With redox-sensitive fluorescene probes DCFH-DA and DHR123, the formation of cytosolic and intramitochondrial reactive oxygen species (ROS) inside immature rat cerebellar granule cells during the apoptosis induced by nitric oxide donor S-nitroso-N-acetyl-pennicillamine (SNAP) was monitored by laser confocal scanning microscopy. The cytosolic and intramitochondrial ROS increase significantly after 0.5 mmol/L SNAP treatment for 1 h. Pre-treatment with the nitric oxide scavenger hemoglobin can effectively inhibit the formation of cytosolic and intrarnitochondrial ROS and protect neurons from apoptosis. Adding glutathione can also protect neurons from apoptosis, and the cytotoxity of nitric oxide increases significantly while the synthesis of glutathione is inhibited. The results indicated that ROS might be involved in NO-induced apoptosis in neural cells and glutathione might be the endogenesis antioxidant to protect neurons from oxidative injury.

Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.
Biomass ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Culture Media ; chemistry ; metabolism ; Eriobotrya ; chemistry ; growth & development ; metabolism ; Triterpenes ; analysis ; metabolism

Objective To make the identification of medicinal herbs in Salvia L. quickly and accurately. Methods In this work,DNA barcoding and chemical fingerprint were compared for the identification of herbs in Salvia L. First, the nucleotide sequences of the internal transcribed spacer region two amplified from 48 medicinal plants in Salvia L., and three other groups of medicinal plants in Lamiaceae were sequenced. A molecular phylogeny was constructed using the minimum evolution and maximum parsimony methods according to their sequence diversity. Second, the water-solution bioactive components and lipid soluble components were tested by HPLC. Then a chemical phylogeny was built using HPLC fingerprint data. Comparing the molecular and chemical phylogenetic trees revealed many similarities. Results DNA barcoding was sequencing based and could therefore provide more accurate results within a shorter time especially in large-scale studies. Conclusion The results show that ITS2 region is a novel DNA barcode for the authentication of the species in Salvia L. This is the first work to show the relationship between DNA barcoding and chemical components.

Objective:To compare the accuracy of visual qualitative assessment and semi-quantitative analysis for 18F-florbetaben ( 18F-FBB) β-amyloid (Aβ) imaging in the diagnosis of Alzheimer′s disease (AD) and to explore their clinical application value. Methods:From January 2019 to October 2019, 17 patients (8 males, 9 females, age (74.1±8.5) years) with mild/moderate-stage clinically probable AD and 17 cognitive normal control (NC; 9 males, 8 females, age (64.5±6.3) years) were prospectively enrolled in this study. All patients underwent dynamic 18F-FBB PET/CT brain imaging in the Chinese PLA General Hospital. Visual qualitative assessment and semi-quantitative analysis methods were used to analyze PET brain imaging results. The difference of standardized uptake value ratio (SUVR) between the two methods was analyzed by using independent sample t test. The consistency of the two methods and clinical results was analyzed by Kappa test. The cut-off value of SUVR for the diagnosis of AD was determined by receiver operating characteristic (ROC) curve. Results:The sensitivity, specificity and accuracy of visual qualitative assessment to diagnose AD were 14/17, 16/17 and 88.2% (30/34). The global SUVR of NC and AD group were 1.09±0.85 and 1.75±0.25 ( t=-10.263, P<0.001), and the composite SUVR were 1.16±0.57 and 1.89±0.15 ( t=-10.789, P<0.001), respectively. The cut-off value of SUVR for the diagnosis of AD was 1.47, with the sensitivity of 15/17, the specificity of 16/17 and the accuracy of 91.2%(31/34). The visual qualitative assessment and semi-quantitative analysis had good consistency with clinical diagnosis results with Kappa value of 0.765 and 0.824 respectively (both P<0.001). Conclusion:The visual qualitative assessment and semi-quantitative analysis methods used in 18F-FBB Aβ imaging to diagnose AD patients show high accuracy and can provide effective value for clinical diagnosis, but the visual qualitative assessment method is concise and easy to grasp, which is worth further promotion and use in clinical.

