Essential Oil from Alpiniae Zerumbet Fructus Alleviates High Glucose-induced HUVEC Injury via TXNIP Signaling Pathway / 中国实验方剂学杂志

Objective:To study the protective effect of essential oil from Alpiniae Zerumbet Fructus （EOAZF） against high glucose （HG）-induced injury of human umbilical vein endothelial cells （HUVECs） <italic>in vitro</italic>， so as to provide experimental evidence for the treatment of diabetes-induced cardiovascular diseases with EOAZF. Method:The cells were divided into the normal group， model group （25 mmol·L^-1 glucose）， positive control group （100 mg·L^-1 vitamin C）， and the low- （0.25 μg·L^-1）， medium- （1 μg·L^-1）， and high-dose （4 μg·L^-1） EOAZF groups. The HUVECs were damaged by HG. The secretion amounts of malondialdehyde （MDA）， nitric oxide （NO）， and endothelin-1 （ET-1） in HUVECs of different groups were measured to assess the protective effect of EOAZF against HG-induced injury. The effects of EOAZF on the apoptosis and reactive oxygen species （ROS） generation of HUVECs damaged by HG were detected by Annexin V-fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） staining and dichloro-dihydro-fluorescein diacetate （DCFH-DA） assay. The protein and mRNA expression levels of thioredoxin interacting protein （TXNIP） and thioredoxin 1 （Trx-1） were determined by Western blot and Real-time polymerase chain reaction （Real-time PCR）， followed by the measurement of total intracellular Trx-1 activity with insulin disulfide reduction method. Result:The comparison with the control group revealed that the proliferation of HUVECs in the model group was significantly inhibited and their shape was damaged. Compared with the model group， EOAZF protected HUVECs against HG-induced injury in a concentration-dependent manner. The secretion amounts of MDA and ET-1 （<italic>P</italic><0.05） in the model group were increased in contrast to those in the control group， while the NO level was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at all the three concentrations， especially at 4 μg·L^-1， obviously reduced the secretion of MDA and ET-1 （<italic>P</italic><0.05）， but elevated NO after HG induction （<italic>P</italic><0.05）. The cell apoptosis assay and ROS detection results demonstrated that the apoptosis and ROS level in the model group were higher than those in the control group （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly lowered the ROS level and apoptosis （<italic>P</italic><0.05） of HUVECs damaged by HG. The Western blot assay and Trx-1 activity detection uncovered that the protein and mRNA expression levels of TXNIP in the model group were significantly up-regulated as compared with those in the control group （<italic>P</italic><0.05）， whereas the Trx-1 activity was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly down-regulated the mRNA and protein （<italic>P</italic><0.05） expression levels of TXNIP and enhanced the total Trx-1 activity （<italic>P</italic><0.05） in HUVECs， thus suppressing the oxidative stress. Conclusion:EOAZF exerts the protective effects against HG-induced injury in HUVECs by improving the endothelial function and reducing intracellular ROS and apoptosis. Its efficacy in anti-oxidative stress may be related to the down-regulation of mRNA and protein expression levels of TXNIP and the enhancement of Trx-1 activity.

