Fluorescent Quantitative Reverse Transcription Polymerase Chain Reaction Research Articles

Schizophrenia plausible protective effect of microRNA-137 is potentially related to estrogen and prolactin in female patients.

Schizophrenia (SCZ) is a serious chronic mental disorder. Our previous case-control genetic association study has shown that microRNA-137 (miR-137) may only protect females against SCZ. Since estrogen, an important female sex hormone, exerts neuroprotective effects, the relationship between estrogen and miR-137 in the pathophysiology of SCZ was further studied in this study. Genotyping of single-nucleotide polymorphism rs1625579 of miR-137 gene in 1,004 SCZ patients and 896 healthy controls was conducted using the iMLDR assay. The effect of estradiol (E2) on the miR-137 expression was evaluated on the human mammary adenocarcinoma cell line (MCF-7) and the mouse hippocampal neuron cell line (HT22). The relationships between serum E2, prolactin (PRL), and peripheral blood miR-137 were investigated in 41 SCZ patients and 43 healthy controls. The miR-137 and other reference miRNAs were detected by real-time fluorescent quantitative reverse transcription-PCR. Based on the well-known SNP rs1625579, the distributions of protective genotypes and alleles of the miR-137 gene were not different between patients and healthy controls but were marginally significantly lower in female patients. E2 upregulated the expression of miR-137 to 2.83 and 1.81 times in MCF-7 and HT22 cells, respectively. Both serum E2 and blood miR-137 were significantly decreased or downregulated in SCZ patients, but they lacked expected positive correlations with each other in both patients and controls. When stratified by sex, blood miR-137 was negatively correlated with serum E2 in female patients. On the other hand, serum PRL was significantly increased in SCZ patients, and the female patients had the highest serum PRL level and a negative correlation between serum PRL and blood miR-137. The plausible SCZ-protective effect of miR-137 may be female specific, of which the underlying mechanism may be that E2 upregulates the expression of miR-137. This protective mechanism may also be abrogated by elevated PRL in female patients. These preliminary findings suggest a new genetic/environmental interaction mechanism for E2/miR-137 to protect normal females against SCZ and a novel E2/PRL/miR-137-related pathophysiology of female SCZ, implying some new antipsychotic ways for female patients in future.

SNHG15 promotes chemoresistance and glycolysis in colorectal cancer.

Long noncoding RNAs (lncRNAs) play an important role in tumor progression. Small nucleolar RNA host gene 15 (SNHG15) is a lncRNA that has been confirmed to play an oncogenic role in multiple cancer types. However, its role in glycolysis and chemoresistance in colorectal cancer (CRC) is unclear. The expression of SNHG15 in CRC was analyzed using data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases by bioinformatics methods. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to evaluate cell viability. Cell sensitivity to 5-fluorouracil (5-FU) was detected by CCK-8. Glucose absorption and lactate production were used to evaluate the impact of SNHG15 on glycolysis. RNA-seq, real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) and Western blotting (WB) were used to reveal the potential molecular mechanism of SNHG15 in CRC. SNHG15 was upregulated in CRC tissues compared with paired noncancerous tissues. Ectopic SNHG15 expression increased proliferation, 5-FU chemoresistance, and glycolysis in CRC cells. In contrast, SNHG15 knockdown inhibited CRC proliferation, 5-FU chemoresistance and glycolysis. Multiple pathways, including apoptosis and glycolysis, were potentially regulated by SNHG15 based on RNA-seq and pathway enrichment analyses. RT-qPCR and WB experiments confirmed that SNHG15 promoted the expression of TYMS, BCL2, GLUT1 and PKM2 in CRC cells. In conclusion, SNHG15 promotes 5-FU chemoresistance and glycolysis in CRC by potentially regulating the expression of TYMS, BCL2, GLUT1 and PKM2 and appears to be a new target for cancer therapy.

Effects of Krüppel-like factor 4 on inflammatory response and organ injury in septic mice

Effects of Krüppel-like factor 4 on inflammatory response and organ injury in septic mice

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Effects of collagen type ⅩⅦ α1 on epidermal stem cells in aging skin and the microRNA intervention mechanism

Copy number variation of EIF4A2 loci related to phenotypic traits in Chinese cattle.

