1.Optimization of the establishment protocol of a mouse model of MBA/TPA-induced skin cancer
Jiahui SUN ; Hui SHAN ; Xiaoyu PAN ; Yuchao GU
Acta Laboratorium Animalis Scientia Sinica 2017;25(1):31-35
Objective To induce skin cancer in BALB/c mice using DMBA as initiator and TPA as tumor promot-er. Through optimizing the doses and frequencies of DMBA administration to establish a stable skin cancer model with less time and causing less skin damage. Methods Shaving the back of mice to expose a piece of skin around 2 cm × 2 cm. The mice were divided into a blank control group and four treatment groups randomly. These four groups were given 1, 2, 4, 7 times 100μg/100μL DMBA/acetone, respectively, in the first week, and twice 4μg/100μL TPA/acetone per week in the next 11 weeks. The body weight changes, time of tumor formation and number of tumors formed were recorded during the experiment. The mice were sacrificed at 12th week and samples of tumor tissue and adjacent normal skin tissue were taken for pathological examination using HE staining. Results Tumors were observed at the 7th week in the group with once DMBA administration in the first week and at the 4th week in the group with twice DMBA administration in the first week. Skin cancers were formed also in the group with 4?time DMBA administration in the first week, however, with signif?icantly more severe skin damages. The mice receiving DMBA everyday in the first week died at the 3th week. Conclusions The best induction protocol for skin cancer in BALB/c mice should be twice DMBA in the first week followed by twice TPA
2.Investigation for the Mechanism of Vascular Smooth Muscle Cell Calcification Induced by Calcium and Phosphorus in Experimental Rats
Cuiting QIU ; Anlin LV ; Huan LI ; Xiaoyu JIANG ; Xiaolei MA ; Shan LI ; Xian GUO
Chinese Circulation Journal 2015;(1):64-67
Objective: To explore the effect of oxidative stress injury on the mechanism of vascular smooth muscle cell (VSMC) calciifcation induced by calcium and phosphorus in experimental rats.
Methods: The VSMC calcification was induced by incubating the cells with calcium chloride (CaCl2) andβ-sodium glycerophosphate (β-GP) for 8 days, and the cells were divided into 4 groups: ① Control group, ② Calcification group,③ Calciifcation+H2O2 group, ④ Calciifcation+catalase group. The calcium nodule formation and calcium deposition in VSMC were detected by Alizarin red staining and o-cresolphthalein complexone method, the reactive oxygen species (ROS) was detected by DCFH-DA probe staining and the protein expression of Runx2 was examined by Western blot analysis.
Results: Compared with Control group, Calciifcation group showed the higher ROS production, more calcium nodule and calcium deposition, higher Runx2 protein expression;while compared with Calciifcation group, the above indexes were even higher in Calciifcation+H2O2 group, P<0.05. The ROS production, calcium nodule, calcium deposition and Runx2 protein expression were lower in Calciifcation+catalase group than those in Calciifcation group and Calciifcation+H2O2 group, but still higher than that in Control group. The protein expression of Runx2 was similar between Calciifcation+catalase group and Control group, P>0.05.
Conclusion: CaCl2 andβ-GP treatment may induce VSMC calciifcation via activating ROS-Runx2 signal pathway in experimental rats.
3.Mechanism Underlying Increased Expression of a Member of the Serine/Threonine Kinase Family (Citron kinase) Induced by HIV-1 Infection.
Jiwei DING ; Zeyun MI ; Jianyuan ZHAO ; Jinming ZHOU ; Xiaoyu LI ; Shan CEN
Chinese Journal of Virology 2015;31(4):388-394
Human immunodeficiency virus (HIV)-1 infection changes transcriptional profiles and regulates. the factors and machinery of the host that facilitate viral replication. Our previous study suggested that the serine/threonine kinase citron kinase (citK) promotes HIV-1 egress. To ascertain if HIV-1 infection affects citK expression in primary cells, peripheral blood mononuclear cells were infected with vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV-1 vector NL4-3-luc viruses, which resulted in remarkably increased expression of citK. citK overexpression led to a more than two-fold increase in HIV-1 production, whereas a significant decrease was observed when citK was depleted in CD4+ T cells. Infection with HIV-1 pseudoviruses induced increases in the mRNA and protein levels of citK by 2. 5- and 2. 7-fold in HEK293T cells, respectively. By cloning the 5-kb promoter of citK into a luciferase reporter system and transfecting the construct into HEK293T cells, enhanced luciferase activity was observed during HIV-1 infection. Taken together, these data demonstrate that HIV-1 infection upregulates citK expression at the transcriptional level, and thereby renders the host more susceptible to invasion by HIV-1.
CD4-Positive T-Lymphocytes
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virology
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Cloning, Molecular
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Gene Expression Regulation, Enzymologic
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HEK293 Cells
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HIV-1
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physiology
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Humans
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Intracellular Signaling Peptides and Proteins
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genetics
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Protein-Serine-Threonine Kinases
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genetics
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Up-Regulation
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Virus Replication
4.Establishment and application of a high-throughput screening assay for premature activation of HIV-1 precursors.
