Effects of arctigenin on the proliferation, apoptosis, and invasion of oral squamous cell carcinoma HSC-3 cells by regulating the Notch/Hes-1 signaling pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究牛蒡子苷元（ARC）通过调控Notch/Hes-1信号通路对口腔鳞状细胞癌（OSCC）HSC-3细胞增殖、凋亡和侵袭的影响及其机制。方法：使用不同质量浓度的ARC处理人HSC-3细胞，CCK-8法检测ARC对细胞增殖活力的影响，以选择适宜的药物浓度。将HSC-3细胞分为对照组、ARC-L组（10 mg/L ARC）、ARC-M组（20 mg/L ARC）、ARC-H组（40 mg/L ARC）和ARC-H+Jagged1/FC组（40 mg/L ARC+1.2 μg/mL Jagged1/FC）。采用EdU法检测细胞增殖能力，划痕愈合实验、Transwell实验和流式细胞术分别检测细胞的迁移、侵袭能力及细胞周期和细胞凋亡率，WB法检测增殖（c-Myc、cyclin D1）、凋亡（BAX、Bcl-2、survivin）、EMT（E-cadherin、vimentin、Snail）及Notch/Hes-1通路（Notch 1、Hes-1、NICD）相关蛋白的表达水平。结果：与0 mg/L相比，10~80 mg/L的ARC均能显著降低HSC-3细胞增殖活力（均P<0.05）。与对照组相比，ARC-L组、ARC-M组和ARC-H组HSC-3细胞EdU阳性率、划痕愈合率、侵袭细胞数、S期和G2/M期细胞占比及c-Myc、cyclin D1、Bcl-2、survivin、vimentin、Snail、Notch 1、Hes-1和NICD蛋白表达均显著降低（均P<0.05），细胞凋亡率、G0/G1期细胞占比及BAX、E-cadherin的蛋白表达均显著升高（均P<0.05），且呈浓度梯度依赖性。同时使用Notch激动剂Jagged1/FC，则可部分逆转ARC对HSC-3细胞增殖、迁移、侵袭、凋亡及相关蛋白表达的作用（均P<0.05）。结论：ARC可能通过抑制Notch/Hes-1信号通路抑制OSCC细胞HSC-3增殖和侵袭并促进细胞凋亡。

Effects of specific knockdown of ribosomal protein S5 in hepatic stellate cells on liver fibrosis / 药学实践杂志

Objective To observe the effect of specific knockdown of hepatic stellate cells (HSC) ribosomal protein S5 (RPS5) on liver fibrosis in rats. Methods The glial fibrillary acidic protein (GFAP) promoter-driven RPS5 shRNA adenovirus was established, and AdGFa2-shRPS5 and its control AdGFa2 shNC were used to transfect primary rat HSCs and hepatocytes, respectively. RPS5 was determined by Western-blot and Real Time PCR, α-SMA and type I collagen expression; the rat liver fibrosis model was established by dimethyl nitrosamine (DMN) and bile duct ligation (BDL), and intrahepatic HSC was specifically knocked down by tail vein injection of adenovirus of RPS5 levels. The pathological changes of liver tissue sections were analyzed by HE staining; the content of hydroxyproline, sections of Sirius red and Masson staining were used to evaluate collagen deposition; immunohistochemical staining was used to detect the expression of α-SMA and RPS5. Results AdGFa2-shRPS5 specifically knocked down the expression level of RPS5 in HSC and increased the expression of α-SMA and type I collagen in vitro. The in vivo results showed that in two animal models of chronic liver injury, specific knockdown of RPS5 expression in HSCs promoted HSC activation, increased the deposition of extracellular matrix, and promoted liver fibrosis. Conclusion RPS5 is essential for HSC activation and liver fibrosis, which could be a potential target for the treatment of liver fibrosis.

