BACKGROUND

Prohibitin 1 (PHB) is a potential target for the treatment of urothelial carcinoma of the bladder (UCB). FL3 is a newly synthesized agent that inhibits cancer cell proliferation by targeting the PHB protein; however, the effect of FL3 in UCB cells remains unexplored.

METHODS

FL3 was identified to be a potent inhibitor of UCB cell viability using CCK-8 (cell counting kit-8) assay. Then a series of in vitro and in vivo experiments were conducted to further demonstrate the inhibitory effect of FL3 on UCB cell proliferation and to determine the underlying mechanisms.

RESULTS

FL3 inhibited UCB cell proliferation and growth both in vitro and in vivo. By targeting the PHB protein, FL3 inhibited the interaction of Akt and PHB as well as Akt-mediated PHB phosphorylation, which consequently decreases the localization of PHB in the mitochondria. In addition, FL3 treatment resulted in cell cycle arrest in the G2/M phase, and this inhibitory effect of FL3 could be mimicked by knockdown of PHB. Through the microarray analysis of mRNA expression after FL3 treatment and knockdown of PHB, we found that the mRNA expression of the growth arrest and DNA damage-inducible alpha (GADD45α) gene were significantly upregulated. When knocked down the expression of GADD45α, the inhibitory effect of FL3 on cell cycle was rescued, suggesting that FL3-induced cell cycle inhibition is GADD45α dependent.

CONCLUSIONS

Our data provide that FL3 inhibits the interaction of Akt and PHB, which in turn activates the GADD45α-dependent cell cycle inhibition in the G2/M phase.

The CCK-B/gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B/gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B/gastrin receptor in the known CCK-B/gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B/gastrin receptor antibody (alpha-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified alpha-CCKBR-Ser antibody was shown to be specific for the CCK-B/gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B/gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.

BACKGROUND

Fine particulate matter (PM2.5) is a major risk factor for the development and progression of atherosclerosis. Proliferation and infiltration of vascular smooth muscle cells (VSMCs) from the blood vessel media into the intima is a crucial step in the pathophysiology of atherosclerosis. Puerarin, a natural extract from Radix Puerariae, possesses significant anti-atherosclerosis properties. However, the underlying molecular mechanisms responsible for the effect of puerarin on the VSMCs proliferation induced by PM2.5 remain unclear. The present study was designed to examine the effect of puerarin on PM2.5-induced VSMCs proliferation, and to explore the p38 mitogen-activated protein kinase (p38 MAPK) signal mechanism involved.

METHODS

VSMCs viability was measured by CCK-8 assay, VSMCs proliferation was assessed by BrdU immunofluorescence, the levels of superoxide dismutase (SOD) and malonaldehyde (MDA) were assayed by colorimetric assay kits, the levels of nitric oxide (NO) and endothelin-1 (ET-1) were determined by nitrate reductase method and radioimmunoassay, the levels of vascular cell adhesion molecule-1 (VCAM-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were measured by ELISA. The protein expressions of phospho-p38 MAPK (p-p38 MAPK) and proliferating cell nuclear antigen (PCNA) in the VSMCs were subjected by Western blot.

RESULTS

Compared to the PM2.5-treated cells, in addition to inhibiting the PM2.5-induced VSMCs proliferation, puerarin also down-regulated the protein expressions of p-p38 MAPK and PCNA, decreased the levels of ET-1, VCAM-1, IL-6, TNF-α and MDA, increased the levels of NO and SOD. Moreover, the anti-proliferative effects of puerarin were significantly enhanced by the co-incubation of puerarin with SB203580, a selective inhibitor of p38 MAPK, as compared to the puerarin-treated cells.

CONCLUSIONS

These results suggest that puerarin might suppress the PM2.5-induced VSMCs proliferation via the inhibition of the p38 MAPK signaling pathway.

