV71）. Methods GES⁃1 cells were cultured in vitro and divided into infected group（EV71 infected at a MOI of 3 and control group（no virus infected）. The morpho⁃logical changes of EV71 infected cells were observed by inverted microscope. The level of VP1 in GES⁃1 cells infected with EV71 was detected by Western blot；CCK⁃8 assay was used to detect the viability of GES⁃1 cells infected with EV71；Nuclear staining with DAPI was used to observe the morphological changes of nuclear apoptosis infected with EV71. GES⁃1 cells were divided into control group（without virus infection），infection group and AM intervention group with final concentration of 1，2. 5，5 and 10 μg／mL，respectively. Western blot was used to detect the effect of AM intervention on the expression of apoptosis⁃related proteins Caspase⁃3，PARP and autophagy⁃related proteins LC3 and P62 in GES⁃1 cells infected withEV71. CCK⁃8 method was used to detect the effect of AM intervention on the viability of GES⁃1 cells infected with EV71. Results GES⁃1 cells were round，shrunken with nuclear pyknosis and uneven size；VP1 level increased（t = 41. 56，P < 0. 01），cell viability decreased（t = 19. 07，P < 0. 01），Caspase⁃3 and PARP proteins were cut off（t = 35. 29 and 3. 648， P < 0. 01 and 0. 021 8，respectively），LC3Ⅱ／LC3Ⅰ ratio increased（t = 10. 16，P = 0. 000 5）and P62 protein was degraded（t = 68. 68，P < 0. 01）；AM inhibited the degradation of Caspase⁃3，PARP and P62 proteins induced by EV71 （t = 52. 66，59. 60 and 40. 22，respectively，each P < 0. 01）and increased the ratio of LC3Ⅱ／LC3Ⅰ（t = 5. 521，P = 0. 005 3），andreducedtheinhibitoryeffectofEV71ontheviabilityofGES⁃1cells（t =4. 420，P =0. 0115）. Conclusion EV71 infection induced apoptosis of GES⁃ 1 cells and AM intervention inhibited EV71 induced apoptosis by inhibiting EV71 induced autophagy.

Kallikrein-related peptidase 7 is a potential target for the treatment of pancreatic cancer.

Pancreatic cancer is one of the deadliest cancers with very poor prognosis, and the five-year survival rate of the patients is less than 5% after diagnosis. Kallikrein-related peptidases (KLKs) belong to a serine protease family with 15 members that play important roles in cellular physiological behavior and diseases. The high expression level of KLK7 in pancreatic cancer tissues is considered to be a marker for the poor prognosis of this disease. In this work, we set out to investigate whether KLK7 could be a target for the treatment of pancreatic cancer. Short hairpin RNAs (shRNAs) were designed and constructed in lentivirus to knock down KLK7 in pancreatic cancer cell line PANC-1, and the real time cellular analysis (RTCA) was used to evaluate cell proliferation, migration and invasion abilities. Small molecules inhibiting KLK7 were discovered by computer-aided drug screening and used to inhibit PANC-1 cells. Our results confirmed that KLK7 is significantly up-regulated in pancreatic cancer tissue, and knocking down or inhibiting KLK7 efficiently inhibited the proliferation, migration and invasion of pancreatic cancer cells. This study suggested that KLK7 could be a potential chemotherapy target for treatment of pancreatic cancer, which would provide us a novel strategy for the treatment of this disease.

A comparative study on postoperative mortality prediction of SFLI scoring system and Child-Pugh classification in patients with hepatocellular carcinoma.

PURPOSE: In our previous study, we have established the clinical significance of the SFLI (scoring formula of liver injury), the purpose of this study was to compare the SFLI system and the Child-Pugh grading system in the prediction of postoperative mortality in patients with hepatocellular carcinoma (HCC). METHODS: 114 patients with HCC who underwent surgical treatment were enrolled. According to the requirement of the indicators for the Child-Pugh classification, various indices (including albumin [ALB], total bilirubin [TBIL], prothrombin time [PT], ascites, and hepatic encephalopathy) were considered in these patients before surgery, and then Child-Pugh grading was performed. Similarly, the serum biochemical markers including ALB, pre-albumin (PA), TBIL, serum creatining (SCR), international normalized ratio (INR), alanine transminase (ALT), aspartate transaminase (AST), γ-glutamyl transpeptidase (ggr;-GT), alkaline phosphatase (ALP), PT, activated partial thromboplastin time (APTT), and thrombine time (TT) were collected before surgery for SFLI analysis. The predicted postoperative mortality rates of these two scoring models and their diagnostic efficacy were analyzed and compared. RESULTS: According to the Child-Pugh grading system, in level A, B and C were 75, 35, and 4 cases respectively, and the corresponding mortality rates were 1.3% (1/75), 17.1% (6/35) and 75% (3/4). Meanwhile, according to the SLFI classification, the number of patients in the grade I, I+, II, and III were 36, 29, 28, and 21, respectively, and the corresponding mortality rates were 0, 0, 14.3% (4/28), and 28.6% (6/21), respectively. The patient mortality rate increased significantly with increasing grading (p<0.01). These two classification methods were further compared using ROC analysis, in which the area under the curve (AUC) for the Child-Pugh method was 10.2% with a 95% confidence interval (95% CI) 17-18, and the AUC of SFLI was 88.2% with a 95% CI 80-96. CONCLUSION: The SFLI scoring system is very useful in the assessment of liver function and postoperative mortality, and its grading standard is much better than the traditional Child-Pugh classification in many aspects.