Vitamin D receptor (VDR) mediates the quiescence of activated hepatic stellate cells (aHSCs) by regulating M2 macrophage exosomal smooth muscle cell-associated protein 5 (SMAP-5) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

An effective therapeutic regimen for hepatic fibrosis requires a deep understanding of the pathogenesis mechanism. Hepatic fibrosis is characterized by activated hepatic stellate cells (aHSCs) with an excessive production of extracellular matrix. Although promoted activation of HSCs by M2 macrophages has been demonstrated, the molecular mechanism involved remains ambiguous. Herein, we propose that the vitamin D receptor (VDR) involved in macrophage polarization may regulate the communication between macrophages and HSCs by changing the functions of exosomes. We confirm that activating the VDR can inhibit the effect of M2 macrophages on HSC activation. The exosomes derived from M2 macrophages can promote HSC activation, while stimulating VDR alters the protein profiles and reverses their roles in M2 macrophage exosomes. Smooth muscle cell-associated protein 5 (SMAP-5) was found to be the key effector protein in promoting HSC activation by regulating autophagy flux. Building on these results, we show that a combined treatment of a VDR agonist and a macrophage-targeted exosomal secretion inhibitor achieves an excellent anti-hepatic fibrosis effect. In this study, we aim to elucidate the association between VDR and macrophages in HSC activation. The results contribute to our understanding of the pathogenesis mechanism of hepatic fibrosis, and provide potential therapeutic targets for its treatment.

Anti-pyroptosis effect of Albiziae Cortex-Tribuli Fructus combination on hepatic stellate cell line LX2: based on network pharmacology / 中国中药杂志

Based on network pharmacology, molecular docking, and in vitro experimental verification, this study aims to explore the effect of Albiziae Cortex-Tribuli Fructus combination on HSC-LX2 pyroptosis. Specifically, the targets of Albiziae Cortex, Tribuli Fructus, and hepatic fibrosis were retrieved from an online database and CNKI, and "drug-component-target" network and "drug-component-target-disease" network were constructed. Protein-protein interaction(PPI) network was established based on STRING. Metascape was employed for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and the mechanism of Albiziae Cortex-Tribuli Fructus combination against liver fibrosis was predicted. Molecular docking was used to verify some of the results of network pharmacology, and in vitro experiment was carried out to further verify the above conclusions. According to the results of network pharmacological analysis, 25 active components and 439 targets of Albiziae Cortex-Tribuli Fructus combination and 152 anti-liver fibrosis targets were screened out, including nucleotide-binding oligomerization domain and leucine-rich-repeat-and pyrin-domain-containing 3(NLRP3) and caspase-1. The key targets were involved in 194 KEGG pathways in which the NOD-like receptor signaling pathway topped. The binding common targets were related to pyroptosis. The results of in vitro experiment showed that the pair-containing serum reduced the proliferation rate of HSC-LX2 and the content of reactive oxygen species(ROS), interleukin-18(IL-18), and interleukin-1β(IL-1β)(P<0.05). Western blot and qRT-PCR suggested that the protein and gene expression of NLRP3, caspase-1, α-smooth muscle actin(α-SMA), and gasdermin D(GSDMD) in HSC-LX2 increased after AngⅡ stimulation, and the expression decreased after the intervention of pair-containing serum(P<0.05). In summary, the pair-containing serum can inhibit the classic pathway of pyroptosis, which may be the anti-liver fibrosis mechanism. This is consistent with the predicted results of network pharmacology.

