[Explorations about the correlation between biological changes of meninges in periodontitis mice and cognitive impairment via single-cell RNA sequencing].

Objective: To clarify the potential correlation between biological changes of meninges in periodontitis mice and cognitive impairment by analyzing the biological changes of meninges in periodontitis mice using single-cell RNA sequencing. Methods: Thirty C57BL/6 mice were divided into two groups by using random number table method (15 mice in each group). Mice in the control group were locally administered 2% carboxyl methyl cellulose (CMC) without Porphyromonas gingivalis (Pg) on both buccal sides. A mixture of Pg and 2% CMC was applied on both buccal sides in the experimental group mice three times a week, lasting for 16 weeks in total. The absorption of alveolar bone, locomotor activity and cognitive function, the activation of microglia and astrocytes in the cortex were observed and assessed. The mRNA expression levels of Occludin in meninges and brain were detected in two groups. Single-cell RNA sequencing data of meninges were processed by uniform manifold approximation and projection (UMAP). Differential genes expressions of endothelial cells were processed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In addition, real-time fluorescence quantitative PCR (RT-qPCR) was used to verify the expressions of Atf3 and Apold1. Results: Methylene blue staining found the distances of buccal and palatal cement-enamel junction-alveolar bone crest (CEJ-ABC) in experimental mice [(185.60±17.60), (206.90±13.37) µm] increased significantly compared with the control group [(135.33±9.57), (163.05±14.98) µm] (t=5.02, P=0.002; t=4.37, P=0.005). Open field experiment showed the total distance and average speed of mice in the experimental group [(971.88±164.57) cm; (3.25±0.55) cm/s] were not statistically significant compared with the control group [(914.24±278.81) cm; (3.05±0.93) cm/s] (t=0.65, P=0.525; t=0.65, P=0.520). The recognition index of the experimental group [(48.02±16.92) %] was lower than the control group [(66.27±17.90) %] (t=2.40, P=0.027) by novel object recognition tests. Compared with the control group [(63.56±11.88) %], the alternation of experimental group [(50.99±14.17) %] was significantly decreased in Y maze tests (t=2.33, P=0.030). Immunohistochemistry results showed microglia and astrocytes were activated in the cortex of experimental mice. Compared with the control group (1.02±0.25, 1.04±0.31), the relative mRNA expressions of Occludin decreased significantly in the meninges and brain of periodontitis mice, respectively (0.61±0.10, 0.64±0.20) (t=3.47, P=0.010; t=2.66, P=0.024). By single-cell RNA sequencing, meninges cells were divided into 11 types, such as endothelial cells, fibroblasts, immune cells and so on. Endothelial cells were the main cell types in meninges [the control group: 26.47% (1 589/6 004), the experimental group: 26.26% (807/3 073)]. Compared with the control group [5.56% (334/6 004)], the percentage of granulocytes increased in the periodontitis mice [11.65% (358/3 073)]. Using clustering analysis to further focus on endothelial cells, GO enrichment analysis revealed differential genes were mainly related to angiogenesis, cell adhesion, apoptosis and so on. KEGG enrichment analysis revealed that differential genes were related to signaling pathways of interleukin-17, relaxin and so on. The relative mRNA expressions of Atf3 and Apold1 in meninges of periodontitis mice (0.42±0.24, 0.54±0.27) were significantly lower than the control group (1.03±0.26, 1.02±0.23) (t=3.88, P=0.005; t=3.02, P=0.017). Conclusions: The mice chronically infected with W83 occurred memory impairment, neuroinflammation and changes of barrier function. In the meninges of periodontitis mice, there were infiltration of immune cells and down-regulation expressions of Atf3 and Apold1 by single-cell RNA sequencing. Meningeal immunity and changes of barrier function may play an important role in the cognitive impairment caused by periodontitis.

[Evaluation of anticoagulant effect and predicted dose of low molecular weight heparin in hemodialysis by anti-â ©a factor activity].

