Establishment of an optimized CTC detection model consisting of EpCAM, MUC1 and WT1 in epithelial ovarian cancer and its correlation with clinical characteristics.

Objective: Emerging studies have demonstrated the promising clinical value of circulating tumor cells (CTCs) for diagnosis, disease assessment, treatment monitoring and prognosis in epithelial ovarian cancer. However, the clinical application of CTC remains restricted due to diverse detection techniques with variable sensitivity and specificity and a lack of common standards. Methods: We enrolled 160 patients with epithelial ovarian cancer as the experimental group, and 90 patients including 50 patients with benign ovarian tumor and 40 healthy females as the control group. We enriched CTCs with immunomagnetic beads targeting two epithelial cell surface antigens (EpCAM and MUC1), and used multiple reverse transcription-polymerase chain reaction (RT-PCR) detecting three markers (EpCAM, MUC1 and WT1) for quantification. And then we used a binary logistic regression analysis and focused on EpCAM, MUC1 and WT1 to establish an optimized CTC detection model. Results: The sensitivity and specificity of the optimized model is 79.4% and 92.2%, respectively. The specificity of the CTC detection model is significantly higher than CA125 (92.2% vs. 82.2%, P=0.044), and the detection rate of CTCs was higher than the positive rate of CA125 (74.5% vs. 58.2%, P=0.069) in early-stage patients (stage I and II). The detection rate of CTCs was significantly higher in patients with ascitic volume ≥500 mL, suboptimal cytoreductive surgery and elevated serum CA125 level after 2 courses of chemotherapy (P<0.05). The detection rate of CTCEpCAM + and CTCMUC1+ was significantly higher in chemo-resistant patients (26.3% vs. 11.9%; 26.4% vs. 13.4%, P<0.05). The median progression-free survival time for CTCMUC1+ patients trended to be longer than CTCMUC1- patients, and overall survival was shorter in CTCMUC1+ patients (P=0.043). Conclusions: Our study presents an optimized detection model for CTCs, which consists of the expression levels of three markers (EpCAM, MUC1 and WT1). In comparison with CA125, our model has high specificity and demonstrates better diagnostic values, especially for early-stage ovarian cancer. Detection of CTCEpCAM+ and CTCMUC1+ had predictive value for chemotherapy resistance, and the detection of CTCMUC1+ suggested poor prognosis.

Application of a TLR overexpression cell model in pyrogen detection.

Pyrogens are components derived from microorganisms that induce complex inflammatory responses. Current approaches to detect pyrogens are complex and difficult to replicate, thus there is a need for new methods to detect pyrogens. We successfully constructed a pyrogen-sensitive cell model by overexpressing Toll-like receptor (TLR)2, TLR4, MD2, and CD14 in HEK293 cells. Since the cytokine IL-6 is specifically released upon stimulation of the TLR2 and TLR4 signaling pathways in response to pyrogen stimulation, we used it as a read out for our assay. Our results show that IL-6 is released in response to trace amounts of pyrogens in our cell model. Pyrogen incubation times and concentrations were explored to determine the sensitivity of our cell model, and was found to be sensitive to 0.05 EU/ml of LPS and 0.05 ug/ml of LTA after stimulation for 5 hr. Our TLR overexpressing cell model, with IL-6 as readout, could be a new method for in vitro testing of pyrogens and applicable for evaluating the safety of drugs.

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes.

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.

Analysis of Circulating Tumor Cells in Ovarian Cancer and Their Clinical Value as a Biomarker.

