[The efficacy and side effects of rigosertib combined with chemotherapy in KRAS mutant colorectal cancer mice].

Objective: To investigate the effect of rigosertib (RGS) combined with classic chemotherapy drugs including 5-fluorouracil, oxaliplatin, and irinotecan in colorectal cancer. Methods: Explore the synergy effects of RGS and 5-fluorouracil (5-FU), oxaliplatin (OXA), and irinotecan (IRI) on colorectal cancer by subcutaneously transplanted tumor models of mice. The mice were randomly divided into control group, RGS group, 5-FU group, OXA group, IRI group, 5-FU+ RGS group, OXA+ RGS group and IRI+ RGS group. The synergy effects of RGS and OXA on KRAS mutant colorectal cancer cell lines in vitro was detected by CCK-8. Ki-67 immunohistochemistry and TdT-mediated dUTP nick-end labeling (TUNEL) staining were performed on the mouse tumor tissue sections, and the extracted tumor tissue was analyzed by western blot. The blood samples of mice after chemotherapy and RGS treatment were collected, blood routine and liver and kidney function analysis were conducted, and H&E staining on liver sections was performed to observe the side effects of chemotherapy and RGS. Results: The subcutaneously transplanted tumor models were established successfully in all groups. 55 days after administration, the fold change of tumor size of OXA+ RGS group was 37.019±8.634, which is significantly smaller than 77.571±15.387 of RGS group (P=0.029) and 92.500±13.279 of OXA group (P=0.008). Immunohistochemical staining showed that the Ki-67 index of tumor tissue in control group, OXA group, RGS group and OXA+ RGS group were (100.0±16.8)%, (35.6±11.3)%, (54.5±18.1)% and (15.4±3.9)%, respectively. The Ki-67 index of OXA+ RGS group was significantly lower than that in control group (P=0.014), but there was no significant difference compared to OXA group and RGS group (OXA: P=0.549; RGS: P=0.218). TUNEL fluorescence staining showed that the apoptotic level of OXA+ RGS group was 3.878±0.547, which was significantly higher than 1.515±0.442 of OXA group (P=0.005) and 1.966±0.261 of RGS group (P=0.008). Western blot showed that the expressions of apoptosis related proteins such as cleaved-PARP, cleaved-caspase 3 and cleaved-caspase 8 in the tumor tissues of mice in the OXA+ RGS group were higher than those in control group, OXA group and RGS group. After the mice received RGS combined with chemotherapy drugs, there was no significant effect on liver and kidney function indexes, but the combined use of oxaliplatin and RGS significantly reduced the white blood cells [(0.385±0.215)×10(9)/L vs (5.598±0.605)×10(9)/L, P<0.001] and hemoglobin[(56.000±24.000)g/L vs (153.333±2.231)g/L, P=0.001] of the mice. RGS, chemotherapy combined with RGS and chemotherapy alone did not significantly increase the damage to liver cells. Conclusions: The combination of RGS and oxaliplatin has a stronger anti-tumor effect on KRAS mutant colorectal cancer. RGS single agent will not cause significant bone marrow suppression and hepatorenal injury in mice, but its side effects may increase correspondingly after combined with chemotherapy.

The effect of manganese and iron on mediating resuscitation of lactic acid-injured Escherichia coli.

Lactic acid can induce sublethal injury of E. coli through oxidative stress. In this study, we investigated changes in SOD activity, CAT activity, GSH production and ROS production during sublethal injury and resuscitation of E. coli. Then, the effect of manganese and iron during resuscitation were studied. Both cations (≥1 mmol l-1 ) significantly promoted the resuscitation of sublethally injured E. coli induced by lactic acid and shortened the repair time (P < 0·05). Conversely, addition of N,N,N',N'-tetrakis (2-pyridylmethyl) which is a metal chelator extended the repair time. Compared with minA, manganese and iron significantly improved SOD activity at 40, 80 and 120 min and decreased ROS production at 40 and 80 min, thereby recovering injured E. coli quickly (P < 0·05). The deletion of sodA encoding Mn-SOD, sodB encoding Fe-SOD or gshA/gshB encoding GSH significantly strengthened sublethal injury and extended the repair time (P < 0·05). It meant these genes-related oxidative stress played important roles in the acid resistance of E. coli and recovery of sublethal injury. Therefore, manganese and iron can promote the recovery of lactic-injured E. coli by the way of increasing SOD activity, scavenging ROS, and relieving oxidative stress.

[Analysis of pathogen monitoring results of infectious diarrhea in Beijing Traditional Chinese Medicine Hospital Affiliated to Capital Medical University from 2016 to 2019].

