RESUMO
Methionine is indispensable for growth and meat formation in pigs. However, it is still unclear that increasing dietary sulphur-containing amino acid (SAA) levels using different methionine sources affects the growth performance and meat quality of barrows and gilts. To investigate this, 144 pigs (half barrows and half gilts) were fed the control (100% SAA, CON), DL-Methionine (125% SAA, DL-Met)-supplemented, or OH-Methionine (125% SAA, OH-Met)-supplemented diets during the 11-110 kg period. The results showed that plasma methionine levels varied among treatments during the experimental phase, with increased plasma methionine levels observed following increased SAA consumption during the 25-45 kg period. In contrast, pigs fed the DL-Met diet had lower plasma methionine levels than those fed the CON diet (95-110 kg). Additionally, gilts fed the DL-Met or OH-Met diets showed decreased drip loss in longissimus lumborum muscle (LM) compared to CON-fed gilts. OH-Met-fed gilts had higher pH45min values than those fed the CON or DL-Met diets, whereas OH-Met-fed barrows had higher L45min values than those fed the CON or DL-Met diets. Moreover, increased consumption of SAA, regardless of the methionine source, tended to decrease the shear force of the LM in pigs. In conclusion, this study indicates that increasing dietary levels of SAA (+25%) appeared to improve the meat quality of gilts by decreasing drip loss and increasing meat tenderness.
Assuntos
Suplementos Nutricionais , Metionina , Suínos , Animais , Feminino , Metionina/farmacologia , Dieta/veterinária , Carne , Sus scrofa , Racemetionina/farmacologia , Ração Animal/análise , Composição CorporalRESUMO
BACKGROUND: Chondroitin sulfate (CS) is found in humans' cartilage, bone, cornea, skin, and arterial wall. It consists of the foundation substance in the extracellular matrix (ECM) of connective tissue. The oral supplement form of CS is clinically used in treating osteoarthritis (OA). METHODS: Cell migration was observed by the transwell assay. The EMT, Akt/IKK/IκB pathways, TIMPs, collagen and MMPs in cell lysate were determined by Western blotting. The expression of MMP activity was determined by gelatin zymography. The production of reactive oxygen species (ROS) was determined by using a fluorescence spectrophotometer. RESULTS: In the current report, we demonstrated that CS can increase the cell proliferation and migration of chon-001 chondrocytes. Treatment with CS induced the epithelial-mesenchymal transition and increased the expression of type II collagen and TIMP-1/TIMP2 and inhibited the expressions and activities of metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2). The phosphorylation of Akt, IκB kinase (IKK), IκB and p65 was decreased by CS. CS treatment resulted in ß-catenin production and XAV939, a ß-catenin inhibitor, and inhibited the cell proliferation by CS treatment. In addition, also significantly induced intracellular ROS generation. Treatment with antioxidant propyl gallate blocked cell migration induced by CS. CONCLUSION: We demonstrated that CS induced cell proliferation and migration of chondrocytes by inducing ß-catenin and enhancing ROS production. Moreover, our studies demonstrated that CS can increase the activity of chondrocytes and help patients with osteoarthritis to restore cartilage function.
Assuntos
Condrócitos , Osteoartrite , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/farmacologia , Humanos , Interleucina-1beta/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , NF-kappa B/metabolismo , Osteoartrite/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , beta Catenina/metabolismoRESUMO
Dioscin presents extents of pharmacological activities on several diseases, but its effect and mechanism on diabetes cognitive dysfunction (DCD) remains unclear. Herein, we conducted a series of pharmacological evaluation assays of purinergic receptor P2X7 (P2X7R) with dioscin. We uncovered that dioscin presented a clearly protective effect on diabetes cognitive dysfunction via a methylglyoxal-treated PC12 cell model and streptozocin (STZ)-induced rat models. Additionally, it found that P2X7R and NLRP3 inflammasome signals were activated in diabetes cognitive dysfunction via in vivo and in vitro detection. Moreover, it was demonstrated that P2X7R regulated NLRP3 inflammasome signals in methylglyoxal-treated PC12 cells. Meanwhile, it was showed that dioscin-induced anti-diabetes cognitive dysfunction effect was accompanied with an inhibition of P2X7R/NLRP3 signal. A deeper mechanical study indicated that an overexpression of P2X7R further enhanced the protective effect of dioscin. Whilst, an inhibition of P2X7R abolished the protective effect of dioscin. These results suggested that dioscin protected type 2 diabetes cognitive dysfunction through, at least partially, regulating the P2X7R/NLRP3 signal pathway. Our findings further indicate the great value of dioscin on preventing type 2 diabetes cognitive dysfunction.
Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Disfunção Cognitiva , Diabetes Mellitus Tipo 2 , Diosgenina/análogos & derivados , Modelos Animais de Doenças , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Ratos , Receptores Purinérgicos P2X7/metabolismo , Transdução de SinaisRESUMO
Dioscin, one natural product, has various pharmacological actions. However, its effects on methotrexate (MTX)-induced hepatorenal damages still remain unknown. In the present study, the data manifested that dioscin restored the viabilities of L-02 and NRK-52E cells, reduced ALT, AST, Cr, BUN levels, and ameliorated histopathological changes of liver and kidney. Besides, dioscin decreased ROS levels in cells, and adjusted SOD, MDA, GSH and GSH-Px levels in rats. Dioscin reduced the expression levels of miR-145-5p which directly targeted Sirt5, and then regulated the expression levels of SOD1, Nrf2, Gst, Keap1, HO-1, GCLC and NQO1. MiR-145-5p mimic in cells deteriorated ROS levels and decreased Sirt5 expression to accentuate oxidative stress by regulating the expression levels of SOD1, Nrf2, Keap1, which were all reversed by dioscin. Moreover, MTX-induced hepatorenal damage were worsened in mice by Sirt5 siRNA or miR-145-5p agomir, which were also alleviated by dioscin. Dioscin relieved MTX-induced hepatorenal damages through regulating miR-145-5p-medicated oxidative stress, which should be considered as one effective drug to treat the disorder in future.
Assuntos
MicroRNAs , Fator 2 Relacionado a NF-E2 , Animais , Diosgenina/análogos & derivados , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Rim/metabolismo , Fígado , Metotrexato/toxicidade , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , RatosRESUMO
Pulmonary arterial hypertension (PAH) is a progressive and lethal cardiopulmonary. Pulmonary vascular remodeling (PVR) caused by excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) is the chief pathological feature of PAH. Dioscin is a natural product that possesses multiple pharmacological activities, but its effect on PAH remains unclear. In this study, effect of dioscin on vascular remodeling in PAH was assessed in hypoxia-induced PASMCs, hypoxia-induced and monocrotaline (MCT)-induced rats. Western blot, Real-time PCR and siRNA transfection tests were applied to evaluate the possible mechanisms of dioscin. In vitro experiments, results showed dioscin markedly inhibited the proliferation and migration, and promoted apoptosis of hypoxic PASMCs. In vivo, dioscin significantly decreased the right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI), and improved pulmonary vascular stenosis in rats induced by hypoxia or MCT. Molecular mechanism studies showed that dioscin significantly reduced the expression of growth factor receptor-bound protein 2 (GRB2). Subsequently, dioscin reduced the expressions of Ras, Cyclin D1, CDK4, c-Fos, PCNA and p-ERK to inhibit proliferation and migration of PASMCs, inhibited p-PI3K and p-AKT levels and increased Bax/Bcl2 ratio to promote cell apoptosis. GRB2 siRNA transfection in PASMCs further confirmed that the inhibitory action of dioscin in PAH was evoked by adjusting GRB2/ERK/PI3K-AKT signal. Taken together, our study indicated that dioscin attenuates PAH through adjusting GRB2/ERK/PI3K-AKT signal to inhibit PASMCs proliferation and migration, and promote apoptosis, and dioscin may be developed as a therapeutic strategy for treating PAH in the future.