BACKGROUND/AIMS: Monitoring the appearance and progression of tumors are important for improving the survival rate of patients with ovarian cancer. This study aims to examine circulating tumor cells (CTCs) in epithelial ovarian cancer (EOC) patients to evaluate their clinical significance in comparison to the existing biomarker CA125. METHODS: Immuomagnetic bead screening, targeting epithelial antigens on ovarian cancer cells, combined with multiplex reverse transcriptase-polymerase chain reaction (Multiplex RT-PCR) was used to detect CTCs in 211 samples of peripheral blood (5 ml) from 109 EOC patients. CTCs and CA125 were measured in serial from 153 blood and 153 serum samples from 51 patients and correlations with treatment were analyzed. Immunohistochemistry was used to detect the expression of tumor-associated proteins in tumor tissues and compared with gene expression in CTCs from patients. RESULTS: CTCs were detected in 90% (98/109) of newly diagnosed patients. In newly diagnosed patients, the number of CTCs was correlated with stage (p=0.034). Patients with stage IA-IB disease had a CTC positive rate of 93% (13/14), much higher than the CA125 positive rate of only 64% (9/14) for the same patients. The numbers of CTCs changed with treatment, and the expression of EpCAM (p=0.003) and HER2 (p=0.035) in CTCs was correlated with resistance to chemotherapy. Expression of EpCAM in CTCs before treatment was also correlated with overall survival (OS) (p=0.041). CONCLUSION: Detection of CTCs allows early diagnose and expression of EpCAM in CTC positive patients predicts prognosis and should be helpful for monitoring treatment.

Neuroprotective Effect of Anethole Against Neuropathic Pain Induced by Chronic Constriction Injury of the Sciatic Nerve in Mice.

Neuropathic pain is an intractable disease with few definitive therapeutic options. Anethole (AN) has been confirmed to possess potent anti-inflammatory and neuroprotective properties, but its effect on neuropathic pain has not been reported. The present study was designed to investigate the antinociceptive effect of AN on chronic constriction injury (CCI)-induced neuropathic pain in mice. AN (125, 250, and 500 mg/kg) and pregabalin (40 mg/kg) were intragastric administered for 8 consecutive days from the 7th day post-surgery. Behavioral parameters were measured on different days, namely, 0, 7, 8, 10, 12, and 14, from CCI operation. Additionally, electrophysiological and histopathological changes were analyzed on the 14th day. Afterward, immunofluorescence and Western blot were utilized to examine the activation of glial cells and the expression of inflammatory cytokines, respectively. AN treatment of CCI mice considerably alleviated hyperalgesia and allodynia, ameliorated abnormal sciatic nerve conduction, and restored injured sciatic nerves in a dose-dependent manner. Furthermore, AN suppressed the activation of glial cells, down-regulated pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL-6, and IL-1ß), and up-regulated the anti-inflammatory cytokine (IL-10). These assays first indicated that AN exerted an antinociceptive effect on CCI-induced neuropathic pain, and might be attributed to the anti-inflammatory and neuroprotective activities of AN.

Betaine Attenuates Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats via Inhibiting Inflammatory Response.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. Several studies have demonstrated that betaine possesses outstanding anti-inflammatory effects. However, whether betaine exerts protective effects on PAH by inhibiting inflammatory responses in the lungs needs to be explored. To test our hypothesis, we aimed to investigate the effects of betaine on monocrotaline-induced PAH in rats and attempted to further clarify the possible mechanisms. METHODS: PAH was induced by monocrotaline (50 mg/kg) and oral administration of betaine (100, 200, and 400 mg/kg/day). The mean pulmonary arterial pressure, right ventricular systolic pressure, and right ventricle hypertrophy index were used to evaluate the development of PAH. Hematoxylin and eosin staining and Masson staining were performed to measure the extents of vascular remodeling and proliferation in fibrous tissue. Monocyte chemoattractant protein-1 (MCP-1) and endothelin-1 (ET-1) were also detected by immunohistochemical staining. Nuclear factor-κB (NF-κB), tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) were assessed by Western blot. RESULTS: This study showed that betaine improved the abnormalities in right ventricular systolic pressure, mean pulmonary arterial pressure, right ventricle hypertrophy index, and pulmonary arterial remodeling induced by monocrotaline compared with the PAH group. The levels of MCP-1 and ET-1 also decreased. Western blot indicated that the protein expression levels of NF-κB, TNF-α, and IL-1ß significantly decreased (p < 0.01). CONCLUSION: Our study demonstrated that betaine attenuated PAH through its anti-inflammatory effects. Hence, the present data may offer novel targets and promising pharmacological perspectives for treating monocrotaline-induced PAH.

