Establishment and validation of bioactivity measurement method for recombinant human midkine / 药学学报

To establish and optimize a method for the detection of recombinant human midkine (rhMK) activity and verify its methodology, cell counting kit-8 (cck-8) method was used to measure the proliferation activity of rat knee chondrocytes. The specificity, accuracy, precision, linearity and robustness of the method were also verified in this study. The established method was proven to have good specificity because the buffer of rhMK and recombinant human interleukin-1 receptor antagonist have no obvious active effect; the recoveries of the samples with relative activities of 50%, 75%, 100%, 125%, 150% were in the range of 80.0% to 124.0% by statistical analysis, the relative standard deviations (RSD) of relative potency were all within 20%, the linear correlation coefficient, R2 ≥ 0.98, suggesting that the accuracy, precision and linearity of the method were good; the robustness correlation coefficient, R2 ≥ 0.92 and the ratio of maximum to minimum of sigmoidal dose-response were no less than 1.5, indicating that robustness of the methods was good. In conclusion, a bioactivity measurement method for rhMK was established and fully validated in this study and it provides a reliable method for the bioactivity analysis of rhMK routine samples during the development. This study was approved by the Animal Care and Use Committee of Shanghai Model Organisms Center, Inc. (approval number: 2019-0008-06).

Chemo-photothermal immunotherapy for eradication of orthotopic tumors and inhibition of metastasis by intratumoral injection of polydopamine versatile hydrogels

Cancer remains one of the leading causes of death globally and metastasis always leads to treatment failure. Here, we develop a versatile hydrogel loading photothermal agents, chemotherapeutics, and immune-adjuvants to eradicate orthotopic tumors and inhibit metastasis by combinational therapy. Hydrogel networks were synthesized via the thiol-Michael addition of polydopamine (PDA) with thiolated hyaluronic acid. PDA acted as a cross-linking agent and endowed the hydrogel with excellent photothermal property. Meanwhile, a chemotherapeutic agent, doxorubicin (DOX), was loaded in the hydrogel via π‒π stacking with PDA and an immune-adjuvant, CpG-ODN, was loaded via electrostatic interaction. The release of DOX from the hydrogel was initially slow but accelerated due to near infrared light irradiation. The hydrogels showed remarkably synergistic effect against 4T1 cancer cells and stimulated plenty of cytokines secreting from RAW264.7 cells. Moreover, the hydrogels eradicated orthotopic murine breast cancer xenografts and strongly inhibited metastasis after intratumoral injection and light irradiation. The high anticancer efficiency of this chemo-photothermal immunotherapy resulted from the strong synergistic effect of the versatile hydrogels, including the evoked host immune response. The combinational strategy of chemo-photothermal immunotherapy is promising for highly effective treatment of breast cancer.

Targeting a cryptic allosteric site of SIRT6 with small-molecule inhibitors that inhibit the migration of pancreatic cancer cells

SIRT6 belongs to the conserved NAD+-dependent deacetylase superfamily and mediates multiple biological and pathological processes. Targeting SIRT6 by allosteric modulators represents a novel direction for therapeutics, which can overcome the selectivity problem caused by the structural similarity of orthosteric sites among deacetylases. Here, developing a reversed allosteric strategy AlloReverse, we identified a cryptic allosteric site, Pocket Z, which was only induced by the bi-directional allosteric signal triggered upon orthosteric binding of NAD+. Based on Pocket Z, we discovered an SIRT6 allosteric inhibitor named JYQ-42. JYQ-42 selectively targets SIRT6 among other histone deacetylases and effectively inhibits SIRT6 deacetylation, with an IC50 of 2.33 μmol/L. JYQ-42 significantly suppresses SIRT6-mediated cancer cell migration and pro-inflammatory cytokine production. JYQ-42, to our knowledge, is the most potent and selective allosteric SIRT6 inhibitor. This study provides a novel strategy for allosteric drug design and will help in the challenging development of therapeutic agents that can selectively bind SIRT6.

Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy

Parkin, an E3 ubiquitin ligase, plays a role in maintaining mitochondrial homeostasis through targeting damaged mitochondria for mitophagy. Accumulating evidence suggests that the acetylation modification of the key mitophagy machinery influences mitophagy level, but the underlying mechanism is poorly understood. Here, our study demonstrated that inhibition of histone deacetylase (HDAC) by treatment of HDACis activates mitophagy through mediating Parkin acetylation, leading to inhibition of cervical cancer cell proliferation. Bioinformatics analysis shows that Parkin expression is inversely correlated with HDAC2 expression in human cervical cancer, indicating the low acetylation level of Parkin. Using mass spectrometry, Parkin is identified to interact with two upstream molecules, acetylase acetyl-CoA acetyltransferase 1 (ACAT1) and deacetylase HDAC2. Under treatment of suberoylanilide hydroxamic acid (SAHA), Parkin is acetylated at lysine residues 129, 220 and 349, located in different domains of Parkin protein. In in vitro experiments, combined mutation of Parkin largely attenuate the interaction of Parkin with PTEN induced putative kinase 1 (PINK1) and the function of Parkin in mitophagy induction and tumor suppression. In tumor xenografts, the expression of mutant Parkin impairs the tumor suppressive effect of Parkin and decreases the anticancer activity of SAHA. Our results reveal an acetylation-dependent regulatory mechanism governing Parkin in mitophagy and cervical carcinogenesis, which offers a new mitophagy modulation strategy for cancer therapy.

Effect of Different Extracts and Monomers of Lepidium meyenii on Proliferation of Mouse Splenic Lymphocyte and Induction of IL-2 and TNF-α / 中国实验方剂学杂志

Objective:To explore effects of different extracts and monomers of <italic>Lepidium meyenii </italic>（Maca） on the proliferation of mouse splenic lymphocytes and induction of interleukin-2 （IL-2） and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） by observing their immunomodulatory effects. Method:An octadecylsilyl （ODS） column was used to enrich the methanol extract of <italic>L. meyenii</italic> in stages to obtain six fractions and three monomers. Different groups of extracts and monomers of <italic>L. meyenii </italic>at different doses were set up. Cell counting Kit-8 （CCK-8） was used to detect the effect on the proliferation of mitogen-free， concanavalin A （Con A）-induced， and lipopolysaccharides （LPS）-induced mouse splenic lymphocytes. Enzyme-linked immunosorbent assay （ELISA） was used to determine the levels of IL-2 and TNF-<italic>α</italic>. Result:<italic>L. meyenii </italic>extracts Fr₃ and Fr₆， and monomers <italic>N</italic>-benzyl hexadecanamide and 1，2-dihydro-4-carboxaldehyde-3-benzyl-<italic>N</italic>-hydroxypyridine slightly promoted the proliferation of Con A-induced T lymphocytes and LPS-induced B lymphocytes （<italic>P</italic><0.01） as compared with the conditions in the model group. <italic>L. meyenii</italic> extracts and monomers significantly induced the secretion of IL-2 and TNF-<italic>α</italic> by splenic lymphocytes （<italic>P</italic><0.01）. Conclusion:<italic>L. meyenii</italic> extracts and monomers can achieve immunological enhancement by promoting the secretion of IL-2 and TNF-<italic>α</italic>， and facilitate the proliferation of splenic lymphocytes. The active components are presumedly macamides and pyridine alkaloids， and the specific mechanism still needs to be further explored.

Discovery of novel diarylamides as orally active diuretics targeting urea transporters

Urea transporters (UT) play a vital role in the mechanism of urine concentration and are recognized as novel targets for the development of salt-sparing diuretics. Thus, UT inhibitors are promising for development as novel diuretics. In the present study, a novel UT inhibitor with a diarylamide scaffold was discovered by high-throughput screening. Optimization of the inhibitor led to the identification of a promising preclinical candidate,

Cholesterol-associated lysosomal disorder triggers cell death of hematological malignancy: Dynamic analysis on cytotoxic effects of LW-218