Quan ZHANG ; Xiaoyu LI ; Zhenlong LIU ; Pingping JIA ; Xiaolu WEI ; Lixun ZHAO ; Jiandong JIANG ; Shan CEN
Acta Pharmaceutica Sinica 2010;45(2):247-52
Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).
5.Establishment and application of a screening anti-HIV-1 drug model targeted nuclear trafficking of virus RNA.
Zhenlong LIU ; Xiaoyu LI ; Quan ZHANG ; Pingping JIA ; Liang YANG ; Xiaolu WEI ; Jiandong JIANG ; Shan CEN
Acta Pharmaceutica Sinica 2010;45(2):257-62
The HIV-1 Rev protein facilitates nuclear export of unspliced and singly spliced viral transcripts containing RRE RNA through the CRM1 export pathway. Inhibition of Rev-mediated RNA nuclear export can arrest HIV-1 transcriptional process, which clearly, reveals a target for anti-HIV drug development. In this work, a cell-based assay has been established for screening anti-HIV compounds targeting the Rev-mediated RNA nuclear export. This assay utilized a codon-optimized green fluorescent protein (GFP) as reporter gene, which expression is in a Rev-dependent manner. Any compound that inhibits the Rev-mediated RNA nuclear export is identified by reducing emission of GFP. The Z' score of this model is 0.8220. Three thousands compounds were screened and the positive rate was 9.3% with a cutoff at 50% inhibition. IMB7C7, one of the positive compounds, efficiently inhibits viral production from HIV-1 infected cells.
6.Effects of oligomeric grape seed proanthocyanidins on isoproterenol-induced cardiac remodeling in rats
Youmei ZUO ; Shan GAO ; Jianfei CAO ; Xiaoyu LIU ; Hongjian YU ; Ye ZHANG
Acta Pharmaceutica Sinica 2010;45(5):565-70
The purpose of this study is to evaluate the effect of oligomeric grape seed proanthocyanidins (GSP) on isoproterenol (ISO)-induced cardiac remodeling in rats. ISO was given subcutaneously (5 mg x kg(-1), sc, 7 days) to induce cardiac remodeling in rats. Therapeutic groups were given GSP (50, 100, and 150 mg x kg(-1)) after ISO treatment. After 2 weeks intervention, heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), rate of rise of left ventricular pressure (+/- dp/dt(max)) were examined. The myocardial hypertrophy index was expressed as heart weight/body weight (HW/BW) and left ventricle weight/body weight (LVW/BW), the histological changes were investigated by HE and Van Gieson stain. SOD activity and MDA content in serum, contents of hydroxyproline (Hyp) in the left ventricular tissue were assayed by xanthinoxidase method, thiobarbituric acid (TBA) method and alkaline hydrolysis method, respectively. After the onset of ISO-treatment, GSP therapy potently improved cardiac function, inhibited myocardial hypertrophy, improved cardiac pathology change, decreased the myocardial cross-section area (CSA), collagen volume fraction (CVF) and perivascular circumferential collagen area (PVCA), reduced the content of Hyp in the left ventricular tissue, inhibited the decrease of SOD activity and increase of MDA content in serum. GSP possess protective effect against ISO induced cardiac remodeling in rats, this may be related to reducing the oxidative stress and improving antioxidant capacity.
7.The neural mechanism of left hemiparalexia and left hemialexia in reading Chinese characters
Chunlei SHAN ; Tong WANG ; Meixia YU ; Xuchu WENG ; Benyan LUO ; Zhiqiang ZHANG ; Xiaoyu ZHAO ; Zhisu LV
Chinese Journal of Physical Medicine and Rehabilitation 2011;33(1):29-33
Objective To investigate the underlying neural mechanism of left hemiparalexia and left hemialexia in reading Chinese characters. Methods A patient with reading disorders caused by brain infarctions at the left ventralis medialis occipitotemporal lobe and the splenium of the corpus callosum was studied. A series of neuropsychological tests, such as reading Chinese characters presented in the central foveal field or in the left and right half of the foveal field, were conducted with the patient, and neuroimaging techniques including high spatial resolution 3D-MRI and diffusion tensor tractography (DTT) were used to examine whether or not there were lesions of the neural pathway. Results The patient showed left hemiparalexia, which was characterized by making substitution or omission mistakes, mostly in the left parts of Chinese characters, and also left hemialexia(alexia for characters presented in left visual field). 3D-MRI demonstrated infarctions in the left ventral mesial occipitotemporal area and in the left side of the splenium of the corpus callosum. The left lateral mid-fusiform cortex, which has been identified as the visual word form area(VWFA), was almost intact. DTT indicated the major forceps fibers running through the splenium were all disconnected due to the infarction of the left splenium. Conclusion As a result of disruption of the splemium-major forceps pathway, visual character information in the left visual field which is initially projected to the right occipital cortex cannot be transferred from the right visual cortex to the left VWFA. This mechanism of left hemiparalexia and left hemialexia in reading Chinese characters is similar to that in reading English words.