Trans-cinnamaldehyde protected PC12 cells against oxygen and glucose deprivation/reperfusion (OGD/R)-induced injury via anti-apoptosis and anti-oxidative stress.

Ischemia stroke is the major cause of mortality and permanent neurological disability with little definitive therapeutic options. This cerebral ischemic injury leads to the oxidative stress and eventually cell death. We hypothesized that treatment of this condition with the trans-cinnamaldehyde(TC) could protect cells from ischemic and reperfusion injury. Oxygen and glucose deprivation/reperfusion (OGD/R) was used as an in vitro model of hypoxic ischemic injury in present study. MTT was used to evaluate the protective effects of TC. Next, we tested whether TC reduced the production of reactive oxygen species (ROS). Besides, experiments were performed to determine whether or not the mitochondrial membrane potential was affected. Furthermore, the inhibiters of NO and PI3 K were used to determine the initial mechanisms. TC treatment improved cell viability, reduced intracellular ROS, and increased MMP. Further, the inhibition of NO or PI3 K significantly reduced TC's protective effects. These findings suggest that TC might be a promising agent for ischemic stroke.

Exposure of tumor-associated macrophages to apoptotic MCF-7 cells promotes breast cancer growth and metastasis.

Tumor-associated macrophages (TAMs) have been found to be associated with the progression and metastasis of breast cancer. To clarify the mechanisms underlying the crosstalk between TAMs and cancer stem cells (CSCs) in breast cancer recurrence and metastasis, we used a co-culture model of macrophages and apoptotic human breast cancer cell line MCF-7 cells to investigate the effects of TAMs on MCF-7 in vitro and in vivo. Macrophages co-cultured with apoptotic MCF-7 had increased tumor growth and metastatic ability in a nude mouse transplantation assay. The macrophages exposed to apoptotic cells also induce an increase in the proportion of CD44+/CD24- cancer stem-like cells, as well as their proliferative ability accompanied with an increase in mucin1 (MUC1) expression. During this process, macrophages secreted increased amounts of interleukin 6 (IL-6) leading to increased phosphorylation of signal transducers and activators of transcription 3 (STAT3), which likely explains the increased transcription of STAT3 target genes such as TGF-ß1 and HIF-1α. Our results indicate that when cancer cells endure chemotherapy induced apoptosis, macrophages in their microenvironment can then activate cancer stem cells to promote cancer growth and metastasis by secreting IL-6, which activates STAT3 phosphorylation to regulate the transcription of its downstream target genes.

Role of glucokinase in the subcellular localization of glucokinase regulatory protein.

Glucokinase (GCK) is the rate-limiting enzyme of liver glucose metabolism. Through protein-protein interactions, glucokinase regulatory protein (GCKR) post-transcriptionally regulates GCK function in the liver, and causes its nuclear localization. However the role of GCK in regulating GCKR localization is unknown. In the present study, using in vitro and in vivo models, we examined the levels of GCK and GCKR, and their subcellular localization. We found that total cellular levels of GCKR did not vary in the in vivo models, but its subcellular localization did. In animals with normal levels of GCK, GCKR is mainly localized to the nuclei of hepatocytes. In seven-day old rats and liver-specific Gck gene knockout mice (animals that lack or have reduced levels of GCK protein), GCKR was found primarily in the cytoplasm. The interaction of GCK with GCKR was further examined using in vitro models where we varied the levels of GCK and GCKR. Varying the level of GCK protein had no effect on total cellular GCKR protein levels. Taken together, our results indicate that GCK is important for the localization of GCKR to the nucleus and raises the possibility that GCKR may have functions in addition to those regulating GCK activity in the cytoplasm.

Drug-repurposing identified the combination of Trolox C and Cytisine for the treatment of type 2 diabetes.

