Methylation analysis of Rb, P16, MGMT, and hMLH-1in mucoepidermoid carcinomas. A pilot study

Mucoepidermoid carcinoma (MC) is the most common malignant epithelial neoplasm in the salivary glands. This neoplasm has varying proportions of mucous, epidermoid, intermediate, columnar, and clear cells. MCs have been associated with CRTC1-MAML2 genes; however, their pathogenesis is uncertain. Recently, epigenetic changes have been considered a possible aetiologic factor. To identify the methylation state of RB, P16, MGMT, and hMLH genes in the three severity grades of MC were used five MCs and one healthy minor salivary gland as a control group (CG) obtained from the Pathology and Oral Medicine Laboratory and analyzed using MS-PCR to compare the presence or absence of methylation in promotor regions. The Kruskal- Wallis test was performed, with p≤0.05 considered significant. CG was employed as the normalizer of methylation levels. All assays were performed in triplicate. The mean age of our population was 52.6±18.6 years old; the total population was female and included 2 low grade, 2 intermediate grade, and 1 high grade levels of severity. When comparing the methylation status of the three histopathological grades of MC against the control, statistically significant differences were observed in Rb-M, MGMT-M, and hMLH-1-NM for high-grade severity, with p values of 0.03, 0.05, and 0.04, respectively. Methylation is a possible mechanism for pathogenesis processing of high-grade MC. However, a larger sample population is necessary to validate this finding.

El carcinoma mucoepidermoide (CM) es la neoplasia epitelial maligna más frecuente de glándulas salivales. Esta neoplasia tiene proporciones variables de células mucosas, epidermoides, intermedias, cilíndricas y claras. Los CM se han asociado con los genes CRTC1-MAML2; sin embargo, su patogenia es incierta. Recientemente, los cambios epigenéticos se han considerado un posible factor etiológico. Para identificar el estado de metilación de los genes RB, P16, MGMT y hMLH en los tres grados de severidad de CM se utilizaron cinco CM y una glándula salival menor sana como grupo control (GC) obtenidos del Laboratorio de Patología y Medicina Oral y analizados mediante MS-PCR para comparar la presencia o ausencia de metilación en regiones promotoras. Se realizó la prueba de Kruskal-Wallis, considerándose significativa una p≤0,05. Se empleó GC como normalizador de los niveles de metilación. Todos los ensayos se realizaron por triplicado. La edad media de nuestra población fue de 52,6 ± 18,6 años; la población total era femenina e incluía 2 niveles de severidad de grado bajo, 2 de grado intermedio y 1 de alto grado. Al comparar el estado de metilación de los tres grados histopatológicos de CM contra el GC, se observaron diferencias estadísticamente significativas en Rb-M, MGMT-M y hMLH-1-NM para severidad de alto grado, con valores de p de 0.03, 0.05, y 0,04, respectivamente. La metilación es un posible mecanismo para el procesamiento de patogénesis de CM de alto grado. Sin embargo, se necesita una población de muestra más grande para validar este hallazgo.

Protective effects of ginsenosides Rg1 and Rb1 against cognitive impairment induced by simulated microgravity in rats / 中国药理学与毒理学杂志

OBJECTIVE Microgravity exerts several negative effects on the learning and memory of astro-nauts during space flight.Rg1 and Rb1,the key steroidal components of ginseng,have shown potent neuroprotec-tive effects with a high safety profile.The object of the current study is to investigate the influence of Rg1 and Rb1 on simulated microgravity-induced memory and learning dysfunction in the hindlimb suspension(HLS)rat model.METHODS The HLS rats were orally administered Rg1(30 and 60 μmol·kg-1)or Rb1(30 and 60 μmol·kg-1)for four weeks.The Morris water maze test(MWM)and reward operating conditioning reflex test(ROCR)were conducted to evaluate spatial and associative learning and memory.After the behavior tests,the serum and the prefrontal cortex(PFC)were dissected to measure the mechanism.RESULTS Rg1 and Rb1 treatment amelio-rated the cognitive deficits of HLS-exposure rats in MWM and ROCR,reduced reactive oxygen species generation and increased antioxidant enzyme activity.Rg1 and Rb1 also assisted in the recovery of mitochondrial complex Ⅰ(NADH dehydrogenase)activities and Mfn2,and decrea-sed Drp-1 expression.Furthermore,Rg1 and Rb1 reduced the ratio of Bax/Bcl-2 and the expression of cleaved-cas-pase 3,cytochrome c,increased the levels of SYN,PSD95 and activated BDNF-TrkB/PI3K-Akt pathway in the PFC.CONCLUSION Rg1 and Rb1 treatment attenuated cog-nitive deficits induced by HLS,mitigated mitochondrial dysfunction,attenuated oxidative stress,inhibited apopto-sis,and increased the synaptic plasticity,which was partly mediated by the modulation of the BDNF-TrkB/PI3K-Akt signaling.