BackgroundGenerally, copy number variation (CNV) is a large‐scale structural variation between 50 bp and 1 kb of the genome. It can affect gene expression and is an important reason for genetic diversity and phenotypic trait diversity. Studies have shown that the eukaryotic translation initiation factor 4A2 (EIF4A2) gene plays an essential role in muscle development in both humans and pigs. However, the influence of bovine EIF4A2’s copy number change on phenotypic traits has not been reported.ObjectivesTo detect the tissue expression profile of the EIF4A2 gene in adult cattle and individuals’ CNV type of variation. Then, we explored the correlation between EIF4A2‐CNV and growth traits in Chinese cattle breeds.MethodsReal‐time fluorescent quantitative reverse transcription PCR (qRT‐qPCR) was used to determine the expression profile of the EIF4A2 gene. Real‐time fluorescent quantitative PCR (qPCR) was used to detect the CNV type of bovine populations. Then, SPSS 26.0 was used for association analysis.ResultsIn this study, a total of 513 individuals in four cattle breeds (Qinchuan cattle [QC], Yunling cattle [YL], Pinan cattle [PN] and Jiaxian cattle [JX]) were detected for EIF4A2 gene's CNV. The results showed that EIF4A2‐CNV has an essential impact on hip width (HW) and rump length (RL) in QC, heart girth (HG), chest depth (CD) and RL in YL and HW in PN. However, it had no significant effect on JX.ConclusionsThe above results suggest that EIF4A2 gene's CNV can be used as a molecular marker for cattle breeding, which is helpful to accelerate the breeding of superior beef cattle breeds.

Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice

Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice

Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice

Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice

The role and mechanism of lncRNA C9ORF139 targeting miR-24-3P/TAOK1 in regulating the proliferation of acute myeloid leukemia cells

Objective: To investigate the role and mechanism of long non-coding RNA (lncRNA) C9ORF139 targeting micro RNA(miR)-24-3P/TAOK1 in regulating the proliferation of acute myeloid leukemia (AML) cells. Methods: AML cells HL-60 and THP-1 were purchased from the Chinese Academy of Sciences and divided into 4 groups:group A was negative control group (siNC group), group B was interference C9ORF139 group (siC9ORF139 group), group C was siC9ORF139+miR-24-3p inhibitor group, and group D was miR-24-3P+TAOK1 overexpression group (oe-TAOK1 group). Real-time fluorescence quantitative reverse transcription PCR was used to detect the expression levels of AML cell lines of HL-60 and THP-1 in four groups. Cell Counting Kit-8 assay was performed to measure cell proliferation. Flow cytometry was applied to analyze cell apoptosis. Transwell test was applied to detect cell migration and invasion ability. Western blot was used to detect p-serine/threonine kinase (p-raf) and p-mitogen activation proteinkinase (p-MEK), p-extracellular regulatory protein kinase (p-ERK) expression. The luciferase reporter gene plasmid was constructed to verify the binding ability of C9ORF139,miR-24-3P and TAOK1.Nude mice were inoculated with subcutaneous tumor cells of HL-60 (group A) and HL-60 (group B). Results: After the C9ORF139 gene was knocked down and cultured for 120 h, The cell proliferation ability (0.62±0.02, 0.82±0.02), migration ability (0.22±0.03, 0.05±0.01), invasion ability (0.20±0.02, 0.13±0.03) of group B were all lower than that of group A (1.30±0.02, 1.83±0.07; 0.99±0.02, 0.99±0.02; 1.00±0.01, 1.00±0.01) (all P<0.05). When co-transfected with miR-24-3 inhibitor, cell proliferation ability, migration ability and invasion ability were all higher in group B (all P<0.05). When co-transfected with miR-24-3P and oe-TAOK1 plasmid, cell proliferation ability, migration ability and invasion ability were all higher than group B (all P<0.05).When the C9ORF139 gene in the cells was knocked down, the apoptosis level of group B (28.56±8.07, 17.74±1.91) were higher than those of group A (0.31±0.27, 2.49±0.33)(all P<0.05); when co-transfected with miR-24-3P inhibitor, the apoptosis level (2.34±0.09, 3.06±0.06) were lower than those in group B (all P<0.05); when co-transfected with miR-24-3P and oe-TAOK1 in the plasmid group, the apoptosis level (2.16±1.29, 4.80±0.37) were also lower than those of group B (all P<0.05). In HL-60 and THP-1 cells, when C9ORF139 was not mutated, the luciferase activity of miR-24-3P group was lower than that of the miR-NC group (P<0.05). When the binding site with miR-24-3p in C9ORF139 sequence was mutated, the luciferase activity in miR-24-3p group was equivalent to that in miR-NC group (P>0.05).When TAOK1 was not mutated; the luciferase activity of miR-24-3P group was lower than that of group A (P<0.05). When the binding site with miR-24-3p in TAOK1 sequence was mutated, the luciferase activity in miR-24-3p group was equivalent to that in miR-NC group (P>0.05).When the C9ORF139 gene in HL-60 cells was knocked down and cultured for 72 h, the phosphorylation expression levels of Raf, MEK and ERK molecules in group B were significantly lower than those in group A (all P<0.05). By day 14, the tumor volume in the group A was greater than the tumor cell volume in the group B [(284.49±57.61) vs (125.70±18.64) mm3, P=0.017]. The tumor weight of HL-60 in group A was heavier than that of group B [(847.80±159.36) vs (408.40±113.16) mg, P=0.001]. Conclusions: LncRNA C9ORF139 regulates TAOK1 by sponging miR-24-3P to promote the proliferation, invasion and migration of acute myeloid leukemiacell.In vivo experiments have confirmed that the expression of C9ORF139 can promote the growth of subcutaneous tumors in AML nude mice.

