OBJECTIVETo construct a short hairpin (sh)RNA targeting aquaglyceroporin 9 (AQP9) that effectively silences gene expression in liver cells in order to investigate of the role of AQP9 in nonalcoholic fatty liver disease (NAFLD) pathogenesis using an in vitro cell model system.

METHODSSmall interfering (si)RNAs were designed against the human gene sequences encoding AQP9 (NCBI GenBank Accession No. AB008775) and unrelated control sequences, synthesized, annealed to form double-strands, and inserted into the pGenesil- 1 shRNA-expression plasmid. The silencing effects of the four pshRNA-AQP9 constructs (a-d) and the pshRNA-negative control construct were investigated by transfecting into the L02 human normal liver cell line and detecting expression of AQP9 mRNA and protein (relative to beta-actin) by reverse transcription-PCR and western blotting. The NAFLD cell model was established by treating L02 cells with oleic acid to induce fatty degeneration. After transfecting the NAFLD cell model with various constructs, the effects on NAFLD-related features were investigated by staining with Oil Red O (to detect lipid droplets) and performing enzymatic assays (to quantitate triglyceride (TG), free fatty acid (FFA) and glycerol content). The significance of intergroup differences was assessed by analysis of variance test.

RESULTSOf the four pshRNA-AQP9 constructs, pshRNA-AQP9a produced the most robust silencing effect on AQP9 mRNA (25.1 - 1.2% vs. untransfected: 39.3 +/- 1.7% and pshRNA-negative control: 39.4 +/- 1.5%, P < 0.01) and protein (25.4 - 2.0% vs. untransfected: 35.1 +/- 1.9% and psh-RNA-negative control: 35.6 +/- 2.3%, P < 0.01). Oleic acid-induced L02 cells showed enhanced AQP9 mRNA and protein expression, and increased intracellular content of lipid, TG, FFA, and glycerol, which were significantly reduced by pshRNA-AQP9a transfection (all P <0.05).

CONCLUSIONThe new pshRNA-AQP9a construct can efficiently reduce AQP9 expression in cultured human liver cells and relieve steatosis-related features in an NAFLD cell model, pshRNA-AQP9a represents a novel tool for studying the role ofAQP9 in NAFLD pathogenesis and its potential as a gene therapy strategy.

Aquaporins ; genetics ; Cell Line ; Fatty Liver ; genetics ; Gene Expression ; Genetic Vectors ; Hepatocytes ; metabolism ; Humans ; Non-alcoholic Fatty Liver Disease ; Plasmids ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering

Adiponectin ; Fatty Liver ; Humans

Objective TALE-TFs were adopted to provide a new way in detection of the expression result ofβ-ca-sein gene promoter-interesting gene expression cassettes in mouse fibroblasts.Methods TALE-TFs of eukaryotic expres-sion plasmid and expression cassette withβ-casein gene promoter and red fluorescent protein reporter gene were co-nucleo-fected into mouse fibroblasts by Amaxa nucleofector.Results and Conclusion β-casein gene promoter was activated by artificial TALE-TFs in the mouse fibroblasts.The way is a new expression verification system instead of mammary epithelial cells with fibroblasts.

Animals ; Drugs, Chinese Herbal ; chemistry ; Humans ; Liver Cirrhosis ; drug therapy ; Phytotherapy ; Plants, Medicinal ; chemistry

OBJECTIVETo investigate the expression and alteration (including homozygous deletion and mutation) of MTS1 gene in precancerous lesions and squamous cell carcinomas (SCC) of oral mucosa, and to analyse the function of MTS1 gene alteration in oral mucosal carcinogenesis.

METHODSThe expression of p16 protein produced by MTS1 gene was examined with immunohistochemical SP method in 10 normal oral mucosas, 30 precancerous lesions (10 mild, 10 moderate and 10 severe dysplasia respectively) and 45 squamous cell carcinomas (SCCI18, SCCII 19, SCCIII 8). The deletion and mutation of exon1 and exon2 of MTS1 gene were examined with methods of PCR and SSCP in these same samples.

RESULTSAll the precancerous lesions had p16 protein expression and no alteration of MTS1 gene. In SCC, the positive rate of p16 protein was 60.0% with 72.2% in SCCI, 57.9% in SCCII, 37.5% in SCC III, and there were no significant difference among the three groups by chi2 test (P>0.05). Gene homozygous deletion of exon1 and/or exon2 was detected in 10 cases, and gene mutation in 4 cases. The whole rate of gene alteration was 31.1% (14/45). The MTS1 gene alteration rate was 27.8% in SCCI, 31.6% in SCCII, 37.5% in SCC III and there was also no significant difference among the three groups by chi2 test (P>0.05). In SCC with local lymph nodes metastasis, MTS1 alteration rate was 57.1%, while in SCC with no lymph nodes metastasis was 8.3%, and there was significant difference by chi2 test (P<0.05).

