Effect Mechanism of Total Flavonoid of Mori Cortex Combined with Total Saponins of Anemarrhenar Rhizoma on Hyperlipidemia Rats with Osteoporosis / 中国实验方剂学杂志

Objective:To explore the improvement effect of total flavonoids of Mori Cortex combined with total saponins of Anemarrhena Asphodeloide on hyperlipidemia rats with osteoporosis and its possible mechanism. Method:The 40 SPF male SD rats were adaptively fed for 7 days， and then randomly divided into normal group， model group， calcitriol group （45 ng·kg^-1）， total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloide 1∶2 group （0.6 g·kg^-1+0.4 g·kg^-1） and 2∶1 group （1.2 g·kg^-1+0.2 g·kg^-1）. Except for the normal group， rats in the other groups were fed with high fat for 9 weeks， the normal group and the model grouotal flavonoids of total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloip were given normal saline by gavage， and the other groups were given corresponding drugs by gavage， after 12 weeks of administration， except for the normal group ， the other groups were given intramuscular injection of glucocorticoids at the same time. After 22 weeks of administration， the weight of rats with total flavonoids from Mori Cortex combined with total saponins of Anemarrhena Asphodeloide was measured. Serum levels of total cholesterol （TC）， triglyceride （TG）， low density lipoprotein cholesterol （LDL-C）， osteocalcin （BGP） and bone alkaline phosphatase （BALP） were determined by biochemical assay. Hematoxylin-eosin （HE） staining to observe the pathological changes of rat tibia. Real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） was used to detect the expression levels of peroxisomal proliferators activate the receptor gamma（PPAR<italic>γ</italic>） and Runt-related transcription factor 2 （Runx2） mRNA in rat bone tissue， immunofluorescence was used to detect the expression of PPAR<italic>γ</italic> and Runx2 in rats. Result:Compared with normal group， the body mass of rats in model group was significantly increased （<italic>P</italic><0.01）， and the contents of TC， TG， and LDL-C in the serum were significantly increased （<italic>P</italic><0.01）. Compared with model group， the body weight of rats in thet total flavonoids of Mori Cortex and total saponins of Anemarrhena Asphodeloide 1∶2 group and 2∶1 group were significantly reduced （<italic>P</italic><0.01）， and the contents of TC， TG， and LDL-C in the serum were significantly reduced （<italic>P</italic><0.01）， the content of BGP and BALP increased （<italic>P</italic><0.01）. HE staining results showed that compared with the normal group， the tibia fat vacuoles of the model group increased， and the number of osteoblasts decreased， compared with the model group， the total flavonoids of the Mori Cortex and the flavonoids-total saponins of Anemarrhena Asphodeloide 1∶2 group and 2∶1 group decreased in tibia fat vacuoles and increased the number of osteoblasts， the results of immunofluorescence and Real-time PCR showed that， compared with normal group， the expression of Runx2 in the model group decreased and the expression of PPAR<italic>γ</italic> increased （<italic>P</italic><0.01）. Compared with model group， the total flavonoids of Mori Cortex-total saponins 1∶2 group and the total flavonoids of Mori Cortex-total saponins 2∶1 Group up-regulated the expression of Runx2 and down-regulated the expression of PPAR<italic>γ </italic>（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:The total flavonoids of Mori Cortex combined with the total saponins of Anemarrhena Asphodeloide up-regulated Runx2 and down-regulated the expression of PPAR<italic>γ</italic> mRNA and protein， thereby affecting the metabolism of TG and TC in the blood， achieving a therapeutic effect on osteoporosis， provides experimental basis for the clinical prevention and treatment of hyperlipidemia with osteoporosis.