Effects and mechanism of Lactococcus lactis thermo-sensitive hydrogel on the wound healing of full-thickness skin defects in diabetic mice

Effects and mechanism of Lactococcus lactis thermo-sensitive hydrogel on the wound healing of full-thickness skin defects in diabetic mice

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

Mechanism of maggot debridement therapy in promoting wound angiogenesis in patients with diabetic foot ulcer

Study on mechanisms of interleukin-17A regulating the expressions of interleukin-1β and interleukin-23 in mouse keratinocytes

Objective: To investigate the mechanisms of interleukin-17A (IL-17A) regulating the expressions of IL-1β and IL-23 in mouse keratinocytes (KCs). Methods: Primary KCs were isolated from the skin of 400 newborn male and female wild type C57BL/6 mice and cultured in 24-well plates with Roswell Park Memorial Institute 1640 medium containing fetal bovine serum in the volume fraction of 10% for the following experiments. (1) The cells were divided into phosphate buffer solution (PBS) control group and IL-17A stimulation group according to the random number table (the same grouping method below), which were cultured with 10 μL PBS or 10 μL IL-17A in the mass concentration of 100 ng/mL for 6 hours, respectively. The expression levels of IL-1β and IL-23 mRNA in cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), with 3 samples in each group. (2) The cells were divided into dimethyl sulfoxide (DMSO) control group, IL-17A+ DMSO group, IL-17A+ nuclear factor κB (NF-κB) inhibitor group, IL-17A+ signal transduction and activator of transcription 3 (STAT3) inhibitor group, IL-17A+ extracellular signal-regulated kinase 1 (ERK1) inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ c-Jun N-terminal kinase (JNK) inhibitor group. The reagents were added to cells in corresponding groups respectively and cultured for 6 hours. The volume of each reagent was 10 μL, the mass concentration of IL-17A was 100 ng/mL, and the molarity concentrations of NF-κB, STAT3, ERK1, ERK2, JNK signal pathway inhibitors PDTC, S3I-201, SCH772984, SCH772984, SP600125 were 5 μmol/L, 100 μmol/L, 4 nmol/L, 1 nmol/L, and 10 μmol/L, respectively. The expression levels of IL-1β mRNA and IL-23 mRNA in cells were detected by real-time fluorescence quantitative RT-PCR, with 3 samples in each group. (3) The cells were grouped and treated the same as those in experiment (1). The levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation were detected by Western blotting, with 3 samples in each group. Data were statistically analyzed with two-tailed Student t test, one-way analysis of variance, t test, and Bonferroni correction. Results: (1) After culture of 6 hours, compared with those in PBS control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A stimulation group were significantly increased (t=13.46, 6.72, P<0.01). (2) After culture of 6 hours, the expression levels of IL-1β and IL-23 mRNA in cells in DMSO control group, IL-17A+ DMSO group, IL-17A+ NF-κB inhibitor group, IL-17A+ STAT3 inhibitor group, IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group were 1.00±0.11, 4.01±0.32, 0.32±0.06, 1.76±0.43, 3.62±0.24, 3.80±0.43, 4.26±0.74 and 1.03±0.29, 4.08±0.34, 4.76±0.38, 4.70±0.21, 1.06±0.42, 0.92±0.21, 0.39±0.05, respectively. Compared with those in DMSO control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A+ DMSO group were significantly increased (t=9.24, 12.60, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-1β mRNA was significantly decreased in cells in IL-17A+ NF-κB inhibitor group and IL-17A+ STAT3 inhibitor group (t=11.34, 6.91, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-23 mRNA was significantly decreased in cells in IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group (t=12.44, 13.03, 15.21, P<0.01). (3) After culture of 6 hours, compared with those in PBS control group, the levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation in cells in IL-17A stimulation group were significantly increased. Conclusions: IL-17A promotes the transcription of IL-1β in mouse KCs through the phosphorylation of NF-κB and STAT3 pathways and IL-23 through the phosphorylation of ERK and JNK pathways.