Expansion of cord blood stem cells in fibronectin-coated microfluidic bioreactor

ABSTRACT Background: Hematopoietic stem/progenitor cell transplantation is the main treatment option for hematological malignancies and disorders. One strategy to solve the problem of low stem cell doses used in transplantation is pre-transplant expansion. We hypothesized that using fibronectin-coated microfluidic channels would expand HSPCs and keep self-renewal potential in a three-dimensional environment, compared to the conventional method. We also compared stem cell homing factors expression in microfluidic to conventional cultures. Materials and methods: A microfluidic device was created and characterized by scanning electron microscopy. The CD133+ cells were collected from cord blood and purified. They were subsequently cultured in 24-well plates and microfluidic bioreactor systems using the StemSpan serum-free medium. Eventually, we analyzed cell surface expression levels of the CXCR4 molecule and CXCR4 mRNA expression in CD133+ cells cultured in different systems. Results: The expansion results showed significant improvement in CD133+ cell expansion in the microfluidic system than the conventional method. The median expression of the CXCR4 in the expanded cell was lower in the conventional system than in the microfluidic system. The CXCR4 gene expression up-regulated in the microfluidic system. Conclusion: Utilizing microfluidic systems to expand desired cells effectively is the next step in cell culture. Comparative gene expression profiling provides a glimpse of the effects of culture microenvironments on the genetic program of HSCs grown in different systems.

Gamma radiation induced changes in expression of heat shock proteins (Hsc70 and Hsp83) in the dengue vector Aedes aegypti (L.)

We aimed to assess the effect of gamma radiation on the expression of heat shock proteins Hsc70 and Hsp83 in Aedes aegypti. Adult males were irradiated with 50Gy of gamma radiation, and changes in the expression of proteins in SDS-PAGE gel bands corresponding to molecular weights ~60–75kDa and ~80–95kDa were analyzed at two different time points 6 and 12-hour post-irradiation, using a temporal mass spectrometry based semi-quantitative analysis. A 2-3-fold increase was observed in both proteins Hsc70 and Hsp83, at both time points. In addition, the experiment also revealed the overexpression of several other molecules such as Arginine Kinase - known to be upregulated in certain insects during stress, Esterase B1- implicated in insecticide resistance, and also down-regulation of the 26S proteasome non-ATPase regulatory subunit 1 and ubiquitin-activating enzyme E1 - both known to be involved in ubiquitin-mediated protein degradation. The results taken together with existing data on Hsp83 and Hsc70, indicate that these proteins may enhance the survival of Ae. aegypti following gamma radiation and could serve as molecular markers for the detection of radiation-induced stress.

Effects of HDAC6 regulation of HSC70 acetylation level on bone loss in osteoporosis after oophorectomy / 中华内分泌外科杂志

Objective:To investigate the effects of histone deacetylase 6 (histone deacetylase 6, HDAC6) on oopherectomy (OOX) induced osteoporosis (OP) bone loss by binding to the promoter region of heat-shock protein 70 (HSC70) and regulating it’s acetylation.Methods:OP mouse model was established by using OOX methods. Then the mice were divided into sham operation group, OOX group, OOX+shHDAC6 group, OOX+shNC group and OOX+shHDAC6+shHSC70 group. The micro-CT system and Western blot experiment were used to detect the bone microscopic parameters of the mouse right femur and the protein expression levels of osteoblast-specific transcription factors. In vitro experiments, Westwen blot, alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining were used to determine the effects of HDAC6 and HSC70 on the osteogenic differentiation of MC3T3-E1 cells. QRT-PCR was used to detect the expression levels of HDAC6 and HSC70 in tissue or cells. The relationship between HDAC6 and HSC70 was analyzed by ChIP experiment.Results:Compared with sham group, the expression of bone mineral density (BMD) , trabecular bone number (Tb. N) , trabecular thickness (Tb.th) and bone volume fraction (BV/TV) in the right femur of OOX group mice were decreased, the expression of TB. Sp was increased, protein expression of OSX and RUNX2 was increased. At the same time, compared with sham group (1±0.11) , the expression of HDAC6 was increased in OOX group (2.33±0.19) ( t=10.56, P<0.001) . Compared with pcDNA3.1-NC group, the protein level of Osterix (OSX) and runt-related transcription factor 2 (RUNX2) , ALP activity and mineralized area in pcDNA3.1-HDAC6 group were decreased (all P<0.05) . ChIP analysis showed that compared with the pcDNA3.1-NC group (5.26±0.47) , the acetylation level of the HSC70 promoter region in the pcDNA3.1-HDAC6 group (2.37±0.21) was significantly reduced ( t=9.72, P<0.001) . Compared with pcDNA3.1-HDAC6 group, the expression of OSX and RUNX2, ALP activity and mineralization were increased in pcDNA3.1-HDAC6+ pcDNA3.1-HSC70 group (all P<0.05) . Compared with OOX+shHDAC6 group, the expression of OSX and RUNX2 protein, BMD, Tb.N, Tb.th and BV/TV were decreased but the expression of Tb. Sp was increased in OOX+ shHDAC6+ shHSC70 group. Conclusions:HDAC6 regulates the acetylation level of HSC70 and then affects OOX-induced OP bone loss. Inhibition of HDAC6 can significantly improve OP bone loss.