The integrity of lysosomes is of vital importance to survival of tumor cells. We demonstrated that LW-218, a synthetic flavonoid, induced rapid lysosomal enlargement accompanied with lysosomal membrane permeabilization in hematological malignancy. LW-218-induced lysosomal damage and lysosome-dependent cell death were mediated by cathepsin D, as the lysosomal damage and cell apoptosis could be suppressed by depletion of cathepsin D or lysosome alkalization agents, which can alter the activity of cathepsins. Lysophagy, was initiated for cell self-rescue after LW-218 treatment and correlated with calcium release and nuclei translocation of transcription factor EB. LW-218 treatment enhanced the expression of autophagy-related genes which could be inhibited by intracellular calcium chelator. Sustained exposure to LW-218 exhausted the lysosomal capacity so as to repress the normal autophagy. LW-218-induced enlargement and damage of lysosomes were triggered by abnormal cholesterol deposition on lysosome membrane which caused by interaction between LW-218 and NPC intracellular cholesterol transporter 1. Moreover, LW-218 inhibited the leukemia cell growth

A homogenous nanoporous pulmonary drug delivery system based on metal-organic frameworks with fine aerosolization performance and good compatibility

Pulmonary drug delivery has attracted increasing attention in biomedicine, and porous particles can effectively enhance the aerosolization performance and bioavailability of drugs. However, the existing methods for preparing porous particles using porogens have several drawbacks, such as the inhomogeneous and uncontrollable pores, drug leakage, and high risk of fragmentation. In this study, a series of cyclodextrin-based metal-organic framework (CD-MOF) particles containing homogenous nanopores were delicately engineered without porogens. Compared with commercial inhalation carrier, CD-MOF showed excellent aerosolization performance because of the homogenous nanoporous structure. The great biocompatibility of CD-MOF in pulmonary delivery was also confirmed by a series of experiments, including cytotoxicity assay, hemolysis ratio test, lung function evaluation,

Synthesis of a new type of carbon dots by pyrolysis of human hair using motor oil as a dispersant and their biological effects / 中草药

Objective: New type of nano-carbon dots were found after pyrolysis of human hair using motor oil as a dispersant and the biological effect of these carbon dots was evaluated by animal experiments. Methods: High-temperature pyrolysis was used to carbonize human hair and motor oil, and the carbonized products were extracted, filtered, and dialyzed to obtain a new type of water-soluble substance, carbon dots, named JYRF-CDs. JYRF-CDs were characterized using transmission electron microscopy (TEM) and high-resolution TEM, as well as ultraviolet-visible, fourier transform infrared, fluorescence spectroscopy and X-ray photoelectron spectroscopy (XPS). CCK-8 toxicity test using RAW264.7 cells was used to evaluate the safety of JYRF-CDs and the biological effects of the JYRF-CDs were evaluated by mouse ear swelling experiments and mouse acetate writhing experiments. Results: These JYRF-CDs were nearly spherical and well separated from each other, with a size distribution range of 1.8-3.6 nm, the CDs had a lattice spacing of 0.219 7 nm. The results of cytotoxicity experiments showed that JYRF-CDs had low toxicity, and the results of animal experiments showed that JYRF-CDs had good anti-inflammatory and analgesic effects. Conclusion: In this study, new type of carbon dots, JYRF-CDs, were discovered after pyrolyzing human hair with motor oil as a dispersant for the first time. Taking JYRF-CDs as a breakthrough, the material base of carbonization products after pyrolysis of human hair by high-temperature pyrolysis using motor oil as a dispersant was more clearly explained, providing a new method for the research of nano compounds.

Construction of Salviae Miltiorrhizae Radix et Rhizoma-polydopamine nanoparticles and its protective effect on oxidative damage in cardiomyocytes induced by H2O2 / 中草药

Objective: Using polydopamine (PDA) as a carrier to construct a Salviae Miltiorrhizae Radix et Rhizoma (SMRR) nano-delivery system (PDA-SMRR), which can load a large number of SMRR water-soluble components and better exert antioxidation and antistress effect. Methods: PDA-SMRR nanoparticles (PDA-SMRR) were prepared, and the prescription process was investigated and optimized by single-factor experiments. The particle size, potential, and morphology of the nanoparticles were examined by a laser particle size analyzer and a transmission electron microscope. The drug loading and cumulative release rate were analyzed by dialysis. The cardiomyocytes of neonatal rats were extracted and cultured. The CCK-8 experiment was used to investigate the biological safety of PDA-SMRR and verify the protective effect of PDA-SMRR on oxidative stress-induced cardiomyocytes. Results: The optimal drug loading process was pH value 3.5, drug loading time was 12 h, drug loading temperature was room temperature, and PDA-SMRR was successfully prepared. The morphology and size of the nanoparticles were regular and uniform. The particle size and Zeta potential were (459.2 ± 4.5) nm, (3.01 ± 0.3) mV; In vitro release experiments indicated that SMRR was released slowly by the delivery system. CCK-8 experiments showed that PDA-SMRR had good biological safety and nanoparticles can reduce damaged cardiomyocytes caused by oxidative stress. Conclusion: PDA-SMRR can be used as a multi-component medicine depot for SMRR, with high drug loading and sustained release effect, which can effectively reduce the damage of oxidative damage on myocardial cells.