Discovery of biomarkers related to abnormal lipid metabolism in liver and serum and intervention mechanism of ginsenoside Rb_1 in hyperlipidemia rats based on non-targeted metabolomics / 中国中药杂志

Through the non-targeted metabolomics study of endogenous substances in the liver and serum of hyperlipidemia rats, the biomarkers related to abnormal lipid metabolism in hyperlipidemia rats were found, and the target of ginsenoside Rb_1 in improving hyperlipidemia was explored and its mechanism was elucidated. The content of serum biochemical indexes of rats in each group was detected by the automatic biochemical analyzer. The metabolite profiles of liver tissues and serum of rats were analyzed by HPLC-MS. Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to compare and analyze the metabolic data in the normal group, the hyperlipidemia group, and the ginsenoside Rb_1 group, and screen potential biomar-kers. The related metabolic pathways were further constructed by KEGG database analysis. The results showed that hyperlipemia induced dyslipidemia in rats, which was alleviated by ginsenoside Rb_1. The non-targeted metabolomics results showed that there were 297 differential metabolites in the liver tissues of hyperlipidemia rats, 294 differential metabolites in the serum samples, and 560 diffe-rential metabolites in the hyperlipidemia rats treated by ginsenoside Rb_1. Perillic acid and N-ornithyl-L-taurine were common metabolites in the liver and serum samples, which could be used as potential biomarkers for ginsenoside Rb_1 in the improvement of hyperlipidemia. As revealed by pathway enrichment in the liver and serum, ginsenoside Rb_1 could participate in the metabolic pathway of choline in both the liver and serum. In addition, ginsenoside Rb_1 also participated in the ABC transporter, alanine, aspartic acid, and glutamate metabolism, protein digestion and absorption, β-alanine metabolism, taurine and hypotaurine metabolism, caffeine metabolism, valine, leucine, and isoleucine biosynthesis, arachidonic acid metabolism, and methionine and cysteine metabolism to improve dyslipidemia in rats.

Lack of methylation changes in GJB2 and RB1 non-coding regions of cochlear implant patients with sensorineural hearing loss

Objective@#Recent advances in epigenetic studies continue to reveal novel mechanisms of gene regulation and control, however little is known on the role of epigenetics in sensorineural hearing loss (SNHL) in humans. We aimed to investigate the methylation patterns of two regions, one in RB1 and another in GJB2 in Filipino patients with SNHL compared to hearing control individuals. @*Methods@#We investigated an RB1 promoter region that was previously identified as differentially methylated in children with SNHL and lead exposure. Additionally, we investigated a sequence in an enhancer-like region within GJB2 that contains four CpGs in close proximity. Bisulfite conversion was performed on salivary DNA samples from 15 children with SNHL and 45 unrelated ethnically-matched individuals. We then performed methylation-specific real-time PCR analysis (qMSP) using TaqMan® probes to determine percentage methylation of the two regions. @*Results@#Using qMSP, both our cases and controls had zero methylation at the targeted GJB2 and RB1 regions. @*Conclusion@#Our study showed no changes in methylation at the selected CpG regions in RB1 and GJB2 in the two comparison groups with or without SNHL. This may be due to a lack of environmental exposures to these target regions. Other epigenetic marks may be present around these regions as well as those of other HL-associated genes.

Role of Immunohistochemistry and serology in subclassifying the Inflammatory Bowel Disease cases diagnosed as Inflammatory Bowel Diseases-unclassified on colonic biopsies

Background: Inflammatory bowel diseases (IBDs) mainly consist of Crohn's Disease (CD) and Ulcerative Colitis (UC). These two categories have overlapping histopathological features and sometimes it is difficult to diagnose them into distinct category and such biopsies are categorised as Inflammatory Bowel Disease (IBD-U). Recently, there has been an increase in interest to discover new biomarkers of IBD to differentiate UC and CD and predict their prognosis. Method: In the present study, 273 non-neoplastic colonic biopsies with clinicoendoscopic features of IBD were studied and categorized into UC (88; 32.3%) and CD (03; 1.1%) but a major chunk remained in category of IBD-U (182; 66.6%). 161 (58.9%) of these biopsies were then subjected to IHC for RB protein and ?-catenin and Serology for pANCA and ASCA was done in only 85 (31.13%) of these selected cases for identification of UC and CD on colonic biopsies. Result: 161 biopsies that were subjected to IHC analysis included 57 cases of UC, 03 cases of CD, and rest 101 cases of IBD-U. Out of 101 cases of IBD-U, 87 (86.13%) cases were reclassified as UC (61; 60.3%) and CD (14; 13.86%) on the basis of results of IHC and Serology. Conclusion: The two major tools IHC for ?-catenin and RB protein and the assay of serum ASCA and p-ANCA along with proper history and clinical presentation can act as a good adjunct to conventional H and E in subclassifying cases of IBD-U into UC and CD.