CONCLUSIONSMTS1 gene alteration is not an early event in the carcinogenesis of oral mucosa and can not be used as a biology mark to examine oral precancerous lesions. MTS1 gene may play a certain role in the progression of oral squamous cell carcinomas.

Carcinoma, Squamous Cell ; chemistry ; genetics ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Genes, p16 ; Humans ; Lymphatic Metastasis ; Mouth Neoplasms ; chemistry ; genetics ; pathology ; Mutation ; Precancerous Conditions ; genetics

It is a common understanding that turbidity and precipitation of traditional Chinese medicine are easy to occur in the process of decocting. At present, our research group found that the cause of "multi-phase of traditional Chinese medicine decoction" mainly came from the interaction between the effective components of traditional Chinese medicine, especially the interaction of acid and base components. For example, the Liquorice and Rhizoma chinensis was a supramolecular system formed by a large number of active components in the decoction (>30%), and could stably exist in the decoction system. In this study, the supramolecular part was extracted, and the morphology of the supramolecular part was characterized by scanning electron microscopy and dynamic light scattering. It was observed that the supramolecular particles were uniform in size and regular in shape. The main components of supramolecular sites were identified by liquid mass spectrometry (LC-MSⁿ). The results of UV and IR spectra showed that the chemical components of Liquorice and Rhizoma chinensis in the co-decocting process collided with each other, and weak bonds were formed between the functional groups of the molecules, which then induced the aggregation to form supramolecules. Thereafter, Through the diarrhea model of mice, sensory evaluation and antibacterial activity evaluation found that Liquorice and Rhizoma chinensis decocted together enhanced the antibacterial activity of Rhizoma, and compatibility "reconcile" Rhizoma "big bitter cold" property compared with single decoction group and interval administration group. All animal experiments were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine, and the relevant regulations of Beijing University of Chinese Medicine on experimental animals were strictly followed. In this study, supramolecular chemistry method was used to preliminarily discuss the scientific connotation of "increasing efficiency and decreasing toxicity" of Liquorice and Rhizoma chinensis combined decoction from three perspectives of "property, efficacy and taste", and provide new ideas for the basic research of "reconcile" compatibility of Liquorice.

Based on the interaction between supramolecule of traditional Chinese medicine and enterobacteria, the material basis of Rhei Radix et Rhizoma and Coptidis Rhizoma was explored. Scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to characterize the morphological differences of Rhubarb single decoction, Coptis single decoction and Rhubarb and Coptis co-decoction. An in vitro antibacterial model (E. coli, E. faecium and B. subtilis) was established to evaluate the damage effect of the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma on enterobacteria. Ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the changes of chemical components of single decoctions and co-decoctions. The co-decoction of Rhei Radix et Rhizoma and Coptidis Rhizoma was turbid after decocting. The spherical particles of 300-400 nm were observed under SEM, and the co-decoction was more uniform and stable than that of single decoction. The interaction between supramolecules formed after the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma and enterobacteria was significantly different from that of single decoction. In the process of interaction between supramolecules and enterobacteria, the spherical state was maintained, and the medicinal ingredients in Coptidis Rhizoma or Rhei Radix et Rhizoma were blocked, which could effectively alleviate the damage to enterobacteria. This study provided a reference for subsequent studies on the regulation of intestinal flora homeostasis by the combination of Rhei Radix et Rhizoma and Coptidis Rhizoma.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide ; toxicity ; Carcinogens ; toxicity ; Cell Line, Tumor ; DNA Damage ; drug effects ; Dose-Response Relationship, Drug ; HSP70 Heat-Shock Proteins ; biosynthesis ; Humans

OBJECTIVETo assess the effect and adverse reaction of total glucosides of paeony capsule (TGPC) in combining with citirizine for the treatment of chronic urticaria.

METHODSA total of 120 patients were assigned to two groups by lottery, 65 in the treated group and 55 in the control group. They all were orally treated with citirizine tablet 10 mg per day, but to the treated group, additional 0.2 g TGPC was given three times per day, the therapeutic course for both groups was 4 weeks. The effectiveness of treatment was observed, and the changes of total symptom score, serum levels of interleukin-4 (IL-4), and immunoglobulin E (IgE) were measured before and after treatment. Moreover, a follow-up was carried out one month after ending the treatment.

