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1.
Int. j. morphol ; 40(3): 728-734, jun. 2022. ilus, tab
Article in English | LILACS-Express | LILACS | ID: biblio-1385653

ABSTRACT

SUMMARY: The main objective of this study was to analyze by real-time quantitative polymerase chain reaction (RT-qPCR) the expression patterns of the myosin heavy chain (MHC) isoforms (MHC-I, MHC-IIa, MHC-IIx) in the sphenomandibularis portion of the temporalis muscle. We expected to find differences between the sphenomandibularis and the other portions of the temporalis that could be related to the functional characteristics of the sphenomandibularis identified by electromyography. We dissected the right temporalis muscle of ten adult human individuals (five men and five women). Samples of the anterior and posterior temporalis and of the sphenomandibularis portion were obtained from each dissected muscle. These samples were analyzed by RT-qPCR to determine the percentages of expression of the MHC-I, MHC-IIa and MHC-IIx isoforms. No significant differences were identified between the anterior and the posterior temporalis in the expression patterns of the MHC-I, MHC-IIa and MHC-IIx isoforms. However, there were significant differences between the sphenomandibularis and the anterior temporalis. Specifically, the sphenomandibularis portion had a higher percentage of expression of the MHC-I isoform (P=0.04) and a lower percentage of expression of the MHC-IIx isoform (P=0.003). The pattern of expression that we observed in the sphenomandibularis reflects a greater resistance to fatigue, a lower contraction speed, and a lower capacity of force generation in the sphenomandibularis compared to the anterior temporalis. These characteristics are consistent with electromyographic findings on the functional differences between these two portions.


RESUMEN: El principal objetivo de este estudio fue analizar mediante real-time quantitative polymerase chain reaction (RT-qPCR) los patrones de expresión de las isoformas de la cadena pesada de la miosina (MHC-I, MHC-IIa y MHC-IIx) en la porción esfenomandibular del músculo temporal. Se esperó encontrar diferencias entre el esfenomandibular y las otras porciones del músculo temporal que se pudieran relacionar con las características funcionales del esfenomandibular, identificadas mediante electromiografía. Para obtener estos resultados, se diseccionó el músculo temporal derecho en diez humanos adultos (cinco hombres y cinco mujeres) y se obtuvieron muestras de la porción anterior y posterior del músculo temporal y de su porción esfenomandibular. Estas muestras fueron analizadas mediante RT-qPCR para determinar los porcentajes de expresión de las isoformas MHC-I, MHC- IIa y MHC-IIx. No se identificaron diferencias significativas de los patrones de expresión entre la porción anterior y la porción posterior del músculo temporal, pero sí que se observaron diferencias significativas entre la porción anterior del músculo temporal y su porción esfenomandibular. Concretamente, la porción esfenomandibular presentó un mayor porcentaje de expresión de la isoforma MHC-I (P=0.04) y un menor porcentaje de expresión de la isoforma MHC-IIx (P=0.003). El patrón de expresión que hemos observado en la porción esfenomandibular del músculo temporal refleja una mayor resistencia a la fatiga, una velocidad de contracción más lenta y una menor capacidad de generar fuerza si se compara esta porción con la porción anterior del músclo temporal. Estas características son consistentes con las diferencias funcionales que presentan estas dos porciones, que han sido descritas mediante electromiografía.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Temporal Muscle/metabolism , Myosin Heavy Chains/metabolism , Sphenoid Bone , RNA, Messenger/metabolism , Immunohistochemistry , Protein Isoforms , Electromyography , Real-Time Polymerase Chain Reaction
2.
Article in Chinese | WPRIM | ID: wpr-936373

ABSTRACT

OBJECTIVE@#To investigate the protective effect of fucoxanthin (FX) against diabetic cardiomyopathy and explore the underlying mechanism.@*METHODS@#Rat models of diabetes mellitus (DM) induced by intraperitoneal injection of streptozotocin (60 mg/kg) were randomized into DM model group, fucoxanthin treatment (DM+FX) group and metformin treatment (DM+ Met) group, and normal rats with normal feeding served as the control group. In the two treatment groups, fucoxanthin and metformin were administered after modeling by gavage at the daily dose of 200 mg/kg and 230 mg/kg, respectively for 12 weeks, and the rats in the DM model group were given saline only. HE staining was used to examine the area of cardiac myocyte hypertrophy in each group. The expression levels of fibrotic proteins TGF-β1 and FN proteins in rat hearts were detected with Western blotting. In the cell experiment, the effect of 1 μmol/L FX on H9C2 cell hypertrophy induced by exposure to high glucose (HG, 45 mmol/L) was evaluated using FITC-labeled phalloidin. The mRNA expression levels of the hypertrophic factors ANP, BNP and β-MHC in H9C2 cells were detected using qRT-PCR. The protein expressions of Nrf2, Keap1, HO-1 and SOD1 proteins in rat heart tissues and H9C2 cells were determined using Western blotting. The DCFH-DA probe was used to detect the intracellular production of reactive oxygen species (ROS).@*RESULTS@#In the diabetic rats, fucoxanthin treatment obviously alleviated cardiomyocyte hypertrophy and myocardial fibrosis, increased the protein expressions of Nrf2 and HO-1, and decreased the protein expressions of Keap1 in the heart tissue (P < 0.05). In H9C2 cells with HG exposure, fucoxanthin significantly inhibited the enlargement of cell surface area, lowered the mRNA expression levels of ANP, BNP and β-MHC (P < 0.05), promoted Nrf2 translocation from the cytoplasm to the nucleus, and up-regulated the protein expressions its downstream targets SOD1 and HO-1 (P < 0.05) to enhance cellular antioxidant capacity and reduce intracellular ROS production.@*CONCLUSION@#Fucoxanthin possesses strong inhibitory activities against diabetic cardiomyocyte hypertrophy and myocardial fibrosis and is capable of up-regulating Nrf2 signaling to promote the expression of its downstream antioxidant proteins SOD1 and HO-1 to reduce the level of ROS.


