[Expression of Cyclooxygenase-2 in Patients With Snow-White Sign of Advanced Colorectal Adenomas].

Objective To analyze the expression of cyclooxygenase-2 (COX-2) in the patients with snow-white sign of advanced colorectal adenoma (ACA) and explore its clinical significance.Method Western blotting was employed to determine the expression of COX-2 in the adenoma tissue and the normal tissue adjacent to the adenoma tissue (>5 cm away from the distal end of the adenoma tissue) of 40 ACA patients with snow-white sign and 40 ACA patients without snow-white sign.Results The appearance of snow-white sign in ACA patients was associated with patient age (P=0.001) and not associated with sex,smoking history,drinking history,ethnic groups,family history of colorectal cancer,abdominal pain,diarrhea,constipation,fecal occult blood,or tumor markers (all P>0.05).Snow-white sign mainly appeared in the ACA patients with multiple adenomas (P=0.004),large adenomas (P=0.006),adenomas in distal colon (P=0.015),protruding polyps (P=0.044),and late-stage pathology (P=0.010).The occurrence of snow-white sign showed no difference in the ACA patients with different results of Japan NBI Expert Team classification (P=0.502).The expression of COX-2 in the adenoma tissue was higher than that in the adjacent normal tissue in the patients with and without snow-white sign (P<0.001,P=0.004).The patients with snow-white sign had higher expression of COX-2 protein in the adenoma tissue than the patients without snow-white sign (P=0.001).The expression of COX-2 protein in the adjacent healthy tissue had no significant difference between the patients with and without snow-white sign (P=0.603).Conclusions Snow-white sign is more like to appear in the ACA patients with young age,multiple and large adenomas,adenomas in distal colon,protruding polyps,and late-stage pathology.Moreover,the expression of COX-2 in the ACA patients with snow-white sign is significantly higher than that in the ACA patients without snow-white sign.The adults with snow-white sign are prone to cancerization than those without snow-white sign.

CIRC_0085323 SILENCING INHIBITS TNF-Α-INDUCED NORMAL HUMAN COLONIC EPITHELIAL CELL INFLAMMATION AND APOPTOSIS THROUGH THE MIR-495-3P/TRAF3 AXIS.

ABSTRACT: Background: Previous data have suggested the involvement of circular RNA (circRNA) in ulcerative colitis (UC) development. However, the role and mechanism of circ_0085323 in UC occurrence have not been reported. Methods: Normal human colonic epithelial cells (NCM460) were treated with TNF-α to simulate UC-like cell inflammation and injury in vitro. The expression of circ_0085323, microRNA-495-3p (miR-495-3p), and TNF receptor-associated factor 3 (TRAF3) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blotting analysis. Cell viability, cell proliferation, and cell apoptosis were investigated by cell counting kit-8 assay, 5-ethynyl-29-deoxyuridine assay, and flow cytometry analysis, respectively. IL-1ß, IL-6, and IL-8 production were analyzed by enzyme-linked immunosorbent assays. Lactate dehydrogenase activity was assessed by a lactate dehydrogenase activity detection assay. The interactions among circ_0085323, miR-495-3p, and TRAF3 were identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Results: Circ_0085323 was overexpressed in the colonic mucosal tissues of UC patients and TNF-α-stimulated NCM460 cells. Circ_0085323 knockdown relieved TNF-α-induced inhibitory effect on the proliferation of NCM460 cells and promoting effects on cell apoptosis and inflammation. Circ_0085323 acted as a miR-495-3p sponge, and the effects of circ_0085323 silencing on TNF-α-induced NCM460 cell injury were attenuated by decreasing miR-495-3p expression. TRAF3 was targeted by miR-495-3p, and circ_0085323 combined with miR-495-3p to regulate TRAF3. TRAF3 depletion not only alleviated TNF-α-induced NCM460 cell damage but also partially revoked the effect of circ_0085323 silencing combined with miR-495-3p depletion on TNF-α-induced NCM460 cell injury. Conclusions: Circ_0085323 knockdown ameliorated TNF-α-induced NCM460 cell injury by regulating the miR-495-3p/TRAF3 axis, which suggested that circ_0085323 might be a therapeutic target for UC.

