This study is to establish an HPLC-DAD-ELSD method for simultaneous determination of 5 flavones and saponins in Rhizoma Anemarrhenae including neo-mangiferin, mangiferin, timosaponin B II, timosaponin B III and timosaponin A III. Samples were analyzed on a Merck Purospher STAR column(4.6 mm x 250 mm, 5 μm). The mobile phase consisted of acetonitrile( A) and 0. 1% formic acid (B) with gradient elution at a flow rate of 1.0 mL · min(-1). The column temperature was set at 40 °C. The DAD detector wavelength was set at 254 nm. The ELSD conditions were as follows: the nebulizing gas flow rate was 2.0 L · min(-1) and temperature of drift tube was 105 °C. The volume was 10 μL. The five compounds were well separated with good linear correlations. The mean recoveries were between 102.0%-104.0%. This method was quick and reliable which provides a foundation for quality control of R. Anemarrhenae.
Anemarrhena ; chemistry ; Chromatography, High Pressure Liquid ; instrumentation ; methods ; Drugs, Chinese Herbal ; analysis ; Flavones ; analysis ; Rhizome ; chemistry ; Saponins ; analysis

The aim of this study was to explore the characteristics of Toll-like receptor expression in mesenchymal stem cells derived from bone marrow of healthy donor (BM-MSCs). BM-MSCs were isolated from bone marrow of healthy donor by Ficoll method. Expressions of CD34, CD45, HLA-DR, CD44 and CD71 in BM-MSCs were detected by flow cytometry. CD71 in BM-MSCs was assayed by immunocytochemistry. The adipocyte and osteoblast induction of BM-MSCs were detected by alizarin red stain and oil red stain respectively. TLR 1 - 10 mRNA levels in BM-MSCs were evaluated by semiquantitative RT-PCR. The results showed that expressions of CD34, CD45 and HLA-DR in BM-MSC were negative while the expressions of CD44 and CD71 were positive. CD71 in BM-MSCs was positive. After induced by osteoblast and adipocyte inductor, BM-MSCs were positive for alizarin red staining and oil red staining respectively. All of TLR 1 - 10 mRNA were found in BM-MSCs with high expression levels of TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and low expression levels of TLR1, TLR5, TLR6, TLR10. In conclusion, different levels of TLR 1 - 10 mRNA were expressed in BM-MSCs of healthy donor.
Bone Marrow Cells ; metabolism ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; metabolism ; RNA, Messenger ; genetics ; Toll-Like Receptors ; metabolism

BACKGROUNDAspermia caused by exogenous testosterone limit its usage in late-onset hypogonadism (LOH) patients desiring fertility. Saikokaryukotsuboreito (SKRBT) is reported to improve serum testosterone and relieve LOH-related symptoms. However, it is unclear whether SKRBT affects fertility. We aimed to examine the effects of SKRBT on spermatogenesis and fertility in aging male mice.

METHODSThirty aging male mice were randomly assigned to three groups. Mice were orally administered with phosphate-buffer solution or SKRBT (300 mg/kg, daily) or received testosterone by subcutaneous injections (10 mg/kg, every 3 days). Thirty days later, each male mouse was mated with two female mice. All animals were sacrificed at the end of 90 days. Intratesticular testosterone (ITT) levels, quality of sperm, expression of synaptonemal complex protein 3 (SYCP3), and fertility were assayed.

RESULTSIn the SKRBT-treated group, ITT, quality of sperm, and expression of SYCP3 were all improved compared with the control group (ITT: 85.50 ± 12.31 ng/g vs. 74.10 ± 11.45 ng/g, P = 0.027; sperm number: [14.94 ± 4.63] × 106 cells/ml vs. [8.79 ± 4.38] × 106 cells/ml, P = 0.002; sperm motility: 43.16 ± 9.93% vs. 33.51 ± 6.98%, P = 0.015; the number of SYCP3-positive cells/tubule: 77.50 ± 11.01 ng/ml vs. 49.30 ± 8.73 ng/ml, P < 0.001; the expression of SYCP3 protein: 1.23 ± 0.09 vs. 0.84 ± 0.10, P < 0.001), but fertility was not significantly changed (P > 0.05, respectively). In the testosterone-treated group, ITT, quality of sperm, and expression of SYCP3 were markedly lower than the control group (ITT: 59.00 ± 8.67, P = 0.005; sperm number: [4.34 ± 2.45] × 106 cells/ml, P = 0.018; sperm motility: 19.53 ± 7.69%, P = 0.001; the number of SYCP3-positive cells/tubule: 30.00 ± 11.28, P < 0.001; the percentage of SYCP3-positive tubules/section 71.98 ± 8.88%, P = 0.001; the expression of SYCP3 protein: 0.71 ± 0.09, P < 0.001), and fertility was also suppressed (P < 0.05, respectively).

CONCLUSIONSKRBT had no adverse effect on fertility potential in aging male mice.

