Association of single nucleotide polymorphisms of IL-1, IL-10 and TNF-β with rheumatoid arthritis in Chinese Han population from Quanzhou

AIM: To explore the genetic characteristics of enrolled rheumatoid arthritis and genetic mechanisms of rheumatoid arthritis (RA) by studying the associations of single nucleotide polymorphisms (SNPs) with rheumatoid arthritis in Chinese Han population from a very high prevalence area of rheumatoid arthritis, Quanzhou. METHODS: A case-control study of 155 rheumatoid arthritis patients (RA group) and 170 normal controls (control group) from Quanzhou were enrolled. All of 5 SNPs were genotyped by allele-specific polymerase chain reaction (PCR) and analyzed by SPSS 19.0. χ²-test was applied to predict Hardy-Weinberg equilibrium, and allele and genotype frequencies between RA group and control group were compared. Logistic regression models were used to analyze SNPs. Link disequilibrium analysis and haplotype analysis were performed with SHEsis software. RESULTS: Total of 1 SNP in control group was confirmed by Hardy-Weinberg equilibrium test (P>0.05), and 1 SNP in RA group was confirmed by Hardy-Weinberg equilibrium test (P>0.05). Allele frequencies of 4 SNPs were significantly different between control group and RA group (P <0.05). CONCLUSION: The SNPs of IL-10 rs1800893, IL-1β rs16944, TNF-β rs2009658 and TNF-β rs1041981 were associated with the incidence of rheumatoid arthritis in Chinese Han population of Quanzhou. Allele G of IL-10 rs1800893, allele G of IL-1β rs16944, allele C of TNF-β rs2009658 and allele C of TNF-β rs1041981 can be used as potential genetic markers for the diagnosis of RA in Quanzhou, Fujian.     

Relationship between monocyte to HDL-C ratio and coronary lesion severity/short-term prognosis in postmenopausal women with normal LDL-C level

AIM To investigate the relationship between monocyte to high-density lipoprotein cholesterol (HDL-C) ratio (MHR) and coronary lesion severity/short-term prognosis in postmenopausal women with coronary atherosclerotic heart diseases (CAD) and normal low-density lipoprotein cholesterol (LDL-C) level. METHODS A cohort of 180 postmenopausal women diagnosed as CAD with LDL-C≤130 mg/dL were enrolled. The patients were divided into 3 groups according to the admission MHR: low MHR group (MHR<0.28, n=54), moderate MHR group (0.28≤MHR≤0.44, n=55), and high MHR group (MHR>0.48, n=71). Hospital medical records and coronary angiography of the postmenopausal women were collected. Dominance coronary system, number of lesions, segments for lesions, total occlusion, trifurcation, bifurcation, aorto-ostial lesion, severe tortuosity, length>20 mm, heavy calcification, thrombus, and diffusely diseased and narrowed segment of coronary arteries were scored by SYNTAX score Ⅰ system. The relationship between MHR and SYNTAX score Ⅰ was assessed by multiple linear regression analysis, while the relationship between MHR and acute cardiogenic pulmonary edema was assessed by logistic regression analysis. RESULTS The incidence of acute myocardial infarction in high MHR group (59.2%, 42 cases) was higher than that in low MHR group (22.2%, 12 cases) and moderate MHR group (32.7%, 18 cases), with statistical significance (P=0.001). The SYNTAX score Ⅰ in high MHR group was significantly higher than that in low MHR group and moderate MHR group (P=0.003). The incidence of acute cardiogenic pulmonary edema within 7 d during admission in high MHR group (22.5%) was higher than that in low MHR group (3.7%) and moderate MHR group (3.6%), with statistical significance (P<0.001). The positive correlation between MHR and SYNTAX score Ⅰ was observed (r=0.196, P=0.008). The result of multiple linear regression indicated that SYNTAX score Ⅰ was affected by MHR (F=7.631, P=0.001). The result of logistic regression analysis indicated that MHR (OR: 7.910, 95% CI: 2.268~27.589, P=0.001) and serum creatinine (OR: 1.017, 95% CI: 1.005~1.029, P=0.006) were independent predictors of acute cardiogenic pulmonary edema. CONCLUSION The MHR was a novel biomarker for coronary lesion severity and incidence of acute cardiogenic pulmonary edema in postmenopausal women with CAD and normal LDL-C level.     