Subject(s)
Animals , Antioxidants/metabolism , Atrial Natriuretic Factor/pharmacology , Cardiomegaly , Diabetes Mellitus, Experimental/metabolism , Fibrosis , Kelch-Like ECH-Associated Protein 1/metabolism , Metformin , NF-E2-Related Factor 2/metabolism , Oxidative Stress , RNA, Messenger/metabolism , Rats , Reactive Oxygen Species/metabolism , Superoxide Dismutase-1/pharmacology , Xanthophylls
3.
Article in Chinese | WPRIM | ID: wpr-936366

ABSTRACT

OBJECTIVE@#To investigate the changes of tetraspanin 8 (TSPAN8) expression levels and its role in lipid metabolism during the development of non-alcoholic fatty liver disease (NAFLD).@*METHODS@#Thirty male C57BL/6J mice were randomly divided into normal diet group and high-fat diet (HFD) group (n=15), and after feeding for 1, 3, and 6 months, the expression levels of TSPAN8 in the liver tissues of the mice were detected with Western blotting. In a HepG2 cell model of NAFLD induced by free fatty acids (FFA), the effect of TSPAN8 overexpression on lipid accumulation was examined using Oil Red O staining and an automated biochemical analyzer, and the mRNA expressions of the key genes involved in lipid metabolism were detected using qRT-PCR.@*RESULTS@#Western blotting showed that compared with that in mice with normal feeding, the expression of TSPAN8 was significantly decreased in the liver tissues of mice with HFD feeding for 3 and 6 months (P < 0.05). In HepG2 cells, treatment with FFA significantly decreased the expression of TSPAN8 at both the mRNA and protein levels (P < 0.01). TSPAN8 overexpression in FFA-treated cells showed significantly lowered intracellular triglyceride levels (P < 0.001) and obviously reduced mRNA expression of fatty acid transport protein 5 (FATP5) (P < 0.01). The expression of FATP5 was significantly increased in FFA-treated cells as compared with the control cells (P < 0.001).@*CONCLUSION@#TSPAN8 is involved in lipid metabolism in NAFLD, and overexpression of TSPAN8 may inhibit cellular lipid deposition by reducing the expression of FATP5.


Subject(s)
Animals , Diet, High-Fat/adverse effects , Fatty Acids, Nonesterified , Lipid Metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/metabolism , RNA, Messenger/metabolism
4.
Article in Chinese | WPRIM | ID: wpr-936364

ABSTRACT

OBJECTIVE@#To investigate the relationship between AML1-ETO (AE) fusion gene and intracellular N6-methyladenosine (m6A) modification pattern in t(8;21) acute myeloid leukemia (AML).@*METHODS@#RNA m6A sequencing was performed in SKNO-1 and AE knockdown SKNO-1 (SKNO-1 siAE) cells using RNA-protein co-immunoprecipitation and high-throughput sequencing (methylated RNA immunoprecipitation sequencing, MeRIP-Seq) to analyze the changes in m6A modification of the entire transcriptome. Transcriptome sequencing (RNA-seq) was performed using high-throughput sequencing. The differentially modified mRNAs were further functionally annotated by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The changes in m6A-related enzyme expressions were detected using real-time PCR.@*RESULTS@#A total of 26 441 genes were identified in AE knockdown AML cells and AE-expressing cells, containing 72 036 m6A peaks. AE knockdown caused a reduction of the number of intracellular m6A peaks from 37 042 to 34 994, among which 1278 m6A peaks were significantly elevated and 1225 were significantly decreased; 1316 genes with newly emerged m6A modification were detected and 1830 genes lost m6A modification after AE knockdown. The differential peaks were mainly enriched in pathways involving cancer and human T-lymphocytic leukemia virus I. RNA-seq results showed that 2483 genes were up-regulated and 3913 genes were down-regulated after AE knockdown. The combined analysis of MeRIP-Seq and RNA-Seq results revealed relatively high expression levels of m6A-modified genes as compared with the genes without m6A modification (SKNO-1: 0.6116±1.263 vs 2.010±1.655, P < 0.0001; SKNO-1 siAE: 0.5528±1.257 vs 2.067±1.686, P < 0.0001). The m6A modified genes located in the 3'UTR or 5 'UTR had significantly higher expression levels than those located in exonic regions (SKNO-1: 2.177± 1.633 vs 1.333 ± 1.470 vs 2.449 ± 1.651, P < 0.0001; SKNO-1 siAE: 2.304 ± 1.671 vs 1.336 ± 1.522 vs 2.394 ± 1.649, P < 0.05). Analysis of RNA-seq data identified 3 m6A-related enzymes that showed significantly elevated mRNA expression after AE knockdown, namely WTAP, METTL14, and ALKBH5 (P < 0.05), but the results of real-time PCR showed that the expressions of WTAP and ALKBH5 were significantly increased while the expression of METTL14 was lowered after AE knockdown (P < 0.05).@*CONCLUSION@#AE knockdown results in differential expressions of m6A-associated enzymes, suggesting that the AE fusion gene regulates the expression of one or more m6A-associated enzymes to control cellular methylation levels.


