The Anti-oxidant Monoterpene p-Cymene Reduced the Occurrence of Colorectal Cancer in a Hyperlipidemia Rat Model by Reducing Oxidative Stress and Expression of Inflammatory Cytokines.

BACKGROUND/AIM: Overexpression of inflammatory cytokines and oxidative stress increase the risk of colorectal cancer (CRC) in obesity and hyperlipidemia. The aim of this study was to investigate whether the monoterpene antioxidant p-cymene would reduce the incidence of CRC in a rat model of hyperlipidemia. MATERIALS AND METHODS: The hyperlipidemic CRC rat model was established by a high-fat diet and dimethyl hydrazine (DMH) induction. All rats received 30 mg/kg DMH to induce CRC, and were then assigned to groups with a normal diet or high-fat diet with/without 30 mg/kg/day p-cymene orally during the entire experimental period. Tumor incidence in each group, and the level of serum inflammatory cytokines and oxidative stress-related markers in intestinal tissues were measured. RESULTS: p-Cymene significantly inhibited CRC occurrence in hyperlipemic rats (p=0.024) by reducing the expression of serum inflammatory cytokines (interleukin-1 by 54.5%; interleukin-6 by 28.3%; adiponectin by 26.3%; cyclo-oxygenase-2 by 48.4%) and intestinal oxidative-stress cytokines (total antioxidant capacity by 30.4%; superoxide dismutase by 30.3%; malondialdehyde by 47.1%). CONCLUSION: p-Cymene has clinical potential to reduce the incidence of CRC in hyperlipemia.

Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay.

The first case of African swine fever (ASF) outbreak in China was reported in a suburban pig farm in Shenyang in 2018. Since then, the rapid spread and extension of ASF has become the most serious threat for the swine industry. Therefore, rapid and accurate detection of African swine fever virus (ASFV) is essential to provide effective strategies to control the disease. In this study, we developed a rapid and accurate ASFV-detection method based on the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay. By combining recombinase polymerase amplification with CRISPR-Cas12a proteins, the DETECTR assay demonstrated a minimum detection limit of eight copies with no cross reactivity with other swine viruses. Clinical blood samples were detected by DETECTR assay and showed 100% (30/30) agreement with real-time polymerase chain reaction assay. The rapid and accurate detection of ASFV may facilitate timely eradication measures and strict sanitary procedures to control and prevent the spread of ASF.

Epigallocatechin gallate inhibits dimethylhydrazine-induced colorectal cancer in rats.

BACKGROUND: Epigallocatechin gallate (EGCG) is a polyhydroxy phenolic compound extracted from tea and its antitumor effect has received widespread attention. We explored the inhibitory effect of EGCG on dimethylhydrazine (DMH)-induced colorectal cancer (CRC) using a rat model, predicted the interaction between EGCG and CRC target genes using a database, and explained the EGCG associated target pathways and mechanisms in CRC. AIM: To understand the inhibitory mechanisms of EGCG on CRC cell proliferation and identify its pharmacological targets by network pharmacology analysis. METHODS: DMH (40 mg/kg, s.c., twice weekly for eight weeks) was used to induce CRC in rats. After model establishment, the rats were administered with EGCG (50, 100, or 200 mg/kg, p.o., once daily for eight weeks) and killed 12 and 20 wk after the start of the experiment. Formation of aberrant crypt foci and tumor was studied by histological analysis. Using network pharmacology analysis, candidate and collective targets of EGCG and CRC were identified, and Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses were used to predict the pathways altered by EGCG. RESULTS: At week 12, high-dose EGCG treatment significantly reduced the tumor formation rate, total number of tumors, cancerous and non-cancerous tumors, tumor volume, ascites formation, and aberrant crypt foci count. At week 20, all three doses of EGCG were effective. Seventy-eight collective targets of EGCG and CRC were identified, of which 28 genes were dysregulated in CRC. Kyoto Encyclopedia of Genes and Genomes and GO analyses showed that the dysregulated genes were enriched in hsa05210 (CRC), hsa04115 (p53 signaling pathway), and hsa04151 (PI3K-Akt signaling pathway), GO:0043124 (negative regulation of I-kappaB kinase/NF-kappaB signaling pathway), GO:0043409 (negative regulation of mitogen-activated protein kinase cascade), and GO:2001244 (positive regulation of intrinsic apoptotic signaling pathway) respectively. CONCLUSION: EGCG inhibits the formation of DMH-induced CRC by regulating key pathways involved in tumorigenesis.