Objective: to analyze the distribution characteristics of major enteropathogens in infectious diarrhea cases attending the intestinal outpatient clinic of Beijing Traditional Chinese medicine hospital, Capital Medical University. Methods: From 2016 to 2019, 588 fecal samples of patients with infectious diarrhea in Beijing Hospital of traditional Chinese Medicine Affiliated to Capital Medical University were collected for microbial isolation, culture, identification and pathogen gene detection. Using VITEK 2 compact full-automatic microbial identification/drug sensitivity analysis system to identify the bacteria isolated from the culture; using serum agglutination test to classify the pure colonies; using multiple fluorescence quantitative PCR amplification technology to detect the gene amplification of the samples. Results: In 2016-2019, the total physical examination rate of pathogen was 39.796%. The top three pathogen were diarrhea Escherichia coli (21.769%, n=128), Salmonella (5.782%, n=34), Vibrio (4.762%, n=28). The difference of positive rates of different pathogens in four years was statistically significant (P=0.021), and the peak of incidence was from July to September. The positive rate of norovirus was 5.612% (n=33), and the highest incidence occurred in May. Conclusion: The pathogen of infectious diarrhea patients in Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University from April to October 2016-2019 is mainly diarrhea Escherichia coli, and the pathogen type of norovirus is GⅡ genome.

KCNQ1 opposite strand/antisense transcript 1 promotes aggressive biological behaviors of cervical cancer cells via regulating microRNA-491-5p and pyruvate kinase M1/2.

Long non-coding RNA (lncRNA) KCNQ1 and opposite strand/antisense transcript 1 (KCNQ1OT1) have been validated to be carcinogenic in several cancers. However, the role of KCNQ1OT1 in regulating the malignant biological behavior and radiotherapy resistance of cervical cancer (CC) remains largely unknown. Quantitative real time-polymerase chain reaction (qRT-PCR) was carried out to detect KCNQ1OT1 and miR-491-5p expression in CC tissues and cells. Pyruvate kinase M1/2 (PKM2) expression was detected by Western blot. CC cell proliferation, movement, migration and invasion were monitored by CCK-8, scratch healing and Transwell assay, respectively. The CC cell colony survival was detected by colony formation assay under different doses of radiation. Dual luciferase reporter gene assay, pull-down assay and RIP assay were employed to verify the targeting relationship between KCNQ1OT1, miR-491-5p and PKM2. In this study, KCNQ1OT1 was significantly up-regulated in CC patient cancerous tissues and cell lines, and its high expression was significantly related to tumor volume increase and poor differentiation. KCNQ1OT1 overexpression significantly promoted CC cell proliferation, metastasis and radioresistance. On the contrary, KCNQ1OT1 knockdown compared to the control group inhibited the above biological behavior of CC cells. The underlying mechanism suggested that KCNQ1OT1 promoted progression and radioresistance of CC by modulating the miR-491-5p/PKM2 axis. In conclusion, KCNQ1OT1 enhances CC cell progression through the miR-491-5p/PKM2 axis.

[The effects and immunologic mechanisms of Helicobacter pylori infection on AOM/DSS induced colitis-associated cancer in mice].

Objective: To investigate the effects and potential mechanisms of Helicobacter pylori (H.pylori) infection on azoxymethane (AOM)/dextran sulfate sulphate (DSS) induced colitis-associated cancer (CAC) in mice. Methods: A total of 60 specific pathogen free C57BL/6J mice were randomly divided into four groups: normal control group (control group, n=9), H. pylori-infected group (Hp group, n=9), AOM/DSS-treated group (AOM/DSS group,n=21) and AOM/DSS-treated with H.pylori infection group (Hp+AOM/DSS group, n=21). Mice were sacrificed on day19, 45 or 85 after AOM/DSS challenge. Histopathological changes in colonic tissues were determined by hematoxylin and eosin staining. Flow cytometry analysis was performed to determine T helper cells 17 (Th17) and regulatory T cells (Treg) in colonic lamina propria. The expression levels of Th17-and Treg-associated cytokines and transcription factors [interleukin (IL)-10, IL-17A, retinoic acid receptor-related orphan receptor γt (RORγt) and forkhead box P3 (Foxp3)] were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Results: There were no histopathological changes in colonic tissues of mice in control group and Hp group. H.pylori colonization reduced the histopathological scores at the stages of colitis (day 19) and dysplasia (day 45), and also decreased tumor load (day 85) in mice treated with AOM/DSS (all P<0.05). Compared with AOM/DSS group, the percentages of CD3(+)CD4(+)IL-17A(+)Th17 and CD3(+)CD4(+)IL-17A(+)Foxp3(+)Treg cells (1.88±0.17 vs 2.07±0.89, 1.06±0.13 vs 1.89±0.23) and the expression levels of RORγt and IL-17A (1.08±0.59 vs 2.35±1.35, 2.96±0.92 vs 7.78±4.57) were decreased in colonic tissues of Hp+AOM/DSS group (all P<0.05). The percentages of CD3(+)CD4(+)CD25(+)Foxp3(+)Treg and CD3(+)CD4(+)IL-10(+)Foxp3(+)Treg cells (20.60±3.39 vs 15.63±2.71, 2.94±0.52 vs 2.14±0.47) and the expression levels of Foxp3 and IL-10 [17.59(13.77,24.87) vs 6.27(4.41,13.36), 3.52(1.59,5.99) vs 1.17(1.15,2.75)] in colonic tissues were higher (all P<0.05) in mice of Hp+AOM/DSS group compared with AOM/DSS group on day 85. Conclusion: H.pylori infection slows the progress from inflammation to tumor in a AOM/DSS induced CAC modal, accompanied with the downregulation of Th17 response and upregulation of Treg response.

Physicochemical properties and cytotoxicity of an experimental resin-based pulp capping material containing the quaternary ammonium salt and Portland cement.