Subject(s)
Adenosine/analogs & derivatives , Humans , Leukemia, Myeloid, Acute/genetics , RNA, Messenger/metabolism , Transcriptome
5.
Article in Chinese | WPRIM | ID: wpr-936362

ABSTRACT

OBJECTIVE@#To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H2O2)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism.@*METHODS@#GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H2O2 (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay.@*RESULTS@#Exposure to H2O2 significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P < 0.05 or 0.01). Treatment of the cells prior to H2O2 exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P < 0.05).@*CONCLUSION@#Hyperoside protects GC-2 cells against H2O2- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.


Subject(s)
Animals , Antioxidants/metabolism , Heme Oxygenase-1/metabolism , Hydrogen Peroxide/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , Male , Mice , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Quercetin/analogs & derivatives , RNA, Messenger/metabolism , Spermatocytes/metabolism , Superoxide Dismutase/metabolism
6.
Article in Chinese | WPRIM | ID: wpr-936351

ABSTRACT

OBJECTIVE@#To explore the effect of inhibiting polyribonucleotide nucleotidyl-transferase 1 (PNPT1) on oxygen-glucose deprivation (OGD)-induced apoptosis of mouse atrial myocytes.@*METHODS@#Cultured mouse atrial myocytes (HL-1 cells) with or without OGD were transfected with PNPT1-siRNA or a negative control siRNA (NC-siRNA group), and the cell survival rate was detected using CCK-8 assay. The expression levels of ACTB and TUBA mRNA were detected with qPCR, and the protein expression of PNPT1 was detected with Western blotting. The apoptosis rate of the treated cells was determined with flow cytometry, the mitochondrial membrane potential was detected using JC-1 kit, and the mitochondrial morphology was observed using transmission electron microscope.@*RESULTS@#With the extension of OGD time, the protein expression levels of PNPT1 increased progressively in the cytoplasm of HL-1 cells (P < 0.05). Transfection with PNPT1-siRNA significantly reduced PNPT1 expression in HL-1 cells (P < 0.05). Exposure to OGD significantly enhanced degradation of ACTB and TUBA mRNA (P < 0.05) and markedly increased the apoptosis rate of HL-1 cells (P < 0.05), and these changes were significantly inhibited by transfection with PNPT1-siRNA (P < 0.05), which obviously increased mitochondrial membrane potential and improved mitochondrial morphology of HL-1 cells exposed to OGD.@*CONCLUSION@#Inhibition of PNPT1 improves mitochondrial damage and reduces degradation of apoptotic-associated mRNAs to alleviate OGD-induced apoptosis of mouse atrial myocyte.


Subject(s)
Animals , Apoptosis , Cell Survival , Glucose/pharmacology , Mice , Myocytes, Cardiac , Oxygen/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism
7.
Article in Chinese | WPRIM | ID: wpr-936334

ABSTRACT

OBJECTIVE@#To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway.@*METHODS@#Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR.@*RESULTS@#Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist.@*CONCLUSION@#Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.


Subject(s)
Animals , Cell Differentiation/drug effects , Endoribonucleases/metabolism , Estradiol/pharmacology , Estrogens/metabolism , Interleukin-10 , Interleukin-6/metabolism , Macrophages, Peritoneal/metabolism , Mice , Phenotype , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/metabolism , Signal Transduction/drug effects , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation/drug effects , X-Box Binding Protein 1/metabolism
8.
Article in Chinese | WPRIM | ID: wpr-936331

ABSTRACT

OBJECTIVE@#To investigate the effect of pirfenidone for reducing urethral stricture following urethral injury in rats and explore the possible mechanism.@*METHODS@#Thirty male SD rats were randomly assigned into negative control group, positive control group and pirfenidone group (n=10). In pirfenidone and positive control groups, the rats were subjected to incision of the posterior urethral cavernous body followed by daily intraperitoneal injection of pirfenidone (100 mg/kg) and an equivalent volume of solvent, respectively. The rats in the negative control group were given intraperitoneal injections of solvent without urethral injury. At two weeks after modeling, retrograde urethrography was performed for observing urethral stricture, and the injured urethral tissues were harvested for HE staining, Masson staining, immunohistochemical staining and Western blotting for detecting the protein expressions of α-SMA and TGF-β1. The mRNA expressions of the inflammatory factors TNF-α, IL-6, and IL-1β were detected using qRT-PCR.@*RESULTS@#The body weight of the rats in pirfenidone group was significantly decreased compared with that in the other two groups (P < 0.05). Retrograde urethrography showed significant narrowing of the urethra in the positive control group but not in the pirfenidone group. HE staining of the injured urethral tissues showed obvious proliferation of urethral epithelial cells with narrow urethral cavity and increased inflammatory cells in positive control group. The pathological findings of the urethra were similar between pirfenidone group and the negative control group. Masson staining revealed obviously reduced collagen fibers and regular arrangement of the fibers in pirfenidone group as compared to the positive control group. Compared with those in the negative control group, the expressions of α-SMA and TGF-β1 were significantly increased in the positive control group, and pirfenidone treatment significantly inhibited their expressions (P < 0.05 or 0.01). Pirfenidone also significantly inhibited the mRNA expressions of TNF-α, IL-6, and IL-1β in the injured urethral tissue (P < 0.05 or 0.01).@*CONCLUSION@#Pirfenidone can prevent urethral fibrosis and stricture after urethral injury possibly by inhibiting the TGF-β1 pathway and inflammatory response.


Subject(s)
Animals , Female , Humans , Interleukin-6/metabolism , Male , Pyridones/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Solvents , Transforming Growth Factor beta1/metabolism , Tumor Necrosis Factor-alpha/metabolism , Urethral Stricture/pathology
9.
Article in Chinese | WPRIM | ID: wpr-936328

ABSTRACT

OBJECTIVE@#To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism.@*METHODS@#Wild-type (WT) mice and systemic CD36 knockout (CD36-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively.@*RESULTS@#CD36-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36-/- mice (P>0.05). In the muscle tissue of CD36-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36-/- mice.@*CONCLUSION@#CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.