Inhibition of ginsenoside Rb 1 on the epithelial-mesenchymal transition of renal tubular epithelial cells by regulating TGF-β1/Smad3 signaling pathway / 中国药房

OBJECTIVE To study the effects of ginsenoside Rb 1（G-Rb1）on epithelial-mesenchymal transition （EMT）of renal tubular epithelial cells and its potential mechanism. METHODS The growth factor β1（TGF-β1）10 ng/mL was used to induce EMT of human renal tubular epithelial cells HK- 2. The morphological changes of HK- 2 cells were observed after treated with 10， 20，30 μmol/L G-Rb1 for 48 h. The transcriptional activities of biovector SBE in human embryonic kidney cell HEK 293 were determined after 24 h treatment with 1.0，2.5，5.0，10，20，30 μmol/L G-Rb1. Effects of above concentration of G-Rb 1 on the viability of HK- 2 cells were determined after 24 h of treatment. mRNA expressions of α-smooth muscle actin （α-SMA），collagen Ⅰ （COL-Ⅰ）and fibronectin （FN）in HK- 2 cells were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. The expressions of α-SMA，Smad3，p-Smad3，COL-Ⅰ，FN and E-cadherin were detected after treated with 10，20，30 μmol/L G-Rb1 for 24 h. RESULTS G-Rb1 of 10-30 μmol/L significantly inhibited TGF-β1-induced EMT in HK- 2 cells and the increase of transcriptional activities of biovector SBE induced by TGF-β1（P＜0.05），but had no effects on relative activities of HK- 2 cells（P＞0.05）. The protein and mRNA expressions of α-SMA，COL-Ⅰ and FN ， the protein expressions of Smad 3 and p-Smad 3 were significantly up-regulated induced by TGF-β1（P＜0.05），while the protein expression of E-cadherin was significantly down- regulated（P＜0.05）；G-Rb1 could effectively reverse aboveprotein or mRNA expressions. CONCLUSIONS G-Rb1 can protect renal tubular epithelial cells from EMT induced byxiezhishen TGF-β1 to a certain extent ，which may be related to inhibiting the activation of TGF-β1/Smad3 signaling pathway.

Quality evaluation of ginsenoside reference substances based on qNMR spectroscopy / 中国中药杂志

The present study established a quality evaluation method for ginsenoside reference substances based on quantitative nuclear magnetic resonance(qNMR) spectroscopy. ~1H-NMR spectra were collected on Bruker Avance Ⅲ 500 MHz NMR spectrometer equipped with a 5 mm BBO probe. The acquire parameters were set up as follows: pulse sequence of 30°, D_1=20 s, probe temperature= 303 K, and the scan number = 32. Dimethyl terephthalate, a high-quality ~1H-qNMR standard, was used as the internal standard and measured by the absolute quantitative method. Methyl peaks of comparatively good sensitivity were selected for quantification, and linear fitting deconvolution was adopted to improve the accuracy of integration results. The qNMR spectroscopy-based method was established and validated, which was then used for the quality evaluation of ginsenoside Rg_1, ginsenoside Re, ginsenoside Rb_1, ginsenoside Rd, and notoginsenoside R_1. The results suggested that the content of these ginsenoside reference standards obtained from the qNMR spectroscopy-based method was lower than that detected by the normalization method in HPLC provided by the manufacturers. In conclusion, the qNMR spectroscopy-based method can ensure the quality of ginsenoside reference substances and provide powerful support for the accurate quality evaluation of Chinese medicine and its preparations. The qNMR spectroscopy-based method is simple, rapid, and accurate, which can be developed for the quantitative assay of Chinese medicine standard references.