Clinical significance of MLH1/MSH2 for stage II/III sporadic colorectal cancer.

BACKGROUND: The development of colorectal cancer (CRC) is a complicated multistep process that involves an accumulation of mutations in tumor suppressor genes and oncogenes. In the process of DNA replication, base mismatch often occurs due to various factors leading to abnormal expression of mismatch repair genes (MMR), among which MLH1 and MSH2 are the most important. Recently, numerous studies indicated that MLH1/MSH2 phenotype is associated with CRC. We wanted to elucidate the role of MLH1/MSH2 in the prediction and prognosis of CRC through long-term clinical observation. AIM: To evaluate the prognostic and predictive significance of MLH1/MSH2 in patients with stage II-III CRC using immunohistochemical analysis and GeneScan. METHODS: Specimens from 681 patients with CRC (395 stage II and 286 stage III, 387 males and 294 females) who underwent curative surgical resection from 2013 to 2016 were tested. Immunohistochemistry was used to analyze MMR status and the microsatellite status of 133 patients was determined by GeneScan analysis. RESULTS: Five hundred and fifty (80.76%) patients were MLH1/MSH2 positive and 131 (19.24%) were negative by immunohistochemistry. MLH1/MSH2-positive tumors were significantly more frequent in the colon than in the rectum, and had poor differentiation and less mucin production (P < 0.05). Patients of different groups did not differ in terms of age, gender, tumor size, tumor stage, lymphocytic infiltration, or circumscribed margin. MLH1/MSH2-negative patients had a more favorable OS than MLH1/MSH2-positive patients (P < 0.001). Univariate and multivariate analyses demonstrated MLH1/MSH2 expression as an independent prognostic and predictive factor for stage II/III CRC. MLH1/MSH2 expression was a strong prognostic factor in all patients [P < 0.001, hazard ratio (HR) = 4.064, 95%CI: 2.241-7.369]. Adjuvant chemotherapy had a greater correlation with survival advantage in MLH1/MSH2-negative patients with stage III disease (P < 0.001, HR = 7.660, 95%CI: 2.974-15.883). However, patients with stage II disease or MLH1/MSH2-positive patients with stage III disease did not benefit from adjuvant chemotherapy. GeneScan analysis demonstrated that among 133 patients, 105 (78.95%) were microsatellite stable, and 28 (21.05%) had microsatellite instability (MSI), including 18 (13.53%) with high MSI and 10 (7.52%) with low MSI. This is consistent with the immunohistochemical results. CONCLUSION: MLH1/MSH2 phenotype constitutes a pathologically and clinically distinct subtype of sporadic CRC. MLH1/MSH2 is an independent prognostic and predictive factor for outcome of stage II-III CRC.

Radiofrequency radiation at 2.856 GHz does not affect key cellular endpoints in neuron-like PC12 cells.

To investigate the potential cytotoxicity of radiofrequency (RF) radiation on central nervous system, rat pheochromocytoma (PC12) cells were exposed to 2.856 GHz RF radiation at a specific absorption rate (SAR) of 4 W/kg for 8 h a day for 2 days in 35 mm Petri dishes. During exposure, the real-time variation of the culture medium temperature was monitored in the first hour. Reactive oxygen species (ROS) production, intracellular Ca2+ concentration, and cell apoptosis rate were assessed immediately after exposure by flow cytometry. The results showed that the medium temperature raised about 0.93 °C, but no significant changes were observed in apoptosis, ROS levels or intracellular Ca2+ concentration after treatment. Although several studies suggested that RF radiation does indeed cause neurological effects, this study presented inconsistent results, indicating that 2.856 GHz RF radiation exposure at a SAR of 4 W/kg does not have a dramatic impact on PC12 cells, and suggests the need for further investigation on the key cellular endpoints of other nerve cells after exposure to RF radiation.