Aging ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Fertility ; drug effects ; Hypogonadism ; drug therapy ; Male ; Mice ; Nuclear Proteins ; analysis ; Sperm Count ; Sperm Motility ; drug effects ; Spermatogenesis ; drug effects ; Testis ; drug effects ; pathology ; Testosterone ; blood

OBJECTIVETo determine the relative resistance to HIV-1 infection of CD4 + T lymphocytes in HIV-exposed seronegative individuals (ESNs) in China.

METHODSHIV primary isolates were obtained from peripheral whole blood of HIV-infected persons. CD4 + T lymphocytes of Chinese ESNs were separated from peripheral blood mononuclear cells with magnetic cell sorting (MACS). The purified CD4 + T lymphocytes were cocultured with HIV primary isolates. The p24 level was detected and the culture medium was refreshed every 3 days within 2 weeks.

RESULTSFor M tropic HIV strains, p24 level was significantly lower in ESN group than in control group (P < 0.05); for some M tropic HIV strains, even no p24 replicated in ESN group. However, T tropic virus strains had no significant difference between these two groups (P > 0.05).

CONCLUSIONCD4 + T lymphocytes of Chinese ESNs may possess relative resistance to M tropic HIV strains, which may be one of the main influencing factors that result in ESN.

Adult ; CD4-Positive T-Lymphocytes ; immunology ; virology ; China ; Female ; HIV ; classification ; isolation & purification ; pathogenicity ; HIV Infections ; virology ; HIV Seronegativity ; immunology ; Humans ; In Vitro Techniques ; Male ; Sexual Partners

Ketamine has been used clinically as an analgesic and anesthetic since 1970. Recently, it has been concerned due to its rapid and effective antidepressant effect. However, the psychiatric adverse reactions caused by ketamine such as addiction and hallucination limit its clinical application, yet the mechanism of the adverse reactions is still unclear. At present, it is generally believed that the pharmacological effects of ketamine are mainly mediated by N-methyl-D-aspartate acid (NMDA) receptors, but increasing numbers of research evidences suggest that the opioid receptor also play an important role in the pharmacological effects of ketamine. This review, based on the relevant literature published publicly in the past 20 years, summarizes the mechanisms of opioid receptors in the pharmacological effects of ketamine, such as anesthesia, analgesia, anti-depressant, anti-addiction and addiction, providing reference for revealing the mechanism of ketamine, and making beneficial exploration for solving the clinical side effects of ketamine.

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.
Apoptosis* ; Aspirin ; Cell Survival ; Chickens* ; Heat Stress Disorders ; Heat-Shock Proteins* ; Hot Temperature* ; HSP90 Heat-Shock Proteins ; In Vitro Techniques* ; Proto-Oncogene Proteins c-akt ; Reactive Oxygen Species ; Transducers

OBJECTIVE: To investigate the changes of creatine kinase-MB (CK-MB) and heat shock protein 60 (HSP 60) in rats without electric marks after electric injury, to identify the relationship of the CK-MB, HSP 60 and the time of electric injuries, and to evaluate the damage to cells after electric injury. METHODS: The animal model of electric injury without electric marks was established by alternating current (voltage 110 V). Automatic biochemistry analyzer was used to detect the serum CK-MB and immunohistochemical staining technology was used to analyze the tissues of myocardium and left lobe of liver. RESULTS: The amount of serum CK-MB was increased when the rats were injuried, and reached the peak at 30min. Then the amount of CK-MB began to decrease and showed a slight downward trend in 3-5 h after electric injury, and leveled off at 6 h. Immunohistochemistry staining also showed the changes of HSP 60 of rats' myocardial cells and hepatic cells regularly after electric injury. CONCLUSION The regular changes of serum CK-MB and tissular HSP 60 in rats can be used to diagnosis electric injury and assess the injury of internal organs after the electric injury without electric marks.
Animals ; Chaperonin 60/metabolism* ; Creatine Kinase, MB Form/metabolism* ; Electric Injuries/complications* ; Immunohistochemistry ; Liver/pathology* ; Myocardium/pathology* ; Rats

This study was aimed to explore the effects of peptidoglycan (PGN) on proliferation and cell cycle of human bone marrow mesenchymal stem cells (MSCs). MSCs were isolated from human bone marrow by density gradient centrifugation. The purity of MSCs with the spindle fibroblastic morphology was identified by microphotography and the phenotypes were detected by flow cytometry (FCM). MSCs incubated with different doses of PGN (1, 10, 20 μg/ml) were used as test groups, and those incubated without PGN were regarded as control group. The isolated and cultured MSCs were inoculated into 96-well plates according to a certain concentration. Cell cycle was measured by flow cytometry after incubated with PGN for 72 hours. The results showed that the cell proliferation index was significantly increased in dose and time dependent manners after MSCs was incubated with PGN. Its effects on the proliferation of MSCs were highest in 10 μg/ml group. Compared with the control group, PGN could significantly decrease proportion of MSCs in G₀/G₁ phase and increase them in S and G₂/M phases (p < 0.05). It is concluded that PGN can promote more MSCs to enter the DNA synthesis phase and proliferate many much MSCs in dose and time dependent manners.
Bone Marrow Cells ; cytology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Humans ; Mesenchymal Stromal Cells ; cytology ; Peptidoglycan ; pharmacology ; Toll-Like Receptor 2

OBJECTIVETo evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 β (IL-1 β)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis.