Clinical significance of expression of tumor immunogenic cell death related molecules / 国际肿瘤学杂志

Tumor immunogenic cell death is a type of regulatory cell death, which is driven by stress including chemotherapy drugs, radiotherapy, oncolytic virus, nano carrier drugs and photodynamic force. It can induce specific immune response to tumor death cell antigen. The further study can provide theoretical basis and new ideas for anti-tumor immunity and clinical immunotherapy of tumor.

Effects of DNMT3A regulating Drp1 on proliferation and migration of activated hepatic stellate cells / 中国药理学通报

Aim To investigate the effects of DNMT3A regulating Drp1 mediated mitochondrial fission on the proliferation and migration of active hepatic stellate cells. Methods HSC-T6 cells were activated by 5 μg·L-1 TGF-β1 for 24 h, and DNMT3A lentivirus infection model was established to silence DNMT3A. The experiment was divided into control group, TGF-β1 group, TGF-β1+LV5-NC group and TGF-β1+ LV5-DNMT3A group. The effects of DNMT3A on related mRNA and protein expression were detected by RT-qPCR and Western blot. The cell proliferation was detected by CCK-8. The effect of DNMT3A on the migration ability of HSCs cells was observed by Wound healing assay and Transwell migration assay. Results Lentivirus infection successfully constructed a DNMT3A silencing model. Compared with the control group, the level of DNMT3A significantly increased, the mRNA and protein levels of the fibrosis markers collagen and α-SMA in the TGF-β1 group significantly increased, and the mRNA and protein levels of the mitochondrial fission marker Drp1 significantly increased; At the same time, the proliferation and migration ability of HSCs cells was significantly improved. Compared with the NC group, the DNMT3A level of the DNMT3A silenced group was significantly reduced, the expressions of collagen I, α-SMA and Drp1 were significantly inhibited, and the proliferation and migration capabilities of HSCs were also significantly inhibited. Conclusions Silencing DNMT3A inhibits the level of Drp1 and inhibits the proliferation and migration of HSCs at the same time. It is suggested suggest that DNMT3A-mediated low level DNA methylation modification may inhibit the occurrence of mitochondrial fission by inhibiting the level of Drp1, thereby inhibiting the activation of HSCs and affecting the occurrence and development of liver fibrosis. ,,,,.

Development and clinical advancement of small molecules for ex vivo expansion of hematopoietic stem cell

Hematopoietic stem cell (HSC) transplantation is the only curative therapy for many diseases. HSCs from umbilical cord blood (UCB) source have many advantages over from bone marrow. However, limited HSC dose in a single CB unit restrict its widespread use. Over the past two decades, ex vivo HSC expansion with small molecules has been an effective approach for obtaining adequate HSCs. Till now, several small-molecule compounds have entered the phase I/II trials, showing safe and favorable pharmacological profiles. As HSC expansion has become a hot topic over recent years, many newly identified small molecules along with novel biological mechanisms for HSC expansion would help solve this challenging issue. Here, we will give an overview of HSC biology, discovery and medicinal chemistry development of small molecules, natural products targeting for HSC expansion, and their recent clinical progresses, as well as potential protein targets for HSC expansion.

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβ plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.

