OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord. One important aspect of ALS pathogenesis is superoxide dismutase 1 (SOD1) mutant-mediated mitochondrial toxicity, leading to apoptosis in neurons. This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1 (G-Rg1) and conditioned medium (CM) on a mutational SOD1 cell model, and to explore the underlying mechanisms. We found that the contents of nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells (hUCMSCs) were exposed to neuron differentiation reagents for seven days. CM or G-Rg1 decreased the apoptotic rate of SOD1^G93A-NSC34 cells to a certain extent, but their combination brought about the least apoptosis, compared with CM or G-Rg1 alone. Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups. Proteins associated with mitochondrial apoptotic pathways, such as Bax, caspase 9 (Cas-9), and cytochrome c (Cyt c), were downregulated. Furthermore, CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B (NF-κB) signaling pathway by reducing the phosphorylation of p65 and IκBα. CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid (BetA), an agonist of the NF-κB signaling pathway. In summary, the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^G93A-NSC34 cells through suppressing the NF-κB/Bcl-2 signaling pathway (Fig. 1 is a graphical representation of the abstract).
Humans ; NF-kappa B/metabolism* ; Ginsenosides/pharmacology* ; Amyotrophic Lateral Sclerosis/genetics* ; Culture Media, Conditioned/pharmacology* ; Superoxide Dismutase-1 ; Neurodegenerative Diseases ; Neurons/metabolism* ; Apoptosis

OBJECTIVE: To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts (CAFs). METHODS: Normal human colorectal fibroblasts (CCD-18Co cells) in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells (HCT116-CM) or Caco-2 cells (Caco-2-CM) alone or in combination with 300 nmol/L ERK inhibitor SCH772984. The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR (RT- qPCR) and immunofluorescence assay, and the changes in cell proliferation, colony formation and migration were assessed with RTCA, colony formation and wound healing assays; Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway. RESULTS: HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers (including α-SMA, FAP, FN and TGF-β) in CCD-18Co cells, and strongly promoted fibroblast transformation into CAFs (P < 0.05). The two conditioned media also promoted the proliferation, colony formation and migration of CCD-18Co cells (P < 0.05) and significantly increased the levels of α-SMA protein and ERK phosphorylation in the cells (P < 0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of α-SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells (P < 0.05). CONCLUSION Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.
Humans ; Cancer-Associated Fibroblasts/metabolism* ; Culture Media, Conditioned/pharmacology* ; MAP Kinase Signaling System ; Caco-2 Cells ; Fibroblasts ; Signal Transduction ; Cell Proliferation ; Cell Line, Tumor ; Colorectal Neoplasms/genetics* ; Cell Movement

BACKGROUND: Progressive lipid loss of adipose tissue is a major feature of cancer-associated cachexia. In addition to systemic immune/inflammatory effects in response to tumor progression, tumor-secreted cachectic ligands also play essential roles in tumor-induced lipid loss. However, the mechanisms of tumor-adipose tissue interaction in lipid homeostasis are not fully understood. METHODS: The yki -gut tumors were induced in fruit flies. Lipid metabolic assays were performed to investigate the lipolysis level of different types of insulin-like growth factor binding protein-3 (IGFBP-3) treated cells. Immunoblotting was used to display phenotypes of tumor cells and adipocytes. Quantitative polymerase chain reaction (qPCR) analysis was carried out to examine the gene expression levels such as Acc1 , Acly , and Fasn et al . RESULTS: In this study, it was revealed that tumor-derived IGFBP-3 was an important ligand directly causing lipid loss in matured adipocytes. IGFBP-3, which is highly expressed in cachectic tumor cells, antagonized insulin/IGF-like signaling (IIS) and impaired the balance between lipolysis and lipogenesis in 3T3-L1 adipocytes. Conditioned medium from cachectic tumor cells, such as Capan-1 and C26 cells, contained excessive IGFBP-3 that potently induced lipolysis in adipocytes. Notably, neutralization of IGFBP-3 by neutralizing antibody in the conditioned medium of cachectic tumor cells significantly alleviated the lipolytic effect and restored lipid storage in adipocytes. Furthermore, cachectic tumor cells were resistant to IGFBP-3 inhibition of IIS, ensuring their escape from IGFBP-3-associated growth suppression. Finally, cachectic tumor-derived ImpL2, the IGFBP-3 homolog, also impaired lipid homeostasis of host cells in an established cancer-cachexia model in Drosophila . Most importantly, IGFBP-3 was highly expressed in cancer tissues in pancreatic and colorectal cancer patients, especially higher in the sera of cachectic cancer patients than non-cachexia cancer patients. CONCLUSION Our study demonstrates that tumor-derived IGFBP-3 plays a critical role in cachexia-associated lipid loss and could be a biomarker for diagnosis of cachexia in cancer patients.
Humans ; Insulin-Like Growth Factor Binding Protein 3/metabolism* ; Culture Media, Conditioned/pharmacology* ; Cachexia/pathology* ; Gastrointestinal Neoplasms ; Somatomedins/metabolism* ; Insulins/metabolism* ; Lipids