Serum miR-20a and miR-486 are potential biomarkers for discriminating colorectal neoplasia: A pilot study.

AIM: Recent advances in circulating microRNAs (miRNAs) as noninvasive biomarkers have provided promising prospect in detecting colorectal cancer (CRC). However, the capability of miRNAs for detecting colorectal neoplasia (CRN, including precancerous lesions and curable stage CRCs) remains unclear. This study aimed to identify the potential of serum miRNAs (miR-20a, miR-486, miR-92a, and miR-135b) selected from the literature for discriminating CRN patients. MATERIALS AND METHODS: The serum samples from 46 CRN patients and 33 healthy controls were analyzed with quantitative reverse transcription-polymerase chain reaction. RESULTS: Serum miR-20a and miR-486 were significantly downregulated in CRN patients compared to that of in healthy controls (fold change = 0.697 and 0.696, P = 0.01 and 0.05, respectively). The serum level of miR-92a was not significantly different between two groups, while miR-135b level in serum was too low to be accurately quantified. In addition, serum miR-486 level was much more downregulated in tubulovillous adenoma and high-grade intraepithelial neoplasia patients than that of in healthy controls. For miR-20a and miR-486, the area under the receiver operating characteristic curve for discriminating CRN patients were 0.676 and 0.629, respectively, while their combined value was 0.698. No significant correlation was observed between miR-20a and miR-486 serum levels with age, gender, location, or lesion size. CONCLUSION: The results suggested that serum miR-20a and miR-486 could be potential noninvasive biomarkers for identifying CRN patients.

Lactobacillus plantarum ameliorates tumour necrosis factor-induced bacterial translocation in Caco-2 cells by regulation of TLR4 expression.

Purpose. Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis. In inflammatory conditions, commensal bacteria exploit transcytotic pathways to cross the intestinal epithelium in a TLR4-dependent manner. The aim of this study was to test the hypothesis that Lactobacillus plantarum ameliorates tumour necrosis factor-induced bacterial translocation by regulation of Toll-like receptor-4 expression.Methodology. L. plantarum strains were investigated to determine their capacity to inhibit the initial adhesion of Escherichia coli B5 to Caco-2 cells. The inhibitory effects of L. plantarum on TNF-α-induced E. coli B5 translocation across Caco-2 cells were studied. Barrier function and integrity were simultaneously assessed by transepithelial electrical resistance, HRP permeability, LDH release and distribution of tight junctional proteins. Expression of TLR4 was assessed by RT-PCR.Results/Key findings. Pretreatment of monolayers with L. plantarum L2 led to a significant decrease in E. coli B5 adhesion and cell internalization (P<0.01). Exposure to TNF-α for six hours caused a significant increase in E. coli B5 translocation across Caco-2 cells, which was uncoupled from increases in paracellular permeability and disruption of tight junction proteins. Manipulations that induced bacterial translocation were associated with a marked increase in TLR4 mRNA expression and IL-8 secretion. L. plantarum L2 significantly abrogated TNF-α-induced bacterial translocation of E. coli B5, and also downregulated expression of TLR4 and IL-8 in intestinal epithelial cells.Conclusion. Live L. plantarum L2 can inhibit TNF-α-induced transcellular bacterial translocation via regulation of TLR4 expression.

Portulaca oleracea extract can inhibit nodule formation of colon cancer stem cells by regulating gene expression of the Notch signal transduction pathway.