Improvement Effects of Sanggenone C on Lipid Accumulation in FFA-induced Human Liver Cancer HepG 2 Cells / 中国药房

OBJECTIVE：To study the improvement ef fects of sanggenone C on lipid accumulation in human liver cancer HepG2 cells induced by free fatty acid （FFA）. METHODS ：HepG2 cells were divided into control group ，model group ， fenofibrate group （10 μmol/L），sangerone C low ，medium and high concentration groups （2，4，8 μmol/L）. Except for control group，other groups were treated with 1 mmol/L FFA to induce lipid accumulation model ，and administration groups were cultured with relevant medium containing drugs. The lipid accumulation was observed by oil red O staining ，and lipid level and triglyceride （TG） content were also determined. Real-time PCR and Western blot assay were adopted to detect the mRNA and protein expression of PPARα，CPT-1，SREBP-1c，FAS，SIRT1 and PGC- 1α in HepG2 cells. RESULTS ：Compared with control group ， the nucleus was atrophied significantly and the volume became smaller ，and the number of lipid droplets was significantly increased；the level of lipid ，TG content ，mRNA and protein expression of SREBP- 1c and FAS were significantly increased （P＜ 0.05 or P＜0.01），mRNA and protein expression of PPARα，CPT-1，SIRT1 and PGC- 1α were decreased significantly（P＜0.01）. Compared with model group ，no obvious nucleus atrophy and normal volume were observed in sangerone C groups ，and the number of lipid droplets was significantly reduced ；the levels of lipid ，TG content ，mRNA and protein expression of PPARα pathway related genes （except for SREBP- 1c protein in saggenone C low concentration group ）were significantly reversed （P＜ 0.05 or P＜0.01）. CONCLUSIONS ：Sangenone C can significantly improve the lipid accumulation of HepG 2 cells，and its mechanism may associated with regulating PPAR α signaling pathway，improving cell lipid oxidation ability and inhibiting lipid synthesis.

Effect of Sanggenone C on MC3T3-E1 Based on Molecular Docking Technology / 中国实验方剂学杂志

Objective::To observe the effect of sanggenone C (SanC) on the proliferation and differentiation of mouse MC3T3-E1 osteoblasts induced by dexamethasone (DEX), and to explore its mechanism. Method::Molecular docking was conducted between SanC and Runt-associated transcription factor 2(Runx2) protein structure obtained by homologous modeling. MC3T3-E1 cells were jointly treated by different concentrations of SanC (8, 16, and 32 μmol·L-1) and 1 μmol·L-1 DEX, and then cell counting kit-8(CCK-8) method was used to detect the effect of SanC on the proliferation of MC3T3-E1 osteoblasts. The alkaline phosphatase (ALP) activity of MC3T3-E1 osteoblasts was determined by reagent kit and the formation of mineralized bone nodules were detected by alizarin red staining. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of Runx2, ALP and Osterix. The protein expression of Runx2 was detected by Western blot. Result::The docking score of SanC and Runx2 was -9.78.As compared with the normal group, DEX group significantly reduced the cell survival rate (P<0.01), and the greatest difference occurred on the seventh day. As compared with DEX group, SanC could significantly promote the cell proliferation of MC3T3-E1 (P<0.01), in which 32 μmol·L-1 SanC had the largest difference in proliferation rate on seventh day. As compared with the normal group, the expression of Runx2, ALP and Osterix mRNA increased to a certain extent in DEX group(P<0.01). As compared with DEX group, the expression levels of Runx2, ALP and Osterix mRNA were up-regulated in different concentration groups of SanC in a dose-dependent manner (P<0.01). As compared with the normal group, the expression of Runx2 protein in DEX group decreased significantly (P<0.05), and as compared with DEX group, the expression of Runx2 protein in cells under the intervention of SanC increased significantly (P<0.01). Conclusion::SanC can promote the proliferation, differentiation and mineralization of MC3T3-E1 osteoblasts, and the mechanism may be related to the up-regulation of Runx2 expression.

Effect of Anemarrhena Asphodeloside BⅡ on Osteoclast Differentiation Based on Molecular Docking / 中国实验方剂学杂志