8.Effect of Vitamin K2 on Theaortic Artery Calcification in Experimental Rats
Xiaoyu JIANG ; Donghai ZHANG ; Anlin LV ; Huan LI ; Cuiting QIU ; Xiaolei MA ; Xian GUO ; Shan LI
Chinese Circulation Journal 2015;(11):1101-1105
Objective: To explore the effects of Vitamin K2 (VK2) on theaortic artery calciifcation and oxidative stress injury in experimental rats.
Methods: A total of 24 rats were divided into 4 groups:①Control group,②6-week calciifcation group,③12-week calciifcation group and④6-week calciifcation + 6-week VK2 group;n=6 in each group. The arterial calciifcation was induced by warfarin (WFN) treatment. The calcium nodule and deposition in rat’s theaortic artery were detected by Alizarin red staining and o-cresolphthalein complexone method, the reactive oxygen species (ROS) were measured by DHE probe staining and the morphological changes of mitochondria in smooth muscle cells were detected by transmission electron microscopy.
Results: Calciifcation nodule formed in both 6-week and 12-week calciifcation groups, the calciifcation deposition and ROS were higher than Control group,P<0.01. Compared with both calcification groups, the above indexes were decreased in 6-week calciifcation + 6-week VK2 group,P<0.01. Both calciifcation groups showed mitochondria swelling with unclear structure and cytoplasm vacuoles degeneration in vascular smooth muscle cells. The vascular smooth muscle cell volumes were similar between Control group and 6-week calcification + 6-week VK2 group, and no cytoplasm vacuoles degeneration was observed.
Conclusion: Warfarin induced aortic calciifcation is related to oxidative stress injury which may cause the ultra-micro structural damage in smooth muscle cells; VK2 may reduce the oxidative stress injury and improve the condition of vessel calciifcation in experimental rats.
9.Establishment and drug susceptibility test of isoniazid resistant Mycobacterium smegmatis.
Pingping JIA ; Lili ZHAO ; Xiaoyu LI ; Quan ZHANG ; Zhenlong LIU ; Xin WANG ; Liyan YU ; Lixun ZHAO ; Shan CEN
Acta Pharmaceutica Sinica 2011;46(4):377-82
With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.
10.Effects of liraglutide on ATF4/CHOP pathway of pancreatic beta cells in rats fed with high-fat diet
Ping WANG ; Xiaoyu GUO ; Yu GAO ; Jian LIU ; Xiujie SHAN ; Rui ZHANG ; Xiaochun GE ; Zengbin FENG ; Guifang LI
Chongqing Medicine 2017;46(27):3755-3758
Objective To observe the effects of liraglutide on GRP78,ATF4,CHOP and TRB3 protein and mRNA expression in rats which fed with high fat diet,and to explore the effect of liraglutide on ATF4/CHOP pathway of pancreas in rats fed with high fat diets.Methods Forty-four male Wistar rats were randomly divided into the control group,high fat group,intervention group 1 and intervention group 2,11 cases in each group.The control group was fed with common food,other 3 groups were fed with high fat diet for 8 weeks.Then the intervention group 1 was given with liraglutide (100 μg · kg-1 · d-1) by subcutaneous injection;the intervention group 2 was given liraglutide (200 μg · kg-1 · d-1) by subcutaneous injection.After 2 weeks medication intervention,the rats were killed.Five rats were taken from each group and performed the hyperinsulinemic englycemic clamp experiment under waking state for calculating the glucose infusion rate (GIR).The fasting blood glucose (FBG),fasting insulin (FINS),serum free fatty acid (FFA),total cholesterol (TC),triglyeeride (TG),low density lipoprotein cholesterol (LDL-C) and high density lipoprotein(HDL) were examined,and islet beta cell function index(HOMA-β) were calculated.PCR and Western blot method were used to detect the expression of pancreas GRP78,ATF4,CHOP and TRB3 protein and mRNA.Results Compared with the control group,the levels of FBG,FFA,TC,FINS,TG and LDL-C in the high fat group were significantly increased,the levels of HDL-C,GIR and HOMA-β were significantly decreased(P<0.05 or P<0.01);compared with the high fat group,the levels of HDL-C,GIR and HOMA-β in the intervention group 2 were increased(P<0.05 or P<0.01),the other indicators were significantly decreased;compared with the intervention group 1,blood FGB,FFA,TC in the intervention group 2 were decreased,while the levels of GIR and HOMA-β were increased(P<0.05).Compared with the control group,expression of GRP78,ATF4,CHOP and TRB3 protein and mRNA in the high fat group were significantly increased;compared with the high fat group,the expression of GRP78,ATF4,CHOP and TRB3 protein and mRNA in the intervention group 1 and 2 were gradually decreased with the liraglutide concentration increase.Conclusion Liraglutide can improve insulin resistance and protect pancreatic beta cells in a concentration-dependent manner,its mechanisms may involve in the pancreatic endoplasmic reticulum ATF4/CHOP pathway.