OBEJECTIVE Gecko has been clinically used in China for many years. It has been proved that the gecko polypeptide mixture(GPM)extracted from gecko could inhibit the growth of multiple types of tumor cells.In order to investigate the possible anti-tumor molecular mechanisms of GPM,we used RNA-seq technology to identify the differentially expressed genes of human hepatocellular carci-noma(HCC)HepG2 cells treated with or without GPM.METHODS The HepG2 cells were treated with different concentration of GPM(0,0.1,0.2,0.3,0.4 mg·mL-1)for 6 h,12 h and 24 h,respectively.MTT assay was used to detect the viability of HepG2 cells. DAPI fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells.Western blot analysis was applied to observe the expres-sion of apoptosis- related proteins and endoplasmic reticulum stress (ERs)-related proteins in HepG2 cells.Flow cytometry was also applied to detect reactive oxygen species(ROS)generation.In this report, we showed that GPM could induce HepG2 cells apoptosis and influence HepG2 cells proliferation in a dose-dependent manner.We applied many analysis methods,including differentially expressed genes analysis,Gene Ontology(GO)enrichment analysis,KEGG pathway enrichment analysis,protein-protein interaction network analysis to screen out possible molecular mechanisms.RESULTS ER-nucleus signaling pathway, cellular response to stress and apoptotic processes were identified the potential anti-cancer molecular biological process of GPM.GPM may also induce apoptosis in HepG2 cells via endoplasmic reticulum stress pathway. The GPM could induce ROS generation and up-regulate ERs-related proteins. CONCLUSION The present study revealed the potential anti-tumor mechanism of GPM.

0. 05) . Conclusion Despite immersing in seawater and in freshwater worsen the ALI after open chest trauma,the ALI induced by seawater-immersion is severer than that caused by freshwater-immersion. It is attributed to higher plasma osmotic pressure and abundance of salts after seawater immersion,which increasing the pulmonary penetration index and aggravating the inflammatory response.

OBJECTIVE In order to investigate the possible anti-tumor molecular mechanisms of gecko polypeptide mixture (GPM). METHODS RNA-seq technology was used to identify the differen?tially expressed genes of human hepatocellular carcinoma (HCC) HepG2 cells treated with or without GPM. The HepG2 cells were treated with different concentration of GPM (0, 0.1, 0.2, 0.3, 0.4 mg·mL-1) for 6 h, 12 h and 24 h, respectively. MTT assay was used to detect the viability of HepG2 cells. DAPI fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells. Western blot analysis was applied to observe the expression of apoptosis-related proteins in HepG2 cells. RESULTS The results showed that GPM could induce HepG2 cells apoptosis and influence HepG2 cells proliferation in a dose-dependent manner. We applied many analysis methods, including differen?tially expressed genes analysis, Gene Ontology (GO) enrichment analysis, KEGG pathway enrichment analysis, protein- protein interaction network analysis to screen out possible molecular mechanisms. ER-nucleus signaling pathway, cellular response to stress and apoptotic processes were identified the potential anti-cancer molecular biological process of GPM. GPM may also induce apoptosis in HepG2 cells via endoplasmic reticulum stress pathway. The mechanism is closely related to ERs, which might be beneficial for clinical therapy of HCC. CONCLUSION GPM can inhibit cells proliferation and induce apoptosis in HepG2 cells. The gene expression profile of GPM in HepG2 cells was obtained. The present study revealed the potential anti-tumor mechanism of GPM.