AIM: To evaluate in vitro the physicochemical properties, cytotoxicity and calcium phosphate nucleation of an experimental light-curable pulp capping material composed of a resin with antibacterial monomer (MAE-DB) and Portland cement (PC). METHODOLOGY: The experimental material was prepared by mixing PC with a resin containing MAE-DB at a 2 : 1 ratio. Cured pure resin containing MAE-DB served as control resin. ProRoot MTA and Dycal served as commercial controls. The depth of cure, degree of monomer conversion, water absorption and solubility of dry samples, calcium release, alkalinizing activity, calcium phosphate nucleation and the cytotoxicity of materials were evaluated. Statistical analysis was carried out using anova followed by Tukey's HSD test (equal variance assumed) or Tamhane test (equal variance not assumed) and independent-samples t-tests. RESULTS: The experimental material had a cure depth of 1.19 mm, and the mean degree of monomer conversion was 70.93% immediately post-cure and 88.75% at 24 h post-cure. The water absorption of the experimental material was between those of MTA and Dycal, and its solubility was significantly less (P < 0.05) than that of Dycal and higher than that of MTA. The experimental material exhibited continuous calcium release and an alkalinizing power between those of MTA and Dycal throughout the test period. Freshly set experimental material, control resin and all 24-h set materials had acceptable cytotoxicity. The experimental material, MTA and Dycal all exhibited the formation of apatite precipitates after immersion in phosphate-buffered saline. CONCLUSIONS: The experimental material possessed adequate physicochemical properties, low cytotoxicity and good calcium phosphate nucleation.

[Clinicopathologic features of drug-induced vanishing bile duct syndrome].

Vanishing bile duct syndrome (VBDS) manifests as progressive destruction and disappearance of the intrahepatic bile duct caused by various factors and cholestasis. VBDS associated with drug-induced liver injury (D-VBDS) is an important etiology of VBDS, and immune disorder or immune imbalance may be the main pathogenesis. According to its clinical symptoms, serological markers, and course of the disease, D-VBDS is classified into major form and minor form, and its clinical features are based on various pathomorphological findings. Its prognosis is associated various factors including regeneration of bile duct cells, number of bile duct injuries, level and range of bile duct injury, bile duct proliferation, and compensatory shunt of bile duct branches. This disease has various clinical outcomes; most patients have good prognosis after drug withdrawal, and some patients may experience cholestatic cirrhosis, liver failure, and even death. Due to the clinical manifestation and biochemical changes are similar to the primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), it need to identify by clinical physician.

[PrepFiler Express BTATM Lysis Buffer Combined with Silicon Microbeads for Rapid DNA Extraction from Bone].

OBJECTIVES: To establish a convenient and rapid method for extracting DNA from bone. METHODS: Fifteen long bone samples were washed and sterilized. The skeletal fragments were obtained by electric drill, and lysed by PrepFiler Express BTA™ lysis buffer. DNA was then manually extracted by silicon microbeads for further analysis. RESULTS: STR genotyping was successfully obtained in 14 out of the 15 samples, and the detection rate was 93.33%. CONCLUSIONS: The method for DNA extraction from bone established in present study is convenient, quick, effective, and with a strong applicability, which is worth spreading and applying.

[Expression and significance of serum microRNA-135a-5p level in colorectal cancer].

Objective: To explore the expression level of serum miR135a-5p and its diagnostic value in colorectal cancer (CRC). Methods: Serum samples were randomly collected from 60 primary CRC patients, 40 patients with intestinal polyps and 50 healthy controls, and the serum concentrations of miR135a-5p, CEA and CA199 were detected. The relationships between serum miR135a-5p level and clinicopathological parameters were analyzed by Mann-Whitney U test. The correlation of serum miR135a-5p level and serum concentrations of CEA or CA199 was analyzed by Pearson's correlation test.Receiver operating characteristic (ROC) curves and area under the curve (AUC) were used to evaluate the diagnostic efficacy of miR135a-5p, CEA and CA199 as diagnostic indicators. Results: The serum level of miR135a-5p in CRC patient was 2.451 (1.107, 4.413), significantly higher than 0.946 (0.401, 1.942) in the patients with intestinal polyps and 0.949 (0.194, 1.415) in the healthy controls (U = 351.0 and U = 313.0, respectively, P<0.001). The serum level of miR135a-5p in CRC patients was associated with both histological differentiation and clinical stage (P<0.05 for both), however, not correlated with the serum concentration of CEA (r2 = 0.023, P = 0.293) or CA199 (r2 = 0.067, P = 0.068). The AUC of serum miR135a-5p level in CRC patients was 0.832 (0.730-0.930) when compared to the patients with intestinal polyps and was 0.875 (0.800-0.950) when compared with the healthy controls. Conclusions: The serum level of miR135a-5p in CRC patients is significantly higher than that in patients with intestinal polyps and healthy controls, and might be an important diagnostic marker of CRC.

[Pedigree survey in a family with hereditary protein S deficiency].

