Objective:To investigate the relationship between the expressions of checkpoint with forkhead-associated and ring finger(CHFR)and metastasis-associated protein 1(MACC1)and the sensitivity of patients with rectal cancer for neoadjuvant concurrent chemoradiotherapy(nCRT).Methods:The medical documents of 166 patients with rectal cancer admitted to First Hospital of Qinhuangdao from March 2017 to February 2022 were collected.All patients only received nCRT before surgery,and the radiotherapy adopted three-dimensional conformal intensity modulated radiotherapy,and chemotherapy adopted Capeox scheme.All patients successfully completed total mesorectal excision after 4-6 weeks of nCRT treatment.Immunohistochemical SP staining method was used to detect the protein expressions of CHFR and MACC1 in rectal cancer and its adjacent tissues.According to the tumor regressive grading(TRG)standard of the Joint Committee on Cancer Staging in the United States,75 patients who were grade 0-2 as TRG after nCRT were included in the nCRT insensitive group,and 91 patients who were grade 3-4 as TRG were included in the nCRT sensitive group.The expression levels of CHFR and MACC1 proteins in cancer tissues before and after treatment between the two groups were compared.And then,the relationship between clinically pathological characteristics of patients and nCRT sensitivity was analyzed,and the influencing factors of nCRT sensitivity were analyzed.The receiver operating characteristic(ROC)curves of them were drawn,and area under curve(AUC)values were calculated,and the predictive values of CHFR and MACC1 for the sensitivity of patients with rectal cancer to nCRT were further analyzed.Results:The CHFR positive expression rate in rectal cancer tissue was significantly lower than that in adjacent tissues of rectal cancer,and the MACC1 positive expression rate in rectal cancer tissue was significantly higher than that in adjacent tissues of rectal cancer(x2=81.373,87.150,P＜0.05),respectively.After 166 patients completed the nCRT treatment,there were 6 cases of TRG grade 0,8 cases of TRG grade 1,61 cases of TRG grade 2,59 cases of TRG grade 3 and 32 cases of TRG grade 4.The sensitivity rate of nCRT was 54.82%(91/166).The CHFR positive expression rate in the nCRT sensitive group was significantly higher than that in the nCRT insensitive group,and the MACC1 positive expression rate in the nCRT sensitive group was significantly lower than that in the nCRT insensitive group(x2=4.613,37.509,P＜0.05).The proportions of T4 stage and N+stage in the nCRT sensitive group were higher than those in the nCRT insensitive group,and the differences were statistically significant(x2=54.432,28.912,P＜0.05),respectively.The expressions of CHFR and MACC1 were respectively independent risk factor affected the sensitivity of patients with rectal cancer to nCRT[OR=2.456(95% CI:1.294-4.563),OR=3.281(95% CI:1.472-6.479),P＜0.05].The sensitivity and specificity of the combined detection of CHFR and MACC1 were respectively 65.89% and 69.46% in predicting the nCRT sensitivity for rectal cancer.The predictive value of the combined detection was higher than that of single CHFR detection and single MACC1 detection(AUC values of them were respectively 0.713,0.564,0.589,P＜0.05),respectively.Conclusion:CHFR and MACC1 are related to the sensitivity of patients with rectal cancer to nCRT,which means patients with high expression of CHFR and low expression of MACC1 are more sensitive to nCRT.Therefore,both of them may be indicators that predict the sensitivity of patients with rectal cancer to nCRT.

Objective:To observe the expression of miRNA in retinal tissue of mice with oxygen-induced retinopathy (OIR), and screen miRNAs related to p21 and retinal neovascularization (RNV) formation.Methods:A experimental study. Forty healthy 7-day-old C57BL/6J mice were randomly divided into normal group and OIR group, with 20 mice in each group. The oxygen induced RNV model was constructed in the OIR group, and no treatment was performed in the normal group. At the age of 17 days, the mice were killed and the RNV of mice was observed by retinal fluorescence; the nuclei of vascular endothelium that broke through the inner limiting membrane of retina were counted under light microscope. The retinal tissues were taken for miRNA chip analysis to detect the differentially expressed miRNAs between the normal group and the OIR group. The resulting differential miRNA target genes were subjected to enrichment analysis based on gene annotation (GO) and Kyoto Encyclopedia of genes and genomes (KEGG); miRNAs and pathways that may be related to p21 were screened through Targetscan, MiRanda and MicroT-CDs database alignment. Independent sample t-test was used for pairwise comparison between groups. Results:Compared with the normal group, the area of nonperfusion area, RNV and the number of vascular endothelial nuclei that broke through the inner limiting membrane of the retina in the OIR group increased significantly, differences were statistically significant ( t=18.800, 9.025; P<0.05). Compared with the normal group, there were 54 miRNAs that were statistically differentially expressed in the OIR group, of which 47 were up-regulated and 7 were down-regulated. A total of 13 miRNAs related to p21 were screened from the alignment results of the three databases with the obtained differential miRNAs. According to the difference multiples, they were miR-7218-5p, miR-322-5p, miR-224-5p, miR-335-5p, miR-329-3p, miR-362-3p, miR-532-5p, miR-20b-5p, miR-20a-5p, miR-195a-5p, miR-423-5p, miR-497a-5p, and miR-129-5p. Differential miRNA target gene enrichment analysis yielded 1 112 go entries and 50 KEGG pathways, of which 50 go entries and 13 KEGG pathways were related to p21. Conclusion:13 miRNAs related to p21 were screened out in the OIR model.