Ulcerative colitis is a chronic inflammatory disease of the colon where intestinal motility is disturbed. Interstitial cells of Cajal (ICC) are required to maintain normal intestinal motility. In the present study, we assessed the effect of tumor necrosis factor-alpha (TNF-α) on viability and apoptosis of ICC, as well as on the expression of stem cell factor (SCF), ghrelin, and substance P. ICC were derived from the small intestines of Swiss albino mice. Cell viability and apoptosis were measured using CCK-8 assay and flow cytometry, respectively. ELISA was used to measure the concentrations of IL-1β, IL-6, ghrelin, substance P, and endothelin-1. Quantitative RT-PCR was used to measure the expression of SCF. Western blotting was used to measure the expression of apoptosis-related proteins, interleukins, SCF, and NF-κB signaling pathway proteins. TNF-α induced inflammatory injury in ICC by decreasing cell viability and increasing apoptosis and levels of IL-1β and IL-6. TNF-α decreased the levels of SCF, ghrelin, and substance P, but had no effect on endothelin-1. TNF-α down-regulated expressions of SCF, ghrelin, and substance P by activating the NF-κB pathway in ICC. In conclusion, TNF-α down-regulated the expressions of SCF, ghrelin, and substance P via the activation of the NF-κB pathway in ICC.

Little information is available on the expression of cholecystokinin (CCK) receptors in the human pancreas, especially in the developing pancreas. We evaluated expression patterns for the CCK receptors in human pancreas at three different ages: fetus, infant, and adult. Expressions of CCK-A and CCK-B receptor messenger RNA (mRNA) were studied in human midtrimester fetus (14-15 weeks' gestation), infant (50 days old), and adult pancreas by reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis. Expression levels of mRNA for both receptors also were evaluated by Northern blot analysis of adult pancreas. Northern blot analysis showed a strong signal for CCK-B receptor mRNA in adult pancreas, but no detectable signal for CCK-A receptor mRNA. However, RT-PCR/Southern blotting showed the presence of CCK-A receptor mRNA in adult pancreas. This was confirmed by sequencing of the complementary DNA (cDNA). RT-PCR/Southern blot analysis also showed CCK-A and CCK-B receptor mRNA expression in fetal and infant pancreas. These results show that the both CCK receptor types are expressed in human pancreas at stages of early gestation, but there is predominant expression of CCK-B receptor in adult pancreas.

BACKGROUND

Cholecystokinin (CCK) and gastrin show a potent influence on gastric secretion and motility, but their role in mucosal integrity has been little studied.

METHODS

In this study the effects of CCK-8, pentagastrin, and duodenal oleate on acute gastric lesions induced by 100% ethanol were studied in rats.

RESULTS

CCK-8 was about 13 times more potent than pentagastrin in protecting the gastric mucosa against ethanol damage. CCK released by duodenal oleate also protected gastric mucosa against this damage. The protective effects of CCK-8 were almost completely abolished by the blockage of CCK-A receptors with loxiglumide, whereas the protective effect of pentagastrin was completely abolished by L-365,260. The protective effects of CCK, pentagastrin, or duodenal oleate against ethanol injury were accompanied by a marked increase in luminal content of somatostatin, suggesting that this peptide is implicated in this protection. The protective activity of CCK and pentagastrin against ethanol injury was accompanied by a significant increase in gastric blood flow. Inhibition of nitric oxide (NO) synthase with NG-nitro-L-arginine methyl ester abolished almost completely both gastric protection and hyperemia induced by CCK and pentagastrin. Addition of L-arginine, but not D-arginine, restored the protective and hyperemic effects of CCK and pentagastrin. Pretreatment with the sulfhydryl blocking agent N-ethylmaleimide also abolished the protective and hyperemic effects of CCK and pentagastrin. The hyperemia, but not the protection, afforded by CCK and pentagastrin was reduced after sensory nerve deactivation with capsaicin.

CONCLUSIONS

Both exogenous and endogenous CCK and pentagastrin exert protective activity against ethanol damage, and this effect is mediated through separate receptors, NO, and sulfhydryl-sensitive pathway.

OBJECTIVE

Long non-coding RNAs (lncRNAs) are thought to play crucial roles in human diseases. However, the function of lncRNAs in hypertrophic scar formation remains poorly understood.