The purpose of this study was to explore the reasonable dose of low molecular weight heparin (LMWH) in hemodialysis (HD) and the strategy of preventing extracorporeal circuit coagulation (ECC). A retrospective case-control study included patients who used LMWH for anticoagulation during maintenance hemodialysis (MHD) in the Hemodialysis Center of Beijing Hospital from December 2020 to January 2021. Basic data such as weight, height, basic kidney disease, dialysis age and anti-Ⅹa factor activity before, during and after dialysis were collected. A total of 46 patients were enrolled in this study, including 5 patients in coagulation group (10.9%) and 41 patients in non-coagulation group (89.1%). The anti-Ⅹa factor activity reached its peak at 0.5 h after the start of HD. The level of anti-Ⅹa factor was incorporated into the receiver operating characteristic curve (ROC curve). The results showed that the area under the ROC curve (AUC) was 0.802 (95% confidence interval: 0.651-0.54, P=0.029), and the cutoff was 0.31 IU/ml (sensitivity 1, specificity 0.683). It is suggested that the body surface area should be used as the basis to estimate the anticoagulant dose of LMWH in HD, and the activity of HD 4 h anti-Ⅹa factor ≤0.31 IU/ml, which is of diagnostic value for ECC. In addition, the results of binary logistic regression analysis showed that dialysis age was an independent risk factor for ECC (OR value 1.319, 95%CI 1.052-1.654, P=0.017). In summary, this study reveals that dialysis age may be a risk factor for ECC and that the activity of HD 4 h anti-Ⅹa factor ≤0.31 IU/ml can be used as a potential diagnostic cut-off point for ECC in HD patients, which provides a scientific basis for monitoring strategies to prevent blood coagulation in HD filters.

[Analysis of efficacy and safety of a new endoscopic anastomosis clip in the treatment of defects after endoscopic full-thickness resection].

Objective: To investigate the efficacy and safety of a new endoscopic anastomosis clip in the treatment of defects after endoscopic full-thickness resection (EFTR). Methods: Retrospective cohort study. Fourteen patients [4 males and 10 females, aged (55.9±8.2) years (45-69 years)] with gastric submucosal tumors underwent EFTR at the First Affiliated Hospital of Soochow University were included from December 2018 to January 2021. Patients were divided into new anastomotic clamp group (n=6) and nylon ring combined with metal clips group (n=8). Preoperative endoscopic ultrasound examinations were required to all patients to evaluate the wound condition. The size of the defect, operation time required for wound closure, success rate of closure, postoperative gastric tube placement time, postoperative hospital stay, incidence of complications, preoperative and postoperative serological indexes were compared between the two groups. All patients were followed up after the operation, among which the general endoscopy was reviewed in the first month after the operation, and the telephone and questionnaire follow-up were used in the second, third, sixth month and one year after the operation to evaluate the therapeutic effect of the new endoscopic anastomosis clip and nylon rope combined with metal clip after the EFTR operation. Results: Both groups successfully completed EFTR and were successfully closed. There was no significant difference between the age, tumor diameter and defect diameter of the two groups (all P>0.05). Compared with the nylon ring combined with metal clip group, the operation time of the new anastomotic clip group was shortened [(5.0±1.8) minutes vs (35.6±10.2) minutes, P<0.001]. The operation time was shortened [(62.2±12.5) minutes vs (92.5±0.2) minutes, P=0.007]. Postoperative fasting time decreased [(2.8±0.8) days vs (4.9±1.1) days, P=0.002]. The hospital stay after operation was also shortened [(5.2±0.8) days vs (6.9±1.5) days, P=0.023]. The total intraoperative bleeding volume decreased [(20.00±5.48) ml vs (35.63±14.75) ml, P=0.031]. The patients in both groups received endoscopic examination 1 month after operation, and there was no delayed perforation and bleeding after operation. There was no obvious symptoms of discomfort. Conclusion: The new anastomotic clamp is suitable for the treatment of full-thickness gastric wall defects after EFTR, and shows advantages of shorter operation, less bleeding, and fewer postoperative complications.

Effect of whole-plant corn silage treated with lignocellulose-degrading bacteria on growth performance, rumen fermentation, and rumen microflora in sheep.