Serum CTRP1 and CTRP7 levels are associated with coronary atherosclerotic heart disease and diabetes mellitus

AIM To investigate the relationship between the serum levels of C1q/TNF-related protein (CTRP) 1 and CTRP7 and coronary atherosclerotic heart disease (CAD) in the patients with or without type 2 diabetes mellitus (T2DM). METHODS Totally 138 cases of participants were selected, and divided into control group (without T2DM or CAD; n=40), T2DM group (with T2DM, but without CAD; n=30), CAD group (with CAD, but without T2DM; n=40), and CAD+T2DM group (with both T2DM and CAD; n=28). The serum levels of CTRP1 and CTRP7 in these participants were measured by ELISA. RESULTS (1) Compared with control group, serum CTRP1 level in CAD group and CAD+T2DM group was significantly increased (P<0.05 or P<0.01), and serum CTRP7 level in CAD, T2DM and CAD+T2DM groups was significantly decreased (P<0.05 or P<0.01). (2) Increased serum CTRP1 level was a risk factor for CAD [odds ratio (OR)=1.136; 95% confidence interval (CI): 1.010~1.278; P=0.034). Sex, hypertension and serum triglyceride level were also correlated with CAD. Decreased serum CTRP7 level was a risk factor for T2DM (OR=0.984; 95% CI: 0.969~0.999; P=0.038). Sex, hypertension and serum high-density lipoprotein cholesterol level were also correlated with T2DM. Increased serum CTRP1 level was a risk factor for CAD+T2DM (OR=1.009; 95% CI: 1.005~1.203; P=0.040). Hypertension was also a risk factor for CAD+T2DM. (3) In CAD group, serum CTRP1 level had certain diagnostic value, and the area under the receiver operating characteristic (ROC) curve (AUC) was 0.670 (95% CI: 0.580~0.760; P=0.001), but serum CTRP7 level had no diagnostic value for CAD. In T2DM group, both serum CTRP1 and CTRP7 levels had diagnostic value, and the AUC values of their ROC curves were 0.607 (95% CI: 0.505~0.710; P=0.032) and 0.665 (95% CI: 0.574~0.756; P=0.001), respectively. In CAD+T2DM group, both serum CTRP1 and CTRP7 levels had diagnostic value, and the AUC values of their ROC curves were 0.666 (95% CI: 0.552~0.781; P=0.007) and 0.632 (95% CI 0.517~0.747; P=0.032), respectively. CONCLUSION Increased serum CTRP1 level and decreased serum CTRP7 level are associated with increased risk of T2DM and CAD. Increased serum CTRP1 level is a risk factor for CAD and CAD+T2DM, while decreased serum CTRP7 level is a risk factor for T2DM. Serum CTRP1 level has certain specificity and sensitivity for the diagnosis of CAD, T2DM and CAD+T2DM, while serum level of CTRP7 has certain specificity and sensitivity for the diagnosis of T2DM and CAD+T2DM.     

Relationships between lncRNA HOTAIR/H19 SNPs and genetic susceptibility to gastric carcinoma/EBV-related gastric carcinoma

AIM To investigate the potential associations between the single nucleotide polymorphisms （SNPs） of long noncoding RNA （lncRNA） H19/HOTAIR and the susceptibility to gastric carcinoma, especially to Epstein-Barr virus （EBV）-associated gastric carcinoma （EBVaGC）. METHODS Peripheral blood samples from 65 cases of EBV-negative gastric carcinoma （EBVnGC）, 50 cases of EBVaGC and 115 cases of healthy people were collected. A total of 4 TagSNPs, H19 rs3024270 and rs3741219, as well as HOTAIR rs4759314 and rs874945, were selected. The Taq-Man MGB allele typing kit was used to detect the genotype of each SNP locus, and the experimental results were statistically analyzed. RESULTS （1） There were significant differences of both genotypic and allelic frequencies at H19 rs3024270 locus between gastric carcinoma group and control group （P<0.05）. Individuals carrying the G allele at H19 rs3024270 locus had significantly low risk of gastric carcinoma （P<0.01）, indicating that the G allele was protective. （2） People with the GG genotype at HOTAIR rs4759314 locus had significantly high risk of gastric carcinoma （P<0.05）. Carrying the G allele increased the risk of gastric carcinoma, which indicated that the risk gene for gastric carcinoma might be the G allele. （3） No significant difference of the genotypic and allelic frequencies at H19 rs3741219 and HOTAIR rs874945 loci between gastric carcinoma group and control group was observed （P>0.05）.（4） The G allele frequency at HOTAIR rs4759314 locus in EBVaGC group was significantly higher than that in EBVnGC group. However, no difference of the other 3 SNPs was found between EBVaGC group and EBVnGC group （P>0.05）. CONCLUSION The SNPs at H19 rs3024270 and HOTAIR rs4759314 loci are related to the risk of gastric carcinoma, but not significantly related to the risk of EBVaGC.     