Subject(s)
Animals , Gene Deletion , Histones/genetics , Insulin , Insulin Receptor Substrate Proteins/metabolism , Insulin Resistance/genetics , Membrane Cofactor Protein/genetics , Mice , Mice, Knockout , Muscles/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/metabolism , Receptor, Insulin/metabolism , Tyrosine/genetics , Up-Regulation
10.
Article in Chinese | WPRIM | ID: wpr-936318

ABSTRACT

OBJECTIVE@#To explore the expression patterns, prognostic implications, and biological role of leukotriene B4 receptor (LTB4R) in patients with acute myeloid leukemia (AML).@*METHODS@#We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database for mRNA expression analyses, survival analyses, Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R. The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN. The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis. A protein-protein interaction network was constructed using STRING. GSEA analyses of the differentially expressed genes (DEGs) were performed based on datasets from TCGA-LAML stratified by LTB4R expression level. We also collected peripheral blood mononuclear cells (PBMCs) from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR. We also examined serum LTB4R protein levels in the patients using ELISA.@*RESULTS@#The mRNA expression level of LTB4R was significantly increased in AML patients (4.898±1.220 vs 2.252±0.215, P < 0.001), and an elevated LTB4R expression level was correlated with a poor overall survival (OS) of the patients (P=0.004, HR=1.74). LTB4R was identified as an independent prognostic factor for OS (P=0.019, HR=1.66) and was associated with FAB subtypes, cytogenetic risk, karyotype abnormalities and NPM1 mutations. The co- expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis, including neutrophil inflammation, lymphocyte activation, signal transduction, and metabolism. The DEGs were enriched in differentiation, activation of immune cells, and cytokine signaling. Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA (P=0.044) and protein (P=0.008) in AML patients, and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2 (r= 0.466, P=0.040).@*CONCLUSION@#LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.


Subject(s)
Humans , Leukemia, Myeloid, Acute/metabolism , Leukocytes, Mononuclear/metabolism , Prognosis , RNA, Messenger/metabolism , Receptors, Leukotriene B4/genetics
11.
Article in Chinese | WPRIM | ID: wpr-936307

ABSTRACT

OBJECTIVE@#To establish an cell model of hyperparathyroidism by isolation, in vitro culture, and identification of parathyroid cells from patients with secondary hyperparathyroidism (SHPT).@*METHODS@#The parathyroid gland tissues obtained from 10 patients with SHPT were dissociated by collagenase digestion for primary culture of the parathyroid cells. Morphological changes and growth characteristics of the cells were assessed by microscopic imaging and cell counting. The mRNA and protein expression levels of parathyroid hormone (PTH), calcium-sensing receptor (CaSR), and glial cells missing 2 (GCM2) in the primary and passaged cells were determined by immunofluorescence, qRT-PCR, and Western blotting.@*RESULTS@#Primary cultures of parathyroid cells were successfully obtained. The cells exhibited a high expression of PTH shown by immunofluorescence assay and had a population doubling time of approximately 71.61 h. PTH secretion in the second-passage (P2) cells was significantly lower than that in the primary (P0) and first-passage (P1) cells (P < 0.001). Despite a significant downregulation of CaSR mRNA (P=0.017) and protein (P=0.006) in P1 cells as compared with P0 cells, no significant differences were found in mRNA and protein expressions of PTH or GCM2 between the two cell generations.@*CONCLUSION@#Primary cultures of parathyroid cells isolated from SHPT patients by collagenase digestion show similar biological properties to the cells in vivo.


Subject(s)
Humans , Hyperparathyroidism, Secondary/metabolism , Parathyroid Glands/metabolism , Parathyroid Hormone , RNA, Messenger/metabolism , Receptors, Calcium-Sensing/metabolism
12.
Article in Chinese | WPRIM | ID: wpr-936283

ABSTRACT

OBJECTIVE@#To construct the regulatory network of survival-related onco-miRNAs and their target genes in hepatocellular carcinoma (HCC) and verify the interactions between the key miRNAs and their targets.@*METHODS@#We screened survival-related miRNAs in HCC in OncomiR and Oncolnc databases, predicted their target genes using miRNet, and conducted survival and expression analysis using GEPIA2 and Ualcan, respectively. The miRNA-target gene co-expression analysis was performed and the miRNA-target network was constructed. Enrichment analysis was performed in Enrichr and protein-protein interaction analysis in STRING database. We tested the effects of transfection with the mimic or inhibitor of hsa-miR-1226-3p or hsa-miR-221-5p on proliferation of HepG2 cells using CCK8 assay and examined the changes in the expressions of the target genes using RT-qPCR. The effect of transfection with hsa-miR-221-5p mimic or inhibitor on protein expressions of the target genes was examined using Western blotting in. A dual luciferase reporter assay was used to test the interaction between hsa-miR-221-5p and its potential target gene GCDH. We further examined the effect of transfection with hsa-miR-221-5p mimic and pEGFP N1-GCDH, alone or in combination, on proliferation, migration and invasion of HepG2 cells.@*RESULTS@#We identified 223 survival-related miRNAs in HCC from OncomiR and 146 miRNAs from Oncolnc with an intersection of 131 miRNAs, and 48 miRNAs were identified as onco-miRNAs in HCC after survival and expression analysis. Twenty-seven eligible target genes were identified after miRNA-mRNA co-expression analysis. The constructed miRNA-target gene network consisted of 25 miRNAs and 27 target genes. The most enriched term was fatty acid metabolism for the target genes. In HepG2 cells, transfection with the mimic or inhibitor of hsa-miR-1226-3p or hsa-miR-221-5p caused significant changes of the mRNA and protein levels of their respective target genes (P < 0.05). The results of dual luciferase reporter assay confirmed the targeting relationship between hsa-miR-221-5p and GCDH gene (P < 0.05). Transfection with hsa-miR-221-5p mimic significantly suppressed the proliferation, migration and invasion of HepG2 cells, but this effect was obviously relieved by co-transformation with pEGFP N1-GCDH (P < 0.05).@*CONCLUSION@#Fatty acid metabolism might be one of the most crucial pathways that mediate the effect of the oncomiRNAs in HCC, and the hsa-miR-221-5p/GCDH axis is an important molecular mechanism for HCC progression.


Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Regulatory Networks , Humans , Liver Neoplasms/pathology , MicroRNAs/metabolism , RNA, Messenger/metabolism
13.
Article in Chinese | WPRIM | ID: wpr-936154

ABSTRACT

OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.


Subject(s)
Adipogenesis/genetics , Bone Marrow Cells/metabolism , Cell Differentiation , Cells, Cultured , Mesenchymal Stem Cells , Osteogenesis , PPAR gamma/pharmacology , RNA, Long Noncoding/genetics , RNA, Messenger/metabolism
14.
Chinese Journal of Stomatology ; (12): 390-396, 2022.
Article in Chinese | WPRIM | ID: wpr-935878

ABSTRACT

Objective: To study the effect of microRNA-126 (miR-126) on the polarization of human monocyte-derived macrophages stimulated by Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS). Methods: Macrophages derived from human myeloid leukemia mononuclear cells were stimulated by Pg-LPS (5 mg/L) and by Pg-LPS (5 mg/L) after 24 h-transfection of miR-126 mimic or negative control RNA for 48 h, respectively. Real-time quantitative-PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting were conducted to detect the changes in miR-126, pro-inflammatory factor tumor necrosis factor-α (TNF-α), anti-inflammatory factors interleukin-10 (IL-10), inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1) and M1 polarization-related pathways such as nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Results: Compared with non-LPS stimulation group (TNF-α: 1.000±0.020, iNOS: 1.125±0.064, miR-126: 1.004±0.113, IL-10: 1.003±0.053, Arg-1: 1.130±0.061), the mRNA levels of TNF-α (3.105±0.278) and iNOS (4.296±0.003) increased significantly (t=6.53, P=0.003; t=42.63, P<0.001, respectively), while miR-126, IL-10 and Arg-1 expressions (0.451±0.038, 0.545±0.004 and 0.253±0.017) decreased significantly (t=7.95, P=0.001; t=7.36, P=0.002; t=11.94, P<0.001, respectively) after Pg-LPS stimulated by human-derived macrophages for 48 h. The protein expression of iNOS, TNF-α, Arg-1 and IL-10 were consistent at mRNA levels. Meanwhile, the expressions of phospho-NF-κB p65 (p-p65), phospho-extracellular signal-regulated kinase (p-ERK) and phospho-p38 MAPK (p-p38) increased significantly, while the expression of Arg-1 decreased significantly. Compared with the negative controls (scramble RNA) (TNF-α: 1.141±0.197, iNOS: 1.173±0.115, IL-10: 1.032±0.138, Arg-1: 0.933±0.044), the mRNA levels of TNF-α (0.342±0.022) and iNOS (0.588±0.085) expressions significantly decreased (t=5.35, P=0.006; t=5.05, P=0.007), while IL-10 (1.786±0.221) and Arg-1 expressions (2.152±0.229) significantly increased (t=3.71, P=0.021; t=6.21, P=0.003) after Pg-LPS stimulation with miR-126 mimic transfection. The relative protein expressions of iNOS, p-p65, p-ERK and p-p38 significantly decreased (t=13.00, P<0.001; t=6.98, P=0.002; t=10.86, P<0.001; t=8.32, P=0.001), while the protein level of Arg-1 significantly increased (t=12.08, P<0.001). Conclusions: Pg-LPS could promote M1 polarization of macrophages. miR-126 might inhibit the effect of Pg-LPS on the M1 polarization of macrophages through down-regulating NF-κB and MAPK signaling pathways.


Subject(s)
Cell Polarity , Humans , Interleukin-10/metabolism , Lipopolysaccharides/pharmacology , Macrophage Activation , Macrophages/drug effects , MicroRNAs/metabolism , NF-kappa B/metabolism , Porphyromonas gingivalis , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/metabolism
15.
Article in Chinese | WPRIM | ID: wpr-941030