Lignification of cellulose limits the effective utilisation of fibre in plant cell wall. Lignocellulose-degrading bacteria secrete enzymes that decompose lignin and have the potential to improve fibre digestibility. Therefore, this study aimed to investigate the effect of whole-plant corn silage inoculated with lignocellulose-degrading bacteria on the growth performance, rumen fermentation, and rumen microbiome in sheep. Twelve 2-month-old male hybrid sheep (Dorper ♂ × small-tailed Han ♀) were randomly assigned into two dietary groups (n = 6): (1) untreated whole-plant corn silage (WPCS) and (2) WPCS inoculated with bacterial inoculant (WPCSB). Whole-plant corn silage inoculated with bacterial inoculant had higher in situ NDF digestibility than WPCS. Sheep in the WPCSB group had significantly higher average daily gain, DM intake, and feed conversion rate than those in the WPCS group (P < 0.05). Furthermore, higher volatile fatty acid concentrations were detected in WPCSB rumen samples, leading to lower ruminal pH (P < 0.05). The WPCSB group showed higher abundance of Bacteroidetes and lower abundance of Firmicutes in the rumen microbiome than the WPCS group (P < 0.05). Multiple differential genera were identified, with Prevotella being the most dominant genus and more abundant in WPCSB samples. Moreover, the enriched functional attributes, including those associated with glycolysis/gluconeogenesis and citrate cycle, were more actively expressed in the WPCSB samples than in the WPCS samples. Additionally, certain glucoside hydrolases that hydrolyse the side chains of hemicelluloses and pectins were also actively expressed in the WPCSB microbiome. These findings suggested that WPCSB increased NDF digestibility in three ways: (1) by increasing the relative abundance of the most abundant genera, (2) by recruiting more functional features involved in glycolysis/gluconeogenesis and citrate cycle pathways, and (3) by increasing the relative abundance and/or expression activity of the glucoside hydrolases involved in hemicellulose and pectin metabolism. Our findings provide novel insights into the microbial mechanisms underlying improvement in the growth performance of sheep/ruminants. However, the biological mechanisms cannot be fully elucidated using only metagenomics tools; therefore, a combined multi-omics approach will be used in subsequent studies.

[The value of soluble thrombomodulin in evaluating endothelial injury in patients with kidney disease].

Objective: To evaluate the value of soluble thrombomodulin (sTM) in evaluating endothelial injury in patients with kidney disease. Methods: One hundred and thirty-three patients who first visited the Department of Nephrology of Beijing hospital for various reasons from September 2020 to January 2021 and 130 healthy people were collected and divided into groups according to age, gender, primary disease, complications and so on. The differences of sTM and serum creatinine in patients with different diseases and renal disease stages were analyzed. Results: For patients with chronic kidney disease (CKD), sTM increased significantly with the decrease of renal function. The level of sTM in patients with CKD stage 1-5 was (0.013±0.007), (0.019±0.010), (0.022±0.008), (0.027±0.008), (0.033±0.006)TU/L, respectively (F=21.005,P<0.05). There was no significant difference in the level of sTM between patients with non-CKD urinary tract infection (0.013±0.009) TU/L and patients with stage 1 CKD (t=1.023, P>0.05). No matter whether the patients were complicated with infection or cardiovascular disease, there was no significant difference in sTM level under the condition of serum creatinine matching (all P>0.05). In 4 patients with acute renal injury, serum creatinine returned to normal after active treatment, but sTM did not decrease significantly. Correlation analysis showed that there was a positive correlation between sTM and serum creatinine (r=0.697, P<0.01). Conclusion: sTM can evaluate the renal function damage of patients with CKD more early, and the level of sTM in patients with renal disease is more related to the degree of endothelial damage.

Dioscin ameliorates methotrexate-induced liver and kidney damages via adjusting miRNA-145-5p-mediated oxidative stress.

Dioscin, one natural product, has various pharmacological actions. However, its effects on methotrexate (MTX)-induced hepatorenal damages still remain unknown. In the present study, the data manifested that dioscin restored the viabilities of L-02 and NRK-52E cells, reduced ALT, AST, Cr, BUN levels, and ameliorated histopathological changes of liver and kidney. Besides, dioscin decreased ROS levels in cells, and adjusted SOD, MDA, GSH and GSH-Px levels in rats. Dioscin reduced the expression levels of miR-145-5p which directly targeted Sirt5, and then regulated the expression levels of SOD1, Nrf2, Gst, Keap1, HO-1, GCLC and NQO1. MiR-145-5p mimic in cells deteriorated ROS levels and decreased Sirt5 expression to accentuate oxidative stress by regulating the expression levels of SOD1, Nrf2, Keap1, which were all reversed by dioscin. Moreover, MTX-induced hepatorenal damage were worsened in mice by Sirt5 siRNA or miR-145-5p agomir, which were also alleviated by dioscin. Dioscin relieved MTX-induced hepatorenal damages through regulating miR-145-5p-medicated oxidative stress, which should be considered as one effective drug to treat the disorder in future.