Identification of Bone Morphometric Protein-Related Hub Genes and Construction of a Transcriptional Regulatory Network in Patients With Ossification of the Ligamentum Flavum.

STUDY DESIGN: Basic science laboratory study. OBJECTIVE: To identify hub genes related to bone morphogenetic proteins (BMPs) in the ossification of the ligamentum flavum (OLF) and analyze their functional characteristics. SUMMARY OF BACKGROUND DATA: The exact etiology and pathologic mechanism of OLF remain unclear. BMPs are pleiotropic osteoinductive proteins that may play a critical role in this condition. MATERIALS AND METHODS: The GSE106253 and GSE106256 data sets were downloaded from the Gene Expression Omnibus database. The messenger RNA (mRNA) and long noncoding RNA expression profiles were obtained from GSE106253. The microRNA expression profiles were obtained from GSE106256. Differentially expressed genes were identified between OLF and non-OLF groups and then intersected with BMP-related genes to obtain differentially expressed BMP-related genes. The least absolute shrinkage selection operator and support vector machine recursive feature elimination were used to screen hub genes. Furthermore, a competing endogenous RNA network was constructed to explain the expression regulation of the hub genes in OLF. Finally, the protein and mRNA expression levels of the hub genes were verified using Western blot and real-time polymerase chain reaction, respectively. RESULTS: We identified 671 Differentially expressed genes and 32 differentially expressed BMP-related genes. Hub genes ADIPOQ , SCD , SCX , RPS18 , WDR82 , and SPON1 , identified through the least absolute shrinkage selection operator and support vector machine recursive feature elimination analyses, showed high diagnostic values for OLF. Furthermore, the competing endogenous RNA network revealed the regulatory mechanisms of the hub genes. Real-time polymerase chain reaction showed that the mRNA expression of the hub genes was significantly downregulated in the OLF group compared with the non-OLF group. Western blot showed that the protein levels of ADIPOQ, SCD, WDR82 , and SPON1 were significantly downregulated, whereas those of SCX and RPS18 were significantly upregulated in the OLF group compared with the non-OLF group. CONCLUSION: This study is the first to identify BMP-related genes in OLF pathogenesis through bioinformatics analysis. ADIPOQ , SCD , SCX , RPS18 , WDR82 , and SPON1 were identified as hub genes for OLF. The identified genes may serve as potential therapeutic targets for treating patients with OLF.

Research on Polycaprolactone-Gelatin Composite Scaffolds Carrying Nerve Growth Factor for the Repair of Spinal Cord Injury.