ABSTRACT

OBJECTIVE@#To investigate the effect of Enterococcus faecium QH06 on TNBS-induced ulcerative colitis (UC) in rats and explore the mechanisms in light of intestinal flora and intestinal immunity.@*METHODS@#Thirty-six male Wistar rats were randomized equally into control group, UC model group, and E.faecium QH06 intervention group. The rats in the latter two groups were subjected to colonic enema with 5% TNBS/ethanol to induce UC, followed by treatment with intragastric administration of distilled water or E.faecium QH06 at the dose of 0.21 g/kg. After 14 days of treatment, the rats were examined for colon pathologies with HE staining. The mRNA and protein expression levels of IL-4, IL-10, IL-12, and IFN-γ in the colon tissues were detected using RT-qPCR and ELISA, and the expression of TLR2 protein was detected with immunohistochemistry and ELISA. Illumina Miseq platform was used for sequencing analysis of the intestinal flora of the rats with bioinformatics analysis. The correlations of the parameters of the intestinal flora with the expression levels of TLR2 and cytokines were analyzed.@*RESULTS@#The rats with TNBS- induced UC showed obvious weight loss (P < 0.01) and severe colon tissue injury with high pathological scores (P < 0.01). The protein expression levels of IFN-γ, IL-12, and TLR2 (P < 0.01) and the mRNA expression levels of IFN-γ, IL-12 and IL-10 (P < 0.05) were significantly increased in the colon tissues of the rats with UC. Illumina Miseq sequence analysis showed that in UC rats, the Shannon index (P < 0.05) ACE (P < 0.01)and Chao (P < 0.05) index for the diversity of intestinal flora both decreased with a significantly increased abundance of Enterobacteriaceae (P < 0.01) and a lowered abundance of Burkholderiaceae (P < 0.05). Compared with the UC rats, the rats treated with E. faecium QH06 showed obvious body weight gain (P < 0.05), lessened colon injuries, lowered pathological score of the colon tissue (P < 0.05), decreased protein expressions of IFN- γ, IL- 12, and TLR2 and mRNA expressions of IFN- γ and IL-12 (P < 0.01 or 0.05), and increased protein expressions of IL- 4 (P < 0.05). The Shannon index ACE (P < 0.05) and Chao (P < 0.05) index of intestinal microflora were significantly increased, the abundance of Enterobacteriaceae was lowered and that of Burkholderiaceae and Rikenellaceae was increased in E.faecium QH06- treated rats (P < 0.01 or 0.05). Correlation analysis showed that IFN-γ was positively correlated with the abundance of Enterobacteriaceae, and IFN-γ was negatively correlated with the abundance of Prevotellaceae, Desulfovibrionaceae, norank_o_Mollicutes_RF39 and Clostridiales_vadinBB60_group; TLR2 was negatively correlated with Clostridiales_vadinBB60_group, norank_o_Mollicutes_RF39 and Prevotellaceae.@*CONCLUSION@#E.faecium QH06 can alleviate TNBS-induced colonic mucosal injury in rats, and its effect is mediated possibly by increasing the abundance of SCFA-producing bacteria such as Prevotellaceae and inhibiting abnormal immune responses mediated by TLR2.


Subject(s)
Animals , Colitis, Ulcerative/drug therapy , Colon/metabolism , Interleukin-10 , Interleukin-12/therapeutic use , Male , RNA, Messenger/metabolism , Rats , Rats, Wistar , Toll-Like Receptor 2/metabolism
16.
Article in Chinese | WPRIM | ID: wpr-941020

ABSTRACT

OBJECTIVE@#To investigate the effect of Chaihu Guizhi Decoction (CHGZD) combined with capecitabine on growth and apoptosis of subcutaneous triple-negative breast cancer xenografts in nude mice and explore the possible mechanism.@*METHODS@#Nude mouse models bearing subcutaneous triple-negative breast cancer xenografts were randomized into 6 groups (n=10) for treatment with distilled water (model group), low (10.62 g/kg), medium (21.23 g/kg) and high (42.46 g/kg) doses of CHGZD, capecitabine (0.2 mg/kg), or the combination of CHGZD (42.46 g/kg) and capecitabine (0.2 mg/k) once daily for 21 consecutive days. The general condition of mice was observed, and after 21-day treatments, the tumors were dissected for measurement of tumor volume and weight and histopathological examination with HE staining. Serum IL-6 levels of the mice were determined with enzyme-linked immunosorbent assay (ELISA), and the expression levels of IL-6, STAT3, p-STAT3, Bax, Bcl-2 and cyclin D1 in the tumor tissues were detected using real-time PCR and Western blotting.@*RESULTS@#Compared with those in the model group, the tumor-bearing mice receiving treatments with CHGZD showed significantly increased food intake with good general condition, sensitive responses, increased body weight, and lower tumor mass (P < 0.01). Compared with capecitabine treatment alone, treatment with CHGZD alone at the medium and high doses and the combined treatment all resulted in significantly higher tumor inhibition rates (P < 0.01), induced obvious tumor tissue degeneration and reduced the tumor cell density. Treatments with CHGZD, both alone and in combination with capecitabine, significantly decreased serum IL-6 level, lowered the mRNA expression levels of IL-6 and STAT3, the protein expressions of IL-6, STAT3 and P-STAT3 (P < 0.05), and the mRNA and protein expressions of Bcl-2 and cyclin D1 (P < 0.05), and increased the mRNA and protein expressions of Bax in the tumor tissues (P < 0.05).@*CONCLUSION@#CHGZD combined with capecitabine can significantly inhibit tumor growth in nude mice bearing triple-negative breast cancer xenografts, the mechanism of which may involve the inhibition of IL-6/STAT3 signaling pathway and regulation of Bax, Bcl-2 and cyclin D1 expressions to suppress tumor cell proliferation and differentiation and induce cell apoptosis.


Subject(s)
Animals , Capecitabine/pharmacology , Cyclin D1/metabolism , Drugs, Chinese Herbal , Heterografts , Humans , Interleukin-6/metabolism , Mice , Mice, Nude , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Triple Negative Breast Neoplasms/drug therapy , bcl-2-Associated X Protein/metabolism
17.
Article in Chinese | WPRIM | ID: wpr-941006

ABSTRACT

OBJECTIVE@#To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP).@*METHODS@#Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4μ8c, rapamycin, or both 4μ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay.@*RESULTS@#Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4μ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP.@*CONCLUSION@#IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.


