BACKGROUND:When the intervertebral disc is under stress, the hydraulic pressure generated inside the nucleus pulposus makes the annulus fibrosus extend outward and expand, and the annulus colagen fibers are stretched so that the extracelular matrix of annulus fibrosus cels is also under the pressure. In the intervertebral disc, aggrecan is the main component of proteoglycans, matrix metaloproteinase-2 is a major enzyme for extracelular matrix degradation, and tissue inhibitor of metaloproteinase is a multifunctional specific inhibition factor for matrix metaloproteinase activity. There is a mutual regulation between the latter two to keep the homeostasis between them.
OBJECTIVE: To investigate the mechanism of cyclic tensile strain in the metabolism of intervertebral disc annulus matrix.
METHODS:Rat anulus fibrosus cels were subjected to 2% or 10% cyclic tensile strain at 1.0 Hz for 2 and 12 hours using Flexcel4000 tension system. Then cels were colected and cultured in conditioned medium for gene and protein detection. Real-time quantitative PCR was used to detect mRNA expression of aggrecan, matrix metaloproteinases-2 and tissue inhibitor of metaloproteinase-2. Gelatin zymography was used to detect matrix metaloproteinases-2 activity.
RESULTS AND CONCLUSION:The use of 2% cyclic tensile strain had no obvious effect on the stress fiber of actin cytoskeleton, whereas actin cytoskeleton was depolymerized in response to 10% cyclic tensile strain. The 2% cyclic tensile strain raised the expression of Aggrecan at 12 hours; whereas raised the matrix metaloproteinases-2 and tissue inhibitor of metaloproteinase-2 at 2 hours, both of which were in homeostasis; matrix metaloproteinases-2 activity had no significant changes. 10% cyclic tensile strain had no effect on the mRNA expression of Aggrecan. No matter stretching 2 or 12 hours, the matrix metaloproteinases-2 was up-regulated, and the tissue inhibitor of metaloproteinase-2 was down-regulated, both of which were not in balance. Moreover, the matrix metaloproteinases-2 activity was not significantly changed. These findings indicate that the mRNA expressions of Aggrecan, matrix metaloproteinases-2 and tissue inhibitor of metaloproteinase-2 alter in response to cyclic tensile strain in rat anulus fibrosus cels, and the tensile strain induces different mechano-responses in the actin cytoskeleton.

AIM: To investigate the effect of c-Myc inhibitor 10058-F4 on human chronic myeloid leukemia ( CML) K562 cells and imatinib-resistant K562/G cells.METHODS: The protein expression of c-Myc was detected by Western blotting .Cell proliferation was evaluated by MTT assay and colony formation assay .PI staining was used to deter-mine the cell cycle distribution .Annexin V-PI staining was applied for apoptosis detection .RESULTS:Imatinib-resistant K562/G cells displayed lower sensitivity to imatinib than K 562 cells with high expression of c-Myc.Treatment with specific c-Myc inhibitor 10058-F4 inhibited the cell proliferation in a dose-and time-dependent manner , and K562/G displayed more sensitivity to 10058-F4 than K562 cells.10058-F4 also induced cell cycle arrest in G 0/G1 phase and induced apoptot-ic cell death in the 2 cells.Importantly, 10058-F4 suppressed the colony formation ability in K 562 and K562/G cells. CONCLUSION:c-Myc is a novel target to overcome imatinib-induced drug resistance , and c-Myc inhibitor provides a new approach in CML therapy .

Aim To study the modulation of KCNQ2/3 potassium cha nn els by extracellular pH.Methods In vitro transcription was used to synthesize cRNA of KCNQ2/3 potassium channels.The cRNA was injected into Xenopus oocytes to express the KCNQ2/3 channel.The modulation of KCNQ2/3 potass ium channels by extracellular pH was studied by two electrodes voltage clamp tec hniques.Results KCNQ2/3 currents were inhibited and current-vo ltage relationship of activation were shifted to the right with decreased extrac ellular pH. pH modulation of KCNQ2/3 currents was voltage dependent,with a more pronounced effect at more negative potentials above the activation threshold (-60 mV). Extracelluar pH also decreased activation and deactivation kinetics of KCNQ2/3 currents.Conclusion KCNQ2/3 channels, known to contr ibute to neuronal excitability, were modulated by extracelluar pH. The profound effects of the extracelluar pH exerted on KCNQ2/3 channel may play an important role during physiology neuronal activity and pathological events such a s epileptic seizures, cerebral ischemia and shock etc.

