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OBJECTIVE To study the effects of Yishen daluo decoction on inflammatory factors and cyclic adenosine monophosphate（cAMP）/protein kinase A （PKA）/cAMP response element binding protein （CREB） signal pathway in experimental autoimmune encephalomyelitis （EAE） model mice by inhibiting the expressions of β-arrestin1， and to explore the mechanism of Yishen daluo decoction in the treatment of EAE. METHODS Sixty mice were randomly divided into normal group， model group， TCM group （Yishen daluo decoction 20 g/kg）， positive control group （prednisone acetate 3.9 mg/kg）， β-arrestin1 siRNA adeno- associated virus （AAV-β） group， AAV-β+TCM group， with 10 mice in each group. Except for normal group， EAE model was made in other groups. AAV-β group and AAV-β+TCM group were injected with AAV-β via tail vein to interfere with the expression of β -arrestin1 protein. Starting from the 8th day of modeling， they were given corresponding drug solution/normal saline intragastrically， once a day， for consecutive 14 days. The neurological function score of mice was detected； the pathological and morphological changes were observed in the brain and spinal cord tissues of mice； the serum levels of inflammatory factors [interleukin-2 （IL-2）， IL-23， interferon-γ （IFN-γ）] in mice were determined； the expressions of β-arrestin1， cAMP， PKA and CREB in brain and spinal cord were detected. RESULTS Compared with normal group， neurological function scores， serum levels of inflammatory factors， and protein expressions of β-arrestin1 in brain and spinal cord were significantly increased （P＜0.05 or P＜ 0.01）； protein expressions of PKA， CREB and cAMP in brain and spinal cord were decreased significantly（P＜0.05 or P＜0.01）. The deep staining of cellular shrinkage and aggregation of inflammatory cells were observed in most neurons of the brain and spinal cord， with varying degrees of demyelinating. Compared with model group， the neurological function scores， pathological changes in brain and spinal cord tissues， and most indicators （except for CREB and cAMP proteins in the brain tissue of AAV-β group） were significantly reversed （P＜0.05 or P＜0.01）.Compared with AAV- β group， the neurological function scores， the levels of IFN-γ in serum and β-arrestin1 in spinal cord were significantly decreased （P＜0.05 or P＜0.01）， PKA and cAMP in brain and spinal cord tissues were significantly increased in AAV- β +TCM group （P＜0.05 or P＜0.01）. CONCLUSIONS Yishen daluo decoction can inhibit the expression of β-arrestin1 in the central nervous system thus activating the cAMP/PKA/CREB signaling pathway， relieving nervous system inflammation， and ultimately alleviates the symptoms of EAE.
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ObjectiveTo investigate the effects of Yishen Daluo prescription （YSDL） on Ras homolog（Rho）/Rho-associated coiled-coil containing protein kinase（ROCK）signaling pathway in mice with experimental autoimmune encephalomyelitis （EAE） based on the silencing of β-arrestin1 gene. MethodSixty C57BL/6 female mice were randomly divided into a blank group， a model group， a virus group， a YSDL group， a virus + YSDL group， and a prednisone acetate group （hormone group）. The EAE model was induced in mice except for those in the normal group. Adeno-associated virus（AAV）solution （150 μL， 1×1011 vg·mL-1） was injected into the tail vein of each mouse in the virus group and the virus + YSDL group on the 4th day of immunization. Drugs were administered on the 8th day of modeling. Specifically， normal saline was given to the mice in the normal group，the model group，and the virus group at 10 mL∙kg-1， prednisone acetate suspension to those in the hormone group at 3.9 g∙kg-1，and YSDL to those in other groups at 20 g∙kg-1 for 14 consecutive days. The mice were weighed and scored every day. The neurological function scores of mice in each group were recorded every day after immunization. Hematoxylin-eosin （HE） staining was used to determine the inflammatory response and lesion location in the brain tissues and spinal cord tissues of mice. The protein expression of β-arrestin1，Ras homolog gene family member A（RhoA）， and Rho-associated coiled-coil forming protein kinase Ⅰ（ROCK Ⅰ） in spinal cord and brain tissues of EAE mice was determined by Western blot. ResultCompared with the model group， the virus group and the virus + YSDL group showed decreased neurological function scores （P<0.01），and the YSDL group also showed decreased neurological function scores（P<0.05）. HE results showed that there was obvious inflammatory reaction in the central nervous system （CNS） of the model group， which was alleviated to varying degrees in other groups compared with the model group. Western blot results showed that compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the spinal cord tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression levels of β-arrestin1， RhoA， and ROCKⅠ in the spinal cord tissues （P<0.01）. Compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the brain tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression level of β-arrestin1 in the brain tissues （P<0.01）， and the virus group and the YSDL group showed decreased protein expression levels of RhoA， and ROCKⅠ in the brain tissues （P<0.05）. Additionally， the virus + YSDL group and the hormone group showed decreased protein expression levels of RhoA and ROCKⅠ in the brain tissues （P<0.01）. ConclusionYSDL can improve the clinical symptoms of EAE mice and improve the inflammatory response of CNS. The mechanism is presumably attributed to the fact that YSDL inhibits the expression of β-arrestin1 in CNS，thereby reducing the expression of Rho/ROCK signaling pathway. Furthermore， YSDL may have a synergistic effect with the inhibition of β-arrestin1 gene expression.