The family of voltage-gated potassium Kv2 channels consists of the Kv2.1 and Kv2.2 subtypes. Kv2.1 is constitutively highly phosphorylated in neurons and its function relies on its phosphorylation state. Whether the function of Kv2.2 is also dependent on its phosphorylation state remains unknown. Here, we investigated whether Kv2.2 channels can be phosphorylated by protein kinase C (PKC) and examined the effects of PKC-induced phosphorylation on their activity and function. Activation of PKC inhibited Kv2.2 currents and altered their steady-state activation in HEK293 cells. Point mutations and specific antibodies against phosphorylated S481 or S488 demonstrated the importance of these residues for the PKC-dependent modulation of Kv2.2. In layer II pyramidal neurons in cortical slices, activation of PKC similarly regulated native Kv2.2 channels and simultaneously reduced the frequency of action potentials. In conclusion, this study provides the first evidence to our knowledge that PKC-induced phosphorylation of the Kv2.2 channel controls the excitability of cortical pyramidal neurons.
Action Potentials ; HEK293 Cells ; Humans ; Protein Kinase C/metabolism* ; Pyramidal Cells/enzymology* ; Shab Potassium Channels/genetics*

Objective:To analyze the expression of miRNA involved in regulating retinal neovascularizationin in retinal tissue of oxygen-induced retinopathy (OIR) mice.Methods:Eighty healthy C57BL/6J mice were randomly divided into control group and OIR group at postnatal day 7(P7). Control group were not received any treatment and then exposed to room air. The OIR group was exposed to (75±2)% oxygen and then under room air at P12. Mice of all groups were euthanized at P17. Retinal neovasculation (RNV) was evaluated by counting the number of pre-retinal neovascular cells and analysing no perfusion area by immunofluorescent staining of the mouse retina.Total RNA was extracted from retinal tissue,and miRNA microarrays was performed to identify differentially expressed miRNA in the two groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed differential microRNA.Results:Compared with the control group,the retinal neovascular tufts and the no perfusion area were both significantly smaller than those in OIR group. The number of pre-retinal neovascular cell nuclei in retinas from control group were obviously lower than those in the retinas from OIR group ( t=9.025, P<0.05). MiRNA microarray analysis showed that 54 miRNA in OIR group showed statistically different expression in control group, 47 miRNA were up-regulated and 7 miRNA were down-regulated. The results of PCR were consistent with the trend of microarray. In GO analysis, 1112 items were significantly different ( P<0.05), and 65 items were significantly different in KEGG analysis of expression profile ( P<0.05). Conclusions:The miRNA expression in retinal tissue of OIR mice is different from that of normal mice, and these miRNA may be involved in the development of RNV. There are 54 miRNA expression differences in retinal tissue of OIR compared with normal mouse retinal tissue.

Objective:To observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.Methods:hRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.Results:Different concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant ( t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant ( t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1 285-3p, hsa-miR-30d-3p, hsa-miR-3 170, hsa-miR-7 976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions. Conclusion:FA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.