OBJECTIVE To explore the role of gecko crude peptides (GCPs) in the proliferation, apoptosis, migration and lymphangiogenesis of human hepatocellular carcinoma cells (HepG2) and human lymphaticendothelial cells (HLECs) in vitro. METHODS The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the anti- proliferative effect of GCPs and siRNA-VEGF-C on HepG2 cells, Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis. The migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay. The protein and mRNA expressions of vascular endo?thelial growth factor-C (VEGF-C) and CXC chemokine receptor-4 (CXCR4) were detected by q-PCR, immunofluorescence, Western blot. The protein expressions of the extracellular signal regulated kinase (ERKI/2), c-Jun N-terminal kinase (JNK), p38-mitogen activated protein kinases (p38 MAPK), serine/threonine kinase (Akt) and phosphatidylinositol- 3- kinase (PI3K) were detected by western blot. The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay. The protein and mRNA expressions of vascular endothelial growth factor receptor-3 (VEGFR-3) and stromal cell-derived factor-1 (SDF-1) were detected by q-PCR, Western blot. RESULTS GCPs and siRNA-VEGF-C inhibited HepG2 proliferation, invasion and migration, and the most obvious inhibitory effect was both synergistic effects. Thus, GCPs suppressed HLECs proliferation, migration and tube-like structure formationin a dose- dependent manner, and had inhibitory effect of tumor- induced lymphangiogenesis in vitro. Additionally, we found that GCPs and siRNA- VEGF- C decreased the expressions of MMP-2, MMP-9, VEGF-C, CXCR4, phospho-ERK1/2, phospho-P38, phospho-JNK and PI3K in HepG2 cells. Moreover, GCPs had a dose-dependent depressive effecton the expressions of VEGFR- 3, SDF- 1 in HLECs. CONCLUSION The low expression of VEGF- C mediated by siRNA-VEGF-C and GCPs inhibit tumor proliferation, invasion and migrationby suppressing the MAPK signaling pathway through reduced levels of VEGF-C, and GCPs inhibit tumor lymphangiogenesis by suppressing the CXCR4/SDF-1 signaling pathway through suppressed VEGF-C/VEGFR-3.

AIM: To assess the degree of oxidative damage during acute myocardial infarction and reperfusion, and to clarify the protective effect of Tongxinluo in mini-swine model. METHODS: Thirty mini-swines were randomized into 5 study groups: sham group, model group, low dose (0.05 g·kg~(-1)·d~(-1)), medium dose (0.2 g·kg~(-1)·d~(-1)) and high dose (0.5 g·kg~(-1)·d~(-1)) of Tongxinluo groups (pretreated with Tongxinluo for 3 d). Animals except in sham group were subjected to 3 h of coronary occlusion followed by 1 h of reperfusion. Concentrations of total antioxidative capability (T-AOC), total superoxide dismutase (T-SOD), reduced glutathione (GSH) and malondialdehyde (MDA) in blood sample and the myocardium were measured. RESULTS: (1) T-AOC, T-SOD and GSH in serum significantly decreased (all P<0.05), while MDA significantly increased (P<0.01) at 3 h after AMI in comparison with those at baseline. Compared to those at 3 h after AMI, the contents of T-AOC, T-SOD and GSH at 1 h after reperfusion significantly decreased (all P<0.01), accompanied by increase of MDA (P<0.01). (2) Compared to those in normal area, levels of T-AOC, T-SOD and GSH in reperfusion myocardium decreased significantly (all P<0.01) and MDA increased significantly (P<0.01). T-AOC, T-SOD and GSH in no-reflow myocardium further decreased (all P<0.01) and MDA increased (P<0.01) as compared to those in reperfusion myocardium. (3) Compared to model group, medium dose of Tongxinluo increased the contents of T-AOC and T-SOD and reduced MDA production in serum at 3 h after AMI (all P<0.05), while medium dose of Tongxinluo increased T-SOD level at 1 h after reperfusion (P<0.05). High dose of Tongxinluo increased the levels of T-AOC and T-SOD and decreased MDA content in serum at 3 h after AMI and 1 h after reperfusion (all P<0.05). (4) The medium dose of Tongxinluo increased T-AOC content (P<0.05) and reduced MDA (P<0.05) in reperfusion myocardium, while high dose of Tongxinluo increased T-AOC, T-SOD and GSH (all P<0.05), reduced MDA (P<0.01) in reperfusion myocardium, and also increased T-AOC, T-SOD (all P<0.05), reduced MDA (P<0.01) in no-reflow area as compared to those in model group. CONCLUSION: Impairment of antioxidant defense system in vivo and imbalance of redox homeostasis in myocardium region might play an important role in the pathogenesis of no-reflow after myocardial acute infarction following reperfusion. Tongxinluo protects myocardium from reperfusion injury by improving antioxidant defense and attenuating oxidative damage.