Study on oil-water distribution of taxifolin and its absorption and transport mechanism in Caco-2 cell model / 中草药

Objective: To prepare the taxifolin and determine its apparent oil-water partition coefficient in different media, and to study the mechanism of absorption and transport of taxifolin in Caco-2 cell model. Methods: Taxifolin was prepared by enzymolysis. HPLC was used to determine the saturated solubility of taxifolin in 37 ℃, different pH buffer solution and water, apparent oil-water distribution coefficient of taxifolin obtained by calculation formula of oil-water distribution coefficient; CCK-8 experiment was used to investigate the safe concentration range of taxifolin in Caco-2 cells, and then the single-layer model of Caco-2 cells was used to study the mechanism of bilateral transmembrane absorption and transport. CCK-8 experiment was used to investigate the safe concentration range of taxifolin in HDMEC cells. The inflammatory model of HDMEC cells induced by lipopolysaccharide was established, and the activity of lactic dehydrogenase was detected by the intervention of floxacin. The activity of lactic dehydrogenase was detected by lactic dehydrogenase kit. Results: The lgP values of taxifolin in the following solvents were 0.29 (0.1 mol/L hydrochloric acid), 0.48 (pH 2.0), 0.46 (pH 5.8), 0.34 (pH 6.8), 0.26 (pH 7.4), and 0.38 (water), respectively; There was no significant toxic effect on Caco-2 cells in the range of 50-500 μg/mL; There was no significant difference in Papp value of bilateral transport between different concentrations of taxifolin in Caco-2 monolayer cell model, and it was less than 1 × 10-6 cm/s and ER was less than 2. There was no significant toxic effect on HDMEC cells in the range of 50-300 μg/mL; After treatment with taxifolin, compared with LPS stimulation group, the activity of LDH in each treatment group was decreased significantly (P < 0.05), and the activity of LDH was decreased significantly in the range of 50-100 μg/mL, and tended to be stable in the range of 100-250 μg/mL. Conclusion: Taxifolin is a kind of drug which is difficult to absorb in the intestine. The mechanism of transmembrane transport is passive transport. It can inhibit the inflammation of hdmec cells induced by LPS and has anti-inflammatory activity.

Effect and mechanism of tanshinone IIA on TGF-β1 induced human skin fibroblast proliferation / 中草药

Objective: To study the effect and mechanism of tanshinone IIA on human skin fibroblasts cell (HSF). Methods: CCK- 8 method was used to determine the effect of different concentrations of TSA on the proliferation of HSF induced by TGF-β1. The plate cloning ability of HSF treated with different concentrations of TSA (2.5, 5, 10 and 20 μmol/L) for 48 h were analyzed by plate clonogenesis assay. The protein expression of TGF-β1/Smad signaling pathway proteins and α-SMA, VEGFA and COL I were further measured by Western blotting. Results: CCK-8 and plate clonognesis assay results showed that TSA significantly inhibited the proliferation and colony forming efficiency of HSF (P < 0.01). Western blotting results revealed that each concentration group of TSA significantly inhibited the protein levels of p-Smad2 and p-Smad3, and down-regulated the ratio of p-Smad2/Smad2 (P < 0.01). The ratio of p-Smad3/Smad3 was significantly decreased in 5, 10 and 20 μmol/L TSA groups. Additionally, the expression levels of α-SMA, VEGFA and COL I in HSF decreased significantly with the increase of TSA concentration (P < 0.01). Conclusion: TSA exhibits the inhibitory effect on proliferation of HSF, and its mechanism may be related to TGF-β1/Smads signaling pathway.