First trimester serum vitamin D, hs-CRP and second trimester uterine artery diastolic notching in predicting gestational hypertension and preeclampsia

Background: Gestational hypertension and preeclampsia is one of the leading causes of maternal and fetal morbidity and mortality. The objective of this study was to study prediction of gestational hypertension/preeclampsia by using first trimester serum vitamin D and hs-CRP and second trimester uterine artery diastolic notching.Methods: It was an observational study conducted in the departments of obstetrics and gynaecology, clinical biochemistry and radiology, Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi, India. All pregnant women with 11 to 14 weeks gestational age attending antenatal clinic between October 2012 and June 2013 were enrolled in the study. A detailed history including history of the duration of sun exposure was taken and a general physical examination including obstetrical examination was done at every visit. Serum sample were taken for hs-CRP and vitamin-D levels at 11-14 weeks. Uterine artery colour doppler study was done between 22-24 weeks for uterine artery diastolic notching. The main outcome measures were development of gestational hypertension/ preeclampsia/ eclampsia.Results: The mean vitamin D levels were significantly lower and mean hs-CRP levels were significantly higher in the hypertensive group as compared to the normotensive group, p=0.001 and p=0.004, respectively. Significant number women who developed hypertension had unilateral (46.2%) or bilateral (20.4%) uterine artery diastolic notching, p=0.005 and p=0.000, respectively. Crude’s odds ratio of uterine artery diastolic notching for prediction of hypertension in pregnancy was high, 9.894, 95% CI, 3.273-29.907 as compared to vitamin D (<13.5 ng/ml) and hs-CRP (>9.15 mg/L), 2.859, 95% CI, 1.418-5.763 and 7.16, 95% CI, 3.33-15.397.Conclusions: Uterine artery diastolic notching in the early second trimester is found to be the best predictor of PE followed by first trimester hs-CRP and vitamin D.

Inhibitory Effects of Protopine on the Proliferation of Human Hepatic Stellate Cells HSC-LX 2 and Its Mechanism Study / 中国药房

OBJECTIVE： To investigate inhibitory effects of protopine on the proliferation of human hepatic stellate cells HSC-LX2 and to explore its mechanism preliminarily. METHODS： MTT assay was used to detect the effects of 25， 50， 100， 200， 400 and 500 μmol/L protopine on the proliferation of HSC-LX2 cells. The inhibitory effect of cell proliferation was calculated. HSC-LX2 cells were divided into control group （1640 medium containing 5％ fetal bovine serum）， protopine low-concentration， medium-concentration and high-concentration groups （100， 200， 400 μmol/L）. After treated for 24 h. The apoptotic rate of the cells was detected by flow cytometry. RT-PCR was used to determine the mRNA expression of α-SMA， Collagen Ⅰ， Collagen Ⅲ， MMP-2 and TIMP-1 in cells. The protein expressions of α-SMA， Collagen Ⅰ， Collagen Ⅲ and MMP-2 were detected by Western blot. RESULTS： The inhibitory rates of 25， 50， 100， 200， 400 and 500 μmol/L protopine on proliferation HSC-LX2 cells were 0， 6.9％， 18.7％， 34.2％， 48.9％， 53.9％， respectively. Compared with control group， mRNA expression of Collagen Ⅰ， TIMP-1 and protein expression of α-SMA were decreased significantly in protopine low-concentration， medium-concentration and high-concentration groups， while protein expression of MMP- 2 was increased significantly， with statistical significance （P＜0.05 or P＜0.01）. Apoptotic rate of HSC-LX2 cells and mRNA expression of MMP-2 were increased significantly in protopine medium-concentration and high-concentration groups， mRNA expression of α-SMA and Collagen Ⅲ， protein expression of Collagen Ⅰ and Collagen Ⅲ were decreased significantly， with statistical significance （P＜0.05 or P＜0.01）. CONCLUSIONS： Protopine can induce the apoptosis of HSC-LX2 cells and inhibit their cell proliferation， and reduce the expression of a-SMA， Collagen Ⅰ， Collagen Ⅲ and TIMP-1， and increase the expression of MMP-2.

