Clinical Observation on Modified Huqianwan in Treatment of Rheumatoid Arthritis Liver-kidney Yin Deficiency Syndrome / 中国实验方剂学杂志

Objective: To explore the clinical efficacy and safety of modified Huqianwan in treatment of rheumatoid arthritis (RA) liver-kidney Yin deficiency syndrome, and investigate its possible mechanism. Method: A total of 184 patients with RA liver-kidney Yin deficiency syndrome were randomly divided into Chinese medicine group (62 cases), western medicine group (57 cases) and integrated Chinese and western medicine group (65 cases) according to the digital table method. The patients in Chinese medicine group were treated with Huqianwan; the patients in western medicine group were treated with methotrexate tablets and leflunomide tablets; and the patients in integrated Chinese and western medicine group received Huqianwan+methotrexate tablets and leflunomide tablets,with a treatment course of 12 weeks in all groups. The pain visual analog scale (VAS), swelling and tenderness scores of 28 joints (DAS28), average hands grip strength, morning stiffness time and liver-kidney Yin deficiency syndrome differentiation of traditional Chinese medicine (TCM) syndrome score were compared between groups before and after treatment. The changes of erythrocyte sedimentation rate (ESR), C reactive protein (CRP), immunoglobulin (Ig) G, tumor necrosis factor-alpha (TNF-α) and rheumatoid factor (RF) were detected in all groups after treatment. Clinical efficacy, and incidence of adverse reactions such as gastrointestinal response, liver injury, leukopenia, serum glutamate oxaloacetic aminotransferase (GOT) and platelet (PLT) level changes were compared between the groups, so as to investigate the efficiency and safety of the different medicines. Result: After 12 weeks of treatment, the total clinical effective rate was 79.0%, 80.7%, and 92.3% respectively in Chinese medicine group, western medicine group, and integrated Chinese and western medicine group; the integrated Chinese and western medicine group was significantly better than the Chinese medicine group and western medicine group (PPPPConclusion: The efficacy in treating RA liver and kidney Yin deficiency syndrome shows no significant difference between modified Huqianwan and methotrexate tablets+leflunomide tablets. In the treatment of RA liver and kidney Yin deficiency syndrome, Huqianwan has fewer adverse reactions. Huqianwan combined with methotrexate tablets+leflunomide tablets is superior to that in methotrexate tablets+leflunomide tablets in treatment of RA liver-kidney Yin deficiency syndrome.

Construction and expression of recombinant adenovirus containing human catalase gene in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro.</p><p><b>METHODS</b>Total RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods.</p><p><b>RESULT</b>High expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity.</p><p><b>CONCLUSION</b>Ad/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.</p>

Identification of differentially expressed genes in rat asthma model by suppression subtractive hybridization technology / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To identify differentially expressed genes related to asthma by using a rat model.</p><p><b>METHODS</b>Total RNA extracted from the asthmatic rats was taken as the tester and the total RNA from the control rats as the driver. Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes. The products of SSH were inserted into pGEM-T Easy vector to establish the subtractive library. The library was amplified through E.coli transformation and positive clones of the transformants were screened. The white clones in selective medium from cDNA library were isolated and digested by EcoR I restriction endonuclease. Thirty-six positive clones were chosen randomly and sequenced. Nucleic acid similarity was subsequently analyzed by comparing with the data from GenBank (NCBI).</p><p><b>RESULTS</b>There were more than 300 white clones in the cDNA library. The clones were sequenced and similarity search (http://www.ncbi.hlm.nih.gov/BLAST) revealed 4 known genes, 2 ESTs without homologous genes and 3 potential new gene fragments.</p><p><b>CONCLUSION</b>The forward-subtracted cDNA library for differentially expressed in the lung of asthmatic rats has been successfully constructed and the interesting candidate genes related to asthma have been identified.</p>

KCNE2 protein S98 phosphorylation in heart of old SHR rats detected by point mutagenesis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the phosphorylation of KCNE2 protein in heart of old SHR rats.</p><p><b>METHODS</b>The membrane proteins from ventricular myocardium of old SHR were extracted, treated with or without alkaline phosphatase and tested binding with Ab2 (an anti-KCNE2 polyclonal antibody) by Western blot. A KCNE2 fusion protein with c-myc was obtained from in vitro translation system and treated with or without alkaline phosphatase. A series of mono- and double-point mutated fusion KCNE2 proteins with c-myc were obtained from an in vitro translation system, and Western blots with Ab2 or anti-myc antibody were performed.</p><p><b>RESULTS</b>After alkaline phosphatase treatment, Ab2 significantly attenuated its binding with KCNE2. In vitro translation system confirmed that after alkaline phosphatase treatment, Ab2 weakened binding ability to KCNE2 while binding to c-myc was not changed. Point mutation experiments showed that serine residue in position 98 of KCNE2 proteins might be phosphorylated.</p><p><b>CONCLUSION</b>KCNE2 protein in heart of old SHR rats is phosphorylated and this phosphorylation takes place in serine residue of position 98.</p>

In vitro study on the genotoxicity of dichloromethane extracts of valerian (DEV) in human endothelial ECV304 cells and the effect of vitamins E and C in attenuating the DEV-induced DNA damages.

To study the genotoxicity of valepotriates in vitro, the degree of DNA damage in human endothelial cell line ECV304 treated with 5-60 microg/mL of dichloromethane extracts of valerian (DEV) was analyzed by the Comet assay. No DNA damage was observed in ECV304 cells after culture for 48 h in the presence of 5,10, and 20 microg/mL of DEV. But a moderate degree of DNA damage was observed in the cells treated with 40 or 60 microg/mL of DEV. Quantitative analyses of DNA damage in the presence of antioxidants vitamin E (VE) and vitamin C (VC) were also carried out. The study revealed that both VE and VC exhibited a biphasic effect, reducing DEV-induced DNA damages at low concentrations but increasing them at high concentrations. We concluded that (1). the observed DNA damage in ECV304 cells induced by high concentrations of DEV was mainly through epigenetic mechanisms, i.e., reactive oxygen species mediated oxidative DNA damage (2). at the low doses, DEV did not appear to have any significant genotoxicity in ECV304 cells, and (3). VE and VC, at proper concentrations, can reduce or eliminate the adverse effects derived from high doses of DEV. This study should serve as scientific guidance for clinical therapy of valerian preparation.

The protective effect and mechanism of soybean oil and its extracts on DNA damage in human ECV304 cells exposed to UV-C.

The degree of DNA damage in the human endothelial cell line ECV304 exposed to UV-C, with or without the presence of soybean oil (SBO), was assessed by the Comet assay. After 5-min exposure to UV-C, the %Tail DNA in the ECV304 cells ranged from 0% to 20% for SBO treatment groups and from 50% to 70% for the control group. The result indicated a strong protective effect of SBO against UV-C-induced DNA damage. To clarity the mechanism of this protective effect of SBO, the methanol extract of SBO (MESO) was analyzed and its capacity against UV-C-induced DNA damage was evaluated. Gas chromatography mass spectrometry (GC-MS) analysis confirmed that MESO contained many antioxidants including n-3-polyunsaturated fatty acid (n-3-PUFA), tocopherols and phytosterols. Comet assay revealed that the MESO was also active in reducing the DNA damage dose-dependently (P<0.0001) vs. control in the ECV304 cells. Therefore, we concluded that these potential antioxidants may be responsible for the scavenge of oxidative radicals induced by UV-C irradiation. This study suggested that dietary SBO, which is abundant of antioxidants, may reduce the content or impact of reactive oxygen species (ROS) and lower the risk of diseases caused by ROS.