To investigate whether Portulaca oleracea extract affects tumor formation in colon cancer stem cells and its chemotherapy sensitivity. In addition, to analyze associated genetic changes within the Notch signal transduction pathway. Serum-free cultures of colon cancer cells (HT-29) and HT-29 cancer stem cells were treated with the chemotherapeutic drug 5-fluorouracil to assess sensitivity. Injections of the stem cells were also given to BALB/c mice to confirm tumor growth and note its characteristics. In addition, the effect of different concentrations of P. oleracea extract was tested on the growth of HT-29 colon cancer cells and HT-29 cancer stem cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The effects of P. oleracea extract on the expression of ß-catenin, Notch1, and Notch2 in the HT-29 cells were studied using reverse transcription polymerase chain reaction and Western blotting. The tumor volume of the HT29 cells was two times larger than that of HT29 cancer stem cells. Treatment with P. oleracea extract inhibited the proliferation of both HT-29 cancer cells and HT-29 cancer stem cells at doses from 0.07 to 2.25 µg/mL. Apoptosis of HT-29 cancer cells and HT-29 cancer stem cells was assessed by flow cytometry; it was enhanced by the addition of P. oleracea extract. Finally, treatment with P. oleracea extract significantly downregulated the expression of the Notch1 and ß-catenin genes in both cell types. The results of this study show that P. oleracea extract inhibits the growth of colon cancer stem cells in a dose-dependent manner. Furthermore, it inhibits the expression of the Notch1 and ß-catenin genes. Taken together, this suggests that it may elicit its effects through regulatory and target genes that mediate the Notch signal transduction pathway.

Apoptosis of Lewis Lung Carcinoma Cells Induced by Microwave via p53 and Proapoptotic Proteins In vivo.

BACKGROUND: Microwave therapy is a minimal invasive procedure and has been employed in clinical practice for the treatment of various types of cancers. However, its therapeutic application in non-small-cell lung cancer and the underlying mechanism remains to be investigated. This study aimed to investigate its effect on Lewis lung carcinoma (LLC) tumor in vivo. METHODS: Fifty LLC tumor-bearing C57BL/6 mice were adopted to assess the effect of microwave radiation on the growth and apoptosis of LLC tumor in vivo. These mice were randomly assigned to 10 groups with 5 mice in each group. Five groups were treated by single pulse microwave at different doses for different time, and the other five groups were radiated by multiple-pulse treatment of a single dose. Apoptosis of cancer cells was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Western blotting was applied to detect the expression of proteins. RESULTS: Single pulse of microwave radiation for 5 min had little effect on the mice. Only 15-min microwave radiation at 30 mW/cm2 significantly increased the mice body temperature (2.20 ± 0.82)°C as compared with the other groups (0.78 ± 0.29 °C, 1.24 ± 0.52 °C, 0.78 ± 0.42 °C, respectively), but it did not affect the apoptosis of LLC tumor cells significantly. Continous microwave radiation exposure, single dose microwave radiation once per day for up to seven days, inhibited cell division and induced apoptosis of LLC tumor cells in a dose- and duration-dependent manner. It upregulated the protein levels of p53, Caspase 3, Bax and downregulated Bcl-2 protein. CONCLUSIONS: Multiple exposures of LLC-bearing mice to microwave radiation effectively induced tumor cell apoptosis at least partly by upregulating proapoptotic proteins and downregulating antiapoptotic proteins. Continuous radiation at low microwave intensity for a short time per day is promising in treating non-small-cell lung cancer.

[Application of a novel method to collect large amount of fecal mucosa in screening colorectal cancer].