BACKGROUND: Drug-induced gene expression dataset (for example Connectivity Map, CMap) represent a valuable resource for drug-repurposing, a class of methods for identifying novel indications for approved drugs. Recently, CMap-based methods have successfully applied to identifying drugs for a number of diseases. However, currently few gene expression based methods are available for the repurposing of combined drugs. Increasing evidence has shown that the combination of drugs may valid for novel indications. METHOD: Here, for this purpose, we presented a simple CMap-based scoring system to predict novel indications for the combination of two drugs. We then confirmed the effectiveness of the predicted drug combination in an animal model of type 2 diabetes. RESULTS: We applied the presented scoring system to type 2 diabetes and identified a candidate combination of two drugs, Trolox C and Cytisine. Finally, we confirmed that the predicted combined drugs are effective for the treatment of type 2 diabetes. CONCLUSION: The presented scoring system represents one novel method for drug repurposing, which would provide helps for greatly extended the space of drugs.

Evolution of glucose utilization: glucokinase and glucokinase regulator protein.

Glucose is an essential nutrient that must be distributed throughout the body to provide energy to sustain physiological functions. Glucose is delivered to distant tissues via be blood stream, and complex systems have evolved to maintain the levels of glucose within a narrow physiological range. Phosphorylation of glucose, by glucokinase, is an essential component of glucose homeostasis, both from the regulatory and metabolic point-of-view. Here we review the evolution of glucose utilization from the perspective of glucokinase. We discuss the origin of glucokinase, its evolution within the hexokinase gene family, and the evolution of its interacting regulatory partner, glucokinase regulatory protein (GCKR). Evolution of the structure and sequence of both glucokinase and GCKR have been necessary to optimize glucokinase in its role in glucose metabolism.

Long term liver specific glucokinase gene defect induced diabetic cardiomyopathy by up regulating NADPH oxidase and down regulating insulin receptor and p-AMPK.

BACKGROUND: The liver-specific glucokinase knockout (gckw/-) mouse experiences long-term hyperglycemia and insulin resistance. This study was designed to evaluate the functional and structural changes in the myocardium of 60 week-old gckw/- mice, and to investigate the effect of rosiglitazone on the myocardium in this model. METHODS: 60 week-old gckw/- mice were randomly divided into 3 groups: gckw/-, gckw/- mice treated with insulin (1 U/kg) and gckw/- mice treated with rosiglitazone (18 mg/kg). Insulin or rosiglitazone treatment was for 4 weeks. Gckw/w litermates were used as controls. Echocardiography, electrocardiogram, biochemical, histopathological, ultrastructural, real time PCR and Western blot studies were performed to examine for structural and functional changes. RESULTS: Long-term liver-specific gck knockout in mice elicits hyperglycaemia and insulin resistance. Compared to age matched gckw/w mice, 60 week-old gckw/- mice showed decreased LV internal dimension, increased posterior wall thickness, lengthened PR and QRS intervals, up-regulated MLC2 protein expression, decreased SOD activity, increased MDA levels and up-regulated Cyba mRNA. Morphological studies revealed that there was an increase in the amount of PAS and Masson positively stained material, as did the number and proportion of the cell occupied by mitochondria in the gckw/- mice. Western blot analysis revealed that the levels of the insulin receptor, Akt, phosphorylated AMPK beta and phosphorylated ACC were reduced in gckw/- mice. These effects were partly attenuated or ablated by treatment with rosiglitazone. CONCLUSIONS: Our results indicate that changes in the myocardium occur in the liver-specific glucokinase knockout mouse and suggest that reduced glucokinase expression in the liver may induce diabetic cardiomyopathy by up regulating NADPH oxidase and down regulating insulin receptor and p-AMPK protein levels. Rosiglitazone treatment may protect against diabetic cardiomyopathy by altering the levels of a set of proteins involved in cardiac damage.

CD206 modulates the role of M2 macrophages in the origin of metastatic tumors.