This study aims to explore the neuroprotective effect of bilobalide(BB) and the mechanisms such as inhibiting inflammatory response in macrophage/microglia, promoting neurotrophic factor secretion, and interfering with the activation and differentiation of peripheral CD4~+ T cells. BB of different concentration(12.5, 25, 50, 100 μg·mL~(-1)) was used to treat the RAW264.7 and BV2 cells for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and cell counting kit-8(CCK-8) were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment. Lipopolysaccharide(LPS) was applied to elicit inflammation in RAW264.7 and BV2 cells, mouse bone marrow-derived macrophages(BMDMs), and primary microglia, respectively. The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA). Spleen monocytes of C57BL/6 female mice(7-8 weeks old) were isolated, and CD4~+ T cells were separated by magnetic beads under sterile conditions. Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs. The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4~+ T cells. In addition, PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed. The cytoto-xicity was determined by lactate dehydrogenase(LDH) assay. The result showed that BB with the concentration of 12.5-100 μg·mL~(-1) had no toxicity to RAW264.7 and BV2 cells, and had no significant effect on the activity of cell model with low inflammation. The 50 μg·mL~(-1) BB was selected for further experiment, and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines. The experiment on CD4~+ T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activation and differentiation of CD4~+ T cells, while the conditioned medium of the experimental group with BB intervention reduced the activation and differentiation of CD4~+ T cells. In addition, BB also enhanced the release of neurotrophic factors from BMDMs and primary microglia. The conditioned medium after BB intervention can significantly reduce the death of PC12 neurons, inhibit neuronal damage, and protect neurons. To sum up, BB plays a neuroprotective role by inhibiting macrophage and microglia-mediated inflammatory response and promoting neurotrophic factors.
Female ; Rats ; Mice ; Animals ; Bilobalides/pharmacology* ; Neuroprotection ; Lipopolysaccharides/toxicity* ; Culture Media, Conditioned/pharmacology* ; Mice, Inbred C57BL ; Macrophages/metabolism* ; Microglia ; Cytokines/metabolism* ; Nerve Growth Factors/pharmacology* ; Inflammation/metabolism*

To determine the effect of andrographolide (Andro) on angiogenesis of human umbilical vein endothelial cells (HUVECs).
 Methods: HUVECs were treated with different concentrations of Andro and the cell viability was detected with Cell Counting Kit-8 (CCK-8). HUVECs were treated with half lethal dose (IC50) of Andro. Matrigel was used to make capillary formation of HUVECs and the effect of Andro on capillary formation was evaluated by calculating the percentage of capillary formation. Moreover, the effects of Andro and the supernatant from cultured A549 tumor cells on capillary formation were evaluated by calculating the percentage of capillary formation. The effect of Andro on the expression of matrix metalloproteinase-9 (MMP-9) was determined with Western blot.
 Results: The cell viability of HUVECs decreased with the increase of Andro concentrations. IC50 was 20 μmol/L. The capillary formation of HUVECs was inhibited when treated with 20 μmol/L Andro for 24 hours. Moreover, Andro was able to antagonize the promotion of the capillary formation induced by the supernatant from cultured tumor cells. Andro could suppress the expression of MMP-9 and antagonize the capillary formation.
 Conclusion: Andro inhibits the capillary formation of HUVECs and can antagonize the promotion of angiogenesis induced by the supernatant from cultured tumor cells.
Capillaries ; drug effects ; Cell Survival ; Collagen ; Culture Media ; Diterpenes ; pharmacology ; Drug Combinations ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Laminin ; Matrix Metalloproteinase 9 ; metabolism ; Neovascularization, Pathologic ; enzymology ; etiology ; prevention & control ; Proteoglycans ; Tumor Cells, Cultured