Objective:To explore the effect of anemarrhena asphodeloside BⅡ (TBⅡ) on the expressions of nuclear transcription factor-κB receptor activator factor ligand （RANKL）, RANK and C-FOS genes during osteoclast differentiation. Method:Molecular docking software LeDock was used to score the docking of TBⅡ with RANKL, RANK and C-FOS. RAW264.7 was treated with soluble RANKL（sRANKL） and divided into control group, sRANKL group (model group), Icariin (Ica) group, low-dose TBIⅡ group (2 μmol·L-1), medium-dose TBⅡ group (4 μmol·L-1), and high-dose TBⅡ group (8 μmol·L-1). The corresponding kit was used to detect iconic enzyme (TRAP) of osteoclast differentiation. Total RNA was extracted by trizol method, Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the expressions of C-FOS, upstream RANKL/RANK and downstream nuclear factor of activated T-cells cytoplasmic 1 (NFATC1), and osteoprotegerin OPG. Result:The molecular docking score were -11.86, -11.38, -12.34 kcal·mol-1, and there might be multiple binding sites between TBII as well as RANKL, RANK and C-FOS. Compared with the control group, the content of TRAP in model group increased significantly (P<0.01), and compared with model group, the content of TRAP in each administration group decreased significantly (P<0.01), and TBⅡ decreased the content of TRAP in a dose-dependent manner. Compared with the control group, the expressions of RANKL, RANK, C-FOS and NFATC1 increased (P<0.01), whereas the expression of OPG decreased (P<0.01) in model group. Compared with model group, the expressions of RANKL, RANK, C-FOS and NFATC1 decreased (P<0.01), while the expression of OPG increased (P<0.01) in each administration group. Conclusion:TBⅡ may inhibit the differentiation of osteoclast precursors into osteoclasts, inhibit osteoclast activity, reduce bone resorption and improve osteoporosis by regulating RANKL/RANK/C-FOS signal pathway.

Establishment and application of visual LAMP detection method of infectious bovine rhinotracheitis virus / 生物工程学报

Three pairs of primers were designed according to the conserved region of IBRV gB gene published in GenBank(GenBank Accession No. DQ006857.1) using the software Primer Explorer V4. The loop mediated isothermal amplification (LAMP) assay was established by optimization of the reaction system and then evaluated through sensitivity and specificity tests. In total 393 clinical specimens were detected for IBRV using the established LAMP assay performed at 65℃ for 50 min, which produced a ladder-like pattern of amplification bands and the detection result could be judged by color change. The sensitivity of the assay was 10 copies/μL plasmid DNA which was 1000 times higher than that by PCR method and equivalent to nested-PCR. There was no cross-reactivity of the assay with bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV) and vesicular stomatitis virus (VSV). The positive rate of 301 nasal swabs and 92 serum specimens were 87.6% and 58.8%, respectively, which meant nasal swab specimen was more suitable for clinical IBRV detection by the method. The IBRV LAMP method established in this study has the advantages of visualization, quickness, specificity and sensitivity and be suitable for rapid detection of clinical IBRV detection on the spot.

Chemical constituents in leaves of Ostryopsis nobilis and their anti-oxidant activities / 中草药

Objective: To study the chemical constituents of Ostryopsis nobilis and their anti-oxidant activities. Methods: The chemical constituents were isolated and purified by various column chromatographic methods. Their structures were identified by physicochemical properties and spectral analyses. Results: Twenty-three compounds were isolated from the 95% ethanol extract and identified as loliolide (1), maslinic acid (2), vanillic acid (3), 3β-(3, 4-dihydroxycinnamoyl)-erythrodiol (4), dammarenediol II 3-caffeate (5), pinoresinol (6), quercetin (7), daucosterol (8), kaempferol (9), 3, 5-dihydroxy-1, 7-bis (4H-hydroxyphenyl) heptane (10), alnusdiol (11), casuarinondiol (12), quercetin-3-O-α-L-arabinoside (13), isoquercetin (14), 2, 3-dihydroxylbenzoic acid (15), isoquercetin-6″-butyl acetate (16), isoquercetin-6″-benzoate (17), 4″-trans-p-coumaroyl-kaempferol-3-O-α-L-rhamnoside (18), 4″-cis-p-coumaroyl-kaempferol-3-O-α-L-rhamnoside (19), maslinic acid-28-O-β-D-glucoside (20), gallic acid (21), betulatetraol (22), and L-chiroinositol (23). Conclusion: All the compounds are isolated from the plants in this genus for the first time. Diarylheptanoid compounds and other ten monomer compounds exhibit the good scavenging activities against 2, 2-diphenyl-1-picryhydrazyl (DPPH), and O. nobilis extracts show moderate anti-oxidant effects.