In Vitro Pharmacokinetics and Pharmacodynamic Evaluation of Jiaojiang Cataplasms / 中国实验方剂学杂志

ObjectiveTo study the in vitro kinetics of Jiaojiang cataplasms and evaluate its pharmacodynamics， so as to provide a feasible basis for the development of this preparation. MethodThe improved Franz diffusion cell was used for the in vitro release in semipermeable membrane and transdermal absorption in in vitro mouse skins. The contents of hydroxy-α-sanshool， 6-gingerol， ginsenoside Rb1 were determined by high performance liquid chromatography （HPLC）， to evaluate the in vitro release and transdermal absorption of Jiaojiang cataplasms. The mobile phase of 6-gingerol and hydroxy-α-sanshool was water-acetonitrile-methanol （2∶1∶1） with the detection wavelength of 280 nm. The mobile phase of ginsenoside Rb1 was acetonitrile-0.1% phosphoric acid aqueous solution （31∶69） with the detection wavelength of 203 nm. A mouse intestinal paralysis model was established， and mice were randomly divided into five groups， namely sham operation group， model group， domperidone group （3.9 mg·kg-1） and high- and low-dose groups of Jiaojiang cataplasms （6.2， 3.1 g·kg-1， measured by crude drug dosage）， to observe the effect of this preparation on gastrointestinal propulsion function. ResultAverage release rates of hydroxy-α-sanshool， 6-gingerol and ginsenoside Rb1 at 24 h were 16.41， 4.23， 4.15 μg∙cm-2∙h-1， the average transdermal rates of them at 24 h were 2.31， 0.64， 0.29 μg∙cm-2∙h-1， their skin retention values were 19.56， 3.59， 1.61 μg， respectively. According to the Ritger-Peppas equation， the release of hydroxy-α-sanshool， 6-gingerol， ginsenoside Rb1 was non-Fick diffusion. The high-dose group of Jiaojiang cataplasms could improve intestinal function of model mice after small intestinal friction injury， and promote intestinal peristalsis and small intestinal propulsion rate （P<0.05）. ConclusionJiaojiang cataplasms has in vitro release and transdermal properties， the in vitro release conforms to Higuchi equation， and transdermal absorption behavior conforms to zero-order kinetic equation， which can improve the postoperative function of the small intestine and the propulsion function of small intestine. It preliminarily indicates that the preparation has certain clinical development value.

Protective Effect of Ginsenosides Rg1 and Rb1 Against Intestinal Epithelial Barrier Injury Induced by Lipopolysaccharide in Vitro / 中国实验方剂学杂志

ObjectiveTo investigate the effects of ginsenoside Rg1 and ginsenoside Rb1 on the release of inflammatory factors of human myeloid leukemia monocytes （THP-1） induced by lipopolysaccharide （LPS） and their protective effects on the inflammatory injury of intestinal epithelial cells （Caco-2） induced by THP-1 cell activation based on the co-culture system of THP-1 and Caco-2. MethodFirstly，the microfluidic chip of co-culture of THP-1 and Caco-2 cells was prepared. In the experiment， a blank group， an LPS group， and drug intervention groups were set up.The cells in the blank group were cultured conventionally. In the LPS group，LPS （1 mg·L-1） was added to the lower THP-1 cells after the upper Caco-2 cells formed a monolayer barrier. On the basis of the LPS group， 33 mg·L-1 ginsenoside Rg1 and 33 mg·L-1 ginsenoside Rb1 were added to THP-1 cells respectively. After the co-culture of THP-1 cells and Caco-2 cells for 24 hours， the fluorescein isothiocyanate （FITC）-Dextran fluorescence value in the lower chip channel was detected by FITC-Dextran tracer method. A blank group， an LPS group，and drug intervention groups were set up in the THP-1 cell experiment. THP-1 cells in the blank group were cultured conventionally. In the LPS group， LPS （1 mg·L-1） was added to THP-1 cells.Ginsenoside Rg1 and ginsenoside Rb1 of the corresponding doses （11，33，100 mg·L-1） were added to the drug intervention groups respectively on the basis of the LSP group. After 24 hours of cell culture， the activity of THP-1 cells was detected by cell counting kit-8 （CCK-8）. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the expression of inflammatory cytokines such as interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and tumor necrosis factor （TNF）-α of THP-1 cells. A blank group， an LPS group， and drug intervention groups were set up in the Caco-2 cell experiment. Caco-2 cells in the blank group were cultured conventionally， and in other groups， the corresponding cell supernatant in the second part of the THP-1 cell experiment was employed in Caco-2 cells. After 24 hours of cell culture，the activity of Caco-2 cells was detected by CCK-8. Real-time PCR was used to detect the expression of IL-6，interleukin-8 （IL-8）， TNF-α， and Occludin of Caco-2 cells. The expression of tight junction protein Occludin in Caco-2 cells was detected by Western blot. ResultBoth ginsenoside Rg1 and ginsenoside Rb1 could effectively protect LPS-induced intestinal epithelial barrier permeability in the co-culture system of THP-1 and Caco-2 cells （P<0.01）. Ginsenosides Rg1 and Rb1 antagonized LPS-induced increased expression of IL-6，IL-1β， and TNF-α in THP-1 cells （P<0.05）. When the supernatant of THP-1 cells treated with ginsenosides Rg1 and Rb1 was co-cultured with Caco-2 cells， the expression of IL-6，IL-8， and TNF-α in Caco-2 cells was significantly reduced （P<0.01）， and the expression of tight junction protein Occludin was up-regulated. ConclusionIn the co-culture system of THP-1 and Caco-2 cells simulating the intestinal epithelial barrier function in vitro，ginsenosides Rg1 and Rb1 play a protective role against LPS-induced intestinal epithelial barrier injury by regulating the release of inflammatory cytokines by THP-1 cells， thereby regulating the inflammatory response and cell barrier integrity of Caco-2 cells.