Objective: To observe the clinical feature of familiar hereditary protein S deficiency(HPSD), and to explore the related gene mutations. Methods: A total of seven family members were enrolled in this study and examined during the June to September 2015. Medical histories of the families were analyzed to detect HPSD according to the diagnostic criteria. PROS1 genes of the proband and her family were analyzed. DNA was extracted from peripheral blood. The 15 exons and their intron-exon boundaries of PROS1 were amplified with PCR, and the PCR products were sequenced and analyzed to identify potential mutations. Medical histories from the family members died prior this study were also obtained. Results: Four out of 7 family members of 2 generations were diagnosed as HPSD. The proband suffered from pulmonary embolism, her elder brother suffered from cerebral infarction and her niece suffered from deep vein thrombosis. A missense mutation at the 1063 bp of cDNA(c.1063C>T)was detected in the exon 10 of PROS1, which resulted in arginine 355 to cysteine replacement in the first ball domain of laminin of the protein S(p.R355C). Conclusion: HPSD is an autosomal dominant genetic disease, patients often suffer from recurring vein thrombosis and pulmonary embolism. A missense mutation(c.1063C>T, p. R355C)of PROS1 was discovered in this Chinese family with HPSD, thus, this mutation might be the genetic basis responsible for these family members with HPSD .

Effect of sinomenine on the expression of rheumatoid arthritis fibroblast-like synoviocytes MyD88 and TRAF6.

The effect of sinomenine (SIN) on the toll-like receptor (TLR) signal transduction pathway as well as the expression of myeloid differentiation factor 88 (MyD88) and tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) was investigated. SIN inhibition of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) proliferation and RA cartilage and subchondral bone destruction was also investigated. RA-FLS were cultured in vitro and the intracellular alkaline phosphatase (ALP) activity was determined in order to obtain the optimal drug concentration. The rate of cell proliferation was determined. Fluorescence quantitative polymerase chain reaction (PCR) was applied to determine the MyD88 and TRAF-6 gene expression and western blot was used to detect the MyD88 and TRAF-6 protein expression. The ALP activity in the SIN groups was lower than that in the control group, among which the 0.5 mM SIN group had the lowest ALP activity (P < 0.01). The rate of RA-FLS proliferation detected by CCK-8 assay in the 0.5-mM SIN group was lower than that in the control group (P < 0.01) and was the highest 4 days after SIN induction. Gene and protein expression of MyD88 and TRAF-6 were downregulated significantly in the 0.5-mM SIN group compared to that in the control group (P < 0.01). SIN effectively inhibited MyD88 and TRAF-6 expression in RA-FLS, which may be one of the important molecular mechanisms involved in RA treatment and prevention of cartilage and subchondral bone destruction.

Ion beam transformation with corn DNA alters proteinase expression in rice seedling roots.

Corn DNA was introduced into dry seeds of rice (cv. 'YuJing-6') by ion beam irradiation. Proteinase activities in rice seedling roots were subsequently analyzed by renaturation electrophoresis at pH 4.5, 7.0, and 8.5. Proteinase activity was more pronounced on gels at higher pH. Irradiation of rice seedling roots caused the loss of some proteinase bands at all pH conditions although a novel 50-kDa band was found at both pH 7.0 and 8.5. No new proteinase activity was detected at pH 4.5. However, novel bands and bands showing stronger activity were observed at pH 7.0 and 8.5. The data indicate that the expression of proteinases in rice seedling roots was altered following low energy ion beam mediated transformation with corn DNA.

Analysis of specific sequences in mutant rice generated by introduction of exogenous corn DNA.

Rice variation induced by the introduction of exogenous DNA has become an important method of improving rice varieties and creating new germplasms. In this study, we transferred maize genomic DNA fragments to the receptor of Nipponbare rice using a modified "pollen-tube pathway" method. Material from mutant rice B1 and B2 were acquired and 14 specific bands were obtained from the material using amplified fragment length polymorphism analysis. From the 14 specific sequences obtained, there were 3791 bp, including 144 base mutations with a base mutation rate of 3.80%. Specific bands resulted from base mutation of selective bases or restriction endonuclease recognition sequences, or insertion or deletion of DNA fragments. The frequency of single-base mutations was significantly higher than that of double-base mutations, three-sequential base mutations, and multiple-sequential base mutations. The site frequency of base substitution (87.04%) was significantly higher than that of base insertion (3.70%) or deletion (9.26%). In all cases of base substitution, the frequency of transition (76.47%) was significantly higher than transversion (23.53%). The above results indicate that transferring foreign-species DNA into rice cells can induce base mutations in the receptor, with base substitutions occurring at the highest frequency, and the dominant type of base substitutions being transition. Preliminary analysis reveals that the molecular mechanism of transferring exogenous DNA into rice causes mutations, which provides theoretical data on biological mutagenesis for further research.

Current status and hotspots evolution in myeloproliferative neoplasm: a bibliometric analysis from 2001 to 2022.