METHODS

In this study, we investigated the expression of lncRNA8975-1 in hypertrophic scar tissues and fibroblasts by quantitative reverse transcription PCR (qRT-PCR). To investigate its function, overexpression and knockdown of lncRNA8975-1 were performed using lentivirus infection and Stealth RNAi transfection, respectively. Cell proliferation was detected by CCK-8 assay. The protein levels of collagens and alpha-smooth muscle actin (α-SMA) were analysed by western blot.

RESULTS

We found that lncRNA8975-1 was overexpressed in hypertrophic scar tissues and dermal fibroblasts. Overexpression of lncRNA8975-1 inhibited cell proliferation and reduced the protein expression levels of COL1A2, COL1A1, COL3A1 and α-SMA in hypertrophic scar fibroblasts, whereas knockdown of lncRNA8975-1 had the opposite effect.

CONCLUSIONS

Our results show that the long non-coding RNA lncRNA8975-1 is upregulated in hypertrophic scar fibroblasts; furthermore, it inhibits fibroblast proliferation and reduces collagen and α-SMA expression. Further studies on the mechanisms regulated by lncRNA8975-1 would lead to a better understanding of the pathogenesis of hypertrophic scar formation.

Prostaglandins of the E type may have a potential role in pancreatic physiology and pathophysiology. Because prostaglandins of the E type inhibit HCl secretion in parietal cells via a specific receptor by inhibition of adenylylcyclase, we studied whether a similar mechanism exists in the exocrine pancreas. Isolated rat pancreatic acini were incubated with various concentrations of secretagogues, such as cholecystokinin-octapeptide (CCK-8), bombesin, carbachol, and vasoactive intestinal peptide (VIP), in the absence or presence of prostaglandin E2 (PGE2), and amylase secretion was measured. For receptor binding studies, acini and pancreatic membranes were incubated with [3H]PGE2 and either unlabeled PGE2 or other types of prostaglandins. PGE2 (10(-13) to 10(-5) M) did not inhibit basal amylase secretion. However, CCK-8-stimulated secretion was significantly inhibited. Stimulation of secretion by bombesin, carbachol, VIP, and secretin was also inhibited by PGE2, but not as pronounced as CCK-8-stimulated secretion. The formation of inositol 1,4,5-trisphosphate induced by CCK-8 was markedly inhibited by simultaneous incubation with PGE2. Furthermore, PGE2 slightly but significantly reduced the CCK-8-induced efflux of 45Ca2+ from prelabeled acini. Intact acini and a membrane fraction bound [3H]PGE2 and this function could be equally competed by either unlabeled PGE2 or PGE1 in contrast to less-related prostaglandins such as PGF2 alpha, PGD2, and prostacyclin. We conclude that prostaglandins of the E type inhibit pancreatic enzyme secretion stimulated by various secretagogues. This function is mediated via specific receptors for PGE. With regard to CCK-8-stimulated secretion this function may be mediated by an inhibition of formation of inositol 1,4,5-trisphosphate.

OBJECTIVE

To investigate the mechanisms of the effects of solanine on human androgen-independent prostate cancer cell line PC-3 in vitro.

METHODS

PC-3 cells were treated with solanine at the concentration of 0, 30, 40 and 50 microg/ml, and the cell activity was measured by CCK-8 at 12, 24 and 48 hours after the treatment. At 24 hours, the cell cycle and apoptosis were detected by flow cytometry and fluorescence microscopy, and the protein expressions of I(kappa)B(alpha) and Bcl-2 determined by Western blot.

RESULTS

Solanine suppressed the growth of PC-3 cells in a dose- and time-dependent manner in vitro, with significant differences among different concentration and time groups (P < 0.05). The cycle of the PC-3 cells was arrested in the S phase (P < 0.05), with a significantly higher rate of apoptosis in the treated groups than in the controls (P < 0.05). The protein expression of I(kappa)B(alpha) was obviously up-regulated and that of Bcl-2 down-regulated in all the solanine concentration groups.

CONCLUSIONS

Solanine has an anti-prostate cancer effect by inhibiting PC-3 cell proliferation, arresting the S phase, inducing cell apoptosis, up-regulating the protein expression of I(kappa)B(alpha) and down-regulating that of Bcl-2.