Exploration of the mechanism of human platelet-rich plasma in regulating and controlling human epidermal stem cells for promoting wound re-epithelialization at transcriptome level

Objective: To analyze target genes of human platelet-rich plasma (PRP) in regulating and controlling human epidermal stem cells (ESCs). Methods: (1) The discarded foreskin tissues were collected from 6 male patients of the First Affiliated Hospital of Army Medical University after urological surgery. The patients aged 5 to 25 years with good health and without urinary system infection. Human ESCs were cultivated using quick attachment method, and were subjected to morphological observation and identification. Venous blood sample in the volume of 40 mL was collected from a female healthy volunteer (aged 29 years) of General Hospital of Southern Theater Command of PLA, and PRP was extracted by second centrifugation method. (2) The successfully cultured primary human ESCs were divided into control group and PRP group according to the random number table, with 3 wells in each group. The cells in control group were not specially treated. In PRP group, PRP was added to the ESC medium to achieve final volume fraction of 2.5% after the cells were adhered for 12 hours. RNA was extracted, and transcriptome sequencing and data analysis of human ESCs of two groups were performed using RNA sequencing technology. Using the false discovery rate less than 0.05 and the fold change more than or equal to 4 as the standard, the differentially expressed genes were screened by Dr. Tom data mining system. Gene ontology (GO) enrichment analysis was performed on the obtained differentially expressed genes to find out the GO entries with significant enrichment. Then Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis was used to further analyze the biological processes or metabolic pathways in which differentially expressed genes might be involved. Finally, the genes related to re-epithelialization and significantly differentially expressed were selected, and the differential expression of genes was verified by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). Data were statistically analyzed with independent-samples t test. Results: (1) The cultured cells were cloned with a paving stone-like shape and positive rate of CD49f of 95.132% and CD71 of 0.006%, which proved that the primary culture of ESCs was successful. (2) The quality control data analysis showed that the selected samples had better quality and higher sequence alignment rate, which met the requirements of sequencing. (3) Sequencing data showed that there were a total of 449 differentially expressed genes between the two groups, including 354 up-regulated genes and 95 down-regulated genes. Further cluster analysis determined that there were 18 significantly up-regulated genes and 5 significantly down-regulated genes between the two groups. GO enrichment analysis and KEGG pathway annotation analysis showed that the significantly differentially expressed genes were mainly enriched in the epidermis construction and keratinization process, which also might be related to interleukin 17 signaling pathway. (4) Keratin 19, keratin 10, and S100A7 genes which were related to the process of re-epithelialization and significantly differentially expressed were selected for verification. Real-time fluorescent quantitative RT-PCR showed that compared with those of control group, the mRNA expressions of keratin 19 and S100A7 of cells in PRP group were significantly increased (t=10.270, 5.690, P<0.01), while the mRNA expression of keratin 10 was significantly decreased (t=7.306, P<0.01), which was consistent with the result of sequencing data. Conclusions: PRP regulates function of human ESCs and promotes wound re-epithelialization involving transcriptional regulation of multiple genes, including keratin 19, keratin 10, and S100A7. In-depth exploration of the possible regulatory network of PRP affecting human ESCs will provide the basis for its subsequent clinical application.

