Objective To explore the effect of wild type or mutant parkin gene expression on the growth of human hepatocellular carcinoma cell line Huh-7. Methods The parkin (wild type or mutant) expression vector and empty vector were transferred into Huh-7 cell lines with LipofectAMINE 2000 reagents. The positive clones that expressed parkin gene stably were chosen by G418 and checked by reverse transcription-polymerase chain reaction (RT-PCR) to check the DNA sequences. The cytobiological behaviors of those positive clones were analyzed by cell proliferation assay and tumorigenesis in nude mice. Results Huh-7 cell lines that expressed wild type or mutant parkin gene stably were successfully established. The growth of wild type parkin-expressed cells was obviously inhibited compared with the control cells transfected with empty vectors(t= 3. 875, P= 0. 031).The volume of tumor formed by wild type parkin-expressing cells in nude mice was also significantly reduced (t=8. 228,P=-0. 003). Mutant parkin gene expression had a slight effect on the growth of Huh-7 cells in vitro and in vivo (P＞0.05). Conclusion The re-expression of wild type parkin gene can favor the malignant phenotype revision of Huh-7 cells. Therefore, it might be a good candidate for tumor suppressor gene associated with HCC.

OBJECTIVETo compare the differences in the therapeutic effect on irritable bowel syndrome (IBS) between acupuncture at Tianshu (ST 25) and Dachangshu (BL 25) and western medication with Trimebutine Maleate.

METHODSForty cases were divided randomly into an acupuncture group and a western medication group, 20 cases in each one. In acupuncture group, acupuncture was applied to Tianshu (ST 25) and Dachangshu (BL 25). Ziwu Daojiu needling technique was adopted, once daily. In western medication group, Trimebutine Maleate capsule was administered, 2 capsules in each time, 3 times per day. The assessment on the therapeutic effect was performed in 4 weeks of treatment in two groups.

RESULTSAs compared with those before treatment, the time of abdominal pain, the frequency of abdominal pain, the morbidity of abnormal stool appearance, the morbidity of defecation abnormality, the morbidity of mucus stool and the score of bloating or abdominal pain on bowel movement were all reduced after treatment in two groups (all P < 0.01). The results in acupuncture group were much more significant than those in western medication group (the total score: 16.70 +/- 2.40 vs 15.70 +/- 3.01, P < 0.01). The total effective rate in acupuncture group was 95.0% (19/20), which was superior to that of 70.0% (14/20) in western medication group (P < 0.05).

CONCLUSIONAcupuncture at Tianshu (ST 25) and Dachangshu (BL 25) may remarkably relieve the clinical symptoms of IBS and its efficacy is superior to that of oral medication with Trimebutine Maleate.

Acupuncture Therapy ; Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Irritable Bowel Syndrome ; drug therapy ; therapy ; Male ; Middle Aged ; Trimebutine ; therapeutic use ; Young Adult

To study the in vitro anti-angiogenesis effect of three curcumin pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin). In the study, the inhibitory effect of the three curcumin pigments on proliferation of HUVEC cells induced by OX-LDL and the effect on migration of HUVEC cells were detected. The effect on neovascularization was observed by chorioallantoic membrane (CAM) test. The effect on cell adhesion factors ICAM-1 and VCAM-1 of HUVECs were tested by Real-time RT-PCR. It was found that the three curcumins could inhibit the proliferation of HUVEC cells induced by OX-LDL within the dosage range 4, 8, 16 mg x L(-1), with a dose-dependence. The proliferative effect of curcumins on HUVECs was greater than the other two derivatives (P < 0.01). All of the three curcumin pigments inhibited the migration of HUVEC cells and the angiogenesis of chick chorioallantoic membrane (CAM). The migration inhibition rate of curcumins at middle and high concentrations was greater than the other two (P < 0.01). All of the three curcumin could down-regulate the expression of VEGF and ICAM-1, and curcumins showed more obvious effect in down-regulating VEGF than demethoxycurcumin and bisdemethoxycurcumin(P < 0.01); Bisdemethoxycurcumin showed the most significant effect in down-regulating ICAM-1 (P < 0.01). All of the three showed no remarkable effect on expression of VCAM-1, and only bisdemethoxycurcumin showed the down-regulating effect (P < 0.05). According to the findings, all of the three curcumin pigments could resist angiogenesis by inhibiting proliferation and migration of endothelial cells and down-regulating the expression of VEGF and adhesion molecules ICAM-1.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Cell Movement ; drug effects ; Cells, Cultured ; Chorioallantoic Membrane ; drug effects ; Curcumin ; analogs & derivatives ; pharmacology ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; RNA, Messenger ; analysis ; Vascular Cell Adhesion Molecule-1 ; genetics ; Vascular Endothelial Growth Factor A ; genetics