Proteolytic Enzymes Screening for Enzymolysis Preparation Technology of Active Parts of Periplaneta americana against Liver Fibrosis / 中国药房

OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and  purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.

Intra-bone marrow injection of fetal liver mononuclear cells to reconstruct the hematopoietic system in mice / 上海交通大学学报（医学版）

Objective:To observe the effect of intra-bone marrow injection (IBMI) of fetal liver mononuclear cell suspension on reconstructing the hematopoietic system in mice. Methods : The fetal liver mononuclear cells from the mice with gestational age of 13.5 days were obtained by Ficoll separation technique, and then the hematopoietic stem cells (HSCs) were obtained by magnetic bead sorting. The fetal liver mononuclear cells were injected into the tibias of 137Cs irradiated mice by IBMI. The recipient mice were observed for reconstitution of the hematopoietic system by routine blood examination, blood smear and bone marrow smear. The donor HSCs in the bone marrow of the recipient mice was subjected to flow cytometry 1 year after transplantation. Results: HSCs accounted for 0.171% in the mouse fetal liver mononuclear cells. One week after transplantation of mouse fetal liver mononuclear cells, blood cells derived from donor cells were detected from peripheral blood; in the third week after transplantation, peripheral blood leukocytes, red blood cells and hemoglobin of recipient mice were restored to the pre-irradiation levels; the percentage of donor-derived blood cells in peripheral blood in the fifth week was stable; the HSCs in the bone marrow of recipient mice were derived from donor cells 1 year after transplantation. Conclusion: The murine fetal liver mononuclear cells can efficiently reconstruct the hematopoietic system of mice by IBMI.

Biological characteristics and gene expression profile analysis of two kinds of mouse fetal liver stromal cells / 上海交通大学学报（医学版）

Objective • To explore the differences in morphology, molecular characteristics and biological functions of different types of mouse fetal liver stromal cells. Methods • E13.5 mouse fetal liver stromal cells were obtained by adherent culture, and different cell types were distinguished by morphology and expression of surface markers, such as CD44, CD29, CD106, CD45 and Sca-1. Then microarray assay was conducted by Mouse Genome 430 2.0 Array and analyzed at P3 or <-2 to identify differential expression genes. Canonical pathway and biological function analyses were performed by using ingenuity pathway analysis (IPA software). Results • Spindle-like (CD45+CD106-CD29+CD44+Sca-1-) and fibroblast-like (CD45-CD106+CD29+CD44+Sca-1-) fetal liver stromal cells were isolated in this study according to the cell morphology. The 1 485 highly-expressed genes in spindle-like cells mainly involved in immune and inflammation-related signaling pathways; while the 3 374 highly-expressed genes in fibroblast-like cells mainly involved in extracellular matrix formation and cellular adhesion. Conclusion • Mouse fetal liver stromal cells have strong heterogeneity in biological characteristics and functions, especially in hematopoietic promoting capability.

Effect of Danggui Shaoyao San Drug-containing Serum on Expression of p-MLCⅡ and MLCⅡ Protein in HSC-T6 Cells Induced by ET-1 / 中国实验方剂学杂志

Objective:To investigate the effect of endothelin-1 (ET-1) on the expression of phosphorylated myosin light chain Ⅱ(p-MLCⅡ)and myosin light chain Ⅱ(MLCⅡ)protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San(DSS)drug-containing serum. Method:After HSC-T6 cells were seeded, DMEM and blank rat serum with final concentrations of 2.5%, 5%, 10%, 15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium(MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group (5%, 10%, 15%) and DSS drug-containing serum group (5%, 10%, 15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), DSS drug-containing serum low (5%), medium (10%) and high dose (15%) groups. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group (10%), model group (10%), DSS drug-containing serum low (5%), medium (10%), high dose (15%) groups and Y-27632 inhibitor group (100 μmol·L-1). Except the blank serum control group, the other groups all received 10 nmol·L-1 ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡ and MLCⅡ in HSC-T6 cells induced by ET-1. Result:Serum concentrations of 5%, 10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group, the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state (PPPPPConclusion:DSS drug-containing serum may down-regulate the expression of p-MLCⅡ and MLCⅡ by down-regulating the content of ET-1 and inhibiting the autocrine of ET-1.