Subject(s)
Animals , Autophagy , Calcium/metabolism , Chondrocytes , Endoplasmic Reticulum/metabolism , Endoribonucleases/pharmacology , Homeostasis , Inositol , Mice , Mice, Knockout , Protein Serine-Threonine Kinases , RNA, Messenger/metabolism , Sirolimus/pharmacology , Tunicamycin/pharmacology
18.
Chinese Journal of Cardiology ; (12): 690-697, 2022.
Article in Chinese | WPRIM | ID: wpr-940908

ABSTRACT

Objective: To investigate the effect and mechanism of sacubitril/valsartan on left ventricular remodeling and cardiac function in rats with heart failure. Methods: A total of 46 SPF-grade male Wistar rats weighed 300-350 g were acclimatized to the laboratory for 7 days. Rats were then divided into 4 groups: the heart failure group (n=12, intraperitoneal injection of adriamycin hydrochloride 2.5 mg/kg once a week for 6 consecutive weeks, establishing a model of heart failure); heart failure+sacubitril/valsartan group (treatment group, n=12, intragastric administration with sacubitril/valsartan 1 week before the first injection of adriamycin, at a dose of 60 mg·kg-1·d-1 for 7 weeks); heart failure+sacubitril/valsartan+APJ antagonist F13A group (F13A group, n=12, adriamycin and sacubitril/valsartan, intraperitoneal injection of 100 μg·kg-1·d-1 APJ antagonist F13A for 7 weeks) and control group (n=10, intraperitoneal injection of equal volume of normal saline). One week after the last injection of adriamycin or saline, transthoracic echocardiography was performed to detect the cardiac structure and function, and then the rats were executed, blood and left ventricular specimens were obtained for further analysis. Hematoxylin-eosin staining and Masson trichrome staining were performed to analyze the left ventricular pathological change and myocardial fibrosis. TUNEL staining was performed to detect cardiomyocyte apoptosis. mRNA expression of left ventricular myocardial apelin and APJ was detected by RT-qRCR. ELISA was performed to detect plasma apelin-12 concentration. The protein expression of left ventricular myocardial apelin and APJ was detected by Western blot. Results: Seven rats survived in the heart failure group, 10 in the treatment group, and 8 in the F13A group. Echocardiography showed that the left ventricular end-diastolic diameter (LVEDD) and the left ventricular end-systolic diameter (LVESD) were higher (both P<0.05), while the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were lower in the heart failure group than in the control group (both P<0.05). Compared with the heart failure group, rats in the treatment group were featured with lower LVEDD and LVESD (both P<0.05), higher LVEF and LVFS (both P<0.05), these beneficial effects were reversed in rats assigned to F13A group (all P<0.05 vs. treatment group). The results of HE staining showed that the cardiomyocytes of rats in the control group were arranged neatly and densely structured, the cardiomyocytes in the heart failure group were arranged in disorder, distorted and the gap between cells was increased, the cardiomyocytes in the treatment group were slightly neat and dense, and cardiomyocytes in the F13A group were featured similarly as the heart failure group. Masson staining showed that there were small amount of collagen fibers in the left ventricular myocardial interstitium of the control group, while left ventricular myocardial fibrosis was significantly increased, and collagen volume fraction (CVF) was significantly higher in the heart failure group than that of the control group (P<0.05). Compared with the heart failure group, the left ventricular myocardial fibrosis and the CVF were reduced in the treatment group (both P<0.05), these effects were reversed in the F13A group (all P<0.05 vs. treatment group). TUNEL staining showed that the apoptosis index (AI) of cardiomyocytes in rats was higher in the heart failure group compared with the control group (P<0.05), which was reduced in the treatment group (P<0.05 vs. heart failure group), this effect again was reversed in the F13A group (P<0.05 vs. treatment group). The results of RT-qPCR and Western blot showed that the mRNA and protein levels of apelin and APJ in left ventricular myocardial tissue of rats were downregulated in heart failure group (all P<0.05) compared with the control group. Compared with the heart failure group, the mRNA and protein levels of apelin and APJ were upregulated in the treatment group (all P<0.05), these effects were reversed in the F13A group (all P<0.05 vs. treatment group). ELISA test showed that the plasma apelin concentration of rats was lower in the heart failure group compared with the control group (P<0.05); compared with the heart failure group, the plasma apelin concentration of rats was higher in the treatment group (P<0.05), this effect was reversed in the F13A group (P<0.05 vs. treatment group). Conclusion: Sacubitril/valsartan can partially reverse left ventricular remodeling and improve cardiac function in rats with heart failure through modulating Apelin/APJ pathways.


Subject(s)
Aminobutyrates/pharmacology , Animals , Apelin/metabolism , Biphenyl Compounds , Collagen/metabolism , Doxorubicin/pharmacology , Fibrosis , Heart Failure/pathology , Male , Myocytes, Cardiac/pathology , RNA, Messenger/metabolism , Rats , Rats, Wistar , Valsartan/pharmacology , Ventricular Function, Left/drug effects , Ventricular Remodeling
19.
Article in English | WPRIM | ID: wpr-939805

