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目的探讨长散在核元件1读码框2基因（L1-ORF2）对GES-1细胞衰老的影响及分子调控机制。方法采用高糖诱导法构建GES-1细胞衰老模型,构建L1-ORF2 si RNA载体,脂质体法将其瞬时转染正常及衰老的GES-1细胞,转染48h后用细胞计数法绘制细胞生长曲线,流式细胞仪分析细胞周期,β-半乳糖苷酶染色检测细胞衰老情况,Western blotting检测转染细胞中L1-ORF2、P53、P21蛋白表达水平。结果成功构建了稳定的GES-1细胞衰老模型及L1-ORF2si RNA载体。与转染了阴性对照载体的细胞相比,转染L1-ORF2 si RNA载体的正常及衰老GES-1细胞的L1-ORF2表达均下降（P〈0.05）。与转染阴性对照载体的衰老GES-1细胞相比,转染L1-ORF2 si RNA载体的衰老GES-1细胞增殖速度变快（P〈0.05）,G0/G1期比例明显减少（34.2% vs 39.3%,P〈0.05）,β-半乳糖苷酶染色比例下降（56% vs 69%,P〈0.05）,而转染阴性对照载体和L1-ORF2 si RNA载体的正常GES-1细胞相比则无明显差异（P〉0.05）。P53蛋白仅在衰老GES-1细胞中有表达,在正常GES-1细胞中无表达,而P21蛋白在正常和衰老的GES-1细胞中均有表达,且后者表达更高（P〈0.05）。与转染阴性对照载体的细胞相比,转染L1-ORF2 si RNA载体的GES-1细胞P53、P21蛋白表达量明显下降（P〈0.05）。结论L1-ORF2 si RNA载体可使正常及衰老的GES-1细胞中L1-ORF2表达下调,促进衰老GES-1细胞的生长和增殖,而对正常GES-1细胞无明显影响。P53、P21蛋白参与了L1-ORF2调控细胞衰老的过程。

Aim： Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan found in traditional Chinese herbs, has been determined to exhibit a variety of pharmacological activities, including anti-tumor, anti-inflammation, neuroprotection, and endurance enhancement. In the present study, we investigated the antioxidation and anti-fatigue effects of arctigenin in rats. Methods： Rat L6 skeletal muscle cell line was exposed to H202 （700 pmol/L）, and ROS level was assayed using DCFH-DA as a probe. Male SD rats were injected with arctigenin （15 mg.kg-1., ip） for 6 weeks, and then the weight-loaded forced swimming test （WFST） was performed to evaluate their endurance. The levels of antioxidant-related genes in L6 cells and the skeletal muscles of rats were analyzed using real-time RT-PCR and Western blotting. Results： Incubation of L6 cells with arctigenin （1, 5, and 20 pmol/L） dose-dependently decreased the H2O2-induced ROS production. WFST results demonstrated that chronic administration of arctigenin significantly enhanced the endurance of rats. Furthermore, molecular biology studies on L6 cells and skeletal muscles of the rats showed that arctigenin effectively increased the expression of the antioxidant-related genes, including superoxide dismutase （SOD）, glutathione reductase （Gsr）, glutathione peroxidase （GPX1）, thioredoxin （Txn） and uncoupling protein 2 （UCP2）, through regulation of two potential antioxidant pathways： AMPK/PGC-1α/PPARα in mitochondria and AMPK/p53/Nrf2 in the cell nucleus. Conclusion： Arctigenin efficiently enhances rat swimming endurance by elevation of the antioxidant capacity of the skeletal muscles, which has thereby highlighted the potential of this natural product as an antioxidant in the treatment of fatigue and related diseases.