Evodiamine-inspired dual inhibitors of histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) with potent antitumor activity

A great challenge in multi-targeting drug discovery is to identify drug-like lead compounds with therapeutic advantages over single target inhibitors and drug combinations. Inspired by our previous efforts in designing antitumor evodiamine derivatives, herein selective histone deacetylase 1 (HDAC1) and topoisomerase 2 (TOP2) dual inhibitors were successfully identified, which showed potent and antitumor potency. Particularly, compound was orally active and possessed excellent antitumor activity in the HCT116 xenograft model (TGI = 75.2%, 150 mg/kg, .) without significant toxicity, which was more potent than HDAC inhibitor vorinostat, TOP inhibitor evodiamine and their combination. Taken together, this study highlights the therapeutic advantages of evodiamine-based HDAC1/TOP2 dual inhibitors and provides valuable leads for the development of novel multi-targeting antitumor agents.

Miltirone induces cell death in hepatocellular carcinoma cell through GSDME-dependent pyroptosis

Pyroptosis is a form of programmed cell death, and recently described as a new molecular mechanism of chemotherapy drugs in the treatment of tumors. Miltirone, a derivative of phenanthrene-quinone isolated from the root of Bunge, has been shown to possess anti-cancer activities. Here, we found that miltirone inhibited the cell viability of either HepG2 or Hepa1-6 cells, and induced the proteolytic cleavage of gasdermin E (GSDME) in each hepatocellular carcinoma (HCC) cell line, with concomitant cleavage of caspase 3. Knocking out switched miltirone-induced cell death from pyroptosis to apoptosis. Additionally, the induction effects of miltirone on GSDME-dependent pyroptosis were attenuated by siRNA-mediated caspase three silencing and the specific caspase three inhibitor Z-DEVD-FMK, respectively. Miltirone effectively elicited intracellular accumulation of reactive oxygen species (ROS), and suppressed phosphorylation of mitogen-activated and extracellular signal-regulated kinase (MEK) and extracellular regulated protein kinases 1/2 (ERK1/2) for pyroptosis induction. Moreover, miltirone significantly inhibited tumor growth and induced pyroptosis in the Hepa1-6 mouse HCC syngeneic model. These results provide a new insight that miltirone is a potential therapeutic agent for the treatment of HCC GSDME-dependent pyroptosis.

The role of APOD in the proliferation and migration of human dental pulp cells / 口腔疾病防治

Objective@#To explore the role of apoliprotein D (APOD) in the proliferation and migration of human dental pulp cells (DPCs) and to provide a basis for the use of APOD to promote pulp regeneration. @*Methods@#APOD expression in human dental pulp cells was inhibited by siRNA. The inhibition effect of APOD was confirmed by qPCR and Western blot. After APOD inhibition, colony formation experiments and CCK8 assays were employed to confirm the proliferation ability of dental pulp cells. Transwell assays were used to verify the cell migration ability after the inhibition of APOD expression.@*Results @# After inhibiting APOD expression, the colony formation rate in the si-apod group was reduced compared with the NC group, and the difference was statistically significant (t=7.624, P=0.002). The CCK8 experiment showed that the OD value in the si-apod group decreased at 3, 5 and 7 d compared with that in the NC group (P < 0.05). Transwell results showed that the number of cell divisions was 57.25 ± 4.03 in the si-apod group and 154.50 ± 8.39 in the NC group, and the difference was statistically significant (t=10.45, P < 0.001).@*Conclusion@# Inhibition of APOD expression in dental pulp cells inhibits their proliferation and migration ability.

Dissolution kinetics of Compound Danshen Tablets based on cell bioelectrical sensing effect / 中草药

Objective: Establishing the model of cell bioelectrical sensing effect of Compound Danshen Tablets to study its dissolution kinetics. Methods: By means of real-time cell-based assay, the in vitro dissolution of Compound Danshen Tablets can be investigated, and then the dissolution kinetics model can also be established. In addition, the result was compared and verified by UV-Vis. Results: The cell line with specific dependence on Compound Danshen Tablets was screened by CCK-8 experiment and RTCA experiment. The dissolution kinetics model of Compound Danshen Tablets based on RTCA technology was established, and the best fitting model was obtained: Weibull model ln{ln[1/(1-Q)] =1.071 4 lnt-3.736 7; Establish a dissolution kinetic model of Compound Danshen Tablets based on UV spectrophotometry to obtain the best fitting model, Weibull model ln{ln[1/(1-Q)]}=1.080 4 lnt-3.723 4; Comparing the two Weibull models, the RTCA fitted model worked better. Conclusion: The application of RTCA in the dissolution kinetics of traditional Chinese medicine compound solid preparations is feasible, Which provides new ideas for traditional Chinese medicines and the quality evaluation of traditional Chinese medicine compunds.