HIV-specific IL-2(+) and/or IFN-γ(+) CD8(+) T cell responses during chronic HIV-1 infection in former blood donors / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 Tcell responses and HIV-1 viral load or CD4 count over the course of infection. In this study, 153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors.</p><p><b>METHODS</b>The patients were stratified into three groups according to CD4 count: CD4≥500 cells/μL; 350 cells/μL≤CD4<500 cells/μL; CD4<350 cells/μL. PBMCs were isolated from the patients' anticoagulated blood samples. IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay.</p><p><b>RESULTS</b>An overall inverse correlation were observed between CD4 count and plasma viral load. Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses, CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/μL, no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/μL to 500 cells/μL, significant correlation was only observed between the response breadth of IL-2+IFN-γ+ CD8 T cells and CD4 count; however, as for patients whose CD4 counts were more than 500 cells/μL, direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+ CD8 T cells and viral load or CD4 count.</p><p><b>CONCLUSIONS</b>Universal consistent inverse correlation was only indentified between CD4 count and viral load. The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata, which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions.</p>

Comparison of the quantities and subset distributions of natural killer cells among different races / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Natural killer (NK) cells play critical roles in host immune defense, while the quantities and subset distributions may vary among different races. To address the difference, we compared these variables among Chinese Han, the Caucasians and the Blacks. The study may provide critical background information for both basic research and clinical investigation.</p><p><b>METHODS</b>Blood samples collected from populations of different races were tested within 12 hours after collection and subsets of NK cells were characterized using flow cytometry.</p><p><b>RESULTS</b>The absolute NK count in the Chinese Han was significantly higher than that in the Caucasian. The Han and Caucasian groups showed higher percentages of cytotoxic subset compared to that of the Black group. The percentage of cytokine-producing subset of Chinese Han group was lower than that of Caucasian and Black groups. Black group had a higher percentage of function-unknown NK subset than that of the Han and Caucasian groups.</p><p><b>CONCLUSION</b>Our data indicated that NK cell count and the distribution of different subsets varied among different races, which should be taken into consideration in related investigations.</p>

A mouse model based on replication-competent Tiantan vaccinia expressing luciferase/HIV-1 Gag fusion protein for the evaluation of protective efficacy of HIV vaccine / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy.</p><p><b>METHODS</b>We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy.</p><p><b>RESULTS</b>Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (< or = 10(6) relative luciferase units (RLU)/mg protein) and HIV-1 Gag (> 3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r(2) = 0.71, P < 0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006 +/- 3141) RLU/mg protein in control group to (1538 +/- 463) RLU/mg protein in vaccine group (P = 0.1969).</p><p><b>CONCLUSIONS</b>The luciferase activity in ovary could represent viral replication in vivo; this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.</p>

Analysis of Epstein-Barr virus BamH I "f" variant in nodal metastasis of nasopharyngeal carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the Epstein-Barr virus (EBV) BamH I "f" variant in primary nasopharyngeal carcinoma (NPC) and its metastases in lymph nodes (LN).</p><p><b>METHODS</b>In situ hybridization was used to detect EBV-encoded small RNA (EBER) expression in 21 paired paraffin-embedded tissue from primary NPC and their lymph node metastases and 22 primary NPC without lymph node metastasis. PCR and restriction fragment length polymorphism (RFLP) assay were used to detect EBV BamH I "f" variant in all cases of NPCs, lymph node metastases and 50 cases of chronic inflammation of nasopharynx from Canton.</p><p><b>RESULTS</b>All cases of NPCs and their lymph node metastases showed EBER expression, indicating a high EBV-positive rate in Cantonese NPC patients. EBV BamH I "f" variant was found in 11 cases (52.4%, 11/21) of primary NPCs with LN metastasis, 12 cases (57.1%, 12/21) of the LN metastases, and 18 cases (81.8%, 18/22) of primary NPCs without LN metastasis. However, of the 50 cases of chronic inflammation of nasopharynx, only one case (2.1%, 1/47) demonstrated BamH I "f" variant. The frequency of BamH I "f" variant in NPC was therefore dramatically higher than that in chronic inflammation of nasopharynx. It is of note that atypical hyperplasia was observed in a few epithelial cells from the case of chronic inflammation of nasopharynx expressing BamH I "f" variant.</p><p><b>CONCLUSIONS</b>The frequency of EBV BamH I "f" variant in NPC is significantly higher than that in chronic inflammation of nasopharynx. It is the first demonstration that the BamH I "f" variant is also present in the LN metastases of NPC. The frequency of BamH I "f" variant in metastatic NPC of the lymph node is almost equal to that of primary NPCs.</p>