Free fatty acid receptors GPR120 and GPR40 are involved in the secretion of gut hormones. GPR120 and GPR40 are expressed in enteroendocrine K cells, and their activation induces the secretion of the incretin glucose-dependent insulinotropic polypeptide (GIP). However, the role of these receptors in fat-induced GIP secretion in vivo and the associated mechanisms are unclear. In this study, we investigated corn oil-induced GIP secretion in GPR120-knockout (GPR120-/-) and GPR40-knockout (GPR40-/-) mice. Oil-induced GIP secretion was reduced by 50% and 80% in GPR120-/- and GPR40-/- mice, respectively, compared with wild-type mice. This was not associated with a significant difference in K-cell number or GIP content in K cells, nor messenger RNA levels of the lipid receptor GPR119, nor bile acid receptors TGR5 and farnesoid X receptor. GPR120-/- and GPR40-/- mice also exhibited substantially decreased levels of cholecystokinin (CCK), a hormone from I cells that promotes bile and pancreatic lipase secretion, and this decrease was associated with impaired gallbladder contraction. Notably, treatment with a CCK analog resulted in recovery of oil-induced GIP secretion in GPR120-/- mice but not in GPR40-/- mice. These results indicate that corn oil-induced GIP secretion from K cells involves both GPR120 and GPR40 signaling pathways, and GPR120-induced GIP secretion is indirectly mediated by CCK.

Ten-eleven translocation 1 (TET1), a widely reported DNA demethylation protein, has been associated with tumorigenesis and metastasis. However, whether TET1 is an oncogene or tumor suppressor gene has been controversial; the mechanism of how TET1 affects cancer progression remains unclear. The current study aims to investigate how TET1 is changed in the tumor microenvironment and to explore the mechanisms of how TET1 affects colon cancer progression. Because hypoxia prevails on solid tumors, we established an important connection between hypoxia and DNA demethylation in tumorigenesis. By qPCR and RNA interference (RNAi) technology, we found that hypoxia increased TET1 expression with a hypoxia-inducible factor-1-alpha (HIF-1α)-dependent manner. By CHIP-qPCR and pyrosequencing technology, we demonstrated that TET1 regulated the target gene expression of HIF-1α through HIF-1α binding to hypoxia-responsive elements (HREs), and HIF-1α binding to HREs depended on CpG methylation levels. By Cell Counting Kit-8 (CCK-8) and transwell assay, we showed that loss of TET1 did not affect cell proliferation but inhibited migration. We also identified two novel gene mutants of TET1 in 120 paired tumor/normal tissue specimens by DNA sequencing and found that TET1 E2082K mutant blocked the TET1-enhanced cell migration. Our results showed that the downregulation of TET1 rescued the abnormally high levels of gene expression resulting from hypoxia in tumors and reduced the migration activity of tumor cells, suggesting a therapeutic role by interference with TET1 in colon cancer treatment. By demonstrating that hypoxia upregulated TET1 and that TET1 drove HIF-1α-responsive genes, we showed that an epigenetic mechanism and tumor microenvironment-driven models coexisted and mutually affected colon cancer.

BACKGROUND Endothelial cell (EC) injury is underlies for the pathogenesis of atherosclerosis (AS). MicroRNAs (miRNAs) have been indicated play important role in modulating AS occurrence and development. However, how miR-328-3p modulates EC injury molecular level for AS remains unclear. MATERIAL AND METHODS MiR-328-3p and forkhead box protein O4 (FOXO4) expression were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell viability was analyzed by Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was utilized to analyze the apoptotic rate. The migration and invasion abilities were measured by Transwell assay. Western blot was applied to examine the expression of C-caspase 3, Beclin, LC3-I, and LC3-II. The levels of interleukin (IL)-1ß, IL-10, and tumor necrosis factor-alpha (TNF-alpha) were tested by enzyme-linked immunosorbent assay (ELISA) assay and western blot. RESULTS MiR-328-3p expression was downregulated in oxidized low-density lipoprotein (ox-LDL) induced human umbilical vein endothelial cells (HUVECs). Overexpressed miR-328-3p obviously alleviated ox-LDL induced inhibition on cell viability, migration and invasion, stimulation on apoptosis, autophagy as well as inflammation in HUVECs. FOXO4 was elevated in ox-LDL HUVECs, and functional assay indicated that FOXO4 aggravated ox-LDL induced HUVECs impairment. In addition, FOXO4 was a target of miR-328-3p in HUVECs; rescue experiments suggested miR-328-3p could protect HUVECs against ox-LDL induced injury via regulating FOXO4. CONCLUSIONS MiR-328-3p protected vascular endothelial cells against ox-LDL induced injury via targeting FOXO4, suggesting a novel insight for atherosclerosis treatment.