Protective effect and mechanism of ginsenoside Rb1 on repairing sciatic nerve injury in mice / 解剖学报

Objective To explore the effect and mechanism of ginsenoside Rb1 on the repair of sciatic nerve injury (SNI) in mice. Methods Seventy-eight adult male Kunming mice were randomly divided into sham group (26), SNI group (26), SNI+Rb1 group (26). The SNI+Rb1 group was given 10 mg/kg ginsenoside Rb1 (i.p.), and the SNI group and the sham group were given the same volume of normal saline. The injury method was established by squeezing the sciatic nerve. Sciatic functional index (SFI) was used to evaluate sciatic nerve function. Growth associated protein 43 (GAP43) immunofluorescent staining was used to detect neural regeneration and repair on day 14, and the structure changes of the myelin sheath of the injured segment were observed under transmission electron microscope. Ki67 and S100β were used to detect the proliferation and migration ability of Schwann cells, and Real-time PCR was used to detect the mRNA expression levels after crush on day 3 and day 7. Results SFI of SNI+Rb1 group was higher than SNI group. The HE result showed that the sciatic nerve was uniform in the SNI + Rb1 group. The result of immunofluorescent staining displayed that Rb1 enhanced GAP43

Application of QAMS for quality evaluation and control of Chinese patent medicines:taking Bufonis Venenum-contained preparations as examples / 中国中药杂志

As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.

Ginsenoside Rb1 Protects Human Umbilical Vein Endothelial Cells against High Glucose-Induced Mitochondria-Related Apoptosis through Activating SIRT3 Signalling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate whether ginsenoside Rb1 (Rb1) can protect human umbilical vein endothelial cells (HUVECs) against high glucose-induced apoptosis and examine the underlying mechanism.@*METHODS@#HUVECs were divided into 5 groups: control group (5.5 mmol/L glucose), high glucose (HG, 40 mmol/L) treatment group, Rb1 (50 µ mol/L) treatment group, Rb1 plus HG treatment group, and Rb1 and 3-(@*RESULTS@#Rb1 ameliorated survival in cells in which apoptosis was induced by high glucose (P<0.05 or P<0.01). Upon the addition of Rb1, mitochondrial and intracellular reactive oxygen species generation and malondialdehyde levels were decreased (P<0.01), while the activities of antioxidant enzymes were increased (P<0.05 or P<0.01). Rb1 preserved the mitochondrial membrane potential and reduced the release of Cyt-c from the mitochondria into the cytosol (P<0.01). In addition, Rb1 upregulated mitochondrial biogenesis-associated proteins (P<0.01). Notably, the cytoprotective effects of Rb1 were correlated with SIRT3 signalling pathway activation (P<0.01). The effect of Rb1 against high glucose-induced mitochondria-related apoptosis was restrained by 3-TYP (P<0.05 or P<0.01).@*CONCLUSION@#Rb1 could protect HUVECs from high glucose-induced apoptosis by promoting mitochondrial function and suppressing oxidative stress through the SIRT3 signalling pathway.

Construction and efficacy evaluation of Rb luciferase reporter gene detection system / 西安交通大学学报(医学版)

【Objective】 To construct the Rb luciferase reporter gene assay system and detect the activation ability of Rb gene for screening the targeted drugs. 【Methods】 The synthetic Rb gene sequence was annealed to form a double-stranded DNA structure and then inserted into the polyclonal site of pGL6-TA. The junction product was transformed into E.coli DH5α competent cells for expanded culture, and the constructed pGL6-Rb-Luc plasmid and pGL6-TA plasmid were transfected into HEK293 cells. The monoclonal cell line HEK293-Rb-Luc with stable expression was screened by G418, and the activation and inhibition of Rb in HEK293-Rb-Luc were tested by serum and CDK4/6 inhibitor Palbociclib. 【Results】 The sequence of Rb reaction elements in pGL6-Rb-Luc was completely correct. The recovery of serum culture significantly increased the luciferase activity in HEK293-Rb-Luc (P<0.001). Compared with 0 nmol/L, 25, 50, 75 and 100 nmol/L, CDK4/6 inhibitor Palbociclib made the inhibition rate of Rb activity rise to 6.90%, 40.23%, 50.57% and 52.07%, respectively (P<0.05). 【Conclusion】 The Rb luciferase reporter gene detection system HEK293-Rb-Luc was successfully constructed, which can effectively detect the activation level of Rb.

Superoxide dismutase 3 as an inflammatory suppressor in A549 cells infected with Mycoplasma pneumoniae

Herein, we found that serum concentration of superoxide dismutase 3 (SOD3) was significantly reduced inchildren with mycoplasma pneumonia (MP) infection. To study the roles of SOD3 in inflammatory regulationof MP infection, human A549 type II alveolar epithelial cells were stimulated with 107 CCU/ml of MP to buildMP infection in vitro. Secretion of pro-inflammatory cytokine interleukin (IL)-8 and tumor necrosis factor(TNF)-a were measured via enzyme-linked immunosorbent assay (ELISA) to assess the inflammatory responseof A549 cells. Levofloxacin (LVFX) was used as an anti-inflammatory drug while recombinant TNF-a wasused as an inflammatory promotor in MP-infected cells. Transcriptional activity of nuclear factor (NF)-rB wasassessed by detecting protein levels of nuclear NF-rB and cytoplasm NF-rB using Western blot analysis. Ourdata suggested that the expression of SOD3 mRNA and protein, as well as content of SOD3 in culturedsupernatant, were time-dependently inhibited in MP-infected A549 cells. However, lentiviruses-mediatedSOD3 overexpression alleviated inflammatory response of MP-infected A549 cells, and prevented the uncleartranslocation of NF-rB, as evidenced by obviously reducing the production of IL-8 and TNF-a in cell culturedsupernatant, as well as decreasing nuclear NF-rB while increasing cytoplasm NF-rB. Inspiringly, SOD3overexpression induced anti-inflammatory effect and the inactivation of NF-rB was similar to that of 2 lg/mlof LVFX, but reversed by additional TNF-a treatment. Therefore, we can conclude that transcriptional activityof NF-jB was the underlying mechanism, by which SOD3 regulated inflammatory response in MP infectionin vitro

UPLC-MS/MS determination of active components from Wuzhuyu Decoction in plasma and brain tissues of nitroglycerin-induced migraine rats / 中国中药杂志

A sensitive and specific ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS) method was deve-loped for analysis of rutaecarpine(Ru), evodiamine(Ev), rutaevine(Rv), limonin(Li), ginsendside Rb_1(Rb_1), ginsendside Re(Re) in rat plasma and brain tissues of nitroglycerin-induced migraine rats. Male healthy Sprague-Dawley(SD) rats were orally given multiple dose of optimized(OS) and un-optimized Wuzhuyu Decoction(UNOS), and their blood samples and brainstem were collected at different time points after injection of nitroglycerin(10 mg·kg~(-1)) into the frontal region. The drug concentrations of the 6 analytes in plasma and brainstem were determined by UPLC-MS/MS method. Subsequently, the main pharmacokinetics parameters of plasma were calculated by using Phoenix WinNolin 5.2.1 software. The methodological test showed that all of analytes in both plasma and brainstem homogenate exhibited a good linearity within the concentration range(r>0.994 7). The intra-day and inter-day accuracy, precision, matrix effect, stability of the investigated components meet the requirements for biopharmaceutical analysis. The developed method was successfully applied in pharmacokinetic studies on abovementioned ingredients in rat plasma and brain stem. The plasma pharmacokinetic parameters of active ingredients in two different Wuzhuyu Decoction group were compared, it was found that Rb_1 had higher t_(1/2), T_(max), C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. Meanwhile, Ev had higher t_(1/2) and T_(max) but lower C_(max), AUC_(0-24 h) and AUC_(0-∞), Ru has higher t_(1/2 )but lower C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. The brain tissue distribution of each component were compared between the two groups, the component with higher content in OS, such as Ru at 30 min and 2 h after administration, Ev at 30 min, Rb_1 at 30 min and Rb_1 at 2 h after administration have lower brain tissue distribution than those in UNOS group, while the component with higher content in UNOS, such as Rv at 30 min, 2 h and 12 h after administration had higher brain tissue distribution than those in OS group.