Brain damage can cause lung injury. To explore the mechanism underlying the lung injury induced by acute cerebral ischemia (ACI), we established a middle cerebral artery occlusion (MCAO) model in male Sprague-Dawley rats. We focused on glia maturation factor β (GMFB) based on quantitative analysis of the global rat serum proteome. Polymerase chain reaction, western blotting, and immunofluorescence revealed that GMFB was over-expressed in astrocytes in the brains of rats subjected to MCAO. We cultured rat primary astrocytes and confirmed that GMFB was also up-regulated in primary astrocytes after oxygen-glucose deprivation (OGD). We subjected the primary astrocytes to Gmfb RNA interference before OGD and collected the conditioned medium (CM) after OGD. We then used the CM to culture pulmonary microvascular endothelial cells (PMVECs) acquired in advance and assessed their status. The viability of the PMVECs improved significantly when Gmfb was blocked. Moreover, ELISA assays revealed an elevation in GMFB concentration in the medium after OGD. Cell cultures containing recombinant GMFB showed increased levels of reactive oxygen species and a deterioration in the state of the cells. In conclusion, GMFB is up-regulated in astrocytes after ACI, and brain-derived GMFB damages PMVECs by increasing reactive oxygen species. GMFB might thus be an initiator of the lung injury induced by ACI.
Animals ; Brain ; metabolism ; pathology ; Brain Ischemia ; complications ; pathology ; Bronchoalveolar Lavage Fluid ; Cell Hypoxia ; physiology ; Cells, Cultured ; Cerebrovascular Circulation ; physiology ; Chromatography, High Pressure Liquid ; Culture Media, Conditioned ; pharmacology ; Disease Models, Animal ; Endothelial Cells ; metabolism ; Gene Expression Regulation ; physiology ; Glia Maturation Factor ; metabolism ; In Situ Nick-End Labeling ; Lung Injury ; etiology ; metabolism ; pathology ; Male ; Neuroglia ; metabolism ; Neurologic Examination ; Peroxidase ; metabolism ; Proteome ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Tandem Mass Spectrometry

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

Guided bone regeneration (GBR) often utilizes a combination of autologous bone grafts, deproteinized bovine bone mineral (DBBM), and collagen membranes. DBBM and collagen membranes pre-coated with bone-conditioned medium (BCM) extracted from locally harvested autologous bone chips have shown great regenerative potential in GBR. However, the underlying molecular mechanism remains largely unknown. Here, we investigated the composition of BCM and its activity on the osteogenic potential of mesenchymal stromal cells. We detected a fast and significant (P < 0.001) release of transforming growth factor-β1 (TGF-β1) from autologous bone within 10 min versus a delayed bone morphogenetic protein-2 (BMP-2) release from 40 min onwards. BCMs harvested within short time periods (10, 20, or 40 min), corresponding to the time of a typical surgical procedure, significantly increased the proliferative activity and collagen matrix production of BCM-treated cells. Long-term (1, 3, or 6 days)-extracted BCMs promoted the later stages of osteoblast differentiation and maturation. Short-term-extracted BCMs, in which TGF-β1 but no BMP-2 was detected, reduced the expression of the late differentiation marker osteocalcin. However, when both growth factors were present simultaneously in the BCM, no inhibitory effects on osteoblast differentiation were observed, suggesting a synergistic TGF-β1/BMP-2 activity. Consequently, in cells that were co-stimulated with recombinant TGF-β1 and BMP-2, we showed a significant stimulatory and dose-dependent effect of TGF-β1 on BMP-2-induced osteoblast differentiation due to prolonged BMP signaling and reduced expression of the BMP-2 antagonist noggin. Altogether, our data provide new insights into the molecular mechanisms underlying the favorable outcome from GBR procedures using BCM, derived from autologous bone grafts.
Biomarkers ; metabolism ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Adhesion ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Culture Media, Conditioned ; pharmacology ; Guided Tissue Regeneration, Periodontal ; methods ; Humans ; Mesenchymal Stem Cells ; metabolism ; Osteoblasts ; metabolism ; Osteogenesis ; drug effects ; Transforming Growth Factor beta1 ; metabolism

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism