1. Effects and mechanism of Rhizoma Paridis total saponin on invasion and migration abilities of human gastric adenocarcinoma cell MKN-45 induced by LiCl
Chinese Traditional and Herbal Drugs 2019;50(13):3134-3139
Objective: To investigate the effect and the possible mechanism of Rhizoma Paridis total saponin (RPTS) on human gastric cancer cell line MKN-45 proliferation, migration and invasion in vitro. Methods: MKN-45 cells were cultured in vitro and treated respectively with indicated concentrations of RPTS (2.5, 5.0, 10.0, 20.0, and 40.0 μg/mL) for 24 h, and cell viability of cell proliferation was detected by MTT assay; The invasive and metastatic ability of MKN-45 treated with indicated concentrations of RPTS (2.5, 5.0, 10.0 μg/mL) was detected by Transwell migration assay and wound healing assay; Elisa assay was employed to detect the concentrations of MMP-9 induced by LiCl after RPTS administration (10, 20, and 40 μg/mL) in the cell supernatant; Western blotting and qRT-PCR were respectively performed to investigate the invasion and migration related protein and mRNA level of VEGF, COX-2, and GSK-3β in RPTS-treated MKN-45 after LiCl stimulation for 24 h. Results: Compared with the control group, RPTS (10, 20, and 40 μg/mL) significantly inhibited the proliferation of MKN-45 cells (P < 0.05 and P < 0.001); RPTS (2.5, 5.0, 10.0 μg/mL) suppressed the invasion and migration of MKN-45 cells (P < 0.05 and P < 0.001); Compared with the model group, RPTS significantly downregulated the expression of MMP-9 in the cell supernatant of MKN-45 cells induced by LiCl (P < 0.05 and P < 0.01), and RPTS also decreased the protein and mRNA expression level of VEGF and COX-2, but it significantly upregulated the expression of GSK-3β at the protein and mRNA level (P < 0.05 and P < 0.001). Conclusion: RPTS play a pivotal role in suppressing the invasion and migration of MKN-45 cells in vitro, and its mechanism may be related to the regulating effects of the Wnt/β-catenin pathway in the human gastric adenocarcinoma cell.
2.High-level Secretion Expression of Human ScFv Against Botulinum Neurotoxin A in Pichia pastoris*
Hui WANG ; Jun YIN ; Xiao-Jun HOU ; Hong-Guang XING ;
Microbiology 1992;0(02):-
The specific ScFv gene against botulinum neurotoxin A (BoNTa)was cloned into pPIC9k. Positive integrators were screened by increasing the dose of G418 in culture and expressed in Pichia pastoris GS115. As a result, engineered recombinant clone were obtained. 26 kD product of interest was seen easily in SDS-PAGE. Expression of human ScFv got the highest level 15% of total secreted proteins during 72~84 h after 1% methanol inducing. Purification of ScFv was finished by two steps: gel filter and ion exchange. Competing ELISA showed that recombinant ScFv could compete with antiserum to specific bind BoNTa.
3.Expression,Purification of PUMA-BH3 Death Domain Peptide in E.coli and Identification of Its Pro-apoptotic Activity
Yu-Wen ZHANG ; Xing-Han LIU ; Hui-Min LIN ; Ji-Hong LI ; Hong-Xing MA ; Yuan-Li LIU ;
China Biotechnology 2006;0(07):-
The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.
4.Study of intermolecular interactions of piroxicam polymorphs
Wen-hui XING ; Cheng XING ; Hong-mei YU ; Zheng-yu FANG ; Li ZHANG ; Ning-bo GONG ; Yang LÜ
Acta Pharmaceutica Sinica 2022;57(7):2171-2176
Piroxicam has polymorphism. Different crystalline forms can exhibit different physicochemical properties and biological activities. Analysis of the intermolecular interactions is essential to reveal the formation mechanism and differences of polymorphs. In this paper, Hirshfeld surface analysis and semi-empirical methods were used to calculate and analyze the intermolecular interactions in seven polymorphic forms of piroxicam. The results show that the Hirshfeld surface analysis method can clearly and intuitively reveal the intermolecular interactions, among which H…H, O…H/H…O and N…H/H…N interactions account for 95% of the total energy. There are differences in the proportion and distribution of the forces of different crystal forms. The energy calculation shows that the lattice energy of the hydrate is significantly lower than that of the anhydrous forms, and in the specific energy distribution, the contribution of the dispersion force is the most prominent. Further interaction energy analysis was found that within the distance of 3.8 Å from the center of the piroxicam molecule, different crystalline forms of piroxicam molecule have different interaction energies with surrounding molecules.