MiR-23a-5p exacerbates intestinal ischemia-reperfusion injury by promoting oxidative stress via targeting PPAR alpha.

MiR-23a-5p is involved in the occurrence and development of some serious diseases, but its effects on intestinal ischemia-reperfusion (II/R) injury is unclear. In this research, the hypoxia/reoxygenation (H/R) model on IEC-6 cells and II/R model in mice were used. The data showed that the ROS level in model group was significantly increased compared with control group. The level of intestinal MPO was increased and serum SOD was decreased in mice compared with sham group. Moreover, the expression levels of miR-23a-5p in model groups were obviously increased in vitro and in vivo, while the expression levels of PPARα, FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2 were significantly decreased. The double luciferase reporter gene assay showed that there was binding site between miR-23a-5p and PPARα. When miR-23a-5p was inhibited or PPARα gene was overexpressed, H/R-caused cell damage was alleviated and ROS level was decreased compared with NC group. PPARα expression level was increased, accompanied by the increased levels of FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2. After enhancing miR-23a-5p expression or silencing PPARα gene, H/R-caused cell damage was further aggravated compared with NC group, and ROS level was increased associated with the decreased levels of FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2. Our study demonstrated that miR-23a-5p exacerbated II/R injury by promoting oxidative stress via targeting PPARα, which should be considered as one new drug target to treat II/R injury.

MicroRNA-142-3p attenuates hepatic ischemia/reperfusion injury via targeting of myristoylated alanine-rich C-kinase substrate.

MiR-142-3p as one key molecule in oncogenesis and inflammation plays crucial roles in hepatic fibrosis, hepatocellular carcinoma and other liver disease. However, there have no literatures to report its effects on hepatic ischemia-reperfusion (HI/R) injury. In the present work, hypoxia reoxygenation (H/R) models on AML12 and HepG2 cells, and ischemia/reperfusion model in mice were established. The methods of real-time PCR, dual luciferase reporter, mimic, inhibitor, agomir, antagomir and siRNA transfection assays were used. The expression levels of miR-142-3p were decreased in model groups in vitro and in vivo compared with control group or Sham group, which directly targeted MARCKS to regulate its expression. Then, MARCKS activated p38/JNK signal, up-regulated NF-κB expression to accelerate inflammation, and inhibited PI3K/AKT signal to promote apoptosis. Moreover, miR-142-3p mimic in vitro and agomir in vivo lowered the expression levels of MARCKS, thereby alleviating apoptosis and inflammation to relieve HI/R injury. Furthermore, miR-142- 3p inhibitor in vitro and antagomir in vivo up-regulated the expression levels of MARCKS to aggravate HI/R damage via promoting inflammation and apoptosis. Consistently, MARCKS siRNA markedly inhibited HI/R injury by restraining apoptosis and inflamm- ation in mice. MiR-142-3p played a considerable part in adjusting HI/R injury by targeting MARCKS, and miR-142-3p/MARCKS should be a new therapeutic target for HI/R injury.

MicroRNA-27a-3p aggravates renal ischemia/reperfusion injury by promoting oxidative stress via targeting growth factor receptor-bound protein 2.