Association analysis between two SNPs in SNCA and PARK16 genes and Parkinson disease in a Han Chinese population in Liaoning area

AIM: To investigate the genotypic frequency of rs3857059 in SNCA gene and rs16856139 in PARK16 gene for determining the potential genetic risk factors of Parkinson disease (PD) in a Han Chinese population in Liaoning area of China. METHODS: The genomic DNA from 213 PD patients and 214 matched controls was amplified in the multiplex PCR system and subsequently genotyped by digestion with endonuclease Pvu II. Genetic parameter and association studies were carried out with SPSS 13.0 and PLINK 1.07 software. RESULTS: We accurately detected all genotypes in the 2 loci with PCR-restriction fragment length polymorphism (RFLP) techniques. The gene frequency of G allele in the rs3857059 locus was higher in PD group than that in control group with statistical significance (χ²= 7.592,P<0.01, OR=0.677, 95% CI=0.517~0.888). The T allele frequency in the rs16856139 locus was lower in PD group than that in control group and statistical result revealed a significant difference (χ²=11.511, P<0.01, OR=0.390, 95% CI=0.227~0.669). CONCLUSION: The 2 SNPs investigated in SNCA and PARK16 genes are likely to play roles as common risk factors for PD disease in the Han Chinese population.     

Effect of anesthesia depth with Narcotrend monitoring on cognitive function after gastrointestinal tumor surgery in elderly patients

AIM: To determine the correlation of anesthesia depth and the regional cerebral oxygen saturation (rSO₂) under the monitoring of Narcotrend (NT), and its predictive value for post-operative cognitive dysfunction (POCD) in senior patients undergoing gastrointestinal tumor surgery. METHODS: From June 2018 to June 2019, 90 elderly patients who were scheduled for gastrointestinal tumor surgery were enrolled. The depth of anesthesia was monitored with Narcotrend index (NTI), and the rSO₂ was evaluated by the near-infrared spectroscopy throughout the intraoperative periods. Cognitive function of the patients was evaluated by using mini-mental state examination (MMSE) 24 h after surgery. RESULTS: The patients (n=25) were diagnosed with POCD at 24 h after surgery. The patients with POCD had significantly longer duration of NTI<35 than that in non-POCD patients (P<0.05). Moreover, there was significantly longer duration of ΔrSO₂>13% to baseline and rSO₂<55%. The duration of NTI<35 was highly correlated with the duration of ΔrSO₂>13% to baseline (r=0.62, 95% CI=0.35~0.89, P=0.004). The serum S100β level in POCD group was significantly higher than that in non-POCD patients, while there was no statistically difference with respect to the serum level of interleukin-6 (IL-6). CONCLUSION: Deep anesthesia was likely to exacerbate the imbalance of cerebral oxygen supply and consumption in elderly surgical patients, which might further cause the injury of central nervous system. Narcotrend-guided anesthesia might have beneficial effects on the prevention of POCD especially in elderly patients.     

Effect of compound of Epimedium, Astragalus and Radix Puerariae on expression of ADAM10 in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin expression knock-down

AIM To explore the effect of compound of Epimedium, Astragalus and Radix Puerariae on the expression of a disintegrin and metalloproteinase 10 （ADAM10） in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin （HAMP） expression knock-down for analyzing the pathogenesis of Alzheimer disease （AD） at cell level. METHODS Hippocampal neuron HT22 cells were cultured in vitro and randomly divided into 7 groups： control group, Aβ group （Aβ_25-35-induced HT22 cells）, RNAi group （HAMP gene was silenced in HT22 cells）, Aβ+RNAi group （HAMP gene expression in Aβ_25-35-induced HT22 cells was silenced）, Aβ+TCM group （Aβ_25-35-induced HT22 cells were treated with Epimedium, Astragalus root and Radix Puerariae effective components）, RNAi+TCM group （HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components） and Aβ+RNAi+TCM group （Aβ_25-35-induced HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components）. The silence efficiency of HAMP siRNA was detected by qPCR and Western blot. The ADAM10 expression in each group was determined by immunofluorescence, qPCR and Western blot. RESULTS The HAMP siRNA-3 sequence had the highest interference efficiency. Compared with control group, the expression levels of ADAM10 in Aβ group, RNAi group and Aβ+RNAi group were decreased （P<0.05）. Compared with Aβ group,the expression levels of ADAM10 in Aβ+RNAi group was also decreased （P<0.05）, and the expression levels of ADAM10 in Aβ+TCM group was increased （P<0.05）. Compared with RNAi group, the expression levels of ADAM10 in Aβ+RNAi group was decreased （P<0.05）, while the expression levels of ADAM10 in RNAi+TCM group was increased （P<0.05）. Compared with Aβ+RNAi group, the expression levels of ADAM10 in Aβ+RNAi+TCM group was increased （P<0.05）. CONCLUSION The effective components of Epimedium, Astragalus and Radix Puerariae compound promotes the expression of ADAM10 in Aβ_25-35-induced HT22 cells, which mechanism may be related to the expression of HAMP.     