Assuntos
Diosgenina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Hipertensão Arterial Pulmonar/tratamento farmacológico , Remodelação Vascular/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diosgenina/farmacologia , Modelos Animais de Doenças , Proteína Adaptadora GRB2/genética , Masculino , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Fosforilação , Hipertensão Arterial Pulmonar/enzimologia , Hipertensão Arterial Pulmonar/patologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/enzimologia , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
MiR-23a-5p is involved in the occurrence and development of some serious diseases, but its effects on intestinal ischemia-reperfusion (II/R) injury is unclear. In this research, the hypoxia/reoxygenation (H/R) model on IEC-6 cells and II/R model in mice were used. The data showed that the ROS level in model group was significantly increased compared with control group. The level of intestinal MPO was increased and serum SOD was decreased in mice compared with sham group. Moreover, the expression levels of miR-23a-5p in model groups were obviously increased in vitro and in vivo, while the expression levels of PPARα, FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2 were significantly decreased. The double luciferase reporter gene assay showed that there was binding site between miR-23a-5p and PPARα. When miR-23a-5p was inhibited or PPARα gene was overexpressed, H/R-caused cell damage was alleviated and ROS level was decreased compared with NC group. PPARα expression level was increased, accompanied by the increased levels of FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2. After enhancing miR-23a-5p expression or silencing PPARα gene, H/R-caused cell damage was further aggravated compared with NC group, and ROS level was increased associated with the decreased levels of FOXO3α, PGC-1α, Nrf2, CAT, NQO1, HO-1 and SOD2. Our study demonstrated that miR-23a-5p exacerbated II/R injury by promoting oxidative stress via targeting PPARα, which should be considered as one new drug target to treat II/R injury.
Assuntos
Sistemas de Liberação de Medicamentos , Mucosa Intestinal/metabolismo , MicroRNAs/administração & dosagem , Estresse Oxidativo/fisiologia , PPAR alfa/biossíntese , Traumatismo por Reperfusão/metabolismo , Animais , Linhagem Celular , Sistemas de Liberação de Medicamentos/métodos , Mucosa Intestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/antagonistas & inibidores , Ratos , Traumatismo por Reperfusão/patologiaRESUMO
MiR-142-3p as one key molecule in oncogenesis and inflammation plays crucial roles in hepatic fibrosis, hepatocellular carcinoma and other liver disease. However, there have no literatures to report its effects on hepatic ischemia-reperfusion (HI/R) injury. In the present work, hypoxia reoxygenation (H/R) models on AML12 and HepG2 cells, and ischemia/reperfusion model in mice were established. The methods of real-time PCR, dual luciferase reporter, mimic, inhibitor, agomir, antagomir and siRNA transfection assays were used. The expression levels of miR-142-3p were decreased in model groups in vitro and in vivo compared with control group or Sham group, which directly targeted MARCKS to regulate its expression. Then, MARCKS activated p38/JNK signal, up-regulated NF-κB expression to accelerate inflammation, and inhibited PI3K/AKT signal to promote apoptosis. Moreover, miR-142-3p mimic in vitro and agomir in vivo lowered the expression levels of MARCKS, thereby alleviating apoptosis and inflammation to relieve HI/R injury. Furthermore, miR-142- 3p inhibitor in vitro and antagomir in vivo up-regulated the expression levels of MARCKS to aggravate HI/R damage via promoting inflammation and apoptosis. Consistently, MARCKS siRNA markedly inhibited HI/R injury by restraining apoptosis and inflamm- ation in mice. MiR-142-3p played a considerable part in adjusting HI/R injury by targeting MARCKS, and miR-142-3p/MARCKS should be a new therapeutic target for HI/R injury.
Assuntos
Apoptose , Hepatopatias/metabolismo , Fígado/metabolismo , MicroRNAs/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Hipóxia Celular , Modelos Animais de Doenças , Células Hep G2 , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/patologia , Hepatopatias/genética , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Substrato Quinase C Rico em Alanina Miristoilada/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Renal ischemia-reperfusion (RI/R) injury with high morbidity and mortality is one common clinical disease. Development of drug targets to treat the disorder is critical important. MiR-27a-3p plays important roles in regulating oxidative stress. However, its effects on RI/R injury have not been reported. In this paper, hypoxia/reoxygenation (H/R) models on NRK-52E and HK-2 cells, and RI/R model in C57BL/6 mice were established. The results showed that H/R in vitro decreased cell viability and increased ROS levels in cells, and RI/R caused renal injury and oxidative damage in mice. The expression levels of miR-27a-3p were up-regulated based on real-time PCR and FISH assays in model groups compared with control groups, which directly targeted Grb2 based on dual luciferase reporter assay and co-transfaction test. In addition, miR-27a- 3p markedly reduced Grb2 expression to down-regulate the expression levels of p-PI3K, p-AKT, Nrf2, HO-1, and up-regulate Keap1 expression in model groups. MiR-27a-3p mimics in vitro enhanced H/R-caused oxidative stress via increasing ROS levels and decreasing Grb2 expression to down-regulate PI3K-AKT signal. In contrary, miR-27a-3p inhibitor in vitro significantly reduced H/R-caused oxidative damage via decreasing ROS levels and increasing Grb2 expression to up-regulate PI3K-AKT signal. In vivo, miR-27a- 3p agomir exacerbated RI/R-caused renal damage by decreasing SOD level and increasing Cr, BUN, MDA levels via suppressing Grb2 expression to down-regulate PI3K- AKT signal. However, miR-27a -3p antagomir alleviated RI/R-caused oxidative damage via increasing Grb2 expression to up-regulate PI3k-AKT signal. Grb2siRNA in mice further enhanced RI/R-caused renal injury by increasing Cr, BUN, MDA levels and decreasing SOD level via inhibiting the expression levels of Grb2, Nrf2, HO-1, and increasing Keap1 expression. Our data showed that miR-27a-3p aggravated RI/R injury by promoting oxidative stress via targeting Grb2, which should be considered as one new drug target to treat RI/R injury.