Effect of salvianolic acid B on apoptosis of liver fibrotic cells based on cleaved caspase-9 protein / 中国药理学通报

Aim: To investigate the effect of salvianolic acid B (Sal B) on liver fibrotic cells in vivo and in vitro from die perspective of apoptosis, as well as the effect on cleaved caspase-9. Methods: Diethylnitrosamine (DEN) was used to induce liver fibrosis in mice for 12 weeks. The padiological changes were detected by HE staining, and the fibrotic lesion area was determined. The cell apoptosis in the fibrotic area was observed by Hoechst 33258 fluorescence staining. The expression of cleaved caspase-9 in fibrotic tissues was detected by Western blot. The apoptotic rate of each group was detected by double standard method AnnexinV-FITC/PI, and the expression of apoptotic protein cleaved caspase-9 in HSC-T6 was detected by Western blot. Results: HE staining suggested that 12 weeks were the period of liver fibrosis in mice. No pseudoplobular structure was formed in group with low and high dose of Sal B, and the degree of fibrosis was lower than that in model group. In the fibrotic lesion area, the fluorescence staining of Hoechst 33258 showed that apoptotic cells significantly increased in group with low Sal B and high dose compared with model group. The results of the AnnexinV-FITC/PI method showed that TGF-β1 inhibited the apoptosis of HSC-T6, and Sal B promoted the apoptosis of HSC-T6 after TGF-β1 intervention and showed a concentration dependence (P <0. 01). Western blot results showed that in fibrotic liver tissues, Sal B increased the expression of cleaved caspase-9 in HSC-T6 cells compared with model group. Compared with TGF-β1 group, Sal B increased cleaved caspase-9 protein expression (P < 0. 01). Conclusions: Sal B can significantly promote apoptosis of liver fibrotic cells in vitro and in vivo, and its pro-apoptosis mechanism may be related to the up-regulation of cleaved caspase-9.

The sequential switch from macroautophagy to chaperone-mediated autophagy in Raji cells induced by starvation / 中国药理学通报

Aim To research the cross-talk and conversion between macroautophagy and chaperone-media-ted autophagy ( CMA) in cultured Burkitt lymphoma Raji cells induced by starvation. Methods The autophagic vacuoles were observed by fluorescence microscopy and confocal laser-scanning microscopy with monodansylcadaverine staining. The expression of autophagy associated-proteins were determined by West-em blot. Results Both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first hours of near baseline. With starvation persisted, CM A progressively increased along with the decline of macroautoph- A gy. Conclusions Macroautophagy and CMA are maximally activated during different stages of starvation. Activation of these two pathways is often sequential. The sequential switch from macroautophagy to CMA might be conducive to the adaption of cancer cells to miscellaneous intracellular or extracellular changes, maintaining their own growth and proliferation.

Decreased rotavirus infection of MA104 cells via probiotic extract binding to Hsc70 and ß3 integrin receptors / Disminución de la infección por rotavirus en células MA104 por la unión de proteínas de extractos probióticos a receptores celulares integrina ß3 y Hsc70 / Diminuição da infecção por rotavírus em células MA104 pela união de proteínas de extratos probióticos a receptores integrina ß3 e Hsc70