Effect of NLRC3 expression knock-down on growth of normal human bronchial epithelial BEAS-2B cells

AIM To investigate the effect of NOD-like receptor family caspase recruitment domain containing 3 (NLRC3) expression knock-down on the viability and apoptosis of normal human bronchial epithelial BEAS-2B cells and its mechanism. METHODS The small interfering RNA (siRNA) fragments of NLRC3 gene were transfected into BEAS-2B cells using Lipofectamine 2000 transfection reagent to knock down the NLRC3 expression. The interference fragment was screened by RT-qPCR. The cell viability was measured by MTT assay. The mitochondrial membrane potential was detected by JC-1 staining. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. The protein expression levels of Bcl-2 and Bax were determined by Western blot. RESULTS The interference segment 3 of NLRC3 gene (siNLRC3-3) displayed the best interference effect on NLRC3 expression in BEAS-2B cells (P<0.01). Knock-down of NLRC3 expression in BEAS-2B cells enhanced the cell viability (P<0.01). Knock-down of NLRC3 increased the mitochondrial membrane potential, and decreased the apoptotic rate (P<0.05). Moreover, knock-down of NLRC3 significantly up-regulated Bcl-2 protein expression and significantly down-regulated Bax protein expression (P<0.01). CONCLUSION Knock-down of NLRC3 expression enhances the viability and inhibits the apoptosis of BEAS-2B cells, which may be related to increase in the expression of Bcl-2 protein and decrease in the expression of Bax protein.     

Genetic variants of IL-33 are associated with clinical phenotypes of inflammatory bowel disease in Han population of southern China

AIM：To investigate whether the single nucleotide polymorphisms (SNPs) of interleukin-33 (IL-33) gene are associated with inflammatory bowel disease (IBD) in the Han population of southern China. METHODS：Eight tag-SNPs were selected from the IL-33 gene using the HapMap database. These tag-SNPs were genotyped in 250 Crohn disease (CD) patients, 115 ulcerative colitis (UC) cases and 622 healthy controls by MALDI-TOF MS assay. RESULTS：No difference of the distribution frequencies of genotypes and alleles between the cases and the controls was observed (P>0.05). Genotype-phenotype analysis suggested that several sites were associated with clinical phenotypes of CD.The T allele of SNP rs10118795 was a protective factor for extra-intestinal manifestation (EIM; P<0.05, OR=0.513, 95% CI： 0.281~0.938), while the CC genotype of SNP rs7025417 (P<0.05, OR=1.363, 95% CI： 1.006~1.846) was a risk factor for EIM. The C allele of rs10118795 decreased the risk for developing perianal lesions (P<0.05, OR=0.480, 95% CI： 0.232~0.994), while the CC genotype of rs10975519 was a risk factor for perianal lesions (P<0.05, OR=2.054, 95% CI： 1.053~4.009). The G allele of rs10975509 increased the risk of upper gastrointestinal CD (P<0.05, OR=3.570, 95% CI： 1.328～9.600), and the A allele of it increased the risk for developing ileocolonic CD (P<0.05, OR=0.613, 95% CI： 0.377~0.996). In the aspect of treatment, the genotypes of rs10118795, rs10975509 and rs7025417 were associated with mucosal healing after infliximab treatment for 30 weeks (P<0.05, P<0.01 and P<0.05). In the UC patients, no significant effect of the selected 8 tag-SNPs on the UC phenotypes was observed. CONCLUSION：Eight polymorphisms of IL-33 do not increase the risk of CD and UC in the Han population of southern China, but some of them have an effect on the clinical phenotypes of CD, and 3 SNPs may be potential markers for prediction of effectiveness of infliximab treatment.     