The action of stem cells is mediated by their paracrine secretions which comprise the secretory profile. Various approaches can be used to modify the secretory profile of stem cells. Creating a hypoxic environment is one method. The present study aims to demonstrate the influence of CoCl₂ in generating hypoxic conditions in a dental pulp stem cell (DPSCs) culture, and the effect of this environment on their secretory profile. DPSCs that were isolated from human permanent teeth were characterized and treated with different concentrations of CoCl₂ to assess their viability by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and proliferation by a cell counting kit (CCK)-8 assay. The gene expression level of hypoxia-inducible factor 1-alpha (HIF-1α) was analyzed by quantitative real time polymerase chain reaction (qRT-PCR) to demonstrate a hypoxic environment. Comparative evaluation of the growth factors and cytokines were done by cytometric bead array. Gene expression levels of transcription factors OCT4 and SOX2 were analyzed by qRT-PCR to understand the effect of CoCl₂ on stemness in DPSCs. DPSCs were positive for MSC-specific markers. Doses of CoCl_2, up to 20 µM, did not negatively affect cell viability; in low doses (5 µM), it promoted cell survival. Treatment with 10 µM of CoCl₂ significantly augmented the genetic expression of HIF-1α. Cells treated with 10 µM of CoCl₂ showed changes in the levels of growth factors and cytokines produced. It was very evident that CoCl₂ also increased the expression of OCT4 and SOX2, which is the modulation of stemness of DPSCs. A CoCl₂ treatment-induced hypoxic environment modulates the secretory profile of DPSCs.

Keywords: cobalt chloride; dental pulp stem cells; hypoxia inducible factor; secretory profile; stemness.

UNASSIGNED

Allergic asthma is a complex genetic disorder that involves interactions between genetic and environmental factors. Usage of PTEN may be a good therapeutic strategy for the management of allergic inflammation. Thus, the present study aims to explore the effects of phosphatase and tensin homolog (PTEN) gene silencing on airway remodeling and proliferation of airway smooth muscle cells (ASMCs) in a mouse model of allergic asthma.

UNASSIGNED

A total of 56 healthy female BABL/c mice (weighing between 16 to 22 grams) were selected and were assigned on random into ovalbumin (OVA; mice were stimulated with OVA to induce allergic asthma), OVA + si-PTEN, normal saline (NS; mice were treated with normal saline) and NS + si-PTEN groups. Masson staining was employed in order to observe lung tissue sections. Immunohistochemical staining was used to detect the expression of α-SMA+. Gene silencing was conducted in the NS + si-PTEN and OVA + si-PTEN groups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting were used to detect the mRNA and protein expressions of PTEN in ASMCs of each group. CCK-8 assay and flow cytometry were performed to determine the cell proliferation rate and cell cycle.

UNASSIGNED

Airway remodeling and changes of smooth muscle layer were found in allergic asthmatic mice with thick airway walls. The expression of alpha smooth muscle actin (α-SMA+) was significantly higher in ASMCs of the OVA, OVA + si-PTEN and NS + si-PTEN groups compared with ASMCs of the NS group. The mRNA and protein expressions of PTEN reduced in the OVA, OVA + si-PTEN and NS + si-PTEN groups. The rate of ASMCs proliferation in OVA, OVA + si-PTEN and NS + si-PTEN groups were significantly higher than the NS group. The proportion of ASMCs in S and G2 stages increased, while the number of cells in the G1 stage decreased after PTEN gene silencing.

UNASSIGNED

These results demonstrated that PTEN gene silencing might promote proliferation of ASMCs and airway remodeling in a mouse model of allergic asthma.