The present study aims to investigate the lignan constituents from Sambucus williamsii and their proliferation effects on osteoblast-like UMR106 cells. Seven compounds were isolated and purified by macroporous resin D101, silica gel, Sephadex LH-20, Toyopearl HW-40, ODS column chromatographies and Preparative HPLC(C-18). Their structures were elucidated by spectroscopic methods as threo-guaiacylglycerol-beta-0-4'-conifery ether (1), lirioresinol A (2), 1-hydroxypinoresinol (3), 5-methoxybalanophonin (4), balanophonin (5), 5-methoxy-trans-dihydrodehydrodiconiferyl alcohol (6), and p-hydroxybenzaldehyde (7). Compounds 3-7 were obtained from this genus for the first time. The proliferation effects of all isolated compounds on osteoblast-like UMR106 cells were determined. Compounds 1-7 (1 x 10(-12)-1 x 10(-7) mol x L(-1)) increased UMR106 cell proliferation to some extent.
Cell Line ; Cell Proliferation ; drug effects ; Lignans ; isolation & purification ; pharmacology ; Osteoblasts ; cytology ; drug effects ; Plant Stems ; chemistry ; Sambucus ; chemistry

Objective To investigate the possible molecular mechanisms of Gecko peptides mixture (GPM) and research to human hepatocellular carcinoma SMMC7721 cells on autophagy with GPM.Methods SMMC7721 cells were put into plates in its logarithmic phase,and they were treated with different concentration of GPM (0,0.04,0.06,0.09,0.14,0.20,0.30,0.45 mg · mL-1) for 24 h,and then detected corresponding indicators with respective methods.The viability of SMMC7721 cells was detected with 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT).The concentration of normal group,GPM low-dose,middle-dose and high-dose experimental groups was separately 0,0.1,0.15,0.22 mg · mL-1,according to the results of MTT.Rapamycin was chosen as the positive control drug,which concentration was 40 μg · mL-1.The autophagy of different concentration GPM on SMMC7721 cells was detected by monodansylcadaverine (MDC) staining.Expression levels of Beclin1 and LC3 in SMMC7721 cells were measured by immunohistochemical method and Western blot assay.Results GPM could significantly inhibit the proliferation of SMMC7721 cells in a dose -dependent manner,and the IC50value was 0.16 mg · mL-1 for 24 h.MDC staining showed that there are plenty of autophagosome in cytoplasm,emerging bright green fluorescent in the fluorescence microscope after treatment with GPM for 6 h.The percentage number of positive cells of total cells number in normal group,control group,GPM low,middle and high-dose experimental groups were (15.70 ± 0.26)％,(63.47 ± 0.54)％,(17.09 ± 0.37)％,(70.66 ±0.56) ％,(78.48 ±0.68) ％,respectively.Compared with the normal group,control group,GPM middle and high-dose experimental groups increased,the differences were statistically significant (P ＜ 0.05).The indicators of LC3 in that five groups were (35.84 ± 0.36) ％,(82.41 ± 0.82) ％,(60.83 ± 0.61) ％,(69.66 ± 0.70) ％,(74.61 ± 0.75) ％,respectively.The differences between each group and normal group were statistically significant (P ＜ 0.05).The grayscale average ratio of Beclin1 and β actin in normal group,control group,GPM low,middle and high-dose experimental groups were (6.87 ± 0.68) ％,(11.26 ± 0.87) ％,(6.46 ± 1.34) ％,(11.58 ± 0.95) ％,(15.82 ± 1.58)％,respectively.Compared with the normal group,control group,GPM middle and high-dose experimental groups increased,the differences were statistically significant (P ＜ 0.05).The indicators of LC3 Ⅱ and βactin in that five groups were (26.70 ± 0.41) ％,(103.17 0.88) ％,(30.29 ± 0.52) ％,(36.32 ± 0.52) ％,(64.34 ± 0.48) ％.The differences between each group and normal group were statistically significant (P ＜ 0.05).Conclusion GPM could have an effect on the autophagy in human hepatocellular carcinoma SMMC7721 cells.The possible mechanism may be GPM induced autophagy of SMMC7721 cells,causing cell death ultimately.