Tumor metastasis is a key factor affecting the life of patients with malignant tumors. For the past hundred years, scientists have focused on how to kill cancer cells and inhibit their metastasis in vivo, but few breakthroughs have been made. Here we hypothesized a novel mode for cancer metastasis. We show that the phagocytosis of apoptotic tumor cells by macrophages leads to their polarization into the M2 phenotype, and that the expression of stem cell related as well as drug resistance related genes was induced. Therefore, it appears that M2 macrophages have "defected" and have been transformed into the initial "metastatic cancer cells", and thus are the source, at least in part, of the distal tissue tumor metastasis. This assumption is supported by the presence of fused cells with characteristics of both macrophage and tumor cell observed in the peripheral blood and ascites of patients with ovarian cancer. By eliminating the expression of CD206 in M2 macrophages using siRNA, we show that the growth and metastasis of tumors was suppressed using both in vitro cell line and with experimental in vivo mouse models. In summary, we show that M2 macrophages in the blood circulation underwent a "change of loyalty" to become "cancer cells" that transformed into distal tissue metastasis, which could be suppressed by the knockdown of CD206 expression.

Differential expression of genes associated with the progression of renal disease in the kidneys of liver-specific glucokinase gene knockout mice.

Liver glucokinase (GCK) deficient mice possess mild renal complications associated with diabetes. To investigate the progression of kidney disease and identify candidate genes involved in the pathogenesis of renal damage, we examined changes in tissue structure and gene expression in the kidneys of liver-specific GCK knockout (gckw/-) mice and age-matched normal wild-type control (gckw/w) mice as they aged. Suppression subtractive hybridization (SSH) was used to identify candidate genes that showed a pattern of differential expression between kidneys of gckw/- and gckw/w mice at 60 weeks of age. Differential expression of the candidate genes was examined by real-time qPCR in liver-specific gckw/- and gckw/w mice at 16, 26, 40, 60, and 85 weeks of age. Among the candidate genes, only glutathione peroxidase-3 (GPX3) was confirmed to show differential expression by qPCR in the 60-week old mice, however two others genes, MALAT1 and KEG, showed significant changes at other ages. This study shows that liver-specific glucokinase deficient mice display changes in kidney morphology by 40 weeks of age, and that renal complication may be correlated with a reduction in GPX3 levels. Since decreased GPX3 mRNA expression was observed at 26 weeks, which is younger than the age when pathological changes can be seen in kidney biopsies, GPX3 may serve as an early marker for kidney damage.

Putative Prevention of XML Injection Against Myocardial Ischemia Is Mediated by PKC and PLA2 Proteins.

Background: Xinmailong (XML) injection is a CFDA-approved traditional Chinese medicine with clinical value for heart failure treatment. The present investigation was aimed to evaluate the potential protective roles of this injection on myocardial ischemia and the underlying molecular mechanism. Methods: In our study, we selected two models of myocardial ischemia rats. Rats were randomly divided into six groups, with saline or XML administrated 4 days before ischemia model establishment. ECG of different time intervals and biochemical parameters of end point were measured. The potential mechanisms of the protective role of XML were explored using system pharmacology and molecular biology approaches. Results: Myocardial ischemia rats demonstrated abnormal ECG and serum levels of cTnT. Pretreatment with XML significantly attenuated these damages, especially the medium doses. GO and KEGG analysis revealed that the 90 putative target genes were associated with pathways of fatty acid absorption/metabolism, inflammation, RAAS, and vascular smooth muscle. Further network pharmacology method identified five main chemical ingredients and potential targets of XML injection for myocardial ischemia. Mechanically, the beneficial effect of XML injection was mediated by the reactive oxygen species (ROS) inhibition and inflammation attenuation via regulating the expression levels of targets of PKC and PLA2. Conclusion: These findings indicate that XML exerts protective effects against myocardial injury, with attenuated ROS production, apoptosis, and inflammation. Therefore, we speculate that XML may be an alternative supplementary therapeutic agent for myocardial ischemia prevention.

Apoptotic SKOV3 cells stimulate M0 macrophages to differentiate into M2 macrophages and promote the proliferation and migration of ovarian cancer cells by activating the ERK signaling pathway.