The mycotoxin deoxynivalenol (DON) generally exists in cereals and affects human and animal health. The aim of this study is to analyze the impacts of DON in naturally contaminated feed on piglet growth performance and intestinal hormone secretion in the short term. We randomly divided 5-week-old piglets into four groups: Control, DON 1,000, DON 2,000 and DON 3,000 groups. Piglets received a feed naturally contaminated with DON (approximately 400, 1,000, 2,000 or 3,000 μg/kg) for 21 days. Body weight showed no significant difference following exposure to DON. The balance of anti-oxidation and oxidation was disrupted by DON after 21 days. The concentration of tumor necrosis factor-alpha (TNF-α) and cyclooxgenase-2 (COX-2) significantly increased (p < .001) in all DON-treated groups. Gut anorexigenic hormone secretion of peptide YY (PYY) and cholecystokinin (CCK) had a time- and dose-dependent relationship with DON exposure; however, there was no effect on orexigenic hormone ghrelin secretion. Changes of histomorphology in the jejunum were observed in DON-treated groups, including villi flattening and fusion, and apical necrosis of villi. These results indicated that DON could suppress piglet growth performance and alter gut hormone secretion in the short term.

BACKGROUND This study aimed to investigate the effects of three-dimensional (3D) printed titanium (3DTi) scaffolds on osteogenic differentiation and new bone formation by 3D cultured adipose tissue-derived stem cells (ADSCs) in vitro, and the effects of bone regeneration in vivo using a full-thickness mandibular defect rat model, and the mechanisms involved. MATERIAL AND METHODS Alpha-beta titanium alloy (Ti6Al4V) 3DTi scaffolds were prepared with Cellmatrix hydrogel and 3D culture medium. ADSCs were impregnated into the 3DTi scaffolds. ADSC viability and proliferation were assessed using the cell counting kit-8 (CCK-8) assay, and alkaline phosphatase (ALP) levels were measured. Real-time polymerase chain reaction (RT-PCR) and Western blot were performed to assess the expression of osteogenesis-related mRNA for RUNX2, OPN, OCN, and IGF-1 genes and proteins. A rat model of full-thickness mandibular defect was evaluated with micro-computed tomography (microCT) scanning, and histochemistry with Alizarin red and von Giesen's stain were used to evaluate osteogenesis. RESULTS ADSC viability and proliferation were not affected by culture with 3DTi scaffolds. Expression of osteogenesis-related mRNA and proteins for RUNX2, OPN, OCN, and IGF-1, expression of ALP, and histochemical findings showed that the use of 3DTi scaffolds enhanced osteogenic differentiation and new bone formation by ADSCs, with upregulation of components of the IGF-1R/AKT/mTORC1 pathway. CONCLUSIONS The 3D culture of ADSCs with 3DTi scaffolds enhanced osteogenic differentiation and new bone formation through the IGF-1R/AKT/mTORC1 pathway. This improved method of osteointegration may have clinical application in the preparation of bone grafts before implantation for improved repair of mandibular bone defects.

Enterostatin, a pentapeptide released from the exocrine pancreas and gastrointestinal tract, selectively inhibits fat intake through activation of an afferent vagal signaling pathway. This study investigated if the effects of enterostatin were mediated through a CCK-dependent pathway. The series of in vivo and in vitro experiments included studies of 1) the feeding effect of peripheral enterostatin on Otsuka Long Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors, 2) the effect of CCK-8S on the intake of a two-choice high-fat (HF)/low-fat (LF) diet, 3) the effects of peripheral or central injection of the CCK-A receptor antagonist lorglumide on the feeding inhibition induced by either central or peripheral enterostatin, and 4) the ability of enterostatin to displace CCK binding in a 3T3 cell line expressing CCK-A receptor gene and in rat brain sections. The results showed that OLTEF rats did not respond to enterostatin (300 microg/kg ip) in contrast to the 23% reduction in intake of HF diet in Long Evans Tokushima Otsuka (LETO) control rats. CCK (1 microg/kg ip) decreased the intake of the HF diet in a two-choice diet regime with a compensatory increase in intake of the LF diet. Peripheral injection of lorglumide (300 microg/kg) blocked the feeding inhibition induced by either near-celiac arterial or intracerebroventricular enterostatin, whereas intracerebroventricular lorglumide (5 nmol icv) only blocked the response to intracerebroventricular enterostatin but not to arterial enterostatin. Enterostatin did not bind on CCK-A receptors because neither enterostatin nor its analogs VPDPR and beta-casomorphin displaced [3H]L-364,718 from CCK-A receptors expressed in 3T3 cells or the binding of 125I-CCK-8S from rat brain sections. The data suggest that both the peripheral and central responses to enterostatin are mediated through or dependent on peripheral and central CCK-A receptors.