OBJECTIVE: In the last 20 years, the field of myeloproliferative neoplasm (MPN) has changed dramatically. This study aims to provide new ideas for the scientific research of MPN by systematically combing the literature. MATERIALS AND METHODS: CiteSpace and VOSviewer were used to carry out a bibliometric analysis of MPN papers to visualize the development process, research hotspots, and cutting-edge trends in clinical practice, mechanisms, and management strategies related to MPN. RESULTS: 1,099 authors from 736 institutions in 113 countries/regions published 11,922 papers in 1,807 academic journals. The United States and Italy were in the leading positions in this research field. Mayo Clinic is the institution with the largest number of publications. Only a few countries and institutions have shown active cooperation. Ayalew Tefferi and Ruben A. Mesa are outstanding contributors to the field. Blood and Leukemia are considered influential journals based on publications and citations. In this field, the research of MPN mainly focuses on the occurrence and progress mechanism of MPN, the clinical significance of non-driving gene mutation, optimization of primary and secondary thromboprophylaxis, clinical research of long-acting interferon and JAK2 inhibitors, and exploration of better therapies for myelofibrosis (primary and secondary) and post-MPN acute myeloid leukemia (AML). CONCLUSIONS: The research is in a stage of rapid development. The collaboration between different institutions or countries (regions) still has room to grow. The hotspot analysis shows that the research of MPN mainly focuses on gene mutation, thrombosis, new drug applications, disease progression, etc.

Comparing the clinical efficacy and safety of second-line targeted therapy and immunotherapy in patients with mid- to advanced stages of hepatocellular carcinoma - A systematic review and meta-analysis of randomized clinical trials.

OBJECTIVE: The aim of this study was to examine the efficacy and safety of second-line immunotherapy and targeted treatment in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: From January 2000 to January 2023, ProQuest, PubMed, Web of Science, Scopus, Embase, and the Cochrane Library databases were searched for randomized controlled trials (RCTs) using immunotherapy or targeted therapy as second-line therapy for mid-to-advanced stages of HCC. Overall survival (OS), progression-free survival (PFS), and adverse events (AEs) are all examples of measures of success. RESULTS: This analysis included twenty Randomized Clinical Trials (RCTs) from phases II and III. Collective data revealed better OS with immunotherapy (HR = 0.79; 95% CI: 0.67, 0.93 vs. 0.85; 95% CI: 0.78, 0.92), while the targeted therapy played a more effective role in PFS (0.67; 95% CI: 0.56, 0.81). Also, the second-line immunotherapy had a lower odds ratio of AEs of grades 3-5 than the targeted therapy did (OR = 1.75; 95% CI = 0.89, 3.46). CONCLUSIONS: Overall, it appears that targeted medication and immunotherapy as a second-line treatment strategy have generally improved substantially, as well as progression-free survival for patients with mid-to-advanced HCC. Although it is difficult to judge their efficiency, the occurrences of AEs were greater in targeted therapy compared to immunotherapy.

Preoperative anemia and complications after total joint arthroplasty: a systematic review and meta-analysis.

OBJECTIVE: The purpose of this review was to collect data from the literature to assess the impact of preoperative anemia on complications after total joint arthroplasty (TJA). MATERIALS AND METHODS: We conducted a literature search on the websites of PubMed, Scopus, CENTRAL, Embase, and Google Scholar for comparative TJA studies reporting complication rates based on the presence of anemia. The last search was conducted on the 15th of May 2022. Studies only on hip and knee replacements were eligible for inclusion. RESULTS: Twelve studies with 1,463,813 patients published between 2012-2022 were included. Meta-analysis indicated that anemic patients had increased risk of mortality (OR: 2.85 95% CI: 1.89, 2.48 I2=83% p<0.00001), wound complications (OR: 2.06 95% CI: 3.51, 2.48 I2=99% p=0.008), cardiac complications (OR: 2.40 95% CI: 1.56, 3.68 I2=98% p<0.0001), respiratory complications (OR: 2.46 95% CI: 1.10, 5.50 I2=100% p=0.03), renal complications (OR: 2.84 95% CI: 1.39, 5.80 I2=99% p=0.004), sepsis (OR: 3.93 95% CI: 1.15, 13.45 I2=99% p=0.03), urinary complications (OR: 2.42 95% CI: 1.27, 4.59 I2=100% p=0.007), and readmission rates (OR: 1.58 95% CI: 1.42, 1.76 I2=66% p<0.00001) as compared to non-anemic patients undergoing TJA. Most results did not change on sensitivity analysis. There were some non-significant results on subgroup analysis based on joint type and definition of anemia. CONCLUSIONS: Our review suggests that preoperative anemia leads to increased morbidity and mortality after TJA. Specifically, anemia increases the risk of wound, cardiac, respiratory, renal, and urinary complications along with a higher incidence of sepsis and readmissions. Results should be interpreted with caution due to the high heterogeneity in the meta-analyses.

[Effects and mechanism of hydrogen peroxide pretreatment with low molarity on oxidative stress induced apoptosis of mouse bone marrow mesenchymal stem cells].