矮化中间砧‘宫藤富士’苹果栽植密度对树体生长、冠层光照和果实产量的影响

2011 年春季定植的矮化中间砧苹果成品苗（3 年根 1 年干的‘宫藤富士’/SH6/平邑甜茶）为试材，设置 7 种不同的栽植密度（株行距分别为 1 m × 3 m、1.5 m × 3 m、2 m × 3 m、0.75 m × 4 m、1 m × 4 m、1.25 m × 4 m 和 1.5 m × 4 m），细纺锤形整枝修剪，自栽植第 2 年，连续 7 年调查 7 种栽植密度对树体生长、冠层光照分布、果实产量和品质的影响。随着树龄的增长，不同栽植密度下树干粗度和总枝量逐年增加，不同处理间树干粗度无显著差异，第 7 年 1 m × 3 m 和 0.75 m × 4 m 两个栽植密度下树体总枝量超过 140 万条 · hm-2，第 8 年均超过 140 万条 · hm-2。栽植前期（第 2 ~ 4 年）各栽植密度树体短枝比例不断增加，长枝比例不断减少，第 5 年各栽植密度枝类组成趋于稳定；综合稳产 3 年（第 6 ~ 8 年）树体的枝类组成数据，4 m 行距的短枝比例明显高于 3 m 行距，长枝比例略低。树体冠层平均相对光照强度由高到低的株行距处理依次为 1.5 m × 4 m（63.87%）、1.25 m × 4 m（61.44%）、2 m × 3 m（61.27%）、1 m × 4 m（59.19%）、0.75 m × 4 m（55.79%）、1.5 m × 3 m（53.67%）和 1 m × 3 m（49.37%）；相同栽植株数下，4 m 行距处理低光效（相对光照强度小于 40%）的区域比例显著小于 3 m 行距。比较前 5 年的累计产量，以行距 4 m 和 1 m × 3 m 的最高。综合稳产 3 年的结果情况，大果率（单果质量 > 200 g 的果实产量占总产量的比例）以 4 m 行距和 2 m × 3 m 的最高。各栽植密度下的果实的可溶性固形物含量、固酸比、果形指数和果实硬度均无显著差异。综上，采用 4 m 行距，1 ~ 1.25 m 株距，树体成形快，稳产后树体结构合理，冠层光照充足，低效光区比例少，前期产量高。     

IL-33-modified BMSCs attenuate acute kidney injury induced by sepsis in rats through MyD88

AIM To investigate the effect of interleukin-33 (IL-33)-modified bone marrow mesenchymal stem cells (BMSCs) on sepsis-induced acute kidney injury (AKI) in rats and the expression of myeloid differentiation factor 88(MyD88). METHODS A septic rat model was established by cecal ligation and puncture. The SD rats (n=80) were randomly divided into control group, model group, negative transfection group (transplanting untransfected BMSCs) and IL-33 transfection group (transplanting BMSCs transfected with IL-33), with 20 in each group. Survival rates of the rats within 72 h in the 4 groups were compared. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured before, and 24, 48 and 72 h after transplantation. The kidney pathological damage was observed by HE staining, and the apoptosis of renal cells was detected by TUNEL method 72 h after transplantation. Western blot was used to detect the protein expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), Toll-like receptor 4 (TLR4) and MyD88. RESULTS The survival rate of the rats in model group was significantly lower than that in control group (P<0.05). The survival rate of the rats in IL-33 transfection group was higher than that in model group and negative transfection group (P<0.05). The levels of SCr and BUN in model group were higher than those in control group (P<0.05). The levels of SCr and BUN in IL-33 transfection group were significantly reduced after transplantation, and were lower than those in model group and negative transfection group (P<0.05). The renal tissue pathological injury score in model group was significantly higher than that in control group (P<0.05). Compared with model group and negative transfection group, the renal tissue pathological injury score in IL-33 transfection group was significantly reduced (P<0.05). The proportion of apoptotic cells in the kidney tissues in model group were higher than that in control group (P<0.05). Compared with model group and negative transfection group, the proportion of apoptotic cells in the kidney tissues in IL-33 transfection group was significantly reduced (P<0.05). The protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in model group were significantly higher than those in control group (P<0.05). Compared with model group and negative transfection group, the protein expression levels of IL-1β, IL-6, TNF-α, TLR4 and MyD88 in IL-33 transfection group were significantly decreased (P<0.05). CONCLUSION IL-33 gene-modified BMSCs significantly improve the renal function of AKI rats with sepsis. The mechanism may be related to IL-33 regulating TLR4/MyD88 signaling pathway and inhibiting renal inflammatory response.     