Objective:To construct the eukaryotic expression plasmid carrying hTERT-P2A-EGFP, and to explore its expression and transfection efficiency in the HEK293FT cells.Methods:The recombinant plasmid was constructed by using pBABE-puro-hTERT and pRRLSIN-cPPT-MSCV-EGFP plasmids.The hTERT,P2A,and EGFP genes were obtained using pBABE-puro-hTERT as template by PCR.And the correct hTERT was inserted into pRRLSIN-cPPT-MSCV-EGFP vector.Then the recombinant plasmid containing hTERT-P2A-EGFP gene was obtained and identified.The HEK293FT cells were transfected by the recombinant plasmid, and the expression of green fluorescence protein(GFP) was observed by fluorescence microscope.Results:The PCR results showed that the fragments of hTERT, P2A, and EGFP were 3 400, 110 and 720 bp.And the length of gene fragment(hTERT-P2A-EGFP)was 4 300 bp by enzyme digestion.The results of sequencing showed that the 1 547 site of the target gene was mutated.Using site-directed mutagenesis, the 1 547 site was successfully mutated.And the target gene sequence was completely identical with the sequence published in GenBank.The recombinant plasmid was transfected into the HEK293FT cells, and GFP was observed in the cells.The results of flow cytometry showed that the transfection efficiency of recombinant plasmid was 44.8%.Conclusion:The recombinant plasmid carrying hTERT-P2A-EGFP gene is successfully constructed, and it can be used for cell transfection.

Objective Colorectal polyp is a precancerous lesion of colorectal cancer .Aim of the study was to explore the risk fac-tors of colorectal polyp malignant transformation .Methods The related information of 75 084 colonoscopies performed from 2003 to 2012 in Southwest Hospital in Chongqing were collected and the relationship between polyp malignant transformation and the pa-tient age ,sex ,polyp location ,size or histological types was analyzed .Results From 2003 to 2012 ,polyps were diagnosed in 14 806 cases of the total 75 084 patients with a 19 .72% detection rate .There were significant difference of the left-side and right-side pol-yp detection rate in different age groups ,and the frequency of polyps distributed in the whole colorectum increased with the increase of age .The rates of epithelial neoplasia and malignant transformation increased with age .At the same time ,malignant transforma-tion rate was significant higher in polyps located in left-side than that in right-side (P<0 .0167) ,in adenoma than that in inflamma-tory hyperplastic polyp (P<0 .01) .The larger diameter and the more villus ,the higher rate of malignant transformation .Conclusion Patient age ,polyp size ,location and histological type could be considered as the significant predictors of colorectal polyp malignant transformation .It may be useful to treat the polyp with endoscopy in patient with age more than 45 and adenoma whose diameter was not less than 1 cm ,located in left-side for prevention of colorectal cancer .

Objective To explore a clean and efficient new method for extraction of Diosgenin. Methods Yield of the total saponins was evaluated to determine the optimal enzymolysis temperature,pH,solid to liquid ratio,dosage of enzyme and enzymolysis time.Using diosgenin yield as an index,solid to liquid ratio,concentration of sulfuric acid and hydrolysis time were optimized in the saponins hydrolysis process via orthogonal experiment. Results The best conditions for the enzyme pretreatment were as follows:the temperature for enzymolysis was 70℃,pH 5.5,solid to liquid ratio was 1:4,dosage of enzyme was 8 mL?kg-1,and extraction time was 24 h.The best conditions of total saponins hydrolysis were as follows:the solid to liquid ratio was 1:4,concentration of sulfuric acid was 2.0 mol?L-1 ,and hydrolysis time was 5 h. Conclusion The new method is environmental friendly and highly efficient,and expected to be applied in industrial production.

Dioscin chemical compositions are the main effective components in clinical commonly used Chinese medicines such as Diaoxinxuekang capsules and Xinnaoshutong capsules etc , which show distinct curative effect on cardiovascular and cerebrovascular diseases.Meanwhile, they are the important raw materials for the synthesis of steroid hormone drugs .The studies on the extraction technology exhibit important significance in the exploration of pharmacological activities of the components , which also are the external requirements for the growing demand of steroid hormone drugs market .In this paper , the relatively mature extraction methods re-searched in recent years were summarized ,and the advantages and disadvantages of the different processes were discussed in order to provide reference for the further studies and application .