Objective: This study was to investigate the mechanism of action of polycaprolactone/gelatin (PCL/GE) composite fiber scaffold with nerve growth factor (NGF) in the recovery of spinal cord injury (SCI). Methods: Sixty female Sprague-Dawley (SD) rats were randomly assigned to the negative control group, the positive control group, the PCL/GE scaffold group, and the collagen-binding structural domain nerve growth factor (CBD-NGF)/PCL/GE scaffold group, with 15 rats in each group. Spinal cord transection was used to establish SCI models in rats. The negative control group received sham surgery, while the other three groups were given spinal cord transection at the tenth thoracic vertebra (T10) segment. The rats in the PCL/GE scaffold group were implanted with a 4 mm PCL/GE composite fiber scaffold, and those in the CBD-NGF/PCL/GE scaffold group were implanted with a CBD-NGF/PCL/GE composite fiber scaffold. The Basso-Beattie-Bresnahan (BBB) locomotor rating scale was used to evaluate the locomotor ability of the hind limbs of the rats, and the amplitude and latency of motor evoked potentials (MEP) were recorded by neurophysiological testing at 12 w postoperatively. The levels of growth-associated protein 43 (GAP43) and neurofilament protein 200 (NF200) in the spinal cord tissue of the injury site were determined using Western Blot at 12 w after surgery. Spinal cord tissues of 2 cm within the injury site, the thoracic segment above the injury site, and the lumbar segment below the injury site were collected from the measurement of axonal transport using fluorescent retrograde tracer fluorogold, and the integrated absorbance (IA) values of FC-positive cells were calculated. Results: After treatment, the negative control rats showed normal locomotion function of the hind limb with the highest BBB scores, while the positive control rats had the lowest BBB scores and showed paraplegia. The scaffold groups exhibited better locomotion function of the hind limb and higher BBB scores than the positive controls, with greater improvement observed in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Compared with the positive controls, the PCL/GE scaffold group and CBD-NGF/PCL/GE scaffold group exhibited significantly shorter latency and increased amplitude of MEP, with more significant changes observed in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Compared with the positive control group, the GAP43 and NF200 levels of spinal cord tissue were significantly elevated in both the PCL/GE scaffold group and the CBD-NGF/PCL/GE scaffold group, and the changes were more pronounced in the CBD-NGF/PCL/GE scaffold group (P < 0.05). The differences in the IA values of FC-positive cells in the spinal cord tissue of the lumbar segment below the injury site among the four groups did not come up to the statistical standard (P > 0.05). Compared with the positive control group, the FC-positive cell IA values of spinal cord tissue in the thoracic segment above the injury area were markedly increased in the PCL/GE scaffold group and the CBD-NGF/PCL/GE scaffold group, and the alterations were more significant in the CBD-NGF/PCL/GE scaffold group (P < 0.05). Conclusion: PCL/GE composite fiber scaffold with NGF significantly improves motor and neurological functions in the hind limbs of SCI rats and promotes the recovery of axonal transport, and the mechanism may be associated with the upregulation of GAP43 and NF200 levels in spinal cord injury site tissues.

NEP1-40-overexpressing Neural Stem Cells Enhance Axon Regeneration by Inhibiting Nogo-A/NgR1 Signaling Pathway.

BACKGROUND: Nogo-66 antagonistic peptide (NEP1-40) offers the potential to improve spinal cord injury (SCI). OBJECTIVE: To explore the effect of NEP1-40 overexpression on neural stem cells (NSCs) regulating the axon regeneration of injured neurons. METHODS: We isolated NSCs from brain tissues of pregnant rat fetuses and used Nestin immunofluorescence to identify them. The NEP1-40 overexpressing NSCs were constructed by transfection with the NEP1-40-overexpressing vector. The expression of NSCs differentiation associated markers, including Tuj-1, GFAP, Oligo2, and MBP, were detected by RT-PCR, western blotting, and immunofluorescence. NeuN immunofluorescence staining was used to measure the number of neurons. And western blotting was used to detect the phosphorylation levels of LIMK1/2, cofilin, and MLC-2 and the protein levels of GAP-43, MAP-2, and APP. RESULTS: The NEP1-40 overexpression promoted the expression level of Tuj-1, Oligo2, and MBP, and increased the number of Tuj-1, Oligo2, and MBP positive cells. NEP1-40-overexpressing NSCs (NEP-NSCs) improved NeuN positive cells of co-culture with injured neurons. And NEP-NSCs also increased the protein levels of axon regeneration indicators (GAP-43, MAP-2) and decreased APP protein level. In addition, the phosphorylation level of LIMK1/2, cofilin, and MLC-2 were markedly decreased in NEP-NSCs. CONCLUSION: NEP1-40 overexpression enhanced the ability of NSCs differentiation into neurons and promoted axon regeneration by inhibiting the Nogo-A/NgR1 signaling pathway. This study provides an alternative gene modified transplantation NSCs for the SCI treatment.

Effect and mechanism of action of SLP-2 on the apoptosis and autophagy of gastric cancer cells.