Activation of repair pathways after neuronal DNA damage induced by bupivacaine

AIM To investigate the activation of related repair pathways after bupivacaine-induced neuronal DNA damage by cDNA gene screening. METHODS The bupivacaine-induced SH-SY5Y neuronal damage and DNA damage model was established. The technique of cDNA microplate array was used to screen the 21 important regulatory factors in the DNA damage repair pathway. Post-analysis of these differentially expressed repair genes for the repair pathway enrichment and distribution was performed. The data were analyzed by GraphPad Prism 6 statistical software to compare differences between groups. RESULTS The viability of SH-SY5Y cells treated with bupivacaine at different concentrations （detected by CCK-8 assay） showed that the IC₅₀ value of bupivacaine was 1.5 mmol/L. The comet assay related index （the comet tail） was increased （P<0.05）, the phosphorylation level of γH2AX protein was increased （P<0.05）, indicating that DNA damage in the SH-SY5Y cells was significantly aggravated after bupivacaine treatment. The results of cDNA microplate assay showed that compared withcontrol group, the differentially expressed genes after bupivacaine treatment were DNA-PKcs, PTEN, NTH1, RAD9, CSB, GADD45, XPD, XPC-HR23B and P53. The analysis showed that these repair genes were mainly concentrated in the following 3 repair mechanisms： base excision repair, nucleotide excision repair, and non-homologous reconstitution. CONCLUSION The repair genes differentially expressed after neuronal DNA damage caused by local anesthetics are mainly concentrated in the pathways of non-homologous end-joining, base excision repair and nucleotide excision repair.     

Significance of TRPM8 protein expression in lung adenocarcinoma

AIM To investigate the significance of transient receptor potential cation channel subfamily M member 8 （TRPM8） protein expression in lung adenocarcinoma. METHODS The tumor samples from 112 cases of patients with lung adenocarcinoma were collected in our hospital, and 4~5 years of follow-up was conducted. The protein expression of TRPM8 was analyzed by immunohistochemical staining, and the correlations between the TRPM8 protein expression and the clinical characteristics including prognosis of the patients with lung adenocarcinoma were investigated. After TRPM8 protein expression was up-regulated in A549 lung adenocarcinoma cells by lentiviral infection, the proliferation of A549 cells was analyzed by CCK-8 assay and colony formation assay, the cell cycle and apoptosis were analyzed by flow cytometry, and the migration and invasion abilities of the cells were measured by scratch experiment and Transwell assay. The TRPM8 protein expression was stably up-regulated in H1299 cells by lentiviral infection, and then the left and right buttocks of the immunodeficient mice were subcutaneously injected with empty vector control cells and TRPM8-overexpressing cells, respectively. The effects of TRPM8 on the growth of H1299 cell-derived xenograft tumor in immunodeficient mice were evaluated. RESULTS The 4~5-year survival rate in the patients with high TRPM8 protein expression was significantly higher than that in the patients with low expression of TRPM8 protein （P=0.017）. The tumor maximum diameter in the patients with high TRPM8 protein expression was significantly smaller than that in the patients with low TRPM8 protein expression （P=0.028）. The viability, the number of colonies and the migration and invasion abilities of TRPM8-overexpressing A549 cells were significantly decreased as compared with empty vector and parental cells （P<0.01）. The results of flow cytometry analysis showed that the proportion of A549 cells at S stage was significantly increased in TRPM8 overexpression group as compared with empty vector group （P<0.01）. The growth rate and the weight of TRPM8-overexpressing H1299 cell-derived xenograft tumor in immunodeficient mice were significantly lower than those in empty vector group （P<0.01）. CONCLUSION TPRM8 is a tumor suppressor in lung adenocarcinoma, and low expression of TRPM8 protein was a poor prognositic indicator of patients with lung adenocarcinoma.     

Role of IP3R in LH-EGFR-induced mouse oocyte meiotic resumption

AIM: To explore the effect of inositol 1, 4, 5-trisposphate receptor (IP₃R) in luteinizing hormone-epidermal growth factor receptor (LH-EGFR)-induced oocyte meiotic resumption. METHODS: Models of mouse cumulus-oocyte complexs (COCs) culture and follicle culture in vitro were generated to study the effects of 2-aminoethyl diphenyl borate (2-APB) and heparin (IP₃R specific inhibitors) on LH/EGF-induced oocyte meiotic resumption and EGF-induced cumulus cell expansion. Real-time PCR was used to detect the mRNA expression of cumulus expansion-related factors. The changes of the intracellular calcium level were monitored using Fluo 3-AM, and the cGMP level was measured by ELISA. RESULTS: The inhibitors of IP₃R, 2-APB and heparin, dramatically reversed EGF-induced oocyte maturation (P<0.05) and decreased cGMP levels in COCs (P<0.05). In addition, 2-APB and heparin reversed EGF-induced cumulus expansion, and significantly inhibited EGF-induced cumulus expansion-related factor expression (P<0.05). The activation of IP₃R increased intracellular calcium level, and the study found that 2-APB and heparin dramatically reversed EGF-induced elevation of calcium level in cumulus cells (P<0.05). Follicular culture in vitro showed that 2-APB and heparin significantly reversed the LH-induced oocyte maturation (P<0.05). CONCLUSION: LH-EGFR signaling pathway increases calcium level in cumulus cells through IP₃R, resulting in meiotic resumption.     