ABSTRACT

OBJECTIVES@#Long-term treatment of olanzapine, the most widely-prescribed second-generation antipsychotic, remarkably increases the risk of non-alcoholic fatty liver disease (NAFLD), whereas the mechanism for olanzapine-induced NAFLD remains unknown. Excessive hepatic fat accumulation is the basis for the pathogenesis of NAFLD, which results from the disturbance of TG metabolism in the liver. Apolipoprotein A5 (ApoA5) is a key regulator for TG metabolism in vivo that promotes TG accumulation in hepatocytes, thereby resulting in the development of NAFLD. However, there are no data indicating the role of apoA5 in olanzapine-induced NAFLD. Therefore, this study aims to investigate the role of apoA5 in olanzapine-induced NAFLD.@*METHODS@#This study was carried out via animal studies, cell experiment, and ApoA5 gene knockdown experiment. Six-week-old male C57BL/6J mice were randomized into a control group, a low-dose group, and a high-dose group, which were treated by 10% DMSO, 3 mg/(kg·d) olanzapine, and 6 mg/(kg·d) olanzapine, respectively for 8 weeks. The lipid levels in plasma, liver function indexes, and expression levels of ApoA5 were detected. HepG2 cells were treated with 0.1% DMSO (control group), 25 μmol/L olanzapine (low-dose group), 50 μmol/L olanzapine (medium-dose group), and 100 μmol/L olanzapine (high-dose group) for 24 h. HepG2 cells pretreated with 100 μmol/L olanzapine were transfected with siRNA and scrambled siRNA (negative control), respectively. We observed the changes in lipid droplets within liver tissues and cells using oil red O staining and fat deposition in liver tissues using HE staining. The mRNA and protein levels of ApoA5 were determined by real-time PCR and Western blotting, respectively.@*RESULTS@#After intervention with 3 and 6 mg/(kg·d) olanzapine for 8 weeks, there was no significant difference in body weight among the 3 groups (P>0.05). Olanzapine dose-dependently increased the plasma TG, ALT and AST levels, and reduced plasma ApoA5 levels (all P<0.05), whereas there was no significant difference in plasma cholesterol (HDL-C, LDL-C, and TC) levels among the 3 groups (all P>0.05). Olanzapine dose-dependently up-regulated ApoA5 protein levels in liver tissues (all P<0.05), but there was no significant change in ApoA5 mRNA expression among groups (P>0.05). In the control group, the structure of liver tissues was intact, the morphology of liver cells was regular, and only a few scattered lipid droplets were found in the cells. In the olanzapine-treated group, there was a large amount of lipid deposition in hepatocytes, and cells were balloon-like and filled with lipid droplet vacuoles. The nucleus located at the edge of cell, and the number of lipid droplets was increased significantly, especially in the high-dose group. Likewise, when HepG2 cells were treated with olanzapine for 24 h, the number and size of lipid droplets were significantly elevated in a dose-dependent manner. Moreover, olanzapine dose-dependently up-regulated ApoA5 protein levels in HepG2 cells (all P<0.05), but there was no significant difference in ApoA5 mRNA expression among groups (P>0.05). Compared with the HepG2 cells transfected with scrambled siRNA, the number and size of lipid droplets in HepG2 cells transfected with ApoA5 siRNA were significantly reduced.@*CONCLUSIONS@#The short-term intervention of olanzapine does not significantly increase body weight of mice, but it can directly induce hypertriglyceridemia and NAFLD in mice. Olanzapine inhibits hepatic apoA5 secretion but does not affect hepatic apoA5 synthesis, resulting in the pathogenesis of NAFLD. Inhibition of apoA5 secretion plays a key role in the development of olanzapine-related NAFLD, which may serve as an intervention target for this disease.


Subject(s)
Animals , Apolipoprotein A-V/genetics , Body Weight , Dimethyl Sulfoxide/metabolism , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/chemically induced , Olanzapine/metabolism , RNA, Messenger/metabolism , RNA, Small Interfering , Triglycerides
20.
Article in English | WPRIM | ID: wpr-939780

ABSTRACT

OBJECTIVE@#To study the protective effect of anthocyanins extracted from Vaccinium Uliginosum (VU) on retinal 661W cells against microwave radiation induced retinal injury.@*METHODS@#661W cells were divided into 6 groups, including control, model [661W cells radiated by microwave (30 mW/cm2, 1 h)] and VU groups [661W cells pretreated with anthocyanins extracted from VU (25, 50, 100 and 200 µg/mL, respectively) for 48 h, and radiated by microwave 30 mW/cm2, 1 h]. After treatment with different interventions, the cell apoptosis index (AI) was determined using Heochst staining; contents of malonaldehyde (MDA), glutataione (GSH), and activity of superoxide dismutase (SOD) were measured. mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1(HO-1) were detected by real time quantitative polymerase chain reaction, and the expression of HO-1 protein was examined by Western blot analysis. Nucleus and cytoplasm were separated and Nrf2 protein expression was further verified by Western blot analysis.@*RESULTS@#There was significant difference in AI among the groups (F=322.83, P<;0.05). Compared with the control group, AI was significantly higher in the model group and was lower in 4 VU-pretreated groups (P<;0.05). Linear regression analysis showed the decline of AI was in a dose-dependent manner with VU treatment (r=0.8419, P<;0.05). The MDA and GSH contents of 661W cells in VU-treated groups were significantly lower than the model group (P<;0.05). Compared with the model group, the SOD activity in the VU-treated groups (50, 100 and 200 µg/mL) was significantly higher (all P<;0.05). The Nrf2 and HO-1 mRNA expressions were slightly increased after irradiation, and obviously increased in 100 µg/mL VU-treated group. After irradiation, the relative expressions of HO-1 and Nrf2 proteins in nucleus were slightly increased (P<;0.05), and the changes in cytoplasm were not obvious, whereas it was significantly increased in both nucleus and cytoplasm in the VU treatment groups.@*CONCLUSIONS@#Anthocyanins extracted from VU could reduce apoptosis, stabilize cell membrane, and alleviate oxidant injury of mouse retinal photoreceptor 661W cells. The mechanism might be through activating Nrf2/HO-1 signal pathway and inducing HO-1 transcription and translation.


Subject(s)
Animals , Anthocyanins/therapeutic use , Blueberry Plants/metabolism , Heme Oxygenase-1/metabolism , Mice , Microwaves , NF-E2-Related Factor 2/metabolism , Oxidative Stress , RNA, Messenger/metabolism , Superoxide Dismutase/metabolism
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