Mechanism of transcriptional regulation of Meox1 by transforming growth factor β (1) and its effect on cell migration of adult human dermal fibroblasts

Objective: To explore the transcriptional regulation mechanism of transforming growth factor β(1) (TGF-β(1)) on Meox1 and its effect on cell migration of adult human dermal fibroblasts (HDF-a). Methods: (1) HDF-a cells were cultured in RPMI 1640 complete medium (hereinafter referred to as routinely cultured). The cells were divided into TGF-β(1) stimulation group and blank control group. The cells in TGF-β(1) stimulation group were stimulated with 10 μL TGF-β(1) in the mass concentration of 1 mg/μL, while the cells in blank control group were stimulated with the equal volume of phosphate buffer solution. After 72 hours in culture, partial cells in both groups were collected for transcriptome sequencing. The genes with differential expression ratio greater than or equal to 2 and P 0.05); the enrichment of Smad4 protein on the promoter of Meox1 in the cells of empty plasmid group was similar to that of Smad4 OE group (t=0.60, P>0.05). (5) Twenty-four hours after scratching, the scratch healing width of cells in siRNA-Meox1 group was narrower than that of negative interference group, while that of Meox1 OE group was wider than that of empty plasmid group. After 24 hours in culture, the number of migration cells in negative interference group was significantly higher than that in siRNA-Meox1 group (t=9.12, P<0.01), and that in empty plasmid group was significantly lower than that in Meox1 OE group (t=8.99, P<0.01). (6) The expression of Meox1 protein in the scar tissue was significantly higher than that in normal skin of patients with hypertrophic scars. Conclusions: TGF-β(1) transcriptionally regulates Meox1 expression via Smad2/3 in HDF-a cells, thus promoting cell migration.

Effects and mechanism of exogenous tumor necrosis factor α on differentiation of mesenchymal stem cells of mice into sweat gland cells in three-dimensional environment

Objective: To explore the effects and molecular mechanism of tumor necrosis factor α (TNF-α) on differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment. Methods: (1) Five 6-8 week-old female C57BL/6 mice were used, with one 1 cm(2) deep partial-thickness to full-thickness scald wound being created on the back of each mouse with a scald apparatus. One day after injury, the full-thickness skin tissue of the wound was taken, and the concentration of TNF-α in the tissue was detected by enzyme-linked immunosorbent assay. (2) Gelatin in the mass of 0.9 g and 0.3 g sodium alginate were mixed and then dissolved in 30 mL phosphate buffer solution to make hydrogel. Full-thickness skin of the planta of 10 male and female one day newborn C57BL/6 mice was ground into dermal homogenate. The mesenchymal stem cells were isolated from femur and tibia of 10 male and female C57BL/6 mice born for 7 days and cultured. A final density of 1.5×10(5) cells/mL of bioink was made of mixture of 8 mL pre-warmed hydrogel, 1 mL mouse foot dermal homogenate, and 1 mL the second or third passage of mesenchymal stem cell suspensions. The three-dimensional bioprinter was used to print 12 cylindrical blocks arranged in a crisscross pattern in petri dish. The printed blocks were cross-linked with 25 g/L calcium chloride solution for 10 min and then cultured for 12 hours by adding a medium for mesenchymal stem cells. Subsequently, the printed blocks were divided into blank control group and TNF-α treatment group according to the random number table, with 6 plates and 6 blocks in each group. Both groups of printed blocks were cultured with fresh sweat gland induction medium, and a final mass concentration of 20 ng/mL TNF-α was added into the medium of TNF-α treatment group. After 6 hours of culture, the mRNA expression of pluripotency marker Nanog in the mesenchymal stem cells of two plates of each group was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), and the protein expression of Nanog in the mesenchymal stem cells of one plate of each group was detected by Western blotting, both with triplicate samples. After 14 days of culture, the mRNA expression of sweat gland cell markers cytokeratin 14 (CK14), CK18, sodium potassium adenosine triphosphatase protein a1 (ATP1a1), and aquaporin 5 (AQP5) was detected by real-time fluorescent quantitative RT-PCR in the mesenchymal stem cells of 2 plates of each group (n=3), and the protein expression distribution of CK14, CK18, ATP1a1, and AQP5 of the mesenchymal stem cells in one plate of each group was detected by immunofluorescence staining. Data were statistically analyzed with independent sample t test. Results: (1) One day after injury, the mass concentration of TNF-α in the scald wound tissue of mouse was (19±3) ng/mL. (2) After 6 hours of culture, the mRNA and protein expression levels of Nanog in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.39±0.04 and 0.36±0.03, respectively, which were significantly lower than 1.00±0.05 and 1.00±0.07 of blank control group (t=16.51, 14.56, P<0.01). (3) After 14 days of culture, the mRNA expression levels of CK18, CK14, ATP1a1, and AQP5 in the mesenchymal stem cells of printed blocks in TNF-α treatment group were 0.38±0.03, 0.42±0.11, 0.23±0.06, and 0.25±0.03, respectively, which were significantly less than 1.00±0.03, 1.00±0.05, 1.00±0.05, 1.00±0.07 of blank control group (t=25.31, 8.31, 17.07, 17.06, P<0.01). (4) After 14 days of culture, the CK18, CK14, ATP1a1, and AQP5 protein were widely distributed in the cytoplasm of mesenchymal stem cells in printed blocks of blank control group, while the distribution of CK18, CK14, ATP1a1, and AQP5 protein in the cytoplasm of mesenchymal stem cells in printed blocks of TNF-α treatment group were significantly reduced in comparison. Conclusions: Exogenous TNF-α inhibits the directional differentiation of mesenchymal stem cells of mice into sweat gland cells in a three-dimensional environment, which may be related to the inhibition of the expression of Nanog mRNA and protein by TNF-α that subsequently results in the down-regulation of multi-directional differentiation potential of mesenchymal stem cells.