Aim To investigate the effect of Salvianol-ic acid A (Sal A)on mice with isoproterenol (ISO)-induced myocardial infraction and its possible mecha-nisms.Methods The mice were subcutaneously in-jected with ISO (8 mg·kg-1 )to induce myocardial in-farction.The myocardial protective effect of Salvianolic acid A was evaluated from mortality rate,electrocardio-gram (ECG),heart function,myocardial infarction in-dex,serum myocardial enzymes and its action mecha-nisms were explored from inflammation,anti-oxidation and cells apoptosis.Results Salvianolic acid A dose-dependently enhanced the heart function of myocardial infarction mice,reduced the heart index,inhibited the myocardial enzyme leakage,showed obvious myocardi-al protection effects.ELISA results showed that Salvi-
anolic acid A could reduce the expression of myocardial inflammatory cytokines such as IL-6(interleukin-6,IL-6),TNF-α(tumornecrosis factor-α,TNF-α).West-ern-blotting confirmed that Salvianolic acid A could in-crease the expression of anti-apoptotic proteins Bcl-2, reduce the expression of apoptosis protein Bax,and raise the phosphorylation level of PI3K and Akt.Con-clusion Salvianolic acid A displays a significant pro-tective effect against isoproterenol-induced myocardial infarction and its mechanism may be related to the in-crease of PI3K/Akt signal pathway and the inhibition of cell apoptosis and inflammatory reaction.

This study is to investigate the effect of total flavonoids of Uygur medicine bugloss (BTF) on rats with myocardial ischemia/reperfusion injury, and to explore the mechanisms by which it acts. Left anterior descending (LAD) coronary artery in rats was occluded for 30 min followed by 4 h reperfusion. Meanwhile, BTF dissolved in saline was administered intraperitoneally at dosage of 10, 30 and 50 mg x kg(-1). Electrocardiograph, infarction index, serum myocardial enzymes and heart function were determined to evaluate the effect of BTF. Some other observations were carried out to explore whether inhibiting inflammation and apoptosis is involved in the mechanisms underlying BTF. Our results showed that in ischemia/reperfusion injured rats BTF could dose-dependently reduce myocardial infarction index and myocardial enzyme leakage, and enhance heart function, indicating that it possesses significant cardio protection. ELISA analysis showed that BTF could decrease the content of myocardial inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha. Western-blotting confirmed that BTF could increase the expression of anti-apoptotic protein Bcl-2 and reduce the expression of proapoptosis protein Bax. Further more, the phosphorylation level of PI3K and Akt was upregulated by BTF treatment. BTF can protect rat against myocardial ischemia/reperfusion injury. Anti-inflammation and inhibition of apoptosis through upregulating PI3K/Akt signal pathway may contribute to the protective effect of BTF.

Objective To investigate the role of renin angiotensin system (RAS) in pathogenesis of nonalcoholic fatty liver disease (NAFLD).Methods Twenty-four Wistar rats were evenly divided into control group and model group.The rats of control group were fed with normal diet,and model group were with high-fat diet.Rats were killed at the eighth week and serum liver function,blood lipid,glucose and insulin were tested.The liver tissues were stained with HE and Picro acid-Sirius red for pathological observation.The liver tissue concentration of angiotensin Ⅱ was determined by ELISA method and the expression of TGF-β1 in liver tissue was examined by immunohistochemistry.Results After eight weeks high fat feeding,weight,liver index,liver function,blood lipids and serum insulin of model group were significantly higher than those of control group (weight:(463.50±22.72) g vs.(404.29±10.32) g; liver index:(3.75±0.21) g vs.(2.66±0.15) g; ALT:(79.8±8.6) U/L vs.(58.8±11.6) U/L; AST:(200.01±51.72) U/L vs.(150.30±37.27) U/L; total cholesterol:(3.67±0.48) mmol/L vs.(1.50±0.23) mmol/L; triglycerides:(2.06±0.40) mmol/L vs.(0.71±0.34) mmol/L; insulin:(17.37±2.89) pmol/L vs.(11.08±2.12) pmol/L),and all the differences were statistically significant (P＜0.01).The histopathological results of model group indicated liver steatosis,inflammatory reaction in part of lobule and portal area and significant fibrosis in part of liver tissue.The liver tissue angiotonin Ⅱ concentration of model group [(32.80 ± 2.81)pg/ml] was higher than that of control group [(22.83 ± 1.75) pg/ml,t =9.559,P＜0.01].The immunohistochemistry results showed that the expression of TGF-β1 of model group was obviously higher than that of control group (Z=-2.540,P =0.011 ).Spearman correlation analysis revealed that the increasing degree of angiotensin Ⅱ concentration was positively correlated with liver steatosis scores (r=0.644,P=0.002) and the expression of TGF-β1 (r=0.470,P=0.037).Conclusion The concentration of angiotensin Ⅱ and TGF-β1 increased in the livers of model rats,which indicated that RAS may participate in the pathogenesis and progress of NAFLD.