OBJECTIVE: To explore the application of a novel device of collecting large amount of fecal mucosa for detecting the DNA methylation and screening colorectal cancer. METHODS: Preoperative complete fecal sample and surgical specimen of 10 patients with colorectal cancer, and complete fecal sample and normal bowel mucosal samples confirmed by colonoscopy of 6 hospitalization cases at The Third Affiliated Hospital, Nanjing University of TCM from March to April 2014 were collected. A self-made bowel mucosa collector (consisting of upper, middle, lower three containers of 1 000 ml volume, with filter screen in each bottom whose pore diameter is 100, 200 and 300 mesh.) was used to collect mucosal exfoliation cells. Fecal DNA kit was applied to extract DNA of exfoliation cells and the concentration and purity of DNA were measured by UV spectrophotometer (A260/A280), meanwhile DNA methylation of fecal fluid and mucosal tissues was detected by bisulfite sequencing pCR(BSP). RESULTS: DNA methylation sequencing showed that FBN1, SPG20, and SNCA genes presented methylation in CpG island in fecal fluid and cancer tissues from 10 colorectal cancer patients, but did not presented methylation in fecal fluid and mucosa from 6 control cases. When fecal amount was below 100 g, collection rate of fecal fluid was 60% to 80%; when fecal amount was over 100 g, collection rate of fecal fluid was unstable. When fecal amount was 50 to 100 g, DNA A260/A280 value was 1.6 to 1.8, and DNA concentration was 5.0 to 56.1 ng/L. CONCLUSION: Collection rate of fecal fluid with this self-made fecal mucosa collector is quite stable when managing fecal amount of 50 to 100 g once, and can obtain higher purity and concentration of DNA, meeting the demand of methylation detection for screening colorectal cancer.

[Feasibility investigation of three cavity clearance in treatment of perianal abscess].

OBJECTIVE: To explore the feasibility of three-cavity clearance (TCC) in the treatment of perianal abscess. METHODS: A retrospective study of patients with perianal abscess in our center from July 2013 to March 2015 were carried out. Clinical data of 25 patients undergoing TCC (TCC group) were analyzed. At the same time, based on matched gender, age and location of abscess, 25 patients undergoing incision and drainage (incision group) and 25 undergoing cutting seton (seton group) were enrolled. Postoperative pain visual analogue scale (VAS) score (the first defecation,1 week later), rate of fistula formation, fecal incontinence(Wexner score) and wound healing were compared among groups. RESULTS: One week after operation, VAS score in seton group was 6.5±1.3, which was significantly higher than 1.3±0.5 in TCC group and 1.2±0.4 in incision group(P<0.01), while there were no significant differences of VAS among groups at the first defecation(P>0.05). Time of wound healing was (45.8±19.9), (49.2±23.1) and (53.5±24.1) days in TCC, incision and seton group respectively, without significant difference(P>0.05). Rate of fistula formation was 48.0% (12/25) in incision group, which was significantly higher than 12.0% (3/25) in TCC group and 12.0%(3/25) in seton group (all P<0.01). There was no patient with faecal incontinence in TCC group and incision group, while 2 patients with fecal incontinence were found in seton group. CONCLUSION: Three-cavity clearance is feasible in treatment of perianal abscess, and can decrease the rate of fistula formation, ameliorate postoperative pain and protect anal function.

Screening miRNAs for early diagnosis of colorectal cancer by small RNA deep sequencing and evaluation in a Chinese patient population.

PURPOSE: This study aims to screen microRNAs (miRNAs), for an early diagnosis of colorectal cancer, by deep sequencing and evaluation of total miRNAs using clinical samples from a Chinese patient population. METHODS: Total small RNAs from normal colonic mucosa, colonic adenomas, and colorectal cancer tissues were prepared for miRNA analysis by deep sequencing. The sequencing data were then analyzed by bioinformatics for candidate diagnostic miRNAs, which were further validated for their up- or downregulation status. RESULTS: Comparison of cancer tissues with normal mucosa identified 99 upregulated and 90 downregulated miRNAs. Comparison of adenomas and normal mucosa found 114 upregulated and 107 downregulated miRNAs. Comparison of cancer and adenoma tissues found 70 upregulated and 27 downregulated miRNAs. Selected up- and downregulated miRNAs were validated for their expressions in 12 cases of patients with cancer and polyps. Specifically, for the upregulated miRNAs, miR-18a-5p and miR-21-3p were significantly upregulated in adenomas and cancer tissues, compared with the normal mucosa; miR-135b-5p, miR-17-5p, miR-182-5p, miR-200a-5p, and miR-200c-3p were significantly upregulated in cancer tissues compared to the normal mucosa, but their differential expression was not significant in adenoma tissues when compared with the normal mucosa. miR-183-5p and miR-96-5p were significantly upregulated in adenoma tissues when compared with normal mucosa, but these differences were not significant in cancer tissues when compared to normal mucosa. For the downregulated miRNAs, miR-133a-3p was significantly downregulated in both adenoma and cancer tissues when compared to normal mucosa; miR-204-5p, miR-125b-5p, miR-139-5p, miR-100-5p, and miR-30a-5p were significantly downregulated in cancer tissues compared to the normal mucosa, but their differential expression was not significant in adenoma tissue compared to normal mucosa. CONCLUSION: The findings of this study show that a number of miRNAs might be important in the diagnosis and prognosis of colorectal cancer in Chinese patients using the method of small RNA deep sequencing. Upregulation of miR-18a-5p and miR-21-3p or downregulation of miR-133a-3p in adenoma and cancer tissues may serve as an index for early screening of colorectal cancer. Other miRNAs, such as miR-135b-5p, miR-17-5p, miR-182-5p, miR-200a-5p, miR-200c-3p, miR-183-5p, and miR-96-5p, which were either up- or downregulated, in cancer tissues, but not in adenoma tissues, have limited significance in early diagnosis. Further study is needed to determine a screening index with diagnostic value.