The effect of total lignans from Fructus Arctii on Streptozotocin-induced diabetic retinopathy in Wistar rats

Ethnopharmacological relevanceFructus Arctii is the dried ripe fruit of Arctium lappa L. (family Asteraceae). It is a well-known Chinese Materia Medica that was included in the Chinese pharmacopoeia because of its traditional therapeutic actions, such as heat removal, detoxification, and elimination of swelling. Since ancient times Fructus Arctii has been used extensively in a number of classical drug formulas to treat type 2 diabetes mellitus. Modern pharmacological studies have shown that certain components of Fructus Arctii have multiple physiological activities on type 2 diabetes and its complications. Aim of the studyWe have reported the inhibitory effect of total lignans from Fructus Arctii (TLFA) on aldose reductase, the key enzyme in the polyol pathway, which is considered to be closely related to the onset of diabetic retinopathy (DR). The present study aimed to observe the preventive and therapeutic effects of TLFA on DR in Streptozotocin (STZ)-induced DR rats. Materials and methodsTLFA was prepared from Fructus Arctii and its content was determined using UV spectrophotometry. The DR model was induced by STZ in Wistar rats. For DR prevention, the animals were gavaged once daily for 9 weeks with TLFA (1.38, 0.69, and 0.35 g/kg/day) as soon as they were confirmed as diabetes models. Pathological changes to retinal tissues and the expression of vascular endothelial growth factor (VEGF) and protein kinase C (PKC) in the retina were detected after TLFA treatment. The effects of TLFA on blood glucose levels and body weight were also observed. For DR treatment, the animals were gavaged once daily for 12 weeks with TLFA (1.38 and 0.69 g/kg/day) at 3 months after they were confirmed as diabetes models. The therapeutic effect was studied using quantitative detection of blood-retina barrier (BRB) breakdown via an Evans Blue leakage assay. ResultsFor DR prevention, after 9 weeks of TLFA administration, histopathological examination of retinal tissue showed that TLFA improved the lesions in the retina. Changes to retinal microstructures such as capillaries, ganglion cells, bipolar cells, and the membrane disk examined by electron microscopy further confirmed that TLFA has a preventive effect on retinopathy. Terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) detection showed that TLFA could inhibit retinal cell apoptosis in the diabetic rats, and fasting blood glucose (FBG) levels of rats in the TLFA-treated groups decreased during the experiment. For DR treatment, after 3 months of administration, the amount of dye leakage in the TLFA-administered groups was reduced by more than 50% compared with that in the model group, which indicated that TLFA has a therapeutic effect on middle and late DR. Messenger RNA (mRNA) expression of VEGF and PKCβ2 in the retina detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) showed that TLFA could inhibit the expression of them, which was consistent with the results of immunohistochemistry (IHC). ConclusionTLFA has a preventive and therapeutic effect on DR. Its mechanism of action on DR is related to inhibiting PKC activation and blocking VEGF elevation.