Effect of blue light and white light on proliferation of ARPE-19 cells detected by CCK-8 / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effects of blue light and white light on the proliferation of human retinal pigment epithelial cell line(ARPE-19)at different times, and lay a foundation for further detecting the changes of related factors during photodamage and further studying the signal transduction mechanism during light damage. <p>METHODS:Well-grown ARPE-19 cells were collected for experimentation. The standard curve of CCK-8 was made to determine the proper cell density of ARPE-19 cells and the reaction time of CCK-8 reagent. The cells were divided into dark group, blue light group and white light group, which were irradiated for 3, 6 and 9h respectively. After 12h of light-repellent treatment, CCK-8 method was used to examine the effects of different light sources on the proliferation rate of ARPE-19 cells at different times. <p>RESULTS: The number of cells per well was selected by CCK-8 standard curve to be 20 000, and the corresponding absorbance value was measured after 4h of the action of CCK-8 solution. The CCK-8 test results showed that the cell proliferation rates of the three groups were significantly different(<i>P</i><0.01). The cell proliferation rate of the blue light group was significantly different(<i>P</i><0.001)at 3, 6 and 9h, and the cell proliferation rate decreased gradually with the extension of the illumination time. The cell proliferation rate of the white light group was significantly different(<i>P</i><0.05)at 3, 6 and 9h; there was a statistically significant difference in the rate of cell proliferation at 3h and 6h in white light(<i>P</i><0.05), however, there was no significant difference in the rate of cell proliferation at 9h illumination compared with 3h and 6h illumination respectively(<i>P</i>=0.253, 0.120). The proliferation rate of cells under white light for 3-6h showed a downward trend, while that of cells under light for 6-9h showed an upward trend. At the same illumination time, the proliferation rate of the cells in the blue and white groups was lower than that in the dark group, and the cell proliferation rate in the blue group was lower than that in the white group. The difference was statistically significant(<i>P</i><0.05). <p>CONCLUSION: The proliferation of ARPE-19 cells was inhibited by light irradiation. The proliferation rate of cells in blue light group was significantly lower than that of white light group. With the increase of light time, the cell proliferation rate decreased.

Slightly acidic electrolyzed water cytotoxicity to oral keratinocyte monolayers / 口腔疾病防治

Objective @#To investigate the cytotoxicity of slightly acidic electrolyzed water (SAEW) on oral keratinocyte monolayers. @*Methods@#TR146 human keratinocyte monolayers were exposed to SAEW pretreated with bovine serum albumin（BSA）. It was divided into 4 groups, BSA 0 mg/mL (SAEW stock solutsion), BSA 0.5 mg/mL, BSA 1 mg/mL and BSA 2 mg/mL. The relative growth rate (RGR) was measured using a CCK-8 assay at 1 min, 5 min, 15 min, 30 min and 1 h, and the survival rate was measured using a Trypan Blue exclusion assay at 1 h. @*Results@#The CCK-8 assay showed significantly different OD values in the SAEW and negative control groups at different times and FAC concentrations (P<0.05). With increasing FAC concentrations and observation times, the RGR in the SAEW group decreased, and the SAEW showed moderate to severe cytotoxic effects. The OD values in the BSA (0.5~2 mg/mL)-pretreated SAEW and negative control groups were not significantly different at different times or FAC concentrations (P > 0.05); the RGRs of the BSA-pretreated SAEW group all approached 100%, and no cytotoxic effects were observed in the BSA-pretreated SAEW group. The Trypan Blue exclusion assay showed significantly different survival rates in the SAEW and negative control groups at different FAC concentrations (P < 0.05). As the FAC concentration increased, the survival rate in the SAEW group decreased, and SAEW showed moderate to severe cytotoxic effects. The survival rates in the BSA-pretreated SAEW and negative control groups were not significantly different at different FAC concentrations (P > 0.05); the survival rates in the BSA-pretreated SAEW group all approached 100%, and no cytotoxic effects were observed.@*Conclusion@#SAEW showed no adverse effects on the viability of dental oral keratinocyte monolayers in vitro in the presence of BSA at concentrations equivalent to that of protein in saliva.