Ten compounds were isolated from the aerial part of Solanum rostratum by means of various chromatographic techniques such as silica gel, Sephadex LH-20, ODS and preparative HPLC. Their structures were identified as dioscin (1), hypoglaucin H (2), hyperin (3), isoquercitrin (4), isorhamnetin-3-O-beta-D-galactopyranoside (5), kaempferol-3-O-beta-D-glucoside (6), smilaxchinoside A (7), 26-O-beta-D-glucopyranosyl-3beta, 20alpha,26-triol-25 (R) -delta5,22-dienofurostan-3-O-alpha-L-rhamnopyranosyl (1 --> 2) -[ alpha-L-rhamnopyranosyl (1 --> 4)] -beta-D-glucopyranoside (8), beta-sitosterol (9), and daucosterol (10), on the basis of physicochemical properties and spectroscopic data analysis. Among them ,compounds 7 and 8 were isolated from the genus Solanum for the first time, and the remaining compounds were obtained from this plant for the first time.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Solanum ; chemistry ; Spectrometry, Mass, Electrospray Ionization

AIMTo explore the relationship between proliferation and hypertrophy of vascular smooth muscle cells and PDGF-AA and PDGFR-alpha expression in SHRs and the role of [Ca2+]i in it.

METHODSExpress difference of PDGF-AA, PDGFR-alpha, PDGFR-beta in SHR/WKY-VSMC was observed by Western blot. The effect of Ca2+ inhibitor (nimodipine) on proliferation, hypertrophy and free Ca2+ concentration of SHR-VSMC induced by PDGF-AA was observed by Western blot, [3H] incorporation and fluorescent digital image technique.

RESULTSPDGF-AA and PDGFR-alpha expression was markedly increased in SHR-VSMC than in WKY-VSMC, but PDGFR-beta was not different in SHR and WKY-VSMC. PDGF-AA-stimulated PCNA expression, [3H] incorporation and [Ca2+]i increasing were observed in SHR-VSMC. Dose-dependent nimodipine-inhibited PCNA expression, [3H] incorporation and [Ca2+]i increasing induced by PDGF-AA also were observed in SHR-VSMC.

CONCLUSIONSSpontaneously expression increasing of PDGF-AA and PDGFR-alpha in spontaneously hypertension rats (SHRs) may be one of the important factors on vascular reactivity and vascular modeling mediated through proliferation and hypertrophy in SHR-VSMC, and [Ca2+]i play an important role in this process.

Animals ; Calcium ; metabolism ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; Platelet-Derived Growth Factor ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Receptors, Platelet-Derived Growth Factor ; metabolism

Objective　To evaluate the role of transfected angiotensinⅡ(Ang Ⅱ) receptor AT1 anti-sense nucleotide (AT1A) in the expression of subtypes of AngⅡ receptor mRNA, synthesis of protein and nucleic acid in cardiomyocytes. Methods　AT1 cDNA sequence (476 bp) was cloned with RT-PCR and reversely inserted into PcDNA3.1 (5.4 kb) to construct an intact plasmid containing AT1A (PAT1A). The plasmid was then transfected into the cultured cardiomyocytes and identified with RT-PCR and Western blot. The synthesis of protein and nucleic acid identified by 3H-Leu and 3H-TdR incorporation, and expressions of AT1 and AT2 mRNA by RT-PCR, were compared between transfected and nontransfected cardiomyocytes after being stimulated with 10-7 mol/L AngⅡ for 24 h. Results　The plasmid PAT1A were successfully constructed. The AT1 mRNA and its protein were expressed significantly less in the transfected cardiomyocytes than in the control (P<0.01). In the transfected cardiomyocytes, AT1 mRNA expression was markedly decreased, but that of AT2 mRNA obviously increased (P<0.01) when compared with the nontransfected cardiomyocytes after stimulation for 24 h with AngⅡ 10-7 mol/L; no significant difference was found in 3H-Leu and 3H-TdR incorporation between them. Conclusion　After the cardiomyocytes was tranfected with AT1A, the expression of AT1 mRNA was markedly suppressed，while AT2 mRNA up-regulated at the same time. Our results indicate that AT1A blocking can not effectively interrupt the Ang Ⅱ-induced synthesis of the protein and nucleic acid in cardiomyocytes.