Objective: To investigate the effects and mechanism of hydrogen peroxide (HP) pretreatment with low molarity on oxidative stress induced apoptosis of mouse bone marrow mesenchymal stem cells (BMSCs). Methods: The experimental research methods were used. BMSCs were isolated and cultured from two 2-week-old male BALB/c mice by the whole bone marrow culture method. The 3rd-7th passages of cells in logarithmic growth phase were used for the experiments after identification. According to the random number table (the same grouping method below), the cells were divided into 0 µmol/L HP group (without HP, the same below), 25 µmol/L HP group, 50 µmol/L HP group, 100 µmol/L HP group, 150 µmol/L HP group, 200 µmol/L HP group, 250 µmol/L HP group, and 300 µmol/L HP group in which cells were treated by the corresponding final molarity of HP, respectively. The apoptosis rate was detected by flow cytometry (n=4) after 24 hours of culture. The cells were divided into 0 µmol/L HP group, 25 µmol/L HP group, 50 µmol/L HP group, and 100 µmol/L HP group in which cells were treated by the corresponding final molarity of HP, respeclively. After 24 hours of culture, the protein expressions of B-lymphoma-2 (Bcl-2) and Bcl-2-related X protein (Bax) were detected by Western blotting, and the Bcl-2/Bax ratio was calculated (n=3). The cells were divided into 0 µmol/L HP group, 25 µmol/L HP group, 50 µmol/L HP group, 100 µmol/L HP group, 200 µmol/L HP group, and 300 µmol/L HP group in which cells were treated by the corresponding final molarity of HP, respectively. After 24 hours of culture, the protein expressions of glycogen synthase kinase-3ß (GSK-3ß) and phosphorylated GSK-3ß (p-GSK-3ß) were detected by Western blotting (n=3). The cells were divided into 0 µmol/L HP group, 50 µmol/L HP group, and 300 µmol/L HP group in which cells were treated by the corresponding final molarity of HP, respeclively, and HP pretreatment group with 50 µmol/L HP being added in advance for 12 h and then 300 µmol/L HP being added. After 24 hours of culture, the morphology and growth of cells were observed by inverted fluorescence microscopy (non-fluorescent condition) and immunofluorescence method, the apoptosis rate was detected by flow cytometry, the protein expressions of Bcl-2, Bax, cysteine aspartic acid specific protease-3 (caspase-3), caspase-9, cleavage caspase-3, cleavage caspase-9, GSK-3ß, and p-GSK-3ß were detected by Western blotting, and the Bcl-2/Bax ratio was calculated, with all the number of samples being 3. Data were statistically analyzed with one-way analysis of variance and Bonferroni test. Results: After 24 hours of culture, compared with that in 0 µmol/L HP group, the apoptosis rate of cells did not change significantly in 25 µmol/L HP group, 50 µmol/L HP group, or 100 µmol/L HP group (P>0.05) but increased significantly in 150 µmol/L HP group, 200 µmol/L HP group, 250 µmol/L HP group, and 300 µmol/L HP group (P<0.01). After 24 hours of culture, compared with that in 0 µmol/L HP group, the Bcl-2/Bax ratio of cells increased significantly in 25 µmol/L HP group and 50 µmol/L HP group (P<0.05 or P<0.01) but decreased significantly in 100 µmol/L HP group (P<0.05). After 24 hours of culture, compared with those in 0 µmol/L HP group, the protein expression of GSK-3ß in cells showed no significant change in 25 µmol/L HP group and 50 µmol/L HP group (P>0.05), the protein expressions of p-GSK-3ß in cells significantly increased in 25 µmol/L HP group and 50 µmol/L HP group (P<0.01), the protein expressions of GSK-3ß and p-GSK-3ß in cells in 100 µmol/L HP group showed no significant change (P>0.05), the protein expressions of GSK-3ß in cells in 200 µmol/L HP group and 300 µmol/L HP group were significantly increased (P<0.05). but the protein expression of p-GSK-3ß in cells in 200 µmol/L HP group and 300 µmol/L HP group was significantly decreased (P<0.05). After 24 hours of culture, the morphology and growth of cells in 0 µmol/L HP group and 50 µmol/L HP group were similar and normal; in contrast, the cells in 300 µmol/L HP group became smaller and round, with the cell protrusions being shorter or disappeared, the nucleus being cavitated, and the cell abscission being increased significantly; the morphology of most cells in HP pretreatment group was normal, with the shedding of cells being less than that in 300 µmol/L HP group, and the morphology of nucleus being normal. After 24 hours of culture, the protein expression of caspase-9 was similar among the four groups (P>0.05). Compared with that in 0 µmol/L HP group, the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells in 50 µmol/L HP group showed no significant changes (P>0.05), the Bcl-2/Bax ratio of cells in 50 µmol/L HP group increased significantly (P<0.05), the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells in 300 µmol/L HP group were significantly increased (P<0.01), while the Bcl-2/Bax ratio of cells in 300 µmol/L HP group was significantly decreased (P<0.05). Compared with those in 300 µmol/L HP group, the apoptosis rate and the protein expressions of cleavage caspase-9, caspase-3, and cleavage caspase-3 of cells were significantly decreased in HP pretreatment group (P<0.05 or P<0.01), while the Bcl-2/Bax ratio of cells was significantly increased in HP pretreatment group (P<0.01). After 24 hours of culture, the protein expressions of GSK-3ß and p-GSK-3ß of cells in 0 µmol/L HP group, 50 µmol/L HP group, 300 µmol/L HP group, and HP pretreatment group were 1.09±0.14, 0.62±0.17, 1.35±0.21, 0.74±0.34, 0.68±0.03, 0.85±0.08, 0.38±0.10, and 0.54±0.09, respectively. Compared with those in 0 µmol/L HP group, the protein expression of p-GSK-3ß of cells was significantly increased in 50 µmol/L HP group (P<0.05) but significantly decreased in 300 µmol/L HP group (P<0.01), while the protein expression of GSK-3ß of cells was significantly increased in 300 µmol/L HP group (P<0.05). Compared with those in 300 µmol/L HP group, the protein expression of GSK-3ß of cells was significantly decreased in HP pretreatment group (P<0.01), while the protein expression of p-GSK-3ß of cells was significantly increased in HP pretreatment group (P<0.01). Conclusions: The molarity of 50 µmol/L may be the optimal molarity of HP to pretreat mouse BMSCs, and 50 µmol/L HP pretreatment can antagonize mitochondrial pathway of oxidative stress induced apoptosis by inhibiting the activity of GSK-3ß.

[Role of interleukin-6 in human umbilical vein endothelial cell to mesenchymal cell transformation].

Objective: To observe the effect of interleukin-6 (IL-6) on the phenotype and function of human umbilical vein endothelial cells (HUVECs) and explore the role of IL-6 in the process of endothelial-to-mesenchymal transition (EndMT). Methods: The experimental research method was used. Fresh umbilical cord discarded after normal maternal delivery was collected. On the second day of the primary cell isolation and cultivation, the cell morphology was observed under inverted phase contrast microscope. HUVECs of the 4th passage were identified by immunofluorescence method, and 2 batches of HUVECs ofthe 3rd to 5th passages were used for the subsequent experiments. The first batch of cells were divided into 6 groups according to the random number table (the same below): blank control group, 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group. The second batch of cells were divided into 4 groups: blank control group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group,and 50 ng/mL IL-6 group; the cells in blank control group was cultured with complete culture medium only, while the cells in the other groups were added with IL-6 of the corresponding final mass concentrations.Cells from the 1st batch were cultured for 72 hours after grouping, the morphology of HUVECS in the 6 groups was observed under inverted phase contrast microscope. At 72 h after grouping culture, the positive expressions of coagulation factor Ⅷ and α vascular smooth muscle actin (α-SMA) in HUVECs in the 6 groups were detected by immunofluorescence method, and the ratio of the number of double positive cells to the number of coagulation factor Ⅷ positive cells (the ratio of double positive cells for short) was calculated, with 6 samples per group; mRNA expression levels of vascular endothelial cadherin and α-SMA of HUVECs in 6 groups were detected by reverse transcription-polymerase chain reaction, with 3 samples per group.Cells from the 2nd batch were cultured 72 hours after grouping, the protein expression levels of vascular endothelial cadherin, α-SMA, and type Ⅰ collagen in the 4 groups were detected by Western blotting, with 3 samples per group. Data were statistically analyzed with one-way analysis of variance and Bonferroni correction. Results: On the 2nd day after isolation and cultivation, the primary cells were in short spindle shape or polygon, cells of the 4th passage were identified as HUVECs by immunofluorescence method. At 72 hours of culture after grouping, the cells from the 1st batch in the 6 groups changed to long spindle shape morphologically along with the increase of IL-6 concentration, the intercellular connections decreased or disappeared with the gap between cells becoming larger. At 72 h after grouping culture, compared with that inblank control group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (P<0.01); compared with that in 5 ng/mL IL-6 group, the ratio of double positive cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly increased (P<0.01); compared with that in 10 ng/mL IL-6 group, the ratio of double positive cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly increased (P<0.01); the ratio of double positive cells in 100 ng/mL IL-6 group was significantly increased compared with those in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group, 50 ng/mL IL-6 group, and 100 ng/mL IL-6 group were significantly decreased (P<0.01 or P<0.05); compared with that in 5 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (P<0.01); compared with that in 10 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (P<0.01); compared with that in 25 ng/mL IL-6 group, the mRNA expression levels of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group and 100 ng/mL IL-6 group were significantly decreased (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the mRNA expression levels of α-SMA of cells in 5 ng/mL IL-6 group, 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, 50 ng/mL IL-6, group, and 100 ng/mL IL-6 group were significantly increased (P<0.05 or P<0.01). Cells from the 2nd batch were cultured for 72 hours after grouping. Compared with 1.391±0.026 in blank control group, the protein expressions of vascular endothelial cadherin of cells in 10 ng/mL IL-6 group (1.185±0.063), in 25 ng/mL IL-6 group (0.717±0.078), and in 50 ng/mL IL-6 group (0.239±0.064) were significantly decreased (P<0.05); compared with that in 10 ng/mL IL-6 group, the protein expressions of vascular endothelial cadherin of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly decreased (P<0.01); compared with that in 25 ng/mL IL-6 group, the protein expression of vascular endothelial cadherin of cells in 50 ng/mL IL-6 group was significantly decreased (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expression levels of α-SMA of cells in 10 ng/mL IL-6 group, 25 ng/mL IL-6 group, and 50 ng/mL IL-6 group were significantly increased (P<0.01); compared with that in 10 ng/mL IL-6 group, the protein expression levels of α-SMA of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (P<0.01). At 72 h after grouping culture, compared with that in blank control group, the protein expressions of type Ⅰ collagen of cells in 25 ng/mL IL-6 group and 50 ng/mL IL-6 group were significantly increased (P<0.05). Conclusions: After IL-6 treatment, the phenotype and function of HUVECS showed the characteristics of mesenchymal cells in a concentration-dependent manner. The inflammatory factor can promote the process of EndMT, and become one of the important factors regulating the mechanism of tissue fibrosis.