UNASSIGNED

To study the transforming growth factor beta (TGF-β) signaling pathway in interactions with estrogen receptor alpha (ERα) signaling pathway mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs).

UNASSIGNED

The subcultured UL cells were used to determine the validation of TGF-β3 for the viability of human UL cells using CCK-8 assay, mRNA expressions of ERα, and c-fos by quantitative reverse transcription polymerase chain reaction method, and expressions of p-Smad3, SnoN, and c-fos proteins by Western blot assay in each treatment group.

UNASSIGNED

Compared with each of EEs or TGF-β3 treatment, slightly decrease in the proliferation rate of UL was detected in the coexistence of each EE with TGF-β3. Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs. Somehow, the expression of p-Smad3 and c-fos proteins significantly decreased in each of E2, bisphenol A (BPA), nonylphenol (NP), and octylphenol (OP) group, as well as the expression of SnoN protein significantly reduced only in BPA and NP groups, followed by TGF-β3 treatment. With the overlaid action of ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group, but slightly increased in E2, BPA, NP, and OP groups. However, compared with the control group, the expression of SnoN and c-fos proteins significantly decreased in the same setting.

UNASSIGNED

Both ERα signaling pathway and TGF-β signaling pathway have different roles in governing UL cell proliferation. The phenolic EEs can be a promoter to the proliferation of UL cells, which is mediated by ERα signaling pathway and cross-talked with TGF-β signaling pathway.

Intravenously administered cholecystokinin octapeptide (CCK) induces oxytocin release from the neurohypophysis in anaesthetised rats. Memory of conditioned taste aversion can be acquired under anaesthesia. The present experiments aimed at investigating whether taste stimuli previously paired with i.v. CCK evoke oxytocin release from the neurohypophysis in urethane-anaesthetised male rats. Sucrose solution (0.75-2.0 M) paired with i.v. CCK or the vehicle was applied to the tongue. After 3 h, sucrose solution was applied again. The second sucrose slightly increased plasma oxytocin concentration in rats that had received the first sucrose solution paired with the vehicle. Plasma oxytocin concentration after the second sucrose application, however, was significantly higher in CCK-injected than in vehicle-injected rats. In rats that received CCK 1 h before the first sucrose application, a second sucrose application did not produce the oxytocin response. The magnitude of the oxytocin response to the second sucrose solution was increased in a manner related to CCK doses. In separate experiments, NaCl solution (0.75 M) paired with CCK or the vehicle was applied to the tongue. The second NaCl solution applied 3 h after the first one facilitated oxytocin release both in the rats that had received CCK or the vehicle. The increase in plasma oxytocin, however, was significantly larger in CCK than in vehicle-injected rats. In rats that had received the first sucrose solution paired with CCK, a second sucrose solution evoked a significantly larger increase in plasma oxytocin concentrations than a testing NaCl solution did. In rats that had received NaCl solution paired with CCK, a testing sucrose solution did not significantly change plasma oxytocin concentrations. These data suggest that the taste stimulus previously paired with i.v. CCK induces oxytocin release from the neurohypophysis in urethane-anaesthetised rats.