Objective To investigate the protective effect of butylphenyphthalein (NBP) on RPE apoptosis induced by H2O2.Methods The human RPE cell line (human ARPE-19 cell line) were used as the experimental cells and were divided as control group, model group, NBP group. Complete medium was used in control group. The model group was stimulated with 200 μmol/L H2O2 for 2 h, and the cells were cultured in complete medium. The NBP group was cultured with 200 μmol/L H2O2 and 1 μmol/L NBP for 2 h. After changing the medium, complete medium was combined with 1 μmol/L NBP to continue the culture of the cells. Cell viability were detected by MTT assay while the morphology of RPE were observed by HE staining. Moreover, Hoechst 33258 was used to detect RPE cell apoptosis. Mitochondrial membrane potential (JC-1) staining were performed to monitor changes in cell membrane potential and the characteristic change of apoptosis in RPE cells. Furthermore, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining were used to analyze the effect of NBP treatment on the expression of ROS. The effect of NBP on the expression of Heme oxygenase-1(HO-1) was analyzed by cellular immunofluorescence and western blotting.Results The results of MTT assay showed that the cells were cultured for 24 and 48 hours, cell viability of control group (t=17.710, 13.760;P<0.0001,<0.0001) and treatment group (t=4.857, 9.225;P=0.0007,<0.0001) were stronger than that of model group, and the difference was statistically significant. HE staining and Hoechst33258 staining showed that compared with the control group, the number of cells in the model group was significantly less, and the cell morphology was incomplete. Compared with the model group, the number of cells in the treatment group was significantly increased, and the cell morphology was better. The results of JC-1 assay showed that the number of apoptotic cells in the model group was significantly higher than that in the control group, and the number of apoptotic cells in the treatment group was significantly lower than that in the model group. DCFH-DA staining showed that the ROS accumulation in the model group was more than that in the control group, and the ROS accumulation in the treatment group was less than that in the model group. Immunostaining observation showed that the HO-1 fluorescence intensity of the cells in the treatment group was significantly higher than that of the control group, and the difference was statistically significant (t=10.270,P=0.0005). Western blot analysis showed that NBP up-regulated the expression level of HO-1 in a time-dependent manner. The relative expression of HO-1 at 4, 8, and 12 h of NBP showed a clear increase trend compared with 0 h, and the difference was statistically significant (F=164.91,P<0.05).Conclusions Oxidative stress injury can down-regulate the viability of RPE cells and induce apoptosis. NBP can increase the antioxidant capacity of RPE cells, reduce cell damage and inhibit cell apoptosis by up-regulating HO-1 expression.

Objective To observe the effects of maternal high fat diet (MHFD) during pregnancy and lactation on intestinal barrier function in offspring mice.Methods C57BL/6 pregnant mice were divided into high fat diet (MHFD) group and normal diet group (MND) randomly and were given high fat diet and normal diet during pregnancy (3 weeks) and lactation (3 weeks) respectively.Both groups of offspring mice were naturally given and bodyweight of pups was monitored at birth and weekly.After weaning,the intestinal permeability of offspring mice was detected by fluorescein isothiocyanate conjugated-dextran method (FITC-D).Immunofluorescence was used to detect the expression of ZO-1 in intestinal tissues.HE staining was used to assess the villus length and crypt depth.The intestinal cell proliferation (expression of Ki-67) and Mucin 2 (MUC2) were assessed by immunohistochemistry.PAS staining was used to evaluate the goblet cells.The expression of inflammatory cytokines including IL-1β,IL-6,and TNF-α in intestinal tissue were measured by real-time PCR.Results At the age of 2 and 3 weeks,the offspring in MHFD group were significantly heavier than those in MND group.HE staining showed no obvious microscopic inflammation in both groups of 3 weeks old offspring mice,however,the relative expression levels of IL-1β (1.95±0.53 vs.1.13±0.15;t =3.65,P=0.005),IL-6 (1.40±0.71 vs.0.73±0.17;t=2.72,P=0.04),and TNF-α (1.63±0.53 vs.1.04±0.12;t=2.64,P=0.02) mRNA were significantly higher in the MHFD group.Compared with the 3 weeks old offspring mice in MND group,MHFD significantly increased the permeability of intestine and decreased the expression of ZO-1 in membrane.The number of Ki-67 positive cells (18.00±4.74 vs.24.60±4.17;t =3.31,P=0.004) in each villus,goblet cells (14.70±2.91 vs.28.10±4.95;t =7.38,P＜0.001) and MUC2 positive cells (20.60± 3.13 vs.30.00±3.33;t=6.50,P＜0.001) in each crypt were significantly lower than those in MND group.Conclusion Maternal high fat diet in early life of offspring mice can induce intestinal low grade inflammation and lead to the disruption of intestinal mucosal barrier in offspring mice,which may be involved in the progeny diseases.

Hereditary spastic paraplegia is a heterogeneous group of genetic neurodegenerative disorders of the nervous system. It is classified into four subtypes based on the mode of inheritance; and among them, most autosomal recessive hereditary spastic paraplegia cases are due to type SPG11 and SPG15 gene mutations. Autosomal recessive hereditary spastic paraplegia cases with SPG30 gene mutation have never been reported in China. Herein, we present our experience with a case of hereditary spastic paraplegia with SPG30 gene mutation in our hospital from North East China. In this patient we detected a missense mutation of c.499 C>T (p.Arg167Cys) in gene KIF1A, a causative gene of type SPG30.