Assuntos
Proteína Adaptadora GRB2 , Nefropatias , MicroRNAs , Estresse Oxidativo , Traumatismo por Reperfusão , Animais , Linhagem Celular , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
BACKGROUND: Our previous study revealed that microRNA-125a-5p plays a crucial role in regulating hepatic glycolipid metabolism by targeting STAT3 in type 2 diabetes mellitus (T2DM). Dioscin, a major active ingredient in Dioscoreae nipponicae rhizomes, displays various pharmacological activities, but its role in T2DM has not been reported. PURPOSE: The aim of this study was to investigate the effect of dioscin on T2DM and elucidate its potential mechanism. METHODS: The effect of dioscin on glycolipid metabolic disorder in insulin-induced HepG2 cells, palmitic acid-induced AML12 cells, high-fat diet- and streptozotocin- induced T2DM rats, and spontaneous T2DM KK-Ay mice were evaluated. Then, the possible mechanisms of dioscin were comprehensively evaluated. RESULTS: Dioscin markedly alleviated the dysregulation of glycolipid metabolism in T2DM by reducing hyperglycemia and hyperlipidemia, improving insulin resistance, increasing hepatic glycogen content, and attenuating lipid accumulation. When the mechanism was investigated, dioscin was found to markedly elevate miR-125a-5p level and decrease STAT3 expression. Consequently, dioscin increased phosphorylation levels of STAT3, PI3K, AKT, GSK-3ß, and FoxO1 and decreased gene levels of PEPCK, G6Pase, SREBP-1c, FAS, ACC, and SCD1, leading to an increase in glycogen synthesis and a decrease in gluconeogenesis and lipogenesis. The effects of dioscin on regulating miR-125a-5p/STAT3 pathway were verified by miR-125a-5p overexpression and STAT3 overexpression. CONCLUSIONS: Dioscin showed potent anti-T2DM activity by improving the inhibitory effect of miR-125a-5p on STAT3 signaling to alleviate glycolipid metabolic disorder of T2DM.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diosgenina/análogos & derivados , Glicolipídeos/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Dieta Hiperlipídica/efeitos adversos , Diosgenina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Células Hep G2 , Humanos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hipoglicemiantes/farmacologia , Resistência à Insulina , Lipogênese/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Ratos Wistar , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Objective: To study the diagnostic and epidemiological features of the first two HIV-2 indigenous cases in Hunan province. Methods: Blood samples from two individuals with "HIV antibody indeterminate" and HIV-2 specific band showed by HIV-1/2 western blotting method, were repeatedly collected and detected under HIV 1+2 strip immunoassay and PCR, in Changsha city, Hunan province, through March to November, 2017. An epidemiological survey was carried out at the same time. Results: Our findings showed that the two cases were sex partners, without histories of sexual contact with foreigners and the source of infection was unknown. Results from the HIV 1+2 antibody confirmation test showed that they were "HIV-2 antibody positive" . Through amplifying and sequencing the gag area of HIV-2 and BLAST, the similarity of HIV-2 strains presented as 98%. The results also showed that there were HIV-2 specific fragments in the two cases. Conclusion: HIV-2 indigenous cases had never been reported in China. These cases had brought new challenge on prevention, diagnosis and treatment of HIV/AIDS in China.