Renal ischemia-reperfusion (RI/R) injury with high morbidity and mortality is one common clinical disease. Development of drug targets to treat the disorder is critical important. MiR-27a-3p plays important roles in regulating oxidative stress. However, its effects on RI/R injury have not been reported. In this paper, hypoxia/reoxygenation (H/R) models on NRK-52E and HK-2 cells, and RI/R model in C57BL/6 mice were established. The results showed that H/R in vitro decreased cell viability and increased ROS levels in cells, and RI/R caused renal injury and oxidative damage in mice. The expression levels of miR-27a-3p were up-regulated based on real-time PCR and FISH assays in model groups compared with control groups, which directly targeted Grb2 based on dual luciferase reporter assay and co-transfaction test. In addition, miR-27a- 3p markedly reduced Grb2 expression to down-regulate the expression levels of p-PI3K, p-AKT, Nrf2, HO-1, and up-regulate Keap1 expression in model groups. MiR-27a-3p mimics in vitro enhanced H/R-caused oxidative stress via increasing ROS levels and decreasing Grb2 expression to down-regulate PI3K-AKT signal. In contrary, miR-27a-3p inhibitor in vitro significantly reduced H/R-caused oxidative damage via decreasing ROS levels and increasing Grb2 expression to up-regulate PI3K-AKT signal. In vivo, miR-27a- 3p agomir exacerbated RI/R-caused renal damage by decreasing SOD level and increasing Cr, BUN, MDA levels via suppressing Grb2 expression to down-regulate PI3K- AKT signal. However, miR-27a -3p antagomir alleviated RI/R-caused oxidative damage via increasing Grb2 expression to up-regulate PI3k-AKT signal. Grb2siRNA in mice further enhanced RI/R-caused renal injury by increasing Cr, BUN, MDA levels and decreasing SOD level via inhibiting the expression levels of Grb2, Nrf2, HO-1, and increasing Keap1 expression. Our data showed that miR-27a-3p aggravated RI/R injury by promoting oxidative stress via targeting Grb2, which should be considered as one new drug target to treat RI/R injury.

Effect and possible mechanisms of dioscin on ameliorating metabolic glycolipid metabolic disorder in type-2-diabetes.

BACKGROUND: Our previous study revealed that microRNA-125a-5p plays a crucial role in regulating hepatic glycolipid metabolism by targeting STAT3 in type 2 diabetes mellitus (T2DM). Dioscin, a major active ingredient in Dioscoreae nipponicae rhizomes, displays various pharmacological activities, but its role in T2DM has not been reported. PURPOSE: The aim of this study was to investigate the effect of dioscin on T2DM and elucidate its potential mechanism. METHODS: The effect of dioscin on glycolipid metabolic disorder in insulin-induced HepG2 cells, palmitic acid-induced AML12 cells, high-fat diet- and streptozotocin- induced T2DM rats, and spontaneous T2DM KK-Ay mice were evaluated. Then, the possible mechanisms of dioscin were comprehensively evaluated. RESULTS: Dioscin markedly alleviated the dysregulation of glycolipid metabolism in T2DM by reducing hyperglycemia and hyperlipidemia, improving insulin resistance, increasing hepatic glycogen content, and attenuating lipid accumulation. When the mechanism was investigated, dioscin was found to markedly elevate miR-125a-5p level and decrease STAT3 expression. Consequently, dioscin increased phosphorylation levels of STAT3, PI3K, AKT, GSK-3ß, and FoxO1 and decreased gene levels of PEPCK, G6Pase, SREBP-1c, FAS, ACC, and SCD1, leading to an increase in glycogen synthesis and a decrease in gluconeogenesis and lipogenesis. The effects of dioscin on regulating miR-125a-5p/STAT3 pathway were verified by miR-125a-5p overexpression and STAT3 overexpression. CONCLUSIONS: Dioscin showed potent anti-T2DM activity by improving the inhibitory effect of miR-125a-5p on STAT3 signaling to alleviate glycolipid metabolic disorder of T2DM.

[Value of RNA oxidation product 8-oxo-Gsn in evaluating renal function in patients with chronic kidney disease].