[Screening and diagnostic value of the molecular markers of DNA methylation in colorectal neoplasma].

OBJECTIVE: To screen the molecular markers of DNA methylation with potential diagnostic value, and to explore their methylation features in Chinese colorectal neoplasma in order to find out ones with higher diagnostic value. METHODS: Tissue samples of colorectal cancer and normal adjacent mucosa(>10 cm distance to tumors) from 10 colorectal cancer patients undergoing operation, and tissue samples of colorectal adenoma from 10 patients undergoing endoscopic resection in our center from June to August 2013 were collected respectively. Methylation status of 8 genes, such as SNCA, MAL, INA, SPG20, FBN1, CNRIP1, TFPI2, OSMR, was detected by BSP and qMSP to screen genes with potential diagnostic valua. ROC curve was drawn to analyze its diagnostic value. RESULTS: BSP measurement showed that the rate of DNA methylation of SNCA, SPG20 and FBN1 was 100% in colorectal cancer and adenoma, while no methylation was found in normal adjacent mucosa. The other 5 genes expressed in different extent in cancer, adenoma and normal adjacent mucosa. Among 10 cancer tissues and normal adjacent mucosa detected by qMSP method, positive SNCA methylation was found in 5 cases and 1 case respectively; positive SPG20 in 8 cases and 1 case respectively; positive FBN1 in 7 cases and 0 cases respectively, whose differences were significant (P=0.070, P=0.003 and P=0.007). The area under curve(AUC) of SNCA, SPG20, and FBN1 methylation for diagnosing colorectal cancer was 0.890, 0.730 and 0.880 respectively. CONCLUSION: SNCA, SPG20 and FBN1 are potential genes with screening value for colorectal neoplasma.

Microwave-Induced Structural and Functional Injury of Hippocampal and PC12 Cells Is Accompanied by Abnormal Changes in the NMDAR-PSD95-CaMKII Pathway.

Recent studies have highlighted the important role of the postsynaptic NMDAR-PSD95-CaMKII pathway for synaptic transmission and related neuronal injury. Here, we tested changes in the components of this pathway upon microwave-induced neuronal structure and function impairments. Ultrastructural and functional changes were induced in hippocampal neurons of rats and in PC12 cells exposed to microwave radiation. We detected abnormal protein and mRNA expression, as well as posttranslational modifications in the NMDAR-PSD95-CaMKII pathway and its associated components, such as synapsin I, following microwave radiation exposure of rats and PC12 cells. Thus, microwave radiation may induce neuronal injury via changes in the molecular organization of postsynaptic density and modulation of the biochemical cascade that potentiates synaptic transmission.

Improvement of spatial memory disorder and hippocampal damage by exposure to electromagnetic fields in an Alzheimer's disease rat model.