5.Clinical therapeutic effect of Linezolid for community acquired MRSA:1 case report and review of the literatures
Hui LI ; Xiaoli JING ; Xin LI ; Xiaoxing LIAO ; Hong ZHAN ; Yan XING ; Chunlin HU ; Hongyan WEI
Clinical Medicine of China 2011;27(7):697-700
Objective To investigate the clinic effect of Linezolid for community acquired methicillin resistant staphylococcus aureus (MRS A) pneumonia. Methods The clinic data of the patient- were collected from the First Affiliated Hospital of Sun Yat-Sen University, in addition, the body temperature and white blood cell counts were obtained as the index of treatment. Results It was proved that Linezolid was effective in treating community acquired MRSA pneumonia and showed well tolerance with few adverse events. Conclusion Linezolid demonstrated good clinical and antibacterial activity but very few adverse reactions in elderly patients with community acquired MRSA pneumonia.
6.Effect of Paridis Rhizoma total saponins on apoptosis of human gastric cancer cell MKN-45 and Fas/FasL signaling pathway.
Hai-yan FANG ; Xiao-yan GONG ; Xing-hui HONG ; Ming-liu HUA ; Jin-ling HUANG
China Journal of Chinese Materia Medica 2015;40(7):1388-1391
OBJECTIVEThe study aimed to test if Paridis Rhizoma total saponins (PRTS) could induce apoptosis of human gastric cancer cell MKN-45.
METHODBased on the previous researches, PRTS was set by different concentrations to treat human gastric cancer cell for 12 h (5, 10, 20 mg x L(-1)). Fluorescent staining methods were adopted to observe apoptotic morphological changes of MKN-45. The apoptosis rates were analyzed by flow cytometry with Annexin V-FITC/PI staining. The enzymatic activities of caspase-3 and caspase-8 were measured by ELISA. The protein levels of Fas and FasL were detected by Western blotting.
RESULTUnder a fluorescence microscope, MKN-45 treated by PRTS was seen typical apoptotic morphological features. PRTS significantly increased the rate of apoptosis. Compared with the control group, there exsited significant differences in apoptosis rate of PRTS concentration of 20 mg x L(-1) (P < 0.01); besides, the enzymatic activities of caspase-3 and caspase-8 were promoted obviously after the effect of PRTS on MKN-45 cells for 12 h (P < 0.01). The protein levels of Fas and FasL in the MKN-45 were upgraded significantly.
CONCLUSIONPRTS can induce apoptosis of human gastric cancer cell MKN-45 , which is concerned with caspase-3 and caspase-8 and upgraded Fas and FasL.
Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Caspase 8 ; genetics ; metabolism ; Cell Line, Tumor ; Drugs, Chinese Herbal ; pharmacology ; Fas Ligand Protein ; metabolism ; Humans ; Magnoliopsida ; chemistry ; Rhizome ; chemistry ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Stomach Neoplasms ; drug therapy ; genetics ; metabolism ; physiopathology ; fas Receptor ; metabolism
7.Prediction of CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains in China
Zhiming FANG ; Hui XING ; Zhefeng MENG ; Kunxue HONG ; Lingjie LIAO ; Xiang HE ; Jianxin Lü ; Yiming SHAO
Chinese Journal of Microbiology and Immunology 2010;30(2):135-139
Objective To predict the CTL epitopes of Tat exon 1 region in HIV-1 CRF07_BC strains, which were prevailing in China. Methods Total of 236 plasma samples were from the 3rd National HIV Molecular Epidemic Survey (NMES3). All the subjects were infected with HIV-1 CRF07_BC viruses. The tat exon 1 region was amplified by reverse transcription reaction and nested polymerase chain reaction (nested-PCR), then the PCR products were sequenced. The distribution of CTL epitopes of this region were predicted by on-line software BIMAS HLA Peptide Binding Predictions and statistics software. Results To-tal of 236 CRF07_BC strains were from 16 provinces, mainly in intravenous drug asers(58.9%)and then sex(25.0%). It was showed that there were 12 CTL epitopes of 236 Tat exon 1 region of CRF07_BC strains mainly located in proline-rich region, cysteine-rich region and core-region. Those epitopes were banded by 5 HLA presenting molecules in genotype(A * 2501 ,A * 2902, B * 15,B * 5301 and Cw * 1203) and 6 HLA presenting molecules in serotype (B53, B58 ,B57 ,A3 ,A68 and Cw12). The frequency of single amino acid substitution was more than 50% in 7 CTL epitopes. Conclusion The CTL epitopes in Tat exon 1 of CRF07 _BC strains were located in different functional regions, and there were some amino acid variations in them.