Aim： Daidzein （4＇,7-dihydroxyisoflavone） is an isoflavone exiting in many herbs that has shown anti-inflammation activity. The aim of this study was to investigate the mechanism underlying its anti-inflammatory action in murine lung epithelial cells. Methods： C57BL/6 mice were intranasally exposed to TNF-α to induce lung inflammation. The mice were injected with daidzein （400 mg/kg, ip） before TNF-α challenge, and sacrificed 12 h after TNF-α challenge, and lung tissues were collected for analyisis. In in vitro studies, murine MLE-12 epithelial cells were treated with TNF-α （20 ng/mL）. The expression of pro-inflammatory chemokine Cxcl2 mRNA and NF-κB transcriptional activity were examined using real-time PCR and a dual reporter assay. Protein poly-adenosine diphosphate-ribosylation （PARylation） was detecyed using Western blotting and immunoprecipitation assays. Results： Pretreatment of the mice with daidzein markedly attenuated TNF-α-induced lung inflammation, and inhibited Cxcl2 expression in lung tissues. Furthermore, daidzein （10 μmol/L） prevented TNF-α-induced increases in Cxcl2 expression and activity and NF-κB transcriptional activity, and markedly inhibited TNF-α-induced protein PARylation in MLE-12 cells in vitro. In MLE-12 cells co-transfected with the PARP-1 expression plasmid and NF-κB-luc （or Cxcl2-luc） reporter plasmid, TNF-α markedly increased NF-κB （or Cxcl2） activation, which were significantly attenuated in the presence of daidzein （or the protein PARylation inhibitor PJ 34）. PARP-1 activity assay showed that daidzein （10 μmol/L） reduced the activity of PARP-1 by ~75%. Conclusion： The anti-inflammatory action of daidzein in murine lung epithelial cells seems to be mediated via a direct interaction with PARP-1, which inhibits RelA/p65 protein PARylation required for the transcriptional modulation of pro-inflammatory chemokines such as Cxcl2.

Aim： To investigate the effects of diosgenin （Dio）, a naturally occurring steroid saponin, on goiter formation in a mouse model of Graves’ disease （GD） and the underlying mechanisms. Methods： Female BALB/c mice were injected with adenovirus expressing the A subunit of thyrotropin receptor to induce GD. The mice were treated with Dio （20, 100 mg·kg-1·d-1, ip） for 12 or 24 d. The serum levels of TT4 and TRAb were examined using radioimmunoassay and electrochemiluminescence. The size and morphology of thyroid glands were examined. Thyrocyte proliferation was determined using BrdU incorporation assay. The expression of proliferation-associated proteins IGF-1, NF-κB, cyclin D1, and PCNA in thyroids was analyzed using immunohistochemistry and real-time PCR. Results： The GD mice showed significantly high serum levels of TRAb and TT4 compared to the normal mice. Treatment of the GD mice with Dio for 24 d dose-dependently reduced the TT4 level and thyroid size, but did not affect the abnormal level of TRAb. Furthermore, Dio treatment dose-dependently reversed the morphological changes and reduced excessive thyrocyte proliferation in thyroids of the GD mice. Dio treatment also dose-dependently reduced the mRNA and protein levels of IGF-1, NF-κB, cyclin D1, and PCNA in thyroids of the GD mice. Conclusion： Dio relieves goiter in a mouse model of GD through the inhibition of thyrocyte proliferation. The mechanisms involve the suppression of IGF-1, NF-κB, cyclin D1, and PCNA expression.

3′-Daidzein sulfonate sodium is a new synthetic water-soluble compound derived from daidzein（an active ingredient of the kudzu vine root）. It has been shown to have a protective effect on cerebral ischemia/reperfusion injury in rats. We plan to study the mechanism of its protective effect. 3′-Daidzein sulfonate sodium was injected in rats after cerebral ischemia/reperfusion injury. Results showed that 3′-daidzein sulfonate sodium significantly reduced mitochondrial swelling, significantly elevated the mitochondrial membrane potential, increased mitochondrial superoxide dismutase and glutathione peroxidase activities, and decreased mitochondrial malondialdehyde levels. 3′-Daidzein sulfonate sodium improved the structural integrity of the blood-brain barrier and reduced blood-brain barrier permeability. These findings confirmed that 3′-daidzein sulfonate sodium has a protective effect on mitochondrial functions after cerebral ischemia/reperfusion injury, improves brain energy metabolism, and provides protection against blood-brain barrier damage.

The androgen receptor （AR） plays a critical role in prostate cancer development and progression. This study aimed to use a computerized docking approach to examine the interactions between the human AR and phytooestrogens （genistein, daidzein, and flavone） and xeno-oestrogens （bisphenol A, 4-nonylphenol, dichlorodiphenyl trichloroethane [DDT], diethylstilbestrol [DES]）. The predicted three-dimensional structure of AR and androgens was established using X-ray diffraction. The binding of four xeno-oestrogens and three phyto-oestrogens to AR was analysed. The steroids estradiol and dihydrotestosterone （DHT） were used as positive controls and thyroxine as negative control. All the ligands shared the same binding site except for thyroxine. The endogenous hormones DHT and 17β-oestradiol showed the strongest binding with the lowest affinity energy （〈 -10 kcal mol-1）. All three phyto- oestrogens and two xeno-oestrogens （bisphenol A and DES） showed strong binding to AR. The affinities offlavone, genistein, and daidzein were between -8.8 and -8.5 kcal mol 1, while that of bisphenol A was -8.1 kcal mol-l and DES -8.3 kcal mol-1. Another two xeno-oestrogens, 4-nonylphenol and DDT, although they fit within the binding domain of AR, showed weak affinity （-6.4 and -6.7 kcal mol 1, respectively）. The phyto-oestrogens genistein, daidzein and flavone, and the xeno-oestrogens bisphenol A and DES can be regarded as androgenic effectors. The xenooestrogens DDT and 4-nonylphenol bind only weakly to AR.