Mechanism of nuclear translocation of hypoxia-inducible factor-1α in influenza A (H1N1) virus infected-alveolar epithelial cells

Mechanism of nuclear translocation of hypoxia-inducible factor-1α in influenza A (H1N1) virus infected-alveolar epithelial cells

Regulation mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice

Objective: To explore the regulatory mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice. Methods: E2F1 gene knockout heterozygotes C57BL/6 mice and wild-type C57BL/6 mice were introduced and self-reproduced. Two weeks after birth, E2F1 gene knockout homozygotes mice and wild-type mice were identified by polymerase chain reaction (PCR). Twelve identified 6-8 weeks old male E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were selected respectively according to the random number table and set as E2F1 gene knockout group and wild-type group. A full-thickness skin defect wound was made on the back of each mouse. On post injury day (PID) 2 and 7, 6 mice in each group were selected according to the random number table and sacrificed, and the wound tissue was excised. The expression of CD68 and CD206 double positive M2 macrophages was observed by immunofluorescence method, and the percentage of CD206 positive cells was calculated. The protein expression of CD206 was detected by Western blotting. The mRNA expression of arginase 1 was detected by real-time fluorescent quantitative reverse transcription PCR (RT-PCR). Wound tissue specimens of the two groups on PID 7 were obtained, and the protein and mRNA expressions of peroxisome proliferator-activated receptor gamma (PPAR-γ) were detected by Western blotting and real-time fluorescent quantitative RT-PCR respectively. The above-mentioned experiments were repeated four times. Three specimens of wound tissue of mice in wild-type group on PID 7 were obtained to detect the relationship between E2F1 and PPAR-γ by co-immunoprecipitation and Western blotting, and this experiment was repeated two times. Data were processed with unpaired t test. Results: The size of PCR products of E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were 227 and 172 bp respectively, which were the same as those of the designed DNA fragments. On PID 2 and 7, the number of CD68 and CD206 double positive M2 macrophages in the wound tissue of mice in E2F1 gene knockout group was more than that of wild-type group, and the percentages of CD206 positive cells in the wound tissue of mice in E2F1 gene knockout group were (0.234±0.032)% and (0.584±0.023)% respectively, which were significantly higher than (0.129±0.017)% and (0.282±0.071)% of wild-type group (t=3.29, 3.54, P<0.05). On PID 2 and 7, the protein expression of CD206 in the wound tissue of mice in E2F1 gene knockout group were 1.00±0.23 and 1.63±0.26 respectively, which were significantly higher than 0.43±0.06 and 0.97±0.08 of wild-type group (t=2.41, 2.45, P<0.05). On PID 2 and 7, the mRNA expressions of arginase 1 in the wound tissue of mice in E2F1 gene knockout group were 0.482±0.105 and 0.195±0.031 respectively, which were significantly higher than 0.163±0.026 and 0.108±0.017 of wild-type group (t=3.04, 2.86, P<0.05). On PID 7, the protein and mRNA expressions of PPAR-γ in the wound tissue of mice in E2F1 gene knockout group were 0.61±0.12 and 0.51±0.13 respectively, which were significantly higher than 0.20±0.04 and 0.20±0.04 of wild-type group (t=3.36, 2.86, P<0.05). On PID 7, detection of the wound tissue of mice in wild-type group showed that PPAR-γ had unidirectional effect on E2F1. Conclusions: E2F1 transcription factor affects the polarization of M2 macrophages by inhibiting the expression of PPAR-γ, thereby inhibiting the healing process of full-thickness skin defect wounds in mice.