BACKGROUND Leukemia cells have strong proliferation and anti-apoptosis capabilities. The purpose of this study was to investigate the effect of silencing the leucine-rich alpha-2-glycoprotein1 (LRG1) gene, which was found to regulate tumor proliferation and apoptosis in acute myeloid leukemia (AML) cell lines. MATERIAL AND METHODS Plasmid interference technique was used to silence the LRG1 gene in the KASUMI-1 cell line. The cell counting kit-8 (CCK-8) assay was used to test the effect of transduction on cell viability. Cell cycle and apoptosis were detected by flow cytometry. Western blot and quantitative real-time polymerase chain reaction (RT-qPCR) were applied to detect the expression levels of proteins and mRNA, respectively. RESULTS KASUMI-1 cells with the CD34⁺CD38⁻ phenotype were sorted by flow cytometry. After transfection of the siLRG1 plasmid, the level of LRG1 expression was downregulated and cell viability was reduced. Silencing of LRG1 gene blocked KASUMI-1 cells in G0/G1 phase and promoted apoptosis. Further experiments found that LRG1 gene silencing significantly downregulated cell cycle-associated proteins and anti-apoptotic proteins, while upregulating pro-apoptotic proteins. Downregulation of LRG1 gene expression also inhibits signal transduction of the JAK-STAT pathway. CONCLUSIONS LRG1 gene silencing regulates the expression of cyclin and apoptosis-related proteins to reduce cell viability and promote apoptosis, probably through inhibition of the JAK-STAT pathway.

OBJECTIVE

We aimed at investigating the expression of long non-coding RNA (lncRNA) Ptprj-as1 and the role of lncRNAPtprj-as1 in inflammatory cells after intracerebral hemorrhage and its potential mechanism.

METHODS

The rat model of intracerebral hemorrhage was established. Expressions of Ptprj-as1 and inflammatory cytokines in inflammatory cells were detected by quantitative Real-time PCR (qRT-PCR). After BV2 cells were transfected with lentivirus, cell proliferation, migrative ability and apoptosis were detected by cell counting kit-8 (CCK-8) assay, transwell chamber and flow cytometry, respectively. Immunofluorescence was used to explore the ratio of M1 and M2 glial cells. The detection of tumor necrosis factor alpha (TNF-α) expression was performed using enzyme-linked immunosorbent assay (ELISA). Moreover, the expressions of key genes in NF-κB pathway were evaluated using Western blot.

RESULTS

Ptprj-as1 was highly expressed in inflammatory tissues caused by intracerebral hemorrhage (ICH). Overexpressed Ptprj-as1 promoted the migration of BV2 cells and expression levels of inflammatory cytokines such as TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6), iNOS and NO. Meanwhile, Ptprj-as1 enhanced the proportion of M1 glial cells, the mechanism of which might be related to the activation of NF-κB pathway.

CONCLUSIONS

Ptprj-as1 activates NF-κB pathway in microglia and promotes the secretion of inflammatory cytokines, which is involved in inflammatory injury caused by intracerebral hemorrhage.

Background: Macrophage M1 polarization plays a pivotal role in inflammatory diseases. Progranulin (PGRN) has potential anti-inflammation action, however, the effect of PGRN on macrophage M1 polarization has been poorly studied. Our study aimed to investigate the effect of PGRN on lipopolysaccharide (LPS)-induced macrophage M1 polarization and clarify the underlying mechanisms.

Methods: RAW264.7 cells were polarized to M1 macrophage by LPS with or without recombinant PGRN (rPGRN) and tumor necrosis factor alpha antibody (anti-TNF-α). A cell counting kit-8 assay (CCK-8), flow cytometry, Quantitative Real-Time PCR assay (q-PCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) were used to determine the effect of different treatments on cell proliferation, expression of surface phenotype marker and expressions and secretion of inflammatory cytokines. The activation of NF-κB/mitogen-activated protein kinase (MAPK) pathways and the nuclear translocation of NF-κB p65 were detected by Western blot and immunofluorescence respectively. THP-1 and primary bone marrow-derived monocytes (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS.

Results: In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF-α and activated phosphorylation of IKKα/β, IкBα, p65, JNK and p38 and the nucleus translocation of NF-кB p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF-α (rTNF-α) significantly boosted TNF-α and iNOS expression vs the control group. Moreover, anti-TNF-α significantly inhibited LPS-induced TNF-α and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF-α and iNOS and secretion of TNF-α was further demonstrated.

Conclusions: PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.

Keywords: Lipopolysaccharide (LPS); MAPK; Macrophage polarization; NF-κB; PGRN.