Objective: To investigate the relationship of DNA oxidative product 8-oxo-dGsn and RNA oxidative product 8-oxo-Gsn with chronic kidney disease (CKD). Methods: Between January 2015 and December 2016, 146 cases of CKD (30, 30, 31, 30 and 25 cases of CKD stage 1-5, respectively) were collected in the Department of Nephrology in Beijing Hospital. Among them, 70 cases were male, accounting for 47.95%. The age distribution ranged from 21 to 88 years, with an average age of (56.43±16.79) years. Their fasting blood and morning urine were collected. The levels of 8-oxo-dGsn and 8-oxo-Gsn in plasma and urine were quantified by isotope-diluted liquid chromatography mass spectrometry (MS)/MS (ID-LC-MS/MS). Results: The urine 8-oxo-Gsn/Cr in patients with CKD stage 1-5 was (3.07±1.07) µmol/mol, (3.42±1.34) µmol/mol, (3.72±1.47) µmol/mol, (3.90±1.93) µmol/mol and (3.75±2.26) µmol/mol, respectively. The urinary 8-oxo-Gsn content in CKD stage 4 patients was significantly higher than those of other 4 stages (P<0.05). The serum/urine ratio of 8-oxo-Gsn was 0.02±0.02, 0.03±0.02, 0.06±0.04, 0.10±0.05 and 0.34±0.03, respectively, and in CKD stage 4 and 5 patients, it increased significantly, especially in CKD stage 5 cases (P<0.05). Expression of 8-oxo-Gsn had a good correlation with renal function[the Spearman 's correlation coefficient: serum 8-oxo-Gsn and serum creatinine was 0.629 (P<0.001); urine/serum 8-oxo-Gsn and eGFR was 0.799 (P<0.001); serum/urine 8-oxo-Gsn and serum/urine creatinine was 0.888 (P<0.001)]. With age increasing, CKD patients showed increased RNA oxidation, and 8-oxo-Gsn increased significantly in patients over 60 years (P<0.05). After multiple linear regression analysis, 8-oxo-Gsn was only associated with serum creatinine (ß=0.656, t=8.275, P<0.001). Conclusions: Our finding indicates that the RNA oxidation occurs in patients with renal disease, and its oxidation increased as the disease progressing. The significant increase in the ratio of plasma and urinary 8-oxo-Gsn is of great importance on evaluating renal function.

The expression research of miR-210 in the elderly patients with COPD combined with ischemic stroke.

OBJECTIVE: We conduct this study to investigate the expression level of miR-210 in elderly patients with COPD combined with ischemic stroke and analyzed its application value as a sensitive diagnostic indicator. PATIENTS AND METHODS: 50 cases of elderly patients with COPD combined with ischemic stroke were selected as group A, 50 cases of elderly patients with COPD were selected as group B, 50 cases of elderly patients with ischemic stroke were selected as group C, and 50 cases of healthy volunteers as group D. Real-time PCR assay for quantification was used to detect the expression level of miR-210 in peripheral blood and receiver operating curve (ROC) was used to analyze the diagnostic sensitivity and accuracy. RESULTS: MiR-210 level of group A was the lowest, followed by group B and group C; group D had the highest levels. The difference was statistically significant (p<0.05). MiR-210 levels decreased with an increasing decline degree of lung function and the difference was statistically significant (p<0.05). After miR-210 diagnosis, COPD sensitivity was 85.6%, the specificity was 72.6%, accuracy (AUC) was 0.821, and 95% CI 0.632-0.924. CONCLUSIONS: The down-regulation of MiR-210 expression in the COPD combined with ischemic stroke can be regarded as a sensitive index in diagnosis of COPD and ischemic stroke.

[Effect of nicotinic acetylcholine receptor α7 subunit gene on liver inflammatory reaction in mice with nonalcoholic steatohepatitis and related mechanisms].

Objective: To investigate the effect of nicotinic acetylcholine receptor α7 (α7nAChR) subunit gene on liver inflammation in mice with nonalcoholic steatohepatitis (NASH) and related mechanisms. Methods: C57BL/6J mice and α7nAChR gene knockout mice were fed for 24 weeks to establish the NASH model, and the mice were sacrificed to isolate and culture the primary liver macrophages. After the treatment with nicotine and endotoxin, ELISA was used to measure the levels of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in supernatant; indirect immunofluorescence assay and Western blot were used to observe the effect on the NF-κB signaling pathway, and quantitative PCR was used to measure the mRNA expression of Toll-like receptor-4 (TLR-4) in macrophages. An analysis of variance was used for comparison of means between multiple groups. Results: The results of ELISA showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher levels of IL-6 and TNF-α in supernatant (IL-6: 1 599±65 pg/ml vs 1 465±45 pg/ml, P < 0.05; TNF-α: 1 567±66 pg/ml vs 1 433±50 pg/ml, P < 0.05). The results of Western blot showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative protein expression of phosphorylated NF-κB and TLR-4 (NF-κB: 69 425±600 vs 51 133±200, P < 0.05; TLR-4: 93 387±684 vs 64 198±630, P < 0.05). The results of indirect immunofluorescence assay showed that the endotoxin+nicotine group of gene knockout NASH mice had a significantly higher fluorescence intensity of NF-κB than the endotoxin+nicotine group of C57 NASH mice. The results of PCR showed that the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative mRNA expression of TLR-4 than the endotoxin+nicotine group of C57 NASH mice (4.13±0.13 vs 2.93±0.14, P < 0.05). Conclusion: The α7nAChR gene knockout can aggravate the degree of inflammatory reaction in NASH, and its mechanism may be related to the fact that the NF-κB signaling pathway cannot be inhibited, which aggravates inflammatory reaction.