Objective Sarcopenia and metabolic syndrome share similar pathophysiological mechanisms. The aim of this study was to investigate the prevalence of sarcopenia among health examination population, and to analyze the relationship between sar-copenia and blood pressure, blood glucose, uric acid and lipids. Methods Physical examination data of 1191 healthy persons in the medical examination center of the hospital from Mar 2011 to Jun 2011 were collected. The weight, skeletal muscle, body fat, body mass index ( BMI) , waist circumference,body fat percentage, waist-hip ratio and visceral fat area were analyzed by human body compositionanalyzer and the prevalence of sarcopenia was observed. At the same time, triglyceride (TG), total cholesterol (TC), high density lipo-protein-cholesterol ( HDL-C ) , low density lipoprotein-cholesterol ( LDL-C) , uric acid and fasting blood glucose were also detected. Results The prevalence rate of sarcopenia of the subjects was 5.21%, and the highest incidence was found in ≥60 years group( 11.11%) . The prevalence rates of overweight and obesity were 33.8% and 10.2%, respectively. The prevalence of sarcopenia is grad-ually higher along with increasing BMI. The prevalence rates of sarcopenia of overweight and obesity subjects were 5.47% and 26.23%, respectively. Compared with the normal control group, the level of weight[(66.34±11.75)kg vs (76.71±12.84)kg ], BMI[(23.37± 3.13) vs (28.05±3.66)], body fat percentage[(25.33±6.06)% vs (36.76±4.47)%], waist circumference[(83.19±9.56)cm vs (95.45±13.74)cm] and visceral fat area[(88.96±29.74)cm2 vs (136.91±25.56)cm2] were higher in the sarcopenia group (P<0.05). Compared with the normal control group, the incidence of systolic blood pressure[(125.59±30.04)mmHg vs (139.39±19.79) mmHg], diastolic blood pressure[(75.82±11.95)mmHg vs (82.34±10.96)mmHg ] TG[(1.56±1.12)mmol/L vs (1.98±1.72)mmol/L] and uric acid[(313.75±83.07)mmol/L vs (335.55±96.07)mmol/L] were higher in the sarcopenia group (P<0.05). Compared with the normal subjects, the detectable rates of abnormal diastolic blood pressure, fasting blood glucose, uric acid, and LDL-C were increased in the sarcopenia, obesity and sarcopenia combined with obesity subjects (P<0.05). The odds ratio of abnormal systolic blood pressure, diastolic blood pressure, uric acid, and LDL-C increased in the sarcopenia, obesity and sarcopenia combined with obe-sity subjects using logistic regression analyses after correction of gender and age. Conclusion The sarcopenia may have some con-nection with metabolic risk factors. Early detection of sarcopenia can help to distinguish people predisposed to metabolic syndrome, and it has important significance for prevention of chronic disease.

Objective To investigate the pathogen distribution and risk factors of pulmonary infection after acute cervical spinal cord injury (ACSCI) in an attempt to offer reference for early antiinfection therapy.Methods The study comprised 223 cases who were admitted from October 2011 to October 2014.There were 149 males and 74 females,at (43.3 ± 13.5) years of age.Species of pathogens identified were gram-positive,gram-negative and mixed.Effects of age,gender,injury types and tracheotomy on pathogen distribution were analyzed.Results Gram-negative infection was found in 114 cases (51.1％),with tracheotomy accounting for 7.0％ of the cases and death accounting for 1.8％ of the cases,and the main causative pathogens were Klebsiella pneumonia,Escherichia coli,Pseudomonas aeruginosa and Acinetobacter baumannii.Gram-positive infection was found in 41 cases (18.4％),with tracheotomy accounting for 12.2％ of the cases and death accounting for 7.3％ of the cases,and the main causative pathogens were Staphylococcus aureus and Streptococcus pneumonia.Mixed infection was found in 68 cases (30.5％),with tracheotomy accounting for 22.1％ of the cases and death accounting for 13.2％ of the cases.Gender had no significant correlation with pathogen distribution.For the cases of complete spinal cord injury and tracheotomy,the ratio of mixed infection increased significantly (P ＜ 0.05).For the cases younger than 30 years,the pathogens were mainly gram-positive bacteria (P ＜ 0.05).Conclusions Main pathogens of pulmonary infection after ACSCI are gram-negative bacteria.The cases younger than 30 years are associated with higher risk of grampositive infection,while the cases with complete injury or tracheotomy are associated with higher risk of mixed infection.