Ovarian cancer has a high rate of recurrence, with M2 macrophages having been found to be involved in its progression and metastasis. To examine the relationship between macrophages and ovarian cancer in the present study, M0 macrophages were stimulated with apoptotic SKOV3 cells and it was found that these macrophages promoted tumor proliferation and migration. Subsequently, the mRNAs and proteins expressed at high levels in these M2 macrophages were examined by RNA­Seq and quantitative proteomics, respectively, which revealed that M0 macrophages stimulated by apoptotic SKOV3 cells also expressed M2 markers, including CD206, interleukin­10, C­C motif chemokine ligand 22, aminopeptidase­N, disabled homolog 2, matrix metalloproteinase 1 and 5'­nucleotidase. The abundance of phosphorylated Erk1/2 in these macrophages was increased. The results indicate that apoptotic SKOV3 cells stimulate M0 macrophages to differentiate into M2 macrophages by activating the ERK pathway. These results suggest possible treatments for patients with ovarian cancer who undergo chemotherapy; inhibiting M2 macrophage differentiation during chemotherapy may reduce the rate of tumor recurrence.

Overexpression of DUSP6 enhances chemotherapy-resistance of ovarian epithelial cancer by regulating the ERK signaling pathway.

Objective: DUSP6 is a negative regulator of the ERK signaling pathway and plays an important role in chemotherapy-resistance. Previously we showed that DUSP6 is overexpressed in ovarian cancer side population (SP) cells that possess cancer stem cell-like properties and are quiescent and chemotherapy-resistant. Here, we explore the effects of DUSP6 on chemotherapy-resistance by examining its regulation of the ERK signaling pathway and G0/G1 cell cycle arrest. Methods: mRNA and protein expression of DUSP6 and G0/G1 cell cycle checkpoint regulating proteins (CyclinD1, CyclinD3 and CyclinE2) was evaluated among ovarian cancer cell lines and tissue samples. Ovarian cancer cells were transiently transfected to overexpress DUSP6. After treatment with cisplatin, cell viability was measured by the MTS assay at 48 hours and the half maximal inhibitory concentration (IC50) for each cell line was calculated. Subcellular localization and cell cycle analysis were determined by using immunofluorescence and FACS, respectively. Results: SKOV3 and OVCAR8 SP cells were shown to express higher levels of DUSP6 and lower levels of CyclinD3 compared with non-SP (NSP) cells (P<0.001). Among 39 ovarian cancer tissue samples, expression of DUSP6 in the chemotherapy-resistant group (12 samples) was higher than in the chemotherapy-sensitive group (27 samples) (P<0.05). While a lower level of expression of CyclinD3 was seen in the chemotherapy-resistant group, it was not statistically different from the chemotherapy-sensitive group. HO8910 cells where shown to have higher IC50 to cisplatin than SKOV3 or OVCAR8 cells, and this correlated with higher levels of DUSP6 expression. Overexpression of DUSP6 in SKOV3 cells led to an increase in cisplatin IC50 values (P<0.05), and also markedly reduced the expression levels of phospho-ERK1/2 and CyclinD3 and to the predominance of cells in the G0/G1 phase. Conclusion: Our findings reveal an enhancement of chemotherapy-resistance and a predominance of cells in G1 cell cycle arrest in DUSP6-overexpressing ovarian cancer cells. This suggests that overexpression of DUSP6 promotes chemotherapy-resistance through the negative regulation of the ERK signaling pathway, increasing the G0/G1 phase ratio among ovarian cancer cells, and leading to cellular quiescence.

[Antitussive activity comparison of three kinds of Stemonae Radix in Chinese Pharmacopoeia].