Although some epidemiological investigations showed a potential association between long-term exposure of extremely low frequency electromagnetic fields (ELF-EMF) and Alzheimer's disease (AD), no reasonable mechanism can explain this association, and the related animal experiments are rare. In this study, ELF-EMF exposure (50 Hz 400 µT 60 d) combined with D-galactose intraperitoneal (50 mg/kg, q.d., 42 d) and Aß25-35 hippocampal (5 µl/unilateral, bilateral, single-dose) injection was implemented to establish a complex rat model. Then the effects of ELF-EMF exposure on AD development was studied by using the Morris water maze, pathological analysis, and comparative proteomics. The results showed that ELF-EMF exposure delayed the weight gain of rats, and partially improved cognitive and clinicopathologic symptoms of AD rats. The differential proteomic analysis results suggest that synaptic transmission, oxidative stress, protein degradation, energy metabolism, Tau aggregation, and inflammation involved in the effects mentioned above. Therefore, our findings indicate that certain conditions of ELF-EMF exposure could delay the development of AD in rats.

Protecting intestinal epithelial cell number 6 against fission neutron irradiation through NF-κB signaling pathway.

The purpose of this paper is to explore the change of NF-κB signaling pathway in intestinal epithelial cell induced by fission neutron irradiation and the influence of the PI3K/Akt pathway inhibitor LY294002. Three groups of IEC-6 cell lines were given: control group, neutron irradiation of 4 Gy group, and neutron irradiation of 4 Gy with LY294002 treatment group. Except the control group, the other groups were irradiated by neutron of 4 Gy. LY294002 was given before 24 hours of neutron irradiation. At 6 h and 24 h after neutron irradiation, the morphologic changes, proliferation ability, apoptosis, and necrosis rates of the IEC-6 cell lines were assayed and the changes of NF-κB and PI3K/Akt pathway were detected. At 6 h and 24 h after neutron irradiation of 4 Gy, the proliferation ability of the IEC-6 cells decreased and lots of apoptotic and necrotic cells were found. The injuries in LY294002 treatment and neutron irradiation group were more serious than those in control and neutron irradiation groups. The results suggest that IEC-6 cells were obviously damaged and induced serious apoptosis and necrosis by neutron irradiation of 4Gy; the NF-κB signaling pathway in IEC-6 was activated by neutron irradiation which could protect IEC-6 against injury by neutron irradiation; LY294002 could inhibit the activity of IEC-6 cells.

Effects of pulsed 2.856 GHz microwave exposure on BM-MSCs isolated from C57BL/6 mice.

The increasing use of microwave devices over recent years has meant the bioeffects of microwave exposure have been widely investigated and reported. However the exact biological fate of bone marrow MSCs (BM-MSCs) after microwave radiation remains unknown. In this study, the potential cytotoxicity on MSC proliferation, apoptosis, cell cycle, and in vitro differentiation were assayed following 2.856 GHz microwave exposure at a specific absorption rate (SAR) of 4 W/kg. Importantly, our findings indicated no significant changes in cell viability, cell division and apoptosis after microwave treatment. Furthermore, we detected no significant effects on the differentiation ability of these cells in vitro, with the exception of reduction in mRNA expression levels of osteopontin (OPN) and osteocalcin (OCN). These findings suggest that microwave treatment at a SAR of 4 W/kg has undefined adverse effects on BM-MSCs. However, the reduced-expression of proteins related to osteogenic differentiation suggests that microwave can the influence at the mRNA expression genetic level.

Activation of VEGF/Flk-1-ERK Pathway Induced Blood-Brain Barrier Injury After Microwave Exposure.