Abstract Probiotic bacteria are microorganisms beneficial to human health, useful to improving biological conditions. Thanks to probiotic bacteria the symptoms of viral infections can be alleviated. Different mechanisms whereby probiotic bacteria exert they antiviral effect have been proposed. The aim of this study was to determine whether probiotic bacteria extracts bind to receptors of host cells susceptible of rotavirus (RV) infection. To accomplish this objective, four probiotic bacterial strains of Lactobacillus spp. and Bifidobacterium spp. were tested. Probiotic extracts were obtained after bacterial growth, cell lysis and centrifugation. Obtained probiotic extracts were used in assays to interfere with adhesion and penetration of a RV strain in the mammal cell line MA104. Furthermore, the interaction between probiotic extracts and MA104 cell receptors was evaluated by co-immunoprecipitation assays using anti-β3-integrins and anti-Hsc70 antibodies. All four probiotic, protein-rich, extracts reduced RV infections in MA104 cells, suggesting a successful antiviral activity mediated by these probiotic extracts. All probiotic extracts significantly exerted their antiviral activity by interfering with RV adhesion on MA104 cell receptors, with proteins in probiotic extracts competitively interacting with cell surface receptors necessary to RV infection. Co-immunoprecipitation assay results showed that proteins in probiotic extracts were able to bind to β3-integrinsand Hsc70, which are two cellular receptors required to viral infection. The most significant contribution of this study is an insight into the mechanisms of probiotic antiviral activity, thus expanding current probiotics fundamental knowledge.

Resumen Las bacterias probióticas son microorganismos con efectos positivos en la salud humana, gracias a las bacterias probióticas los síntomas de infecciones virales pueden mitigarse. Al respecto, varios mecanismos antivirales de las bacterias probióticas han sido propuestos. El propósito de este estudio fue determinar, de manera experimental, si extractos de bacterias probióticas reducen la infección rotavírica al interferir con la unión entre el rotavirus y sus receptores celulares blanco. Extractos de cuatro cepas probióticas de Lactobacillus spp. y Bifidobacterium spp. fueron obtenidos a partir de cultivos bacterianos lisados y centrifugados. Cada uno de los extractos fue usado en experimentos para determinar si estos interfieren con la adhesión y penetración del rotavirus en células de mamífero MA104. Además, la interacción entre extractos probióticos y receptores de las células MA104 fue evaluada con ensayos de co-inmunoprecipitación, usando anticuerpos anti-integrina β3 y anti-Hsc70. Se observó que los cuatro extractos probióticos, ricos en proteínas, redujeron significativamente la infección de rotavirus en las células MA104. También se estableció que la que la actividad antiviral de los extractos probióticos es mediada por la interacción competitiva de sus proteínas con los receptores integrina β3 y Hsc70 de las células MA104, necesarios para iniciar la infección por rotavirus. Estos hallazgos constituyen un aporte al conocimiento de los mecanismos básicos de acción antiviral de las bacterias probióticas.

Resumo Bactérias probióticas são microrganismos com efeitos positivos na saúde humana, úteis na melhora de certas condições biológicas. Gracas a bactérias probióticas os sintomas de uma infecção viral podem ser aliviados. Diferentes mecanismos pelos quais as bactérias probióticas exercem seus efeitos antivirales têm sido propostos. O objetivo de este estudo foi determinar se extratos de bactérias probióticas reduzem a infecção de rotavírus (RV) ao interferir com a união entre o RV e seus receptores celulares alvo. Quatro cepas probióticas de Lactobacillus spp. e Bifidobacterium spp. foram testadas. Os extratos probióticos foram obtidos após o crescimento bacteriano, lise celular e centrifugação. Os extratos probióticos obtidos foram utilizados em ensaios para determinar se interferem com a adesão e penetração de uma cepa de RV em células de mamífero MA104. Adicionalmente, a interação entre os extratos probióticos e os receptores das células MA104 foi avaliada por ensaios de co-imunoprecipitação usando anticorpos anti-integrina β3 e anti- Hsc70. Os quatro extratos probióticos, ricos em proteínas, reduziram as infecções por RV em células MA104, sugerindo uma atividade antiviral mediada por estes extratos. Todos os extratos interferiram na adesão do RV aos receptores de células MA104, sendo que as proteínas presentes nos extratos mostraram uma interação competitiva com os receptores integrina β3 e Hsc70 das células MA104, necessários para iniciar a infecção por RV. Estes resultados contribuem para o conhecimento dos mecanismos básicos de ação antiviral de bactérias probióticas.