OBJECTIVE: To compare the antitussive activity of three kinds of Stemonae Radix specified in the Chinese Pharmacopoeia, including roots of Stemona sessilifolia, S. japonica and S. tuberosa. METHOD: The antitussive activity was determined in mouse after cough induction by ammonia aerosol stimulation and the number of cough in 2 min were detected with codeine as positive control. RESULT: All the decoctions, the total alkaloid fractions and non-alkaloid fractions of S. sessilifolia, S. japonica and three chemical types of S. tuberosa showed significant antitussive effect and exhibited a dose-dependent inhibition of coughing. The ED50 values showed that the antitussive activity strength for both total alkaloid fractions and the decoctions are: S. tuberosa (Type I) > S. sessilifolia > S. japonica. The total alkaloid fractions had more potent atitussive activity than the decoctions and non-alkaloid fractions. The antitussive activity strength for the three chemical types of S. tuberosa is: Type I > Type III > Type II. The samples from different producing areas for the same species of Stemonae Radix had no significant differences in antitussive activity. The result also showed that the honey-processed slice had much stronger antitussive activity than raw slice. CONCLUSION: The antitussive efficacies of Stemonae Radix were influenced by chemical diversity both in same species and among different species, different fractions and processed method.

Exosomes from Macrophages Exposed to Apoptotic Breast Cancer Cells Promote Breast Cancer Proliferation and Metastasis.

Exosomes have recently become the subject of increasing research interest. Interactions between tumor and host cells via exosomes play crucial roles in the initiation, progression and invasiveness of breast cancer. In our study, we used exosomes isolated from a co-culture model of THP-1-derived macrophages exposed to apoptotic MCF-7 or MDA-MB-231 breast cancer cell line cells to investigate their effects on naïve MCF-7 or MDA-MB-231 cells in vitro and in vivo. This post-chemotherapy tumor microenvironment model allowed us to explore possible mechanisms that explain increased proliferation and metastasis of breast cancer seen in some patients. Our results suggest that while exosomes derived from macrophages normally inhibit proliferation and metastasis of MCF-7 or MDA-MB-231 cells, exposure of macrophages to breast cancer cells that have experienced chemotherapy are modified them to promote these processes. Exosomes from macrophages exposed to apoptotic cancer cells have increased amounts of IL-6 that increases the phosphorylation of STAT3, which likely explains the increased transcription of STAT3 target genes such as CyclinD1, MMP2 and MMP9. These observations suggest that the inhibition of exosome secretion and STAT3 signaling pathway activation might suppress the growth and metastasis of malignant tumors, and provide new targets for therapeutic treatment of malignant tumors after chemotherapy.

Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process.

BACKGROUND: A refractory wound is a typical complication of diabetes and is a common outcome after surgery. Current approaches have difficulty in improving wound healing. Recently, non-expanded stromal vascular fraction (SVF), which is derived from mature fat, has opened up new directions for the treatment of refractory wound healing. The aim of the current study is to systematically investigate the impact of SVF on wound healing, including the rate and characteristics of wound healing, ability of fibroblasts to migrate, and blood transport reconstruction, with a special emphasis on their precise molecular mechanisms. METHODS: SVF was isolated by digestion, followed by filtration and centrifugation, and then validated by immunocytochemistry, a MTS proliferation assay and multilineage potential analysis. A wound model was generated by creating 6-mm-diameter wounds, which include a full skin defect, on the backs of streptozocin-induced hyperglycemic mice. SVF or human adipose-derived stem cell (hADSC) suspensions were subcutaneously injected, and the wounds were characterized over a 9-day period by photography and measurements. A scratch test was used to determine whether changes in the migratory ability of fibroblasts occurred after co-culture with hADSCs. Angiogenesis was observed with human umbilical vein endothelial cells. mRNA from fibroblasts, endotheliocyte, and skin tissue were sequenced by high-throughput RNAseq, and differentially expressed genes, and pathways, potentially regulated by SVF or hADSCs were bioinformatically analyzed. RESULTS: Our data show that hADSCs have multiple characteristics of MSC. SVF and hADSCs significantly improved wound healing in hyperglycemic mice. hADSCs improve the migratory ability of fibroblasts and capillary structure formation in HUVECs. SVF promotes wound healing by focusing on angiogenesis and matrix remodeling. CONCLUSIONS: Both SVF and hADSCs improve the function of fibroblast and endothelial cells, regulate gene expression, and promote skin healing. Various mechanisms likely are involved, including migration of fibroblasts, tubulogenesis of endothelial cells through regulation of cell adhesion, and cytokine pathways.