Duplex effect of CCK8 and PGE2 in CVB-attacked human peripheral blood pDC / 中国免疫学杂志

Objective:To investigate the immunomodulation of CCK8 on the Coxsackievirus B ( CVB )-attacked human peripheral blood plasmacytoid dendritic cells(pDC). Methods:Peripheral blood mononuclear cells of healthy volunteers were separated by Ficoll-Hypaque gradient density centrifugation. The pDC was separated and divided into five groups,which were the control group, CVB attacked group,the group of CCK8 treated after CVB attack,the group of PGE2 treated after CVB attack and the group of CCK8+PGE2 treated after CVB attack. 100-time TCID50 of CVB was applied for the attack on pDC. Real-time PCR and Immunofluorescence technique were employed to detect the expression of CCK1R/CCK2R mRNA and protein. Then,the expression levels of costimulatory molecules such as CD80,CD86,HLA-DR ligand,and the chemokine receptor CCR7 were evaluated by Flow Cytometry Analysis. The supernatants of pDCs were collected, and the content of IFN-α was determined by Enzyme-linked Immunosorbent Assay. Results:CCK1R and CCK2R were co-expressed in human peripheral blood pDC,and both were significantly upregulated after CVB attack in vitro. Expression of CD80,CD86,HLA-DR and IFN-α were decreased in the CVB+CCK8 group compared with the CVB group,which suggested that CCK8 may reduce the CVB activation of pDC. Whereas expression of CD80,CD86,HLA-DR,CCR7 and IFN-α were increased in the CVB+PGE2 group compared with the CVB group,which suggested that PGE2 may increase the CVB activation of pDC in vitro. Conclusion:CCK8 repressed the CVB-attacked pDC,while PGE2 activated the CVB-attacked pDC.

Effect of ginsenoside Rg1 on senescence characteristics of leukemic stem cells / 局解手术学杂志

Objective To explore the effect of ginsenoside Rg1 on leukemia stem cells through comparing the biological senescence characteristics of HSCs in the patients with leukemia and healthy people,and provide new ideas and methods for leukemia prevention and treatment.Methods Fifteen cases of normal bone marrow in normal group and sixteen cases of chronic myeloid leukemia in leukemia group were divided into control group and Rg1 group,respectively.The control group used the conventional culture.The Rg1 group used the culture system with 10 μg/mL ginsenoside Rg1,other conditions were the same as control group.The bone marrow mononuclear cell of all groups were extracted after 2 days,and the CD34 +/CD38-cells population was isolated and purified by immunomagnetic adsorption cell sorting(MACS).The purity of the cells and cell cycles phase were detected by flow cytometry.Cell viability was detected by trypan blue staining.The percentage of positive cells was detected by SA-β-gal staining.CCK-8 detected the CD34 +/CD38-proliferation ability of each group.Results The ratio of CD34 +/CD38-cell population was (1.76 ± 0.34) ％ in every 1 × 106 BMNCs before sorting;the proportion of CD34 +/CD38-cell population per 1 × 106 cells after immunomagnetic sorting was (91.15 ± 2.41)％.The positive rate of SA-β-gal staining in human bone marrow CD34 +/CD38-cells of leukemia Rg1 group was significantly higher than that in leukemia control group,the difference was not significant (P ＞ 0.05);meanwhile there was no significant difference between normal control group and normal Rg1 group,but higher than that in leukemia control group,the difference was significant(P ＜ 0.05).CCK-8 results showed that the proliferation of CD34 +/CD38-cells was significantly increased in leukemia control group than those in the other groups.The survival rate of CD34 +/CD38-cells in human bone marrow was 99.1％ in all groups.Cell cycle phase results showed that the G1 arrest of CD34 +/CD38-cells in leukemia control group was significantly lower than those in the other three groups.Conclusion CD34 +/CD38-cells in chronic myeloid leukemia patients may be caused by some chronic myeloid leukemia.Ginsenoside Rg1 can effectively delay the process of aging.