OBJECTIVETo investigate the effects of crypotanshinone (CPT) on the proliferation and apoptosis of DU145 prostate cancer cells as well as on the metadherin expression and the downstream PI3K/AKT signaling pathway in the DU145 cells.

METHODSWe treated DU145 prostate cancer cells with different concentrations of CPT for 24, 48, and 72 hours followed by evaluation of the proliferation and apoptosis of the cells by MTT assay and TUNEL, respectively. We determined the expressions of metadherin protein and mRNA in the DU145 cells by Western blot and RT-PCR respectively at different time points after CPT treatment. We also detected the expressions of the proteins metadherin, AKT, p-AKT, and Bcl-2 in the CPT-treated DU145 cells at 48 hours.

RESULTSCPT significantly inhibited the proliferation of the DU145 cells in a dose- and time-dependent manner (P < 0.05). After treatment with 10 µmol/L CPT for 24, 48, and 72 hours, the apoptosis rates of the DU145 cells were (29.42 ± 4.51), (55.07 ± 5.67) and (70.84 ± 4.66)%, respectively, significantly higher than (3.1 ± 2.48)% in the control group (P < 0.05). The expression of metadherin was remarkably downregulated at the transcription and translation levels (P < 0.05) and the expressions of the AKT signaling pathway and the Bcl-2 protein were markedly inhibited in the DU145 cells after treated with 10 µmol/L CPT for 48 hours (P < 0.05).

CONCLUSIONCPT can inhibit the proliferation and induce the apoptosis of DU145 prostate cancer cells, which may be associated with its suppression of the downstream PI3K/AKT signaling pathway by reducing the expression of metadherin in the DU145 cells.

Apoptosis ; drug effects ; Cell Adhesion Molecules ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Diterpenes, Abietane ; pharmacology ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Humans ; In Situ Nick-End Labeling ; Male ; Neoplasm Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; Time Factors

BACKGROUND:Traditional therapeutic methods of diabetes combined with lower limb artery obturation showed poor outcomes such as drug or intervention.Stem cell transplantation is a new method to treat this disease in recant years,and has obtained good outcomes in the clinic.Evaluating the therapeutic outcomes of stem cell transplantation has great damage and cost much.Thus,color Doppler ultrasound is used to examine non-invasion in the artery of the lower extremity,which shows important clinical value.OBJECTIVE:To evaluate improvement of lower extremity artery lesion following autologous peripheral blood stem cell transplantation using color Doppler ultrasound,and to compare with results of angiography.METHODS:A total of 41 patients diagnosed as having diabetes combined with lower limb artery obturation were included.Following administration of recombinant human granulocyte-colony stimulating factor(rhG-CSF)(150 μg/d)for 5 days,peripheral blood mononuclear cells(PBMNCs)were mobilized and harvested by using a separator at day 5 to prepare stem cell suspension,and which were injected intramuscularly in the diseased extremities(3×109 per limb).The clinical and laboratory findings were monitored every week for 3 months to 1 year following stem cell transplantation.RESULTS AND CONCLUSION:After stem cell transplantation,severe pain lameness,local cool-feeling and ulcer were significantly improved,and ankle-brachial index was increased obviously(P＜0.01).Color Doppler ultrasonography measurement showed that peak systolic velocity in diseased extremities was significantly increased post-transplantation(P＜0.01).Following transplantation,blood flow of the lower limb arterial canal was improved to different extents;collateral vessels were increased surrounding obstructed artery and in muscles.Spiral CT for new collateral vessel formation were showed increased.No related complication or adverse effects were observed in 41 patients during observation.Results indicated that color Doppler ultrasound can exactly rapidly examine and assess blood transport improvement in the lower limbs following stem cell transplantation,which is identical to results from angiography.It also provides reliable evidences for the diagnosis on the transplantation of stem cells for the treatment of diabetes combined with lower limb artery obturation.