Ginsenoside Rb1 improving autophagic flux against myocardial ischemia reperfusion injury in isolated-heart of rat / 解剖学报

Objective To explore the protective effect of ginsenoside Rb1 on the myocardial ischemia / reperfusion (I / R) injury in rats in vitro. Methods Totally 60 adult male SD rats were randomly divided into 6 groups:sham group,I / R group,ginsenosde Rb1 pretreatment groups(at the doses of 1 μmol / L,5 μmol / L,10 μmol / L and 20 μmol / L,respectively), 10 in each group. The Langendorff perfusion system was used to establish I / R model. The Lab Chart electrophysiological system was used to monitor real-time heart function by monitoring heart rate (HR), left ventricular development pressure (LVDP) and left ventricular development pressure (± dp / dtmax). TTC staining method was used to measure myocardial infarct size. The Western blotting were used to assay Beclin 1, LC3, p62 and Lamp 2 expression, respectively. The immunohistochemistry were used to assay Beclin 1 expression. Results Ginsenoside Rbl of all the four different concent rations improved the decrease of LVDP and ± dp / dtmax arising from myocardial I / R injury. Meanwhile, ginsenoside Rbl significantly decreased the area of cardial infarction. Ginsenoside Rb1 (10 μmol / L) precondition group protected the heart most significantly (P<0. 05). The expression of Beclin 1 with I / R increased significantly in the cytoplasm of cardiomyocytes. Moreover, Beclin 1 expression decreased after addition pretreatment with ginsenoside Rb1 (10 μmol / L) (P < 0. 05). Compared with sham group, we found that the autophagic flux was impaired in I / R group which the expression of Beclin 1, LC3 and p62 increased significantly, as well as the expression of Lamp 2 decreased significantly. On the other hand, pretreatment with ginsenoside Rb1 (10 μmol / L) could reverse impaired autophagic flux (P < 0. 05). Conclusion Ginsenoside Rbl demonstrates pharmacological preconditioning effect and protects against myocardial I / R injury by improving damaged-autophagy flux, the dose of 10 μmol / L precondition protectes the heart most significantly.

Comparison of the effect of soft contact lens and rb-bFGF eye drops on promoting postoperative repair of deep corneal foreign body / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the repairment of wound by using soft contact lenses and rb-bFGF eye drops after deep foreign body removal.<p>METHODS: Patients with deep corneal foreign body(72 cases, 72 eyes)were randomly separated into three groups and received surgery to remove the foreign bodys. Patients in group C only accepted levofloxacin eye drops and ofloxacin eye ointment while patients in group A wore soft contact lenses and group B received rb-bFGF eye drops as an extra after operation. The corneal irritation and pain(1, 3, 5d), wound healing(1wk)and relevant factors in visual acuity impairment(1mo)were observed after deep foreign body removal.<p>RESULTS: Corneal irritation and pain scores in group A were significantly lower than that in the other two groups at 1, 3 and 5d after operation. Patients felt less painful in group B than group C at 3 and 5d. Corneal wound healing in groups A and B was significantly higher than group C at 1wk after surgery(<i>P</i>>0.05). The closer corneal foreign body was to the pupil area, the more vision was affected after 1mo operation(<i>r</i>s=0.635, <i>P</i><0.05).<p>CONCLUSION: Soft contact lenses can effectively alleviate eye irritation after deep corneal foreign body removal in early time. Both SCL and rb-bFGF eye drops can accelerate the recovery of corneal wounds. Visual acuity impairment was closely related to the location of foreign body in deep corneal.

Study on the Improvement Effect of Ginsenoside Rb 3 Combined with β-asarone on Vascular Dementia Model Mice and Its Mechanism / 中国药房

OBJECTIVE：To study the improvement effects of ginsenoside Rb 3 combined with β-asarone on vascular dementia （VD）model mice and its mechanism. METHODS ：ICR mice were randomly divided into model group ，ginsenoside Rb 3 group（10 mg/kg），β-asarone group （10 mg/kg），drug combination group （ginsenoside Rb 3 10 mg/kg+β-asarone 10 mg/kg），positive control group（donepezil hydrochloride 1 mg/kg）and Akt inhibitor group （LY294002，1 mg/kg），and sham operation group was set up ， with 10 mice in each group. Except for sham operation group ，VD model was induced by four vessel occlusion method in other groups. After modeling ，sham operation group and model group were given constant volume of normal saline ，Akt inhibitor group was given relevant medicine intraperitoneally ，and other groups were given relevant medicine intragastrically ，twice a day ，for consecutive 30 d. After last administration ，the learning and memory ability of mice was detected by avoiding darkness test. The contents of 4-hydroxydecenoic acid （4-HNE），8-hydroxydeoxyguanosine （8-OHdG） and reactive oxygen species （ROS） in hippocampus was detected by ELISA. RT-PCR assay was used ， to detect the mRNA expression of Bcl- 2 and Bax inhippocampus. The protein expression of Bcl- 2 in cortex wadetected by immunofluorescence method. Western blotting deng- assay was used to detect the protein expression of Bcl- 2 and mz1@126.com Bax in hippocampus. RESULTS ： Compared with sham operation group ，the incubation period of avoiding darkness xiaoyinlanlp@126.com test in model group was shortened significantly ； and the number of errors was increased significa ntly；4-HNE，8-OHdG and ROS contents ，mRNA and protein expression of Bax were increased significantly ，and mRNA and protein expression of Bcl- 2 was decreased significantly （P＜0.01）. Compared with model group，the incubation period of avoiding darkness test was prolonged significantly in ginsenoside Rb 3 group，β-asarone group ，drug combination group and positive control group ，the number of errors was decreased significantly ；4-HNE，8-OHdG，ROS contents ， mRNA and protein expression of Bax were decreased significantly ，and mRNA and protein expression of Bcl- 2 were increased significantly，especially in drug combination group （P＜0.05 or P＜0.01）. But the incubation period of avoiding darkness test was shortened significantly in Akt inhibitor group ，and the number of errors was increased significantly ；4-HNE，8-OHdG，ROS contents，mRNA and protein expression of Bax were increased significantly ，and mRNA and protein expression of Bcl- 2 were decreased significantly （P＜0.05）. CONCLUSIONS ：Ginsenoside Rb 3 combined with β-asarone has a protective effect on VD model mice ，and the effect was better than that of each compound alone. The mechanism of which may be associated with anti-oxidative stress and anti-apoptosis of hippocampus.