8.Impact of broad antigen HLA-Bw4 on HIV-1 disease progression
Jianping CHEN ; Kunxue HONG ; Mingming JIA ; Guoliang REN ; Hongwei LIU ; Hui XING ; Yuhua RUAN ; Yiming SHAO
Chinese Journal of Microbiology and Immunology 2009;29(3):213-217
Objective To explore the impact of broad antigen HLA-Bw4 on disease progression in HIV-1 infected subjects. Methods Three hundred and forty subjects chronically infected with HIV-1 and 69 HIV-1 negative subjects were recruited and HLA-B alleles were typed with sequence-based high resolution typing assay. HLA-Bw genotypes of these HIV-1 infected subjects were determined and their association with CD4+ T cell counts and viral loads were analyzed. Results Sixty-five HLA-B alleles were detected in HIV-1 positive subjects. Subjects with Bw4 (Bw4 homozygotes and Bw4Bw6 heterozygotes ) had higher CD4+ T cell counts ( P = 0. 004 ) and lower plasma viral load ( P = 0.003 ) than subjects without Bw4 ( Bw6 homozygotes). When compared with HIV-1 postive subjects with CD4+ T cell counts above 500 celis/μl, those with CD4+ T cell counts below 500 cells/μl were observed with decreased percentage of Bw4Bw6 heterozygote ( P =0.0002) and increased percentage of Bw6 homozygotes ( P < 0. 0001 ). There is no significant difference in CD4+ T cell counts between Bw4 homozygotes and Bw4Bw6 heterozygote, but lower viral loads were observed in Bw4Bw6 heterozygotes( P = 0. 037 ). Conclusion HLA-Bw4 can confer pretective effects on H1V-1 infected subjects by maintaining higher CD4+ T cell counts and lower viral load, the mechanism behind this effect need further exploration.
9.Association of the C3435T polymorphism in the multidrug resistance gene 1 and response to antiepileptic drug treatment in epilepsy patients
Jun-Chao LU ; Hui-Min REN ; Guo-Xing ZHU ; Liyun YU ; Ding DING ; Zhen HONG ;
Chinese Journal of Neurology 2005;0(09):-
Objective To determine the frequency of polymorphism at exon 26 (C3435T) of muhidrug resistance 1 gene (MDR1) in epileptic patients in the southern Chinese and to study the association of this polymorphism with pharmacoresistance.Methods DNA samples were obtained from 134 patients,of whom 72 were resistant to antiepileptic drug treatment and 62 were responsive to the treatment. Genotypes of the C3435T polymorphism were determined by polymerase chain reaction (PCR) followed by restriction digestion and gel electrophoresis.Genotype and allele frequencies in the drug resistant group were compared to those in the response group by Chi-square analysis.Results Of all 134 patients,33 (24.6%) had CC genotype,72 (53.7%) had CT genotype,and 29 (21.6%) had TT genotype.The frequency of CC genotype was significantly higher in the pharmaeoresistance group (33.3%) than that in the responsive group (14.5%,P=0.012).The frequency of the C allele was also significantly higher in the pharmacoresistance group (57.6%) than that in the responsive group (44.4%,P=0.03).When patients were divided by types of seizure into three groups:generalized seizure group,partial seizure group,and undefined seizure group,the CC genotype and C allele were associated with pharmacoresistance in the partial seizure group.Conclusions In the southern Chinese,the CC genotype and C allele are associated with resistance to the antiepileptic treatment.This finding needs to be verified in studies with larger sample size.
10.Advance of Ser/Thr Kinase Pololike Kinase 1 (Plk1) Research
Fu-Yin XIONG ; Hui-Yuan LIU ; Hong-Xing CHEN ; Ji-Xian DENG ;
China Biotechnology 2006;0(05):-
Pololike kinase 1(Plk1)contain an Nterminal Ser/Thr kinase catalytic domain and a Cterminal region that contains two poloboxes.As a key regulator of multiple steps during cell cycle across eukaryotic species,many proteins interact with Plk1.Plk1 is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types.Plk1 is a potential target for cancer therapy.Some novel smallmolecule inhibitors of pololike kinase 1 provide novel opportunities for cancerdrug discovery,such as BI 2536,ON01910.