The dried succulent stems of Cistanche (Cistanche deserticola Y. C. Ma and Cistanche tubulosa Wight.) are one of the most widely used components of traditional Chinese medicines. However, it is often confused and substituted with the roots of Orobanche pycnostachya, Boschniakia rossica (Cham. & Schltdl.) Standl., Cistanche sinensis Beck, and Cistanche salsa (C. A. Mey.) Beck. In this study, we identified psbA-trnH regions from species and tested their suitable for the identification of the above mentioned taxa. The psbA-trnH sequences showed considerable variations between species and thus were revealed as a promising candidate for barcoding of Cistanche species. Additionally, the average genetic distance of psbA-trnH ranging from 0.077% to 0.743%. In contrast, the intra-specific variation among Cistanche species was found to be significantly different from those of other species, with percentages of variation studied ranged from 0% to 0.007%. The sequence difference between the psbA-trnH sequences of Cistanche species and Orobanche pycnostachya ranged from 0.979% to 1.149%. The distance between the Cistanche species and Boschniakia rossica ranged from 1.066% to 1.224%. Our results suggest that the psbA-trnH intergenic spacer region represent a barcode that can be used to identify Cistanche species and other morphologically undistinguishable species.

The change of kirenol, darutigenol and darutoside in Siegesbeckia and its first to ninth processed products were studied, and the ten fingerprints were compared, which provided the experimental basis for the study of Siegesbeckia processing tech- nology. The samples were analysed by HPLC on a SunFire-C18 column (4.6 mm x 150 mm, 5 μm) with gradient elution of acetonitrile (0.1% formic acid)-water (0.1% formic acid) at a flow rate of 1.0 mL x min(-1). Column temperaturewas 30 °C and the detected wavelength was 215, 320 nm. The calibration curves of kirenol, darutigenol and darutoside were linear in the range of 2.180-26.16, 2.900-34.80, and 1.012-6.072 mg x L(-1), respectively, and the average recoveries were 96.4%, 97.2% and 96.3% wit RSD 2.2%, 1.7% and 2.4%. This method was simple, the result was stable and had good repeatability, recovery and precision. The re- sult was the basis of the chemical contents variation in the processing of Siegesbeckia Herbs and further clarifying the effect of the changing.
Asteraceae ; chemistry ; Chemistry, Pharmaceutical ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Temperature

Animals ; Haptoglobins ; metabolism ; Mesenteric Artery, Superior ; metabolism ; Mesenteric Vascular Occlusion ; blood ; Rabbits ; Serum ; metabolism

OBJECTIVE To observe the effects of glycogen synthase 3β (GSK-3β) in the regula-tion of NLRP3 inflammasome activation after acute myocardial infarction (MI) in Sprague Dawley(SD) rats. METHODS Ligation of the left anterior descending (LAD) in SD rats was used to establish an acute myocardial infarction model. SD rats were randomly divided into 3 groups (n=10, each group):sham group,MI group,and MI+SB group:the GSK-3β inhibitor(SB216763)was given 1 h by intrave-nous injection(0.6 mg·kg-1·d-1)before surgery.The serum and heart tissue were collected to measure lactate dehydrogenase (LDH) and IL-1β content and mRNA and protein levels of NLRP3, ASC, Cas-pase-1,IL-1β and GSK-3β after 2 days and 7 days operation,respectively.RESULTS The serum levels of LDH and IL-1β in the MI group were significantly higher than those in the sham group(P<0.01),and the MI+SB group was obviously lower than the MI group(P<0.01).In addition,mRNA and protein levels of NNLRP3, ASC, Caspase-1, IL-1β and GSK-3β expressions in MI group were clearly increased (P<0.01) compared with those in sham group.These indicators were significantly decreased in SB+MI group (P<0.01). Interestingly, the indicators were all higher at 7 days than 2 days. CONCLUSION GSK-3β inhibition induces cardioprotection via attenuating the activation of NLRP3 inflammasome after the acute myocardial infarction in rats.