Detectione of the contents of ginsenoside Rb1 and astragaloside IV in Weikang granules by HPLC-ELSD / 药学实践杂志

Objective To optimize the extraction method and develop the detection method of ginsenoside Rb1 and astragaloside Ⅳ in Weikang granules. Methods The extraction process of ginsenoside Rb1 and astragaloside Ⅳ in Weikang granules were optimized by single factor investigation, with the contents of ginsenoside Rb1 and astragaloside Ⅳ as optimization indicators. The HPLC-ELSD method was developed for the detection of ginsenoside Rb1 and astragaloside Ⅳ in Weikang granules. Separation was carried out on an XBridge®Shield RP18 column (4.6 mm×250 mm, 5 μm) with a mobile phase consisting of acetonitrile-water（32:68）at the flow rate of 1 ml/min. The column temperature was maintained at 30 ℃. The drift tube temperature was set at 60 ℃, and the carrier gas flow rate was 1.7 SLM. Results The optimized extraction methods of ginsenoside Rb1 and astragaloside Ⅳ in Weikang granules were as the following: methanol reflux extraction for 1.5 h, and n-butanol extraction and ammonia washed for 5 and 2 times, respectively. The HPLC-ELSD method was established to detect the contents of ginsenoside Rb1 and astragaloside Ⅳ. The linear relationship was good (r > 0.9997). The intra-day and inter-day precision was less than 1%. The recovery rates were 95.65% and 100.57%. The stability and repeatability RSD were less than 3%. The contents were 2.8630 mg/g and 0.2576 mg/g. The RSDs were 0.62% and 1.51%, respectively. Conclusion The extraction method of ginsenoside Rb1 and astragaloside Ⅳ in Weikang granules is optimized, and a reliable, accurate and reproducible HPLC-ELSD method for the detection of the contents of ginsenoside Rb1 and astragaloside Ⅳ in Weikang granules is established.

Role of NR2B and CREB in ginsenoside Rbl regulation of methamphetamine-induced conditioned place preference in rats / 中国药理学通报

Aim To study the effect of ginsenoside Rbl on methamphetamine-induced CPP in rats and to explore the role of NR2B/CREB in it. Methods METH(2mg·kg-1, i.p) was administered to establish METH-induced CPP model in rats. 0 ∼3 d was the adaptation stage and 4 ∼ 13 d was the experimental stage. METH (2 mg · kg-1, i. p) or saline (10 mg · kg-1, i. p) was injected every other day. Rb1 (10 mg · kg-1, i.p) or saline was pre-injected lh before injection of METH or saline. After perfusion, the hippocampus was isolated from brain on ice, and the expression levels of NR2B, CREB and p-CREB were detected by Western blot. Results The animal model of METH-induced CPP was successfully established. The rats were pretreated with Rbl (10 mg · kg-1) for 1 h, and the time that the rats stayed in drug-paired was significantly reduced compared with METH group. Western blot results showed that NR2B, p-CREB and p-CREB/CREB significantly increased in METH group and without altering CREB expression levels compared with control group. However, after pre-treated with Rbl, the expression levels of NR2B, p-CREB and p-CREB/CREB decreased compared with METH group. Conclusions METH can significantly induce CPP in rats. Rbl may inhibit METH-induced CPP in rats by regulating NR2B and p-CREB.