Microwaves have been suggested to induce neuronal injury and increase permeability of the blood-brain barrier (BBB), but the mechanism remains unknown. The role of the vascular endothelial growth factor (VEGF)/Flk-1-Raf/MAPK kinase (MEK)/extracellular-regulated protein kinase (ERK) pathway in structural and functional injury of the blood-brain barrier (BBB) following microwave exposure was examined. An in vitro BBB model composed of the ECV304 cell line and primary rat cerebral astrocytes was exposed to microwave radiation (50 mW/cm(2), 5 min). The structure was observed by scanning electron microscopy (SEM) and the permeability was assessed by measuring transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) transmission. Activity and expression of VEGF/Flk-1-ERK pathway components and occludin also were examined. Our results showed that microwave radiation caused intercellular tight junctions to broaden and fracture with decreased TEER values and increased HRP permeability. After microwave exposure, activation of the VEGF/Flk-1-ERK pathway and Tyr phosphorylation of occludin were observed, along with down-regulated expression and interaction of occludin with zonula occludens-1 (ZO-1). After Flk-1 (SU5416) and MEK1/2 (U0126) inhibitors were used, the structure and function of the BBB were recovered. The increase in expression of ERK signal transduction molecules was muted, while the expression and the activity of occludin were accelerated, as well as the interactions of occludin with p-ERK and ZO-1 following microwave radiation. Thus, microwave radiation may induce BBB damage by activating the VEGF/Flk-1-ERK pathway, enhancing Tyr phosphorylation of occludin, while partially inhibiting expression and interaction of occludin with ZO-1.

The relationship between NMDA receptors and microwave-induced learning and memory impairment: a long-term observation on Wistar rats.

UNLABELLED: Abstract Purpose: To investigate whether high power microwave could cause continuous disorders to learning and memory in Wistar rats and to explore the underlying mechanisms. MATERIALS AND METHODS: Eighty Wistar rats were exposed to a 2.856 GHz pulsed microwave source at a power density of 0 mW/cm(2) and 50 mW/cm(2) microwave for 6 min. The spatial memory ability, the structure of the hippocampus, contents of amino acids neurotransmitters in hippocampus and the expression of N-methyl-D-aspartic acid receptors (NMDAR) subunit 1, 2A and 2B (NR1, NR2A and NR2B) were detected at 1, 3, 6, 9, 12 and 18 months after microwave exposure. RESULTS: Our results showed that the microwave-exposed rats showed consistent deficiencies in spatial learning and memory. The level of amino acid neurotransmitters also decreased after microwave radiation. The ratio of glutamate (Glu) and gammaaminobutyric acid (GABA) significantly decreased at 6 months. Besides, the hippocampus showed varying degrees of degeneration of neurons, increased postsynaptic density and blurred synaptic clefts in the exposure group. The NR1 and NR2B expression showed a significant decrease, especially the NR2B expression. CONCLUSIONS: This study indicated that the content of amino acids neurotransmitters, the expression of NMDAR subunits and the variation of hippocampal structure might contribute to the long-term cognitive impairment after microwave exposure.

RKIP Regulates Neural Cell Apoptosis Induced by Exposure to Microwave Radiation Partly Through the MEK/ERK/CREB Pathway.

In the present study, we investigated whether Raf-1 kinase inhibitory protein (RKIP) is important for neural cell apoptosis induced by microwave exposure and explored the role of MEK/ERK/CREB pathway regulated by RKIP in the apoptosis. Differentiated PC12 cells were exposed to continuous microwave radiation at 2.856 GHz for 5 min with average power density of 30 mW/cm(2). RKIP sense and anti-sense recombinant plasmids were constructed and transfected into PC12 cells, respectively. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and caspase-3 activity assay were used to detect cell apoptosis. The results showed that RKIP was downregulated after microwave exposure while the MEK/ERK/CREB signaling pathway was activated excessively. Moreover, the ratio of Bcl-2/Bax decreased, activity of caspase-3 increased, and thus apoptotic DNA fragmentation increased. RKIP overexpression significantly inhibited the phosphorylation of MEK, ERK, and CREB, while RKIP downregulation had the reverse effect. Furthermore, U0126 was found to antagonize the changes caused by RKIP downregulation after exposure to radiation. In conclusion, RKIP plays an important role in the neural cell apoptosis induced by microwave radiation, and the regulation of cell apoptosis by RKIP is partly through the MEK/ERK/CREB pathway. This suggests that RKIP may act as a key regulator of neuronal damage caused by microwave radiation.