To establish quality standards of Euonymus fortunei, and supply scientific evidence for the quality control of Euonymus fortunei. Empirical and microscopic identification methods were adopted to observe morphological and histological characters. The contents of water, total ash, acid-insoluble ash and alcohol-soluble extractive were analysed according to the methods of Chinese Pharmaco- poeia (2010). Dulcitol and reference herbs were used to identify materia medica of Euonymus fortunei by TLC method. The total flavonol glycosides contents were analysed by HPLC method, using quercetin and kaempferol as reference substances. Quercetin and kaempferol were separated on a C18 column (4.6 mm x 250 mm, 5 μm) with methanol-0.1% formic acid(51:49) as the mobile phase and detected at 366 nm. The flavonoid aglycones content was then multiplied by a conversion coefficient, and the result was the total flavonol glycosides content. The macroscopical identification, microscopic features and TLC methods were proper. The average contents of water, total ash, acid-insoluble ash, alcohol-soluble extractive and total flavonol glycosides were 8.76%, 6.48%, 0.31%, 17.48% and 0.211% , respectively. The quality standards established on the basis of the research results were suitable for the quality evaluation of Euonymus fortunei.
China ; Chromatography, High Pressure Liquid ; Chromatography, Thin Layer ; Drugs, Chinese Herbal ; chemistry ; standards ; Euonymus ; anatomy & histology ; chemistry ; Mass Spectrometry ; Quality Control

The eye is a highly protected organ , which makes it a formida-ble task to develop an effective drug delivery approach for ocular disea-ses.Transport of drugs applied by traditional dosage forms such as topical and intravitreal routes of administration is severely restricted to the eye , especially to the posterior segment areas.During the past decades , great progress has been made in ocular drug delivery systems facilitated by new technologies such as microemulsion , nanosuspension , nanoparticle , lipo-some, niosome, dendrimer, contact lens, intraocular implants, ionto-phoresis and microneedle , et al.In this review , recent developments of ocular drug delivery systems are summarized.

Background:The pathogenesis of multiple sclerosis (MS) is mediated primarily by T cells,but most studies of MS and its animal model,experimental autoimmune encephalomyelitis (EAE),have focused on CD4+ T cells.The aims of the current study were to determine the pathological interrelationship between CD4 and CD8 autoreactive T cells in MS/EAE.Methods:Female C57BL/6 mice (n=20) were induced by myelin oligodendrocyte glycoprotein (MOG)35-55 peptide.At 14 days after immunization,T cells were isolated from the spleen and purified as CD4+ and CD8+ T cells by using CD4 and CD8 isolation kits,and then the purity was determined by flow cytometric analysis.These cells were stimulated by MOG35-55 peptide and applied to proliferation assays.The interferon-gamma (IFN-γ) and interleukin (IL)-4 secretion of supernatant of cultured CD4+ and CD8+ T cells were measured by enzyme-linked immunosorbent assays (ELISA).For adoptive transfer,recipient mice were injected with MOG35-55-specific CD8+ or CD4+ T cells.EAE clinical course was measured by EAE score at 0-5 scale and spinal cord was examined by staining with hematoxylin and eosin and Luxol fast blue staining.Results:CD8+CD3+ and CD4+CD3+ cells were 86％ and 94％ pure of total CD3+ cells after CD8/CD4 bead enrichment,respectively.These cells were stimulated by MOG35-55 peptide and applied to proliferation assays.Although the CD8+ T cells had a generally lower response to MOG35-55 than CD4+ T cells,the response of CD8+ T cells was not always dependent on CD4.CD8+ T cell secreted less IFN-γ and IL-4 compared with CD4+ T cells.EAE was induced in wildtype B6 na(i)ve mice by adoptive transfer of MOG35-55-specific T cells from B6 active-induced EAE (aEAE) mice.A similar EAE score and slight inflammation and demyelination were found in naive B6 mice after transferring of CD8+ T cells from immunized B6 mice compared with transfer of CD4+ T cells.Conclusion:Our data suggest that CD8+ autoreactive T cells in EAE have a lower encephalitogenic function but are unique and independent on pathogenic of EAE rather than their CD4+ counterparts.

Objective To observe the effect of splenectomy on mortality and brain water content of rats with brain injury so as to explore novel way for better clinical management of patients with severe traumatic brain injury. Methods Adult male SD rats were randomly divided into three groups, ie, sham operation on brain and spleen (Group A, n = 23), experimental brain trauma & sham operation on spleen (Group B, n =48) and experimental brain injury & splenectomy (Group C, n = 47). Modified Feeney' s method was used to create the animal model of experimental brain trauma, Longa' s scale was applied to evaluate the neurologic defect. Mortality within seven days following brain injury was calculat-ed. In the meantime, the brain water content was detected at days 1 (n = 8), 2 (n = 8), 3 (n = 8) and 7 (n = 7) after brain injury in each group, Results No statistical difference of Longs' s scale was found between Group B and Group C (P > 0.05). The mortalities within seven days after brain injury were 0%, 35.42 and 14.89% in Groups A, B and C respectively, with statistical difference between groups (P<0.05). The brain water content of Groups B and C at days 1, 2, 3 and 7 were (81.98±0.35)% & (81.78±0.41)%, (82.58±0.63)% & (81.81±0.48)% (P<0.05),(82.54±0.54)% & (81.52±0.84)% (P<0.05) and (81.50±0.41)% & (81.21±0.36)% (P>0.05) respectively. Conclusion Splenectomy can effectively reduce brain water content and significantly decrease mortality in rata with brain injury.