Inhibitory Effect of Essential Oil from Fructus Alpinia zerumbet on Endothelial-to-mesenchymal Transition Induced by High Glucose via Nrf2/Notch1 Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the inhibitory effect and the possible mechanism of essential oil from fructus Alpinia zerumbet （EOFAZ） on endothelial-to-mesenchymal transition （EndMT） induced by high glucose （HG）. Method:Human umbilical vein endothelial cells （HUVECs） was cultured in vitro to analyze the pharmacodynamic effects of EOFAZ on EndMT and oxidative stress damage induced by HG. The experiment was set the blank group， HG group （35 mmol·L-1）， EOFAZ low dose group （1 μg·L-1） and EOFAZ high dose group （4 μg·L-1）. After EOFAZ intervention for 2 h， HG was added to incubate for 72 h in order to establish EndMT cell model. Western blot was used to detect the protein expression of vimentin and platelet endothelial cell adhesion molecule （CD31）. Angiogenesis experiment was used to detect the ability of cell migration ability in order to analyze the effect of EOFAZ on EndMT. The changes of reactive oxygen species （ROS） levels were detected by 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence probe and the contents of malondialdehyde （MDA）， superoxide dismutase （SOD） and catalase （CAT） in cells were detected by the kit method to analyze the effect of EOFAZ on oxidative stress. Western blot was used to detect the protein expression levels of nuclear transcription factor E2 related factor 2 （Nrf2） and Notch1. The overexpression of Nrf2 was achieved by adenovirus （AD） transfection and the mechanism of EOFAZ inhibiting EndMT was further analyzed. The experiment was set the blank group， HG group （35 mmol·L-1）， AD-Nrf2 group， EOFAZ group （4 μg·L-1）， AD-Nrf2+EOFAZ group （4 μg·L-1）. The cells were infected with recombinant adenovirus overexpression plasmid of Nrf2 gene for 6 h， then replaced with normal medium for 24 h. After EOFAZ intervention for 2 h， HG was added to co-incubate for 72 h to induce EndMT. Western blot was used to detect the protein expressions of Nrf2， CD31， vimentin， Notch1 and Snail. Result:Compared with the HG group， after treatment with EOFAZ， the protein expression of CD31 was significantly up-regulated （P<0.05）， the protein expression of vimentin was significantly down-regulated （P<0.01）， the ability of cell migration was decreased （P<0.01）， and the contents of ROS and MDA were decreased （P<0.05， P<0.01）， the levels of CAT and SOD were increased （P<0.01）. In addition， EOFAZ could significantly up-regulate the protein expression of antioxidant signal Nrf2 （P<0.01） and down-regulate the protein expression of Notch1 （P<0.01）. High expression of Nrf2 was achieved by stable AD transfection into HUVECs. The results of Western blot showed that， compared with the HG group， the protein expression levels of Nrf2 and CD31 in each treatment group were significantly increased （P<0.01）， while the protein expression levels of vimentin， Notch1 and Snail were down-regulated （P<0.01）. At the same time， compared with the AD-Nrf2 group， the AD-Nrf2+EOFAZ group could further up-regulate the protein expressions of Nrf2 and CD31 （P<0.05， P<0.01）， while decrease the protein expression levels of vimentin， Notch1 and Snail （P<0.01）. Conclusion:EOFAZ ameliorates oxidative stress injury of vascular endothelial cells induced by HG and inhibits EndMT， which is related to Nrf2/Notch1 signaling pathway.

Effect of Oxymatrine in Inhibiting Proliferation and Migration of HT-29 Cells Induced by Insulin / 中国实验方剂学杂志

Objective:To investigate the effect of oxymatrine (OMT) on the proliferation and migration of human colon cancer cell line HT-29 under Type Ⅱ diabetes environment by co-culturing HT-29 with insulin to simulate hyperinsulinemia. Method:The effect of OMT (2, 4, 8 mmol·L-1) on insulin-induced proliferation of HT-29 was detected by methyl thiazolyl tetrazolium (MTT) assay and cloning assay. The morphology change and cell migration were evaluated under microscope and by wound healing assay. The Annexin V/propidium iodide(PI) assay was used to detect the change of insulin-induced HT-29 cell cycle and apoptosis. Western blot was performed to validate the expression of cell cycle-related protein and cell migration protein. Result:Insulin significantly increased growth of HT-29 (P-1 showed a significant inhibitory effect in this model (P0/G1 phase (P1, CDK4 and the up-regulation of p27 by OMT might involve the growth inhibition mechanism. Furthermore, OMT reduced the migration of insulin-induced HT-29 according to wound healing assay(PPPConclusion:OMT can inhibit the proliferation and migration of insulin-induced HT-29 cells. The changes of cell cycle and migration related proteins may be correlated with the mechanism.

Synthesis and cytotoxicity of longistylin C derivatives / 中国天然药物

The present study was designed to identify potent anti-tumor compounds from a series of new longistylin C derivatives. Ten longistylin C derivatives were synthesized and their structures were confirmed by (1)H NMR, MS, and elemental analyses. Their cytotoxicity in vitro against three human cancer cell lines (A549, HepG2, and MCF-7) were evaluated by the MTT assay. Among these compounds, DT-6 and DT-9 displayed much better cytotoxicity against A549, HepG2, and MCF-7 cells, DT-1 exhibited selective cytotoxicity against HepG2, and the structure-activity relationships were investigated. In conclusion, Compounds DT-6 and DT-9 may serve as potential lead compounds for the discovery of new anti-cancer drugs.