LncRNA FOXC2-AS1 regulated proliferation and apoptosis of vascular smooth muscle cell through targeting miR-1253/FOXF1 axis in atherosclerosis.

OBJECTIVE: Atherosclerosis (AS) is the most dangerous factor for human death, which is responsible for coronary heart disease. Growing evidence has showed that long non-coding RNAs (lncRNAs) are involved in the development of AS. In this study, we mainly aimed at investigating the roles of FOXC2-AS1 in AS patients. PATIENTS AND METHODS: RT-PCR was performed to detect the expressions of FOXC2-AS1 and miR-1253 in serum samples of AS patients (n=35) and healthy volunteer (n=35). The correlation between FOXC2-AS1 and miR-1253 was further analyzed. Human vascular smooth muscle cells (VSMCs) were respectively treated with ox-LDL, IL-6, CRP, TNF-α and IL-8 to explore the affecting factors. P-FOXC2-AS1 was constructed and transfected into VSMCs. Cell proliferation abilities were measured by CCK-8 assay. Cell apoptotic rates were measured by flow cytometry (FACS) analysis. Western blot (WB) was performed to detect protein levels of FOXF1, Bcl-2, Bax and Cleaved Caspase3. Finally, luciferase gene reporter assay was performed to prove the relationships between FOXC2-AS1 and miR-1253, miR-1253 and FOXF1. RESULTS: We found that FOXC2-AS1 was significantly upregulated in AS patients, which could be induced by ox-LDL and IL-6 in VSMCs. MiR-1253 was decreased in AS patients, which was negatively correlated with FOXC2-AS1. Furthermore, FOXC2-AS1 overexpression promoted proliferation and inhibited apoptosis in VSMCs. Luciferase gene reporter assay showed that FOXC2-AS1 could bind to miR-1253 in VSMCs and 293 cells. Moreover, miR-1253 overexpression inhibited proliferation and promoted apoptosis of VSMCs. Luciferase reporter assay proved that miR-1253 could target at FOXF1 in VSMCs and 293 cells, which was reported to be associated with cell proliferation and apoptosis in some cancers. Additionally, miR-1253 mimic or GSK343, a FOXF1 inhibitor, was respectively transfected into VSMCs with p-FOXC2-AS1. Results showed that the promoted cell proliferation and inhibited cell apoptosis were reversed as well, confirming that FOXC2-AS1 promoted cell proliferation and inhibited apoptosis via miR-1253/FOXF1 signaling axis in AS patients. CONCLUSIONS: According to the results, we found that FOXC2-AS1 was upregulated in AS patients; furthermore, FOXC2-AS1 overexpression promoted cell proliferation and inhibited cell apoptosis via targeting miR-1253/FOXF1 signaling axis. Our results elucidated a potential mechanism underlying the role of FOXC2-AS1, which might be used as a promising marker and a potential target for AS patients.

[Epidemiological characteristics of human brucellosis in Tongliao city of Inner Mongolia Autonomous Region, 2004-2018].

Objective: To analyze the epidemiological characteristics of human brucellosis (HB), evolution and origin feature of Brucella strains in Tongliao city, Inner Mongolia Autonomous Region during 2004-2018, and to provide evidence for strategy development against the disease. Methods: Data from the reports on HB in Tongliao during 2004-2018 were extracted from the China Information System for Disease Control and Prevention before being analyzed with software Excel 2016. Epidemiologic feature was described, using the number of cases, constituent ratio and related rates. Conventional biotypes methods were used for identification of species/biovars strains while species of six Brucella strains were further verified by AMOS-PCR. Cluster analyze on six Brucella strains were performed with Bio-Numerics 5.0 software and for examining and revealing the genetic characteristics of the related strains. Results: During 2004-2018, a total of 16 704 HB cases were reported, with the incidence rate as 35.41/100 000. The incidence rates appeared as 110.51/100 000 in Jarud Banner and 67.84/100 000 in Kulun flag, which were both higher than the other areas. Most of the cases were reported in the 40-54 year olds, which accounted for 48.75% (8 143/16 704). The number of HB in farmers appeared as 14 873, which counted for 89.04% (14 873/16 704) of all the cases. Male to female ratio of incidence was 2.40∶1. Most of the reported cases appeared between March to May, which accounted for 56.30% (9 405/16 704). Peak of the disease was seen in April. Using the conventional identification method, results showed that the available six strains all belonged to B. melitensis, including three of them as B. melitensis bv.1 and others three strains as B. melitensis bv. 3. Results from the amplified AMOS-PCR showed that all the strains were B. melitensis. The six strains clustered in two MLVA-11 genotypes (111 and 116) and all belonged to the Eastern Mediterranean lineage. Based on the MLVA-16 cluster analysis, results suggested that strains from this study were having close genetic relationship with B. melitensis strains that were from Jilin and Heilongjiang provinces. Conclusions: Human brucellosis identified in Tongliao area was with greater risk in spreading the disease to the vicinity. Our findings indicated that the programs on detection and control of the disease should be strengthened.