Assuntos
Western Blotting/métodos , Anticorpos Anti-HIV/sangue , Infecções por HIV/etnologia , HIV-2 , Comportamento Sexual , Parceiros Sexuais , Adulto , China , Anticorpos Anti-HIV/isolamento & purificação , Infecções por HIV/diagnóstico , HIV-2/imunologia , Humanos , Inquéritos e QuestionáriosRESUMO
The group 2b (G2b) porcine epidemic diarrhea virus (PEDV) that emerged in 2013 has since caused devastating diseases and economic loss. The full-length genome of the G2b Taiwan PEDV-Pintung 52 (PEDV-PT) strain and its intestinal tropism by evaluating the pathological changes in the original PEDV-PT infected field piglet and orally inoculation of either 10, 103, or 105 50% tissue culture infective dose/mL (TCID50/mL) of the plaque-purified PEDV-PT-Passage 5 (P5) in 7-day-old conventional piglets were analyzed. Phylogenetic analysis of the full-length genome indicated that the G2b Taiwan PEDV-PT strain was closely related to the North American G2b PEDV strains. Some pathological features of the G2b Taiwan PEDV-PT infection, including the absence of lesions and antigen signal in the crypt epithelial cells of the jejunum and ileum and in the villus enterocytes of the duodenum and colon, were different from those of infections by the North American G2b PEDV strains. This difference in the intestinal tropism of the G2b Taiwan PEDV-PT strain highlights the importance of studying the pathogenicities of different PEDV variants. Moreover, similar distributions of PEDV antigens and lesions in the G2b Taiwan PEDV-PT infected field piglet and its plaque-purified isolate, PEDV-PT-P5, inoculated piglets indicating that the plaque-purified PEDV-PT-P5 viral stock could facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing the G2b PEDV infection.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/virologia , Animais , Infecções por Coronavirus/virologia , Filogenia , Vírus da Diarreia Epidêmica Suína/classificação , Suínos , Doenças dos Suínos/patologia , Taiwan , Tropismo , Tropismo ViralRESUMO
Coilia ectenes is a commercially important fishery species in China. C. ectenes taihuensis is an endemic and dominant species found in Taihu Lake of China. When compared with C. ectenes, C. ectenes taihuensis lacks anadromous behavior, and can independently grow and reproduce in Taihu Lake. In this study, the mitochondrial DNA control region (D-loop) sequences were employed to investigate the genetic diversity and population structure of C. ectenes taihuensis. Sixty-eight individuals collected from 4 localities in Taihu Lake were examined. Results indicated that in the 887-bp D-loop region, seventy-seven (8.68%) sites were variant, contributing to 53 distinct haplotypes. Although the population haplotype diversity (Hd = 0.971 to 1.000) was generally high, the nucleotide diversity (π = 0.616 to 0.731%) was relatively low among the 4 populations. Additionally, the genetic distances ranged from 0.62 to 0.74% within the populations and from 0.67 to 0.74% between the populations. The neighbor-joining tree indicated that a distinct distribution of phylogenetic structure existed among haplotypes. Analysis of molecular variance and FST statistics suggested that a divergence existed among populations in 4 localities, indicating that gene communication might have occurred among those populations. Furthermore, neutral tests and analysis of mismatch distribution reflected that C. ectenes taihuensis might have undergone a population expansion during the evolution process. Our study showed the population genetic diversity and structure of C. ectenes taihuensis. Results from this study might be helpful in the development and protection of fishery resource within the localities in Taihu Lake in future.