Role and potential mechanism of fecal microbiota transplantation in treatment of chronic hepatitis B

AIM To investigate the effect of fecal microbiota transplantation （FMT） on the treatment of chronic hepatitis B （CHB） and the potential mechanism. METHODS Fifty C57BL/6J mice （6~8 weeks old） were divided into 5 groups： control group, CHB group, entecavir （ETV） group, comprehensive treatment （ETV+FMT, EFMT） group, and blocker （TAK-242+ETV+FMT, EFMT-TAK） group. The mice in each group were given corresponding treatment. The general condition of the mice was observed daily, and fecal specimens were kept every 10 d. The mice were sacrificed after 12 weeks, and the liver tissues and blood samples were collected. HE staining was used for histological scoring. Serum hepatitis B surface antigen （HBsAg） and interleukin-18 （IL-18） levels were measured by ELISA. Toll-like receptor 4 （TLR4） expression was detected by flow cytometry. Intestinal flora diversity was analyzed by high-throughput sequencing. RESULTS （1） Compared with control group, the body weight of the mice in CHB group was significantly reduced （P<0.05）. The body weight loss of the mice in ETV group, EFMT group and EFMT-TAK group was reversed to some extent as compared with CHB group （P<0.05）. （2） The histological score of the mice in CHB group was significantly higher than that in control group （P<0.05）. The score in ETV group was lower than that in CHB group （P<0.05）. The scores in EFMT group and EFMT-TAK group were lower than that in ETV group （P<0.05）, and that in EFMT-TAK group had a further downward trend compared with EFMT group （P<0.05）. （3） Compared with control group, the serum level of HBsAg in the CHB mice was significantly increased （P<0.05） and decreased after ETV treatment （P<0.05）. The HBsAg level in both EFMT group and EFMT-TAK group was significantly lower than that in ETV group （P<0.05）. （4） The IL-18 level in CHB group was significantly higher than that in control group （P<0.05）. After ETV treatment, the IL-18 level was decreased （P<0.05）, and that in both EFMT group and EFMT-TAK group was decreased more than that in ETV group （P<0.05）. （5） TLR4 expression in CHB group was higher than that in control group （P<0.05）, that in ETV group was lower than CHB group （P<0.05）, and that in EFMT group was further decreased （P<0.05）. （6） The heat map analysis at the class level showed that the abundances of Gammaproteobacteria, Deltaproteobacteria and Negativicutes in CHB group were significantly higher than those in control group, and those of Deltaproteobacteria and Negativicutes in EFMT group were close to those in control group. The heat map analysis at the family level indicated that the abundances of Burkholderiaceae, Desulfovibrionaceae and Veillonellaceae in CHB group were significantly higher than those in control group, while those in ETV group and EFMT group gradually approached normal levels. The α diversity index in CHB group was significantly decreased, while the diversity in ETV group was increased, that in EFMT group was further increased, and that in EFMT-TAK group was the highest. CONCLUSION FMT plays an active role in the treatment of CHB. The mechanism may be related to reducing the level of IL-18 and improving the structure and diversity of intestinal flora. The TLR4 signaling pathway is involved.     

Haplotype analysis of Tim-3 gene polymorphisms within asthma susceptible region 5q31-33

AIM: To investigate the relationship between Tim-3 gene polymorphisms within chromosome susceptible region 5q31-33 and asthma. METHODS: The genotypes of 4 SNPs (rs10053538, rs10515746, rs13170556 and rs9313441) in 118 allergic asthma nuclear pedigrees were analyzed by restriction fragment length polymorphism. Family-based association analysis method for transmission disequilibrium test (TDT) was employed for the data processing. Haplotypes and their frequencies were established and analyzed by TRANSMIT software. RESULTS: ① No transmission disequilibrium was found in 4 SNPs of Tim-3 from heterozygous parents onto patients in our 118 trios analyzed by TDT (P>0.05). The 4 SNPs were not associated with asthma. ② The family-based haplotype analysis showed that the haplotypes of Tim-3 constructed by rs10053538, rs13170556, rs9313441 were associated with asthma (2=10.83, P<0.05). The observed numbers of haplotype GGG from parents to the affected offsprings were less than the expected numbers, which showed a significant biased transmission (2=8.24,P<0.01). CONCLUSION: The Tim-3 gene in chromosome 5q31-33 itself or a locus nearby may confer susceptibility to asthma in this Chinese Han population.     