Effect of dual Bt-expression transformation vectors on transgene expression in tobacco.

This study aimed to determine the influence of vector structure on dual Bt gene expression and establish an efficient expression vector using Cry1Ac and Cry3A genes. Four vectors (N4, N5, N10, and S23) were developed and used for genetic transformation of tobacco to obtain insect-resistant transgenic lines. The vectors were constructed using the MAR structure, applying different promoter and enhancer sequences, and changing the transgene open-reading frame sequence. The average Cry1Ac toxalbumin expression quantity was 67 times higher in N5 than in N4 transgenic lines (8.77 and 0.13 µg/g, respectively). In contrast, the average Cry3A toxalbumin expression quantity was 1.5 times higher in N4 than in N5 lines (12.70 and 8.21 µg/g, respectively). The sequences of both Bt genes significantly influenced toxalbumin expression, although upstream Bt genes presented lower expression levels. The average Cry1Ac toxalbumin content was 13 times higher in the transgenic lines of AtADH 5'-non-translated sequence N5 (8.77 mg/g) than in the omega N10 lines (0.67 mg/g). Furthermore, the average Cry1Ac toxalbumin content was 5 times higher in MAR N5 than in non-MAR S23 lines (8.77 and 1.63 mg/g, respectively). The average Cry3A toxalbumin content was 1.3 times higher in N5 than in S23 lines (8.21 and 6.48 mg/g, respectively). Moreover, toxalbumin expression levels differed significantly among the S23-transformed lines. The MAR structure applied on both ends of the genes increased both the level and stability of exogenous gene expression. In conclusion, N5 was the most optimal of the four tested vectors.

Effect of human umbilical cord mesenchymal stem cells on endometriotic cell proliferation and apoptosis.

The objective of this study was to observe the effects of human umbilical cord mesenchymal stem cells (UCMSCs) on the proliferation and apoptosis of endometriotic cells. Endometriotic cells and UCMSCs were primarily cultured in vitro. In the experimental group, a UCMSC and endometriotic cell non-contact co-culture system was established. The control group consisted of 1 x 10(5) endometriotic cells cultured alone. The proliferation and apoptosis of endometriotic cells were respectively detected using the MTT method and flow cytometry. The mRNA expression level of the tensin homologue gene (PTEN) in endometriotic cells was detected by reverse transcription-polymerase chain reaction amplification. Compared with the control group, the proliferation of endometriotic cells in the experimental group was clearly inhibited (P < 0.05) and time-dependent (P < 0.05). In addition, the number of apoptotic cells were significantly increased (P < 0.05), and the amount of cells, which entered S phase from G1 phase, decreased significantly. Furthermore, the mRNA expression level of the PTEN gene in the experimental group was significantly higher than in the control group (P < 0.05). These results suggest that UCMSCs might inhibit the proliferation of human endometriotic cells in vitro and promote their apoptosis by upregulating the expression of PTEN.

Molecular tracing of white muscardine in Asian corn borer using inter-simple sequence repeat (ISSR) analysis.

Beauveria bassiana is a soil fungus that parasitizes arthropod species, and is used to control the Asian corn borer in Northeast China. In this study, B. bassiana was investigated in Xiaoxian County and Baicheng City, and the results were compared with those of Gongzhuling City, where the fungus was not applied. Using the inter-simple sequence repeat (ISSR) molecular marker technique, 198 isolates were extracted from Asian corn borer and other insect cadavers, and soil and air, and two released strains were analyzed to trace the infection source. In Xiaoxian and Baicheng populations, artificially released B. bassiana subpopulations were more abundant than indigenous fungi, and the released strains were the main cause of disease in those areas. Artificial B. bassiana displayed positive effect on overwintering of Asian corn borers in corn straw stacks in Xiaoxian County. Indigenous populations in Gongzhuling City showed higher genetic variation. In summary, we identified a significant correlation between genetic distance and geographic distance (P < 0.01).