RESULTSThe dropped cases were two in the treated group and seven in the control group; so, the study was accomplished on 63 patients in the treated group and 48 patients in the control group. The total effective rate was assessed at 73.02% (46/63) in the treated group, which was significantly higher than 47.92% (23/48) in the control group (P<0.01). After treatment, the total symptom score decreased in both groups, but the decrement in the treated group was more significant (P<0.05). Serum levels of IL-4 and IgE in the treated group lowered significantly, while the changes in the control group were insignificant, so statistical significant differences were shown between groups (P<0.01). A follow-up study showed that the relapse rate in the treated group was 30.00% (6/20), while that in the control group was 90.00% (9/10), and the former was lower than the latter (P<0.01). Adverse reactions, revealed as drowsiness, dizziness, and weakness, were seen in eight cases and seven cases in the two groups, respectively. Besides, mild diarrhea occurred in two cases of the treated group.

CONCLUSIONSThe treatment of TGPC combining citirizine shows definite curative effect in treating chronic urticaria, with low relapse rate and without evident adverse reaction. Its therapeutic effect might be realized by means of regulating patients' immune function. Besides, the medication should be continued for a rather long period to achieve the full effect.

Adolescent ; Adult ; Anti-Allergic Agents ; adverse effects ; therapeutic use ; Capsules ; Cetirizine ; adverse effects ; therapeutic use ; Chronic Disease ; Drug Therapy, Combination ; Female ; Glucosides ; adverse effects ; therapeutic use ; Humans ; Immunoglobulin E ; blood ; Interleukin-4 ; blood ; Male ; Middle Aged ; Paeonia ; chemistry ; Phytotherapy ; Recurrence ; Treatment Outcome ; Urticaria ; blood ; drug therapy ; Young Adult

Licorice-gypsum (gancao-shigao, GC-SG) drug pair was used as the research object, using supramolecular chemistry to explore the scientific connotation of combining herbal medicine GC with insoluble mineral medicine SG in clinical application of traditional Chinese medicine. ① The Tyndall effect, microscopic morphology and particle size of the single and co-decocted of GC and SG were observed, the paste content and conductivity were determined, and the interaction between GC and SG was detected by isothermal titration calorimetry (ITC) and infrared absorption spectroscopy (IR). ② Calcium chloride (CaCl₂), a soluble calcium salt of equal gypsum quality, was used instead of SG with GC for co-decocting to explore the effect of calcium salt content on the water decocting, and the characteristics were combined with the Tyndall effect, microscopic morphology, paste content and conductivity. ITC and IR techniques were used to detect the interaction between the two, and the interaction between them was detected by ITC and IR. The zeta potential and ultraviolet-visible spectrophotometry (UV-vis) of GC-SG and GC-CaCl₂ co-decoction were compared, and the inorganic and organic components in the co-decoction were detected by inductively coupled plasma optical emission spectrometer (ICP-OES) and high performance liquid chromatography (HPLC). The results showed: ① Compared with the liquid phase of single decoction, GC-SG co-decoction had more obvious Tyndall effect, and showed uniform spherical nanoparticles under electron microscope. Physical characterization results such as paste content and conductivity showed that co-decoction promoted the dissolution of each other's components; ITC and IR results showed that there was strong interaction between GC and SG, which preliminatively indicated that GC and SG co-decoction promoted the formation of uniform and stable supramolecular system of traditional Chinese medicine. ② When soluble calcium salt was used to substitute insoluble SG with GC for co-decocting, a stronger but astigmatic light path appeared than single decocting solution, the zeta potential was reduced, and a large number of accumulated polymers were formed. The results of paste content and conductivity showed that the dissolution of the co-decocting component was reduced than the single decocting component. ITC, UV-vis and IR results showed that there was interaction between GC with Ca²⁺ and SG. The formation of polysink indicated that a large amount of soluble calcium salt would destroy the stability of supramolecular Chinese medicine. The results of ICP-OES and HPLC showed that the glycyrrhizic acid (GA) content of the former lower than the latter, which was related to the formation of a large number of polycondensates with the increase of Ca²⁺ concentration and the decrease of the dissolution of GA and other active ingredients. This study indicates that the compatibility of GC and SG can form a uniform and stable supramolecular system of traditional Chinese medicine. Calcium salt, the main component of SG, is taken as the starting point. Excessive soluble Ca²⁺ can promote the aggregation of active ingredients such as GA, so as to reveal the scientific connotation of the compatibility of GC and SG, an insoluble mineral medicine.