Decreased tumstatin-mRNA is associated with poor outcome in patients with NSCLC.

Tumstatin is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. The purpose of this study was to evaluate the correlation between tumstatin-mRNA expression and the clinicopathologic characteristics, tumor angiogenesis, outcome of patients with non-small cell lung cancer (NSCLC). Specimens from 68 patients with NSCLC were recruited in this study. Tumstatin-mRNA expression and protein level in tumor tissues were quantified respectively by quantitative RT-PCR and ELISA. Microvessel density (MVD) was determined by CD34 immunostaining. The correlation of tumstatin-mRNA expression levels with clinicopathologic variables, tumor angiogenesis, and prognosis was analyzed. Tumstatin-mRNA expression levels were decreased in tumor. Tumstatin-mRNA expression level was significantly correlated with its protein level in tumor (r = 0.562; P = 0.001). Tumstatin-mRNA expression levels in poorly differentiated tumor tissues were significantly lower than in well-differentiated tumor tissues (P < 0.004). Furthermore, tumor tumstatin-mRNA expression were also significantly related to tumor pathologic stage (P = 0.032) and MVD (r = -0.77, P < 0.0001). Overall survival (OS) and disease-free survival (DFS) analysis indicated that NSCLC patients with low tumstatin-mRNA expression had poorer OS and DFS than those with high expression (P = 0.015 and 0.037; respectively). Multivariable Cox regression analysis revealed that the tumstatin-mRNA expression could be an independent prognostic indicators in both DFS and OS. Tumstatin-mRNA expression levels are down-regulated in NSCLC tissues. Tumstatin-mRNA expression level correlates with prognosis, which suggests that tumstatin-mRNA is a new potential independent marker of favorable prognosis in NSCLC.

Association of TNF-α-308 and -238 polymorphisms with risk of cervical cancer: a meta-analysis.

Published data on the associations between tumor necrosis factor-alpha (TNF-α) promoter -308G>A and -238G>A polymorphisms and cervical cancer risk are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. Data were collected from MEDLINE and PubMed databases. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were calculated in a fixed/random effect model. 13 separate studies including 3294 cases and 3468 controls were involved in the meta-analysis. We found no association between TNF-α-308G>A polymorphism and cervical cancer in overall population. In subgroup analysis, significantly elevated risks were found in Caucasian population (A vs. G: OR=1.43, 95% CI=1.00- 2.03; AA vs. GG: OR=2.09, 95% CI=1.34-3.25; Recessive model: OR=2.09, 95% CI=1.35-3.25) and African population (GA vs. GG: OR=1.53, 95% CI=1.02-2.30). An association of TNF-α-238G>A polymorphism with cervical cancer was found (A vs. G: OR=0.61, 95% CI=0.47-0.78; GA vs. GG: OR=0.59, 95% CI=0.45-0.77; Dominant model: OR=0.59, 95% CI=0.46-0.77). When stratified by ethnicity, similar association was observed in Caucasian population (A vs. G: OR=0.62, 95% CI=0.46-0.84; GA vs. GG: OR=0.59, 95% CI=0.43-0.82; Dominant model: OR=0.60, 95% CI=0.44-0.83). In summary, this meta-analysis suggests that TNF-α-238A allele significantly decreased the cervical cancer risk, and the TNF-α-308G>A polymorphism is associated with the susceptibility to cervical cancer in Caucasian and African population.

[Clinical analysis of 16 AIDS patients complicated with toxoplasmic encephalitis].