METHODSSerum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 β stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 β-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 β, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction.

RESULTSIL-1 β stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 β, TNF-α, MMP-3 and MMP-13. However, the IL-1 β-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs.

CONCLUSIONZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.

Animals ; Base Sequence ; Blotting, Western ; Caveolins ; metabolism ; Chondrocytes ; drug effects ; enzymology ; metabolism ; DNA Primers ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Profiling ; Interleukin-1beta ; physiology ; MAP Kinase Signaling System ; Male ; RNA, Messenger ; genetics ; Rabbits ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

OBJECTIVETo elucidate the effect of targeted gene modulation of peroxisome proliferator activated receptor gamma (PPARg) on hepatocellular apoptosis in nutritional fibrotic steatohepatitis in mice. C57BL/6J mice were fed with high fat, methionine-choline deficient (MCD) diet for 8 weeks to induce fibrotic steatohepatitis. Mice fed the MCD diet were treated with adenovirus carrying PPARg (Ad-PPARg), adenovirus-beta-galactosidase (Ad-LacZ), Ad-PPARg plus PPARg agonist rosiglitazone, or PPARg antagonist 2-chloro-5-nitro- benzanilide (GW9662), respectively. H and E stain was performed for observation of hepatocellular apoptosis, hepatic steatosis, inflammation and fibrosis in the liver sections. The expression levels of mRNA and protein of PPARg and apoptosis related genes, Fas, Fas Ligand (FasL), B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax) and cysteine-containing aspartate-specific proteases-3 (caspase-3) were detected by real-time RT-PCR and Western blot assay, respectively.

RESULTSMice fed with MCD diet for 8 weeks showed severe hepatic injury including steatosis, hepatocellular apoptosis, inflammatory infiltration and fibrosis, concomitancy with enhanced expression of pro-apoptosis genes, Fas, FasL, Bax and caspase-3 and increased expression of anti-apoptosis gene Bcl-2, by comparing with the control group. The mRNA expression levels of these genes were 3.59+/-0.35 vs 1.11+/-0.37, 4.37+/-1.03 vs 1.09+/-0.33, 4.27+/-0.48 vs 1.03+/-0.10, 4.93+/-0.67 vs 1.12+/-0.24 and 3.95+/-0.34 vs 1.20+/-0.19, and LSD-t values were 2.49, 3.28, 3.25, 3.80 and 2.75, as compared with the control group, P is less than 0.01; the protein expression levels were 1.96+/-0.07 vs 0.45+/-0.07, 0.53+/-0.07 vs 0.22+/-0.02, 1.32+/-0.06 vs 0.59+/-0.03, 1.51+/-0.23 vs 0.36+/-0.09 and 0.57+/-0.01 vs 0.29+/-0.01, and LSD-t values were 1.51, 0.31, 0.73, 1.14 and 0.28, P is less than 0.01. Administration of PPARg agonist rosiglitazone and/or Ad-PPARg significantly ameliorated hepatic steatosis, hepatocellular apoptosis, necro inflammation and fibrosis. These effects were associated with repressed expression of pro-apoptosis genes and up-regulated expression of anti-apoptosis gene. After rosiglitazone treatment, the mRNA expression levels were 3.78+/-0.58, 3.66+/-0.83, 3.04+/-0.37, 2.54+/-0.62 and 4.42+/-0.42, and LSD-t values were 0.18, 0.71, 1.23, 2.39 and 0.46, as compared with MCD group, the P values were 0.627, 0.241, less than 0.01, less than 0.01 and 0.278, the protein expression levels were 1.06+/-0.03, 0.30+/-0.01, 0.70+/-0.05, 1.19+/-0.30 and 0.90+/-0.01, and LSD-t values were 0.90, 0.23, 0.62, 0.31 and 0.34, the P values were less than 0.01, less than 0.01, less than 0.01, 0.122, less than 0.01. After Ad-PPARg treatment, the mRNA expression levels were 2.31+/-0.16, 2.71+/-0.23, 2.52+/-0.27, 1.79+/-0.32 and 5.97+/-0.72, and LSD-t values were 1.28, 1.66, 1.75, 3.13 and 2.02, as compared with MCD group, P is less than 0.05; the protein expression levels were 1.73+/-0.07, 0.43+/-0.04, 1.01+/-0.08, 1.31+/-0.10 and 1.56+/-0.04, and LSD-t values were 0.23, 0.10, 0.30, 0.20 and 0.99, with P values equal 0.009, 0.01, less than 0.01, 0.322 and less than 0.01.

CONCLUSIONSThis study provided evidences for the protective role of activation and overexpression of PPARg in ameliorating hepatocellular apoptosis in mice with hepatic fibrosing steatohepatitis.

Animals ; Apoptosis ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; PPAR gamma ; metabolism