Senegenin has been shown to inhibit neuronal apoptosis,thereby exerting a neuroprotective effect.In the present study,we established a rat model of spinal cord contusion injury using the modified Allen＇s method.Three hours after injury,senegenin（30 mg/g） was injected into the tail vein for 3 consecutive days.Senegenin reduced the size of syringomyelic cavities,and it substantially reduced the number of apoptotic cells in the spinal cord.At the site of injury,Bax and Caspase-3 m RNA and protein levels were decreased by senegenin,while Bcl-2 m RNA and protein levels were increased.Nerve fiber density was increased in the spinal cord proximal to the brain,and hindlimb motor function and electrophysiological properties of rat hindlimb were improved.Taken together,our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.

[ Objective ] We aimed to establish the identification method of gardenoside and sarsasapogenin in Qingwen baidu granules. [ Method] With ethyl ace- tate-acetone-formic acid-water （10：7：2：0.5） as the developer and 10% sulfuric acid ethanol solution as the chromogenic reagent, the samples were heated at 100 ~C until the spots were clearly visible, and the existence of gardenoside was checked under natural light. With toluene-acetone（9：l ） as the developer and 5% vanillin sulfuric acid solution as the chromogenic reagent, the samples were heated at 105 ~C until the spots were clearly visible, and the existence of sarsasapogenin was checked under natural light. [Result] Qingwen baidu granules had the same spots with gardenoside and sarsasapogenin at the same Rfvalue under natural light. [ Conclusion] A TLC method detecting gardenoside and sarsasapogenin in Qingwen baidu granules was established, which had good specificity and repeatability, suitable for rapid detection of gardenoside and sarsasapogenin.

Daidzein, a plant extract, has antioxidant activity. It is hypothesized, in this study, that daidzein exhibits neuroprotective effects on cerebral ischemia. Rat models of middle cerebral artery oc- clusion were intraperitoneally administered daidzein. Biochemical and immunohistochemical tests showed that superoxide dismutase and nuclear respiratory factor 1 expression levels in the brain tissue decreased after ischemia and they increased obviously after daidzein administra- tion; malondialdehyde level and apoptosis-related cysteine peptidase caspase-3 and caspase-9 immunoreactivity in the brain tissue increased after ischemia and they decreased obviously after daidzein administration. Hematoxylin-eosin staining and luxol fast blue staining results showed that intraperitoneal administration of daidzein markedly alleviated neuronal damage in the ischemic brain tissue. These findings suggest that daidzein exhibits neuroprotective effects on ischemic brain tissue by decreasing oxygen free radical production, which validates the afore- mentioned hypothesis.

Aim： To investigate the protective effects of arctigenin （ATG）, a phenylpropanoid dibenzylbutyrolactone lignan from Arctium lappa L （Compositae）, against ER stress in vitro and the underlying mechanisms. Methods： A cell-based screening assay for ER stress regulators was established. Cell viability was measured using MTI- assay. PCR and Western blotting were used to analyze gene and protein expression. Silencing of the CaMKK[3, LKB1, and AMPK（xl genes was achieved by RNA interference （RNAi）. An ATP bioluminescent assay kit was employed to measure the intracellular ATP levels. Results： ATG （2.5, 5, and 10 μmol/L） inhibited cell death and unfolded protein response （UPR） in a concentration-dependent manner in cells treated with the ER stress inducer brefeldin A （100 nmol/L）. ATG （1, 5, and 10 μmol/L） significantly attenuated protein synthesis in cells through inhibiting mTOR-p7OS6K signaling and eEF2 activity, which were partially reversed by silencing AMPKα1 with RNAi. ATG （1-50 μmol/L） reduced intracellular ATP level and activated AMPK through inhibiting complex I-mediated respiration. Pretreatment of cells with the AMPK inhibitor compound C （25 μmol/L） rescued the inhibitory effects of ATG on ER stress. Furthermore, ATG （2.5 and 5 μmol/L） efficiently activated AMPK and reduced the ER stress and cell death induced by palmitate （2 mmol/L） in INS-115 cells. Conclusion： ATG is an effective ER stress alleviator, which protects cells against ER stress through activating AMPK, thus attenuating protein translation and reducing ER load.