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Objective@#To investigate genotypes and drug susceptibility of the 100 strains of Candida glabrata isolated from 100 women (including 50 pregnant women) in order to study the drug-resistance and gene polymorphism, and to investigate the correlation of drug-resistance, gestation and gene polymorphism.@*Methods@#Multilocus sequence typing (MLST) technique was introduced to identify sequences of 6 housekeeping genes from 100 isolates of Candida glabrata. The results were compared with sequence information in MLST databases by Clustalx software to determine a strain allelic profile and sequence type (ST). Drawing the phylogenetic tree by weighted paired group average method and the minimal spanning tree method of MEGA6.0 software, the microevolution and relationship between different strains were analyzed. ATB FUNGUS semi-automatic system was used to test the drug susceptibility. Fisher analysis method was used to analyze the correlation between the genotypes and pregnancy.Ridit analysis method was used to analyze the correlation between the genotypes and drug susceptibility.@*Results@#The 100 isolates belonged to 34 clone sequences. There were 53 isolates belonged to ST-7 ( 26 isolates of pregnancy), 7 isolates belonged to ST-3 (3 isolates of pregnancy), 6 isolates belonged to ST-19 (5 isolates of pregnancy ), 3 isolates belonged to ST-15 (1 isolate of pregnancy), 2 isolates belonged to ST-10 (2 isolates of pregnancy), 1 isolate belonged to the other types of ST. Total of 100 isolates of Candida glabrata were 100% sensitive to fluorocytosine and amphotericin. The effect of itraconazole was poor with the sensitive rate of 20%. The resistance rates of fluconazole and voriconazole were 4% and 1% respectively. All genotypes were sensitive to voriconazole except ST-X1. In the correlation between genotype and itraconazole resistance, ST-7 as the standard group, the Ridit values in the group of ST-15, ST-19 and other types of ST were not included the mean Ridit value of the standard group in itraconazole (0.5). The system evolution tree was built using the neighbor-joining method (NJ) . All genotypes could be divided into 3 groups, as Ⅰ, Ⅱ, Ⅲ. Group Ⅰ had 44 cases, group Ⅱ had 53 cases , group Ⅲ had 3 cases . All the collected clinical strains had small genetic distances during molecular evolution.@*Conclusions@#ST-7 was the dominant genotype in Guiyang. No correlation between different STs and patients′ pregnancy was found. The different drug susceptibility in itraconazole between ST-7, ST-15, ST-19 and other STs were found. The Candida glabrata associated with VVC showed highly discrimative diversity. However, the phylogenic analysis exhibited genetic similarity among the strains studied.(Chin J Lab Med, 2018, 41: 596-600)
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Objective:To establish an ion chromatography method for the determination of methane sulfonic acid in betahistine me-sylate and evaluate the uncertainty in the measurement. Methods: An ion chromatographic column IonPac AS11-HC ( 25 mm × 4. 0 mm,5 μm) was used with 12 mmol·L-1 NaOH as the eluent and an electrical conductivity detector with the suppressor of 30 mV. Results:The results showed that methane sulfonic acid could be detected without any interference. The calibration curve was linear within the range of 10-30 μg·ml-1(r=0.999 9)and the LOQ was 0.116 μg·ml-1. The average recovery was 100.8% (RSD=1. 2%, n=9). Based on the results of experiments, the influencing factors of uncertainty in the measurement were quantitatively eval-uated. The expanded uncertainty was obtained. Conclusion:The method is simple, accurate and selective. It can be used for the de-termination of methane sulfonic acid in betahistine mesylate. Based on the evaluation of uncertainty, the analysis can help reduce the uncertainty in the measurement and improve the accuracy and reliability of the determination.