Assuntos
DNA Mitocondrial/genética , Peixes/genética , Polimorfismo Genético , Animais , China , Evolução Molecular , Genética Populacional , Haplótipos , Lagos , FilogeniaRESUMO
The accumulation of myeloid-derived suppressor cells (MDSCs) has been observed in solid tumors and is correlated with tumor progression; however, the underlying mechanism is still poorly understood. In this study, we identified a mechanism by which tumor cells induce MDSC accumulation and expansion in the bladder cancer (BC) microenvironment via CXCL2/MIF-CXCR2 signaling. Elevated expression of CXCL2 and MIF and an increased number of CD33+ MDSCs were detected in BC tissues, and these increases were significantly associated with advanced disease stage and poor patient prognosis (P<0.01). A positive association was observed between CXCL2 or MIF expression and the number of tumor-infiltrating CD33+ MDSCs (P<0.01). Subsequently, we demonstrated that CD45+CD33+CD11b+HLA-DR- MDSCs from fresh BC tissues displayed high levels of suppressive molecules, including Arg1, iNOS, ROS, PDL-1 and P-STAT3, and stronger suppression of T-cell proliferation. Interestingly, these CD45+CD33+CD11b+HLA-DR- MDSCs exhibited increased CXCR2 expression compared with that in peripheral blood from BC patients or healthy controls (P<0.05). Chemotaxis assay revealed that bladder cancer cell line J82 induced MDSC migration via CXCL2/MIF-CXCR2 signaling in vitro. Mechanistic studies demonstrated that J82-induced MDSC trafficking and CXCR2 expression were associated with increased phosphorylation of p38, ERK and p65. Conversely, inhibition of the phosphorylation of p38, ERK or p65 decreased J82-induced MDSC trafficking and CXCR2 expression. CXCL2/MIF-stimulated activation of the mitogen-activated protein kinase and nuclear factor kappa B pathways in MDSCs was MyD88 dependent. Overall, our results identify the CXCL2/MIF-CXCR2 axis as an important mediator in MDSC recruitment and as predictors and potential therapeutic targets in BC patients.
Assuntos
Quimiocina CXCL2/metabolismo , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Células Mieloides/patologia , Receptores de Interleucina-8B/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Quimiotaxia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Decorin (DCN), a component of the extracellular matrix (ECM), participates in ECM assembly and influences cell proliferation and apoptosis in many mammalian tissues and cells. However, expression and function of DCN in the ovary remain unclear. This study cloned the full-length cDNA of goat DCN obtained from the ovary of an adult goat. Sequence analysis revealed that the putative DCN protein shared a highly conserved amino acid sequence with known mammalian homologs. The tissue distribution of DCN mRNA expression was evaluated by real-time PCR, and the results showed that DCN was widely expressed in the tissues of adult goat. Immunohistochemistry results suggested that DCN protein existed in the granulosa cells and oocytes from all types of follicles and theca cells of antral follicles. Moreover, hCG-induced DCN mRNA expression was significantly reduced by the inhibitors of protein kinase A, PI3K, or p38 kinase (P < 0.05), which are key mediators involved in hCG-induced DCN expression. Overexpression of DCN significantly increased apoptosis and blocked cell cycle progression in cultured granulosa cells (P < 0.05). Western blot analysis also showed that overexpression of DCN upregulated the expression levels of p21 protein (P < 0.05), whereas no effects were observed on the expression of Bax and Bcl-2 and on Bcl-2/Bax ratio (P > 0.05). These findings suggested that DCN regulates the apoptosis and cell cycle of granulosa cells.
Assuntos
Decorina/metabolismo , Regulação da Expressão Gênica/fisiologia , Cabras/fisiologia , Células da Granulosa/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Feminino , FilogeniaRESUMO
Brain-derived neurotropic factor (BDNF) and its high-affinity receptor, tyrosine kinase receptor B, have been assumed to be involved in female reproduction and have recently shown to play an essential role in follicle activation and oocyte maturation. In this study, we analyzed the expression of miR-10b and BDNF in the ovary and discovered that the expression of miR-10b was higher in monotocous goat ovaries than in polytocous goat ovaries, whereas the expression pattern of BDNF in ovary was opposite. Moreover, human chorionic gonadotropin induced rapid and transient expression of BDNF messenger RNA and protein. In contrast, human chorionic gonadotropin upregulated miR-10b expression in a time-dependent manner. The BDNF gene was identified as a direct target of miR-10b using a dual-luciferase reporter assay. Transfection of granulosa cells with miR-10b decreased BDNF messenger RNA and protein levels. MiR-10b overexpression inhibited cell proliferation, whereas BDNF promoted cell proliferation. However, a combined treatment with miR-10b and BDNF promoted cell proliferation, indicating that the reintroduction of BDNF reversed the suppressive effect of miR-10b. These results demonstrate that miR-10b downregulates BDNF expression in granulosa cells by directly targeting the 3' untranslated regions and plays an important role in inhibiting granulosa cell proliferation by targeting BDNF.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Proliferação de Células/efeitos dos fármacos , Cabras , Células da Granulosa/citologia , MicroRNAs/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/análise , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Regulação para Baixo , Feminino , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , MicroRNAs/genética , Ovário/química , Ovário/metabolismo , RNA Mensageiro/análiseRESUMO