Scalp-acupuncture Improves Neurological Function by Regulating Expression of NF-κB p 65 mRNA, IκB mRNA and IL-1 β and TNF-α in Parahippocampal Gyrus of Cerebral Ischemia Rats

To observe the effect of scalp-acupuncture intervention on the expression of parahippocampal factor-κB p 65 mRNA (NF-κB p 65 mRNA), IκB mRNA, interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in rats with cerebral ischemia (CI), so as to investigate its molecular mechanisms underlying improving CI by reducing inflammatory response. A total of 64 SD rats were randomized into normal control, model, medication and scalp-acupuncture groups, with 16 rats in each group. The focal CI model was established by middle cerebral artery occlusion (MCAO). Intraperitoneal injection of Pyrrolidine Dithiocarbamate (100 mg•kg－1•d－1) was administrated for rats in the medication group, once a day for 7 days. For rats of the scalp-acupuncture group, the acupuncture needles were rapidly inserted into bilateral Dingnieqianxiexian (MS 6) and Dingniehouxiexian (MS 7), followed by twirling the needles at 200 cycles/min for 1 min, once again every 10 min during 30 min's needle retention. The treatment was conducted once a day for 7 days. The neurologic deficit score (0－5 points, impaired consciousness, death, etc.) and neurological function score (motor, sensory and sensory tests, 0－10 points) were assessed according to Longa's (1989) and Schäbitz's (2004) methods, respectively. The expression levels of NF-κB p 65 mRNA and IκB mRNA in the parahippocampus gyrus tissue were detected by fluorescence quantitative reverse transcription-PCR, and IL-1 β and TNF-α proteins in the parahippocampus gyrus tissue were detected by immunohistochemistry. After modeling, the neurologic deficit and neurological function scores and the expression levels of NF-κB p 65 mRNA, IL-1 β and TNF-α in the parahip-pocampus were significantly increased in the model group than in the normal group (P<0.01), while the expression of IκB mRNA was considerably down-regulated (P<0.01). Following treatment intervention, the neurologic deficit and neurological function scores as well as NF-κB p 65 mRNA, and IL-1 β and TNF-α protein expression were significantly decreased in both scalp-acupuncture and medication groups compared with the model group (P<0.05, P<0.01), and the decreased expression of IκB mRNA was obviously increased (P<0.05)．. Scalp-acupuncture can improve neurologic function in cerebral ischemic rats, which is related with its effects in up-regulating the expression of IκB to inhibit the dissociation of NF-κB, then decreasing the expression of IL-1 β and TNF-α (reducing inflammatory response) in the parahippocampal gyrus tissue.

Ethanol extract of Rehmannia glutinosa exerts antidepressant-like effects on a rat chronic unpredictable mild stress model by involving monoamines and BDNF.

The dried roots of Rehmannia glutinosa Libosch. (Scrophulariaceae) are of both medicinal and nutritional importance. Our previous study has found that the 80% ethanol extract of R. glutinosa (RGEE) produced antidepressant-like activities in mouse behavioral despair depression models. However, its mechanisms are still unclear. The present study aimed to observe the antidepressant-like mechanisms of RGEE on a rat chronic unpredictable mild stress (CUMS) model by involving monoaminergic neurotransmitters and brain-derived neurotrophic factor (BDNF). CUMS-stressed rats were orally given RGEE daily (150, 300, and 600mg/kg) or fluoxetine hydrochloride (FH) for 3weeks after starting the CUMS procedure. Sucrose preference test was carried out to observe depression-like behavior, and serum and brain tissues were used for neurochemical and fluorescent quantitative reverse transcription PCR analysis. Results demonstrated that CUMS induced depression-like behavior, whereas RGEE and FH administration inhibited this symptom. Furthermore, CUMS caused excessively elevated levels of serum corticosterone (CORT), an index of hypothalamic-pituitary-adrenal (HPA) axis hyperactivity, in a manner attenuated by RGEE and FH administration. RGEE administration also further elevatedmonoamine neurotransmitters and BDNF levels, up-regulated the mRNA expression of BDNF and tropomyosin-related kinase B (TrkB) in hippocampus of rats suffering CUMS. Together, our findings suggest that RGEE can improve CUMS-evoked depression-like behavior, and indicate its mechanisms may partially be associated with restoring HPA axis dysfunctions, enhancing monoamineergic nervous systems, and up-regulating BDNF and TrkB expression.

Interventional effects of BAY11-7082 on lung inflammatory response at the early stage and acute lung injury of rats with severe burns

Interventional effects of BAY11-7082 on lung inflammatory response at the early stage and acute lung injury of rats with severe burns