This study was designed to investigate the effect of stomatin-like protein 2 (SLP-2) on the apoptosis and autophagy of gastric cancer cells and its underlying mechanism. The expression of SLP-2 was detected in human gastric cancer cell lines (AGS, MKN-45 and NCI-N87) and a human gastric epithelial cell line (GES-1) using reverse transcription-quantitative PCR and western blot analysis. SLP-2-specific small interfering RNA (siRNA) was transfected into NCI-N87 cells. Cell Counting Kit-8 was used to detect cell proliferation. Apoptosis rates were measured using flow cytometry. Autophagosomes were observed by transmission electron microscopy. The expression levels of Annexin A2 (ANXA2), ß-catenin, Bcl-2, Bax, Beclin-1 and LC3-II/I were also measured. The results demonstrated that SLP-2 siRNA transfection significantly reduced cell proliferation and increased cell apoptosis. The mitochondria were severely damaged, and a large number of autophagosomes were seen in SLP-2 siRNA-transfected NCI-N87 cells. Furthermore, the expression levels of ANXA2, ß-catenin and Bcl-2 were downregulated, whereas those of Bax, Beclin-1 and LC3-II/I were upregulated following SLP-2 siRNA transfection. In conclusion, SLP-2 silencing can inhibit proliferation and induce apoptosis and autophagy of gastric cancer cells, and this effect may be related to the inhibition of ANXA2/ß-catenin signaling pathway.

α-Mangostin Induces Apoptosis in Human Osteosarcoma Cells Through ROS-Mediated Endoplasmic Reticulum Stress via the WNT Pathway.

α-mangostin has been confirmed to promote the apoptosis of MG-63 cells, but its specific pro-apoptosis mechanism in osteosarcoma (OS) remains further investigation. Here, we demonstrated that α-mangostin restrained the viability of OS cells (143B and Saos-2), but had little effect on the growth of normal human osteoblast. α-mangostin increased OS cell apoptosis by activating the caspase-3/8 cascade. Besides, α-mangostin induced endoplasmic reticulum (ER) stress and restrained the Wnt/ß-catenin pathway activity. 4PBA (an ER stress inhibitor) or LiCl (an effective Wnt activator) treatment effectively hindered α-mangostin-induced apoptosis and the caspase-3/8 cascade. Furthermore, we also found that α-mangostin induced ER stress by promoting ROS production. And ER stress-mediated apoptosis caused by ROS accumulation depended on the inactivation of Wnt/ß-catenin pathway. In addition, α-mangostin significantly hindered the growth of xenograft tumors, induced the expression of ER stress marker proteins and activation of the caspase-3/8 cascade, and restrained the Wnt/ß-catenin signaling in vivo. In short, ROS-mediated ER stress was involved in α-mangostin triggered apoptosis, which might depended on Wnt/ß-catenin signaling inactivation.

Mechanism of tauroursodeoxycholic acid-mediated neuronal protection after acute spinal cord injury through AKT signaling pathway in rats.

OBJECTIVE: To explore themechanism of tauroursodeoxycholic acid- (TUDCA) mediated neuronal protection after acute spinal cord injury (ASCI) in rats. Methods: ASCI rat model was established following modified Allen's weight-drop method and these rats were assigned to sham group (received sham operation), model group (ASCI rats), TUDCA group (ASCI rats received TUDCA treatment), MK2206 group (ASCI rats received AKT inhibitor MK2206 orally) and TUDCA + MK2206 group. Motor function of rats was evaluated using Basso Beattie Bresnahan (BBB) method. Hematoxylin-eosin (H&E) staining was used to detect histopathologic changes in the spinal cord and TUNEL fluorescence staining was used to check apoptosis. Real time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot were employed to detect the production of AKT pathway related factors, apoptosis related factors (Bax, Bcl-2, caspase-3), autophagy related factor Beclin-1 and endoplasmic reticulum (ER) stress related factors (IRE1, Chop, ATF6) in spinal cord of rats. RESULTS: Compared to the rats in the sham group, rats in ASCI group had decreased BBB scores (P<0.05), more significant tissue edema, structural cavity and apoptosis. Compared to rats in sham group, AKT pathway was inactivated in ASCI rats and was activated by TUDCA treatment (P<0.05). Compared to sham group, expressions of ER stress-related factors were increased, apoptosis was largely induced in other four groups, and expression of Beclin-1 was increased in the model group (P<0.05). TUDCA increased the expression of Beclin-1 and Bcl-2, and inhibited the expression of Bax, Caspase-3, and ER stress-related factors, thus suppressing apoptosis (P<0.05). Treatment by MK2206 had contrary effects and protective effects of TUDCA on ASCI rats could be counteracted by MK2206. CONCLUSION: TUDCA can significantly improve the neural damage, enhance neuron autophagy, alleviate ER stress, and inhibit apoptosis in ASCI rats, by activating the AKT signaling pathway.