The paper is to report the development of a high-performance liquid chromatographic/tandem mass spectrometry (HPLC-MS/MS) method for the determination of icaritin (ICT) in rat plasma. After precipitated with acetonitrile from the plasma, ICT was isolated chromatographically on a Dikma C18 column. The mobile phase consisted of acetonitrile-water-acetic acid (72 : 28 : 1.5, v/v/v). Electrospray ionization (ESI) source was applied and operated in the positive ion mode. Multiple reaction monitoring (MRM) mode with the transitions of m/z 387 --> m/z 313 and m/z 331 --> m/z 315 were used to quantify ICT and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 2.5-1,000 ng x mL(-1). The lower limit of quantification was 2.5 ng x mL(-1). The inter- and intra-day precision (RSD) were less than 9.63%, and the accuracy (relative error) was within +/-7.42%. The method was proved to be suitable for the pharmacokinetics of ICT, which offers advantages of high sensitivity and selectivity.
Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; methods ; Epimedium ; chemistry ; Female ; Flavonoids ; administration & dosage ; blood ; isolation & purification ; pharmacokinetics ; Male ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tandem Mass Spectrometry ; methods

The purpose of this research is to study the effect of small molecule compound piceatannol (PIC) on host inflammation in adenine induced chronic kidney disease (CKD) mice, and then to explore its mechanism based on the regulation of gut microbiota. All procedures were approved by the Institutional Animal Care and Use Committee of the Nanjing University of Chinese Medicine. The level of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was detected by enzyme linked immunosorbent assay (ELISA); UPLC-TQ/MS technology was used to monitor the level of proinflammatory uremic toxin indoxyl sulfate (IS) and p-cresol sulfate (PCS); the expression of occludin was tested by Western blot; in vitro anaerobic culture of gut bacteria was used to produce indole; the abundance of gut microbiota was evaluated by 16S rDNA sequencing. The results showed that PIC had no effect on inflammatory infiltration in kidney tissue of CKD mice, but could decrease IL-6 level in blood and IL-6/TNF-α level in colon tissue. PIC did not improve intestinal occludin protein expression in CKD mice; while it could significantly reduce the levels of IS and PCS in blood and liver of CKD mice. Further mechanism studies showed that PIC could inhibit the synthesis of IS precursor indole in gut bacteria. Moreover, PIC could decrease the abundance of gut bacteria which producing uremic toxin, such as reducing the abundance of indole and p-cresol producing gut bacteria. In conclusion, PIC could regulate gut microbiota and inhibit the synthesis of uremic toxin precursor, thereafter reducing the accumulation of IS and PCS in vivo, ultimately relieving the inflammation of CKD mice.

Objective To establish a high performance liquid chroma-tography-tandem mass spectrometry ( UPLC-MS/MS) method to deter-mination the plasma concentration of cefdinir and to study the pharmaco-kinctics and bioequivalence of three kinds of cefdinir formulations in healthy volunteers.Methods In a randomized, 3 way-crossover and self-control study, 24 healthy male volunteers were orally administrated with three kinds of cefdinir formulations ( test sample and reference sam-ple) 100 mg.The concentrations of cefdinir in plasma were determined by LC-MS/MS.The main pharmacokinetic parameters were calculated.Results The method was validated by investigating the accuracy and precision for intra and inter -day runs in a linear concentration from 11.50-2300.00 ng· mL-1.The main pharmacokinetic parameters of test (cefdinir tables A drug, cefdinir dispersible tables B drug ) and reference (cefdinir capsules, C drug ) formulations in plasma were shown as follows:tmax were (3.00 ±0.80),(3.20 ±0.90) and (3.50 ±0.70) h ; Cmax were (548.96 ±184.58 ), ( 607.09 ±236.38 ) and ( 570.18 ±172.37 ) ng· mL-1;t1/2 were ( 2.00 ±0.30),(1.90 ±0.40)and (1.90 ±0.30) h; AUC0-t were ( 2755.35 ±956.10 ) , ( 3037.49 ±1014.42 ) and (2756.43 ±804.06 ) ng · mL-1 · h, respectively.The relative bioavailability F were ( 104.20 ±37.50 )% and (111.50 ±29.70)%.Conclusion The method was proved to be accurate, rapid and sensitive.The three kinds of cefdinir formulations are bioequivalent in healthy volunteers.