Effect of GBV-C/HIV coinfection on HIV/AIDS disease progression and HIV replication / 病毒学报

Several research groups have recently reported that persistent GB virus C (GBV-C) co-infected with human immunodeficiency virus (HIV) leads to slower AIDSs disease progression than HIV-1 infection alone. However, these findings were not confirmed by several other studies. To investigate the association between GBV-C replication and plasma HIV loads and CD4+ T cell counts, 203 HIV-1 positive former blood/plasma donors(FBDs) were enrolled from Fuyang city of Anhui Province in China. Plasma specimens were collected from them and were tested for GBV-C using RT-PCR and ELISA. Out of 203 specimens, 52 (25.6%) cases were positive for GBV-C, including 35 male (67.3%) and 17 female (32.7%) cases. No significant association was identified between GBV-C infection and CD4+ T-cell counts or between GBV-C infection and HIV viral loads. Since all the subjects studied were naive to ART, the influence of therapy on AIDS disease progression was ruled out in this study. Overall, our data indicated that HIV-1 positive male FBDs were prone to be infected, GBV-C coinfection with HIV-1 does not significantly influence HIV/AIDS disease progression during the late stage of chronic HIV-1 infection.

Study on the epidemiological significance related to community-structural difference of the rat plague host and vectors in Western Yunnan,China / 中华流行病学杂志

Objective To study the epidemiological significance of community-structural difference regarding both small mammal and flea communities on Rattus ftavipectus in Dehong and Baoshan areas,Yunnan province,during 1982 to 1996.Methods Methodologies as cluster analysis,communities dominated constitution and species diversity were used for data analysis.Results 75 490 small mammals of 27 species could be divided into three types:Ⅰ.Ruili habitat-communities dominated bv R.flavipectus (54.41%)and Suncus murinus(33.37%): Ⅱ.Longchuan and Yingiiang habitat-communities dominated by R.flavipectus(62.99%),S.murinus(23.25%)and Mus musculus(10.06%);Ⅲ.Baoshan habitat-community dominated by R.flavipectus(48.07%),S.murinus(19.56%)and Crocidura attenuats(14.37%).The captured 61 122 fleas of 11 species on R.flavipectus could be divided into three types: i.Ruili habitat-communities dominated by Xenopsylla cheopis(83.51%)and Lentistivslius ferinus(13.86%);ii.Longchuan and Yingjiang habitats X.cheopis(74.42%)and L.segnis(22.94%); iii.Baoshan habitat-communities dominated by L.segnis(70.62%)and X.cheopis(22.70%).There had been 1471 strains of Y.pestis isolated from the hosts of 7 species and vectors of 5 species in Dehong areas for the ten year period.36 strains of Y.pestis were isolated from host of one species and vectors of 2 species in Baoshan area for ten years.The constitution ratio of R.flavipectus and X.cheopis was higher in Dehong area with average as 58.70%and 78.97%respectively.However.the diversities of species among host and vector communities were low,with average as 1.010 and 0.625.On the contrary,the constitution ratios of R.flavipectus and X.cheopis were lower in Baoshan area.with an average as 48.07%and 22.70%respectively but the diversities of species among host and vector community were higher with the averages as 1.471 and 0.829 respectively.The main dominative flea species from 'group ii'to 'group iii'in the flea community had changed from X.cheopis to L.segnis.Conclusion The species diversity index of host and vector community was higher in Baoshan area,while the constitution ratio of main host and vector community was lower.This findings seemed to be the important factor of the decrease of plague prevalence in Baoshan area.