MeHg Suppressed Neuronal Potency of Hippocampal NSCs Contributing to the Puberal Spatial Memory Deficits.

Hippocampal neurogenesis-related structural damage, particularly that leading to defective adult cognitive function, is considered an important risk factor for neurodegenerative and psychiatric diseases. Normal differentiation of neurons and glial cells during development is crucial in neurogenesis, which is particularly sensitive to the environmental toxicant methylmercury (MeHg). However, the exact effects of MeHg on hippocampal neural stem cell (hNSC) differentiation during puberty remain unknown. This study investigates whether MeHg exposure induces changes in hippocampal neurogenesis and whether these changes underlie cognitive defects in puberty. A rat model of methylmercury chloride (MeHgCl) exposure (0.4 mg/kg/day, PND 5-PND 33, 28 days) was established, and the Morris water maze was used to assess cognitive function. Primary hNSCs from hippocampal tissues of E16-day Sprague-Dawley rats were purified, identified, and cloned. hNSC proliferation and differentiation and the growth and morphology of newly generated neurons were observed by MTT and immunofluorescence assays. MeHg exposure induced defects in spatial learning and memory accompanied by a decrease in number of doublecortin (DCX)-positive cells in the dentate gyrus (DG). DCX is a surrogate marker for newly generated neurons. Proliferation and differentiation of hNSCs significantly decreased in the MeHg-treated groups. MeHg attenuated microtubule-associated protein-2 (MAP-2) expression in neurons and enhanced the glial fibrillary acidic protein (GFAP)-positive cell differentiation of hNSCs, thereby inducing degenerative changes in a dose-dependent manner. Moreover, MeHg induced deficits in hippocampus-dependent spatial learning and memory during adolescence as a consequence of decreased generation of DG neurons. Our findings suggested that MeHg exposure could be a potential risk factor for psychiatric and neurodegenerative diseases.

The involvement of Helicobacter pylori thioredoxin-1 in gastric carcinogenesis.

Helicobacter pylori infection is related to the development of gastric diseases. Various virulence factors are responsible for the pathogenic mechanisms of H. pylori infection. Our previous studies using two-dimensional gel electrophoresis showed that H. pylori thioredoxin-1 (Trx1) is overexpressed in gastric carcinomas. Here, we examined whether H. pylori Trx1 is a novel virulence factor associated with gastric tumorigenesis. We found that Trx1 expression in H. pylori isolated from gastric cancer tissues was significantly higher than that from tissues exhibiting gastritis. In the gastric epithelial cell line GES-1, infection of H. pylori with high Trx1 expression significantly induced cell apoptosis, decreased the expression of cyclin D1 and upregulated p21. However, in the gastric cancer cell line BGC823, high Trx1 expression in H. pylori significantly increased cell proliferation, and upregulated cyclin D1. The effects on cell lines were confirmed using the H. pylori Trx1-knockout mutant strain. Our observations indicate that high Trx1 expression in H. pylori is associated with gastric carcinogenesis. In H. pylori, Trx1 likely participates in the pathogenesis of gastric cancer and H. pylori expressing high levels of Trx1 would be expected to be highly pathogenic in gastric diseases in China.