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Objective: To assess the renal cortical perfusion parameters by the imaging of computed tomography (CT) in patients of essential hypertension (EH) with target organ damage. Methods: A total of 90 subjects with the entire information including 59 EH patients were studied. The EH patients were divided into 2 groups: EH + target organ damage group,n=30 and EH without target organ damage group,n=29. In addition, there was a Control group,n=31 healthy volunteers. All subjects received 128-slice dual-source CT renal perfusion scanning, the quantitative perfusion of renal cortex blood lfow (BF), blood volume (BV), time to peak (TTP) and the mean transit time (MTT) were examined and compared among different groups. Results: There were 90/97 (92.8%) participants eligible for perfusion analysis. Compared to Control group, EH without target organ damage group had the similar parameters of BF, BV, MTT and TTP,P>0.05. While EH + target organ damage group had decreased BF (214.6 ± 36.1) ml/(min?100 ml ) than Control group (262.1 ± 26.6) ml/(min?100 ml ),P0.05. Compared to EH without target organ damage group, the EH + target organ damage group presented decreased BF (214.6 ±3 6.1) ml/(min?100 ml ) vs (268.9 ± 33.1) ml/(min?100 ml ), P Conclusion: CT imaging may evaluate the renal cortical perfusion changes, and especially BF which can relfect the renal perfusion more sensitively than other parameters in EH + target organ damage patients.
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BACKGROUND:Bone marrow mesenchymal stem cells have potential to self-renewal and multi-lineage differentiation. But after a long period of culture in vitro, the proliferation and differentiation capacities of bone marrow mesenchymal stem cells gradual y loss, the mechanism underlying which is not clear now. OBJECTIVE:To observe the expression of autophagy-related gene Beclin-1 in differentiation from human bone marrow mesenchymal stem cells into neuron-like cells in vitro. METHODS:The changes of morphological characteristics of neuron-like cells differentiated from human bone marrow mesenchymal stem cells induced by epidermal growth factor were observed. The expression of neuron-specific enolase and glial fibril ary acidic protein in treated and untreated human bone marrow mesenchymal stem cells were detected using immunocytochemistry. The Beclin-1 protein expressions were detected by western blot before and after induction. RESULTS AND CONCLUSION:After being induced, human bone marrow mesenchymal stem cells presented classical neuron-like morphology;the expressions of neuron-specific enolase and glial fibril ary acidic protein were 78.7%and 8.1%, respectively. The expression of Beclin-1 protein was changed correspondingly during the induction, which increased after 30 minutes of induction and decreased gradual y after 1 hour of induction. Human bone marrow mesenchymal stem cells could be induced into neuron-like cells in vitro by epidermal growth factor. Autophagy-related gene was highly expressed in the induction of early differentiation and the expression gradual y reduced until it remained at a low level during the differentiation.
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This article was aimed to rapidly analyze chemical composition in A loe Barbadensis Mill, and to compare the chemical composition of commercial aloe vera medicinal materials with that of fresh aloe yellow exudate. An opti-mized liquid chromatography-mass spectrometry-ion trap-time-of-flight (LCMS-IT-TOF) method was applied for the analysis of commercial aloe vera medicinal materials and fresh aloe yellow exudates. The Agilent TC-C18 column (4.6 í 250 mm, 5 m) was used. The gradient elution was a solvent system of water(A)-methanol(B). ESI source was operated in both positive and negative ion modes. The results showed that chromones, pyrones, naphthalene deriva-tive, anthrones and anthraquinones were separated successfully, 30 compounds were characterized by the comparison of characteristic MS/MS fragment ions data with the literature. The diagnostic fragmentation patterns of different chemical compositions were also discussed on the basis of EST-IT-TOF MS data. It was concluded that the chemical composition of commercial aloe vera medicinal materials were significantly different from that of fresh aloe yellow ex-udate in terms of types and contents: the former one mainly contains isoaloeresin D and aloin, and few aloesin; but the latter is mainly composed of aloesin and aloin, and the content of aloesin is the highest. The LCMS-IT-TOF analysis can be used to rapidly obtain rich structural information of different chemical compositions, which improves the efficiency of qualitative analysis of chemical composition, and is of great significance to the quality control, eval-uation and the utilization of A loe Barbadensis Mill.
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To investigate the chemical constituents of A. barbadensis, aqueous extract of the plant was subjected to preparative medium pressure liquid chromatography (MPLC). The chemical structures were mainly determined by spectroscopic evidences (UV, IR, HR-MS, 1H NMR, 13C NMR, HSQC, 1H-1H COSY and HMBC) and chemical methods. A new O, O, O-triglucosylated naphthalene derivative, together with two known 6-phenyl-2-pyrone derivatives and four 5-methylchromones, were isolated and identified as 1-((3-((4- O-beta-D-glucopyranosyl)-beta-D-xylopyranosyloxymethyl)-1-hydroxy-8-alpha-L-rhamnopyranosyloxy)naphthalene-2-y])-ethanone (1), 10-O-beta-D-glucopyranosyl aloenin (2), aloenin B (3), aloesin (4), 8-C-glucosyl-(R)-aloesol (5), 8-C-glucosyl-7-O-methyl-(S)-aloesol (6), and isoaloeresin D (7). Compound 1 is a novel naphthalene derivative and named as aloveroside B, compounds 2-3 are isolated from this Aloe species for the first time.