miR-485-5p inhibits viability and migration and invasion abilities of hepatocellular carcinoma Hep3B cells by targeting SOX5

AIM: To investigate the effects of microRNA-485-5p (miR-485-5p) on the viability, migration and invasion abilities of hepatocellular carcinoma cells and the underlying mechanism. METHODS: The expression levels of sex determining region Y-box 5 (SOX5) mRNA and miR-485-5p in the hepatocellular carcinoma Hep3B cells were detected by RT-qPCR with normal hepatocyte THLE-3 as control. Western blot was used to measure the expression levels of SOX5, proliferating cell nuclear antigen (PCNA), Ki67, cyclin D1 and matrix metalloproteinase-2 (MMP-2). The viability of Hep3B cells was measured by MTT assay. The migration and invasion abilities of the Hep3B cells were detected by Transwell assay. Dual-luciferase reporter assay system was applied to verify the relationship between miR-485-5p and SOX5. RESULTS: Compared with the control cells, the expression level of miR-485-5p was decreased in hepatocellular carcinoma cells Hep3B, Huh7 and HCCLM3 (P<0.05), while the expression of SOX5 at mRNA and protein levels were significantly increased (P<0.05). Over-expression of miR-485-5p inhibited the viability, migration and invasion of Hep3B cells. miR-485-5p targeted the 3′-UTR of SOX5 and negatively regulated the expression of SOX5. Knocking-down of SOX5 expression inhibited the viability, migration and invasion of Hep3B cells. Over-expression of SOX5 partially reversed the inhibitory effect of miR-485-5p over-expression on the viability, migration and invasion of Hep3B cells. CONCLUSION: miR-485-5p inhibits the viability, migration and invasion of Hep3B cells by targeting SOX5 gene. miR-485-5p is a potential molecular target for hepatocellular carcinoma.     

Effect of intermittent hypoxia on bladder detrusor cell apoptosis and regulatory mechanism of Alpiniae oxyphyllae Fructus

AIM To investigate the effect of intermittent hypoxia （IH） on bladder detrusor cells apoptosis and calcium channel, and to discuss the regulatory mechanism of Alpiniae oxyphyllae Fructus （AOF）. METHODS IH model of bladder detrusor cells was established by treating the cells with 6 cycles of 5% O₂ for 60 min and 20% O₂ for 30 min. Human bladder detrusor cells were cultured in vitro, randomly divided into 6 groups, each group had 8 holes. P₂X₃ receptor antagonist + IH （A） group, M₃ receptor antagonist + IH （B） group, β₃ receptor antagonist + IH （C） group, AOF + IH （D） group, saline + IH control （NC） group and air simulation control （AC） group were set up. The cells density and morphology were identified by the methods of counting chamber and immunofluorescence light microscopy （LM） after interventions. The apoptosis was analyzed by flow cytometry. Calcium channel expression was detected by patch clamp. RESULTS （1） Compared with the cells in AC group, the cells density and activity were significantly increased in NC group （P<0.05）; some cells appeared protrusions, turned round and blur in cell borders. （2） The results of immunofluorescence for detecting α-SMA protein expression showed that, compared with the cells in group AC, the mean absorbance （MA） in group NC was significantly increased （F=3.25, P<0.05）; compared with the cells in group NC, that in group A and group D was both decreased significantly （P<0.05）. （3） Compared with the cells in group AC, the apoptotic rate was significantly decreased in group NC （P<0.05）; Compared with the cells in group NC, the apoptotic rates in group A and group D were both significantly increased （P>0.05）. （4） Compared with the cells in group AC, calcium ion channel expression was significantly decreased （P<0.05）. Compared with the cells in group NC, calcium ion channel expression in AOF （100 mg/L） and AOF （50 mg/L） group was significantly increased （P<0.05）. CONCLUSION IH regulates bladder detrusor cells proliferation and apoptosis through P₂X₃ bladder nerve receptors, high or moderate dose of AOF may change calcium channel and play a protective role in IH induced cell damage.