Tissue inhibitor of metalloproteinase 1 (TIMP1) belongs to a group of endogenous inhibitors that control the activity of matrix metalloproteinases and other metalloproteinases. TIMP1 is ubiquitously expressed and implicated in many physiological and pathologic processes. In this study, the full-length complementary DNA of goat (Capra hircus) Timp1 was cloned from adult goat ovary for the first time to better understand the regulatory role of TIMP1. The putative TIMP1 protein shared a high amino acid sequence identity with other species. Real-time polymerase chain reaction results showed that Timp1 was widely expressed in adult goat tissues, and messenger RNA expression was higher in the ovary than in other tissues; meanwhile, increasing expression of Timp1 was also discovered during the process of follicle growth and corpus luteum. We then investigated Timp1 expression patterns in different types of ovarian follicular cells from goats. In small or large antral follicles, Timp1 expression was higher (P < 0.05) in theca cells than in granulosa cells, cumulus cells, and oocytes. Increasing expression of Timp1 in theca and granulosa cells was observed as the variation of the follicle size. Immunohistochemical analyses further revealed the presence of the TIMP1 proteins in follicles at all antral stages of development. The most intense staining for TIMP1 was observed in the theca cells and granulosa cells of large antral follicles and corpus luteum. Timp1 was highly (P < 0.05) induced in granulosa cells in vitro after treatment with the luteinizing hormone agonist, human chorionic gonadotropin. Treatments with forskolin, phorbol 12-myristate 13-acetate, or phorbol 12-myristate 13-acetate + forskolin could also stimulate Timp1 messenger RNA expression. The effects of human chorionic gonadotropin were reduced (P < 0.05) by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase, indicating that Timp1 expression could be adjusted by luteinizing hormone-initiated activation of these signaling mediators. Our results suggested that TIMP1 may be involved in regulating ovarian follicle development and ovulation.
Assuntos
Cabras , Células da Granulosa/química , Inibidor Tecidual de Metaloproteinase-1/química , Inibidor Tecidual de Metaloproteinase-1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Clonagem Molecular , Células do Cúmulo/química , DNA Complementar/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Hormônio Luteinizante/farmacologia , Dados de Sequência Molecular , Oócitos/química , Especificidade de Órgãos , Folículo Ovariano/crescimento & desenvolvimento , Filogenia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Alinhamento de Sequência , Células Tecais/química , Inibidor Tecidual de Metaloproteinase-1/fisiologiaRESUMO
Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions of the prolactin receptor (PRLR) gene by DNA sequencing and PCR-RFLP. Two SNPs (c.1457G>A and c.1645G>A) were identified that caused amino acid variations p.Ser485Asn and p.Val548Met respectively. Statistical results indicated that the c.1457G>A and c.1645G>A SNPs were significantly associated with litter size in Boer and Guanzhong goat breeds. Further analysis revealed that combined genotype C4 (GGGG) and haplotype G-G were better than the others for litter size in both goat breeds. These results might contribute to goat genetic resources and breeding.
Assuntos
Cabras/genética , Tamanho da Ninhada de Vivíparos/genética , Mutação de Sentido Incorreto , Receptores da Prolactina/genética , Animais , Cruzamento , Éxons , Feminino , Frequência do Gene , Genótipo , Haplótipos , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Característica Quantitativa Herdável , Análise de Sequência de DNARESUMO
Ovarian-specific promoter 1 (OSP-1) is a retrovirus-like element isolated from the complementary DNA library of rat that has been thought to be specifically expressed in ovary. To exploit this promoter in dairy goat ovary granulosa cells (GCs), OSP-1 from rat was used to construct the reporter vector pOSP-1-EGFP, in which egfp coding for enhanced green fluorescent protein (EGFP) was used as a reporter to examine the activity of OSP-1 in GCs. EGFP was successfully expressed in dairy goat GCs transfected with pOSP-1-EGFP. Reverse transcriptase-polymerase chain reaction analysis confirmed the tissue-specific transcription of EGFP messenger RNA in dairy goat GCs transfected with pOSP-1-EGFP. We concluded that OSP-1 promoter from rat can specifically drive foreign gene expression in dairy goat GCs. Thus, we obtained a tissue-specific regulation element and provided a potential tool for the research of regulation and development of the ovary in dairy goats.