Sixteen AIDS patients complicated with toxoplasmic encephalitis (TE) were retrospectively analyzed between August 2008 to August 2009 with a mean age of (37.0 +/- 11.6) years. The most common clinical symptoms were headache (68.8%, 11/16) and fever (62.5%, 10/16), and 6 with Babinski sign (37.5%). 81.3%(13/16) were with CD4+ cells < 200/mm. Both sera and CSF showed 62.5% (10/16) TOXO-IgG positive by ELISA. CT and MRI scan demonstrated bilateral and multiple lesions with marked peripheral edema effect, and an enhanced scanning showed small finger ring as the major feature. 15 patients got improved by either oral sulphadiazine tablets or sulphadiazine tablets plus clindamycin capsule, 10 cases received combined HAART treatment, and 1 case died with septic shock.

Development of an ELISA for quantification of tumstatin in serum samples and tissue extracts of patients with lung carcinoma.

BACKGROUND: Tumstatin, an angiogenesis inhibitor with anti-tumor activity in mice, is the bioactive NC1 domain of Col IVa3, the potential significance of tumstatin as an endogenous angiogenesis inhibitor in human is not yet completely understood. This study aimed to develop an enzyme-linked immunoassay (ELISA) for tumstatin to accurately measure its concentrations in biological samples. METHODS: Recombinant tumstatin as an immunogen was expressed by E.coli. The purified tumstatin was injected into mice for antibody generation. An ELISA was developed with the monoclonal antibodies. RESULTS: The detection limit of the ELISA was 1.4ng/ml, and the intra- and inter- assay CVs were within 10%. Recovery of tumstatin added to sera was 92.7-115%. Patients with metastases had serum tumstatin levels 50% lower than patients without metastases (P=0.039). Tumstatin levels in poorly differentiated tumor tissues were significantly lower than in nontumorous tissues and well-differentiated tumor tissues (P<0.001). CONCLUSIONS: The development of a highly sensitive and reliable ELISA method capable of quantifying tumstatin in human serum samples and tissue extracts is reported. This assay for tumstatin in biological samples may be helpful in evaluating the therapeutic and/or prognostic value of tumstatin in cancer patients.

[Treatment of acute myocardial infarction with autologous bone marrow stem cells mobilization combined with recombinant growth factor in rat].

OBJECTIVE: To observe the regeneration cardiomyocytes and neovascularization after mobilizing autologous bone marrow stem cells by granulocyte colony stimulating factor (G-CSF) alone or G-CSF combined with recombinant human growth hormone (rhGH) in Wistar rats with acute myocardial infarction (AMI). METHODS: AMI rat model was reproduced by liquid nitrogen cryoinjury. The rats were randomly divided into six groups: mobilization group (N group, n=8), sham operation group (SO group, n=6), myocardial infarction group (MI group, n=8), G-CSF group (G group, n=8), rhGH group (GH group, n=8) and G-CSF combined with rhGH group (GG group, n=8). White blood cell (WBC) count and mononuclear cells proportion (MNC%) in peripheral blood were determined with ABX blood cell analyzer to estimate bone marrow stem cells mobilization. Four weeks after intervention, the rats were sacrificed, their respective body and heart weight were obtained, and the hearts were harvested for hematoxylin and eosin (HE) and immunohistochemical examination. RESULTS: (1)Comparing with baseline values, after 6 days administration of G-CSF, the WBC and MNC% increased in N and G groups (both P<0.01); WBC increased (P<0.01) but no difference of MNC% in MI group (P>0.05); WBC and MNC% were significantly higher in G group than those in MI group (all P<0.05). (2)Body and heart weights in GH and GG groups were higher than those in SO, MI and G groups respectively (all P<0.05). The ratio of heart and body weight was higher in GC group than that in MI,G and SO groups (P<0.05). (3)There were no significant differences in infarct size among MI, G, GH, and GG groups (P>0.05). (4)The capillary densities were higher in G, GH and GG groups than those in MI and SO groups; the density in GG group was higher than that in G and GH groups (all P<0.01). (5)BrdU positively stained neonatal cells were observed in G, GH and GG groups. Of them some developed into the endothelial cells. BrdU and cTnI double positive stained cells were observed in G and GG groups, which implied these cells might have differentiated into cardiac myocyte like cells. CONCLUSION: (1)G-CSF can mobilize bone marrow stem cells into peripheral blood in normal and cardiac infarct rats. The mobilized stem cells may enter into the infarct zone and induce the regeneration of cardiac myocyte like cells and vascular endothelial cells. (2)rhGH may promote the regeneration of capillary in the zone of infarction, but does not induce regeneration of cardiac myocyte like cells. (3)The combination of G-CSF with rhGH might promote more capillary regeneration than either of them used alone.