RESUMO
The mTORC1 signaling pathway regulates cell growth and metabolism in a variety of organisms from yeast to human, and inhibition of the mTORC1 pathway has the prospect to treat cancer or achieve longevity. The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700 kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2-GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG-MTM (short stabilization domain and GAP domain-membrane targeting motif) and SSG-TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much-reduced sizes (~1/15 and ~ 1/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down-regulating catabolism and autophagy.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina , Neuropeptídeos , Proteína 2 do Complexo Esclerose Tuberosa , Esclerose Tuberosa , Humanos , Guanosina Trifosfato , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de TumorRESUMO
The balance between anabolism and catabolism is disrupted with aging, with the rate of anabolism being faster than that of catabolism. Therefore, mTOR, whose major function is to enhance anabolism and inhibit catabolism, has become a potential target of inhibition for anti-aging therapy. Interestingly, it was found that the downregulation of the mTOR signaling pathway had a lifespan-extending effect resembling calorie restriction. In addition, the mTOR signaling pathway promotes cell proliferation and has been regarded as a potential anti-cancer target. Rapamycin and rapalogs, such as everolimus, have proven to be effective in preventing certain tumor growth. Here, we reviewed the basic knowledge of mTOR signaling, including both mTORC1 and mTORC2. Then, for anti-aging, we cited a lot of evidence to discuss the role of targeting mTOR and its anti-aging mechanism. For cancer therapy, we also discussed the role of mTOR signaling in different types of cancers, including idiopathic pulmonary fibrosis, tumor immunity, etc. In short, we discussed the research progress and both the advantages and disadvantages of targeting mTOR in anti-aging and anti-cancer therapy. Hopefully, this review may promote more ideas to be generated for developing inhibitors of mTOR signaling to fight cancer and extend lifespan.
Assuntos
Neoplasias , Serina-Treonina Quinases TOR , Humanos , Serina-Treonina Quinases TOR/metabolismo , Envelhecimento , Transdução de Sinais , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proliferação de CélulasRESUMO
BACKGROUND: Alisol A 24-acetate (AA-24-a), one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., exhibits multiple biological activities including hypolipidemic activity. However, its effect on lipid metabolism in adipocytes remains unclear. The present study aimed to clarify the effect of AA-24-a on adipocyte lipolysis and to determine its potential mechanism of action using 3 T3-L1 cells. METHODS: We assayed the release of glycerol into culture medium of 3 T3-L1 cells under treatment with AA-24-a. Protein and mRNA expression and phosphorylation levels of the main lipases and kinases involved in lipolysis regulation were determined by quantitative polymerase chain reaction and western blotting. Specific inhibitors of protein kinase A (PKA; H89) and extracellular signal-regulated kinase (ERK; PD98059), which are key enzymes in relevant signaling pathways, were used to examine their roles in AA-24-a-stimulated lipolysis. RESULTS: AA-24-a significantly stimulated neutral lipolysis in fully differentiated adipocytes. To determine the underlying mechanism, we assessed the changes in mRNA and protein levels of key lipolysis-related genes in the presence or absence of H89 and PD98059. Both inhibitors reduced AA-24-a-induced lipolysis. Moreover, pretreatment with H89 attenuated AA-24-a-induced phosphorylation of hormone-sensitive lipase at Ser660, while pretreatment with PD98059 attenuated AA-24-a-induced downregulation of peroxisome proliferator-activated receptor-γ and perilipin A. CONCLUSIONS: Our results indicate that AA-24-a promoted neutral lipolysis in 3 T3-L1 adipocytes by activating PKA-mediated phosphorylation of hormone-sensitive lipase and ERK- mediated downregulation of expression of perilipin A.
Assuntos
Alisma , Hipolipemiantes/farmacologia , Triterpenos/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Lipólise/efeitos dos fármacos , Camundongos , FitoterapiaRESUMO
BACKGROUND: Saturated fatty acids (SFAs) generally have been thought to worsen insulin-resistance and increase the risk of developing type 2 diabetes mellitus (T2DM). Recently, accumulating evidence has revealed that SFAs are not a single homogeneous group, instead different SFAs are associated with T2DM in opposing directions. Pentadecanoic acid (C15:0, PA) is directly correlated with dairy products, and a negative association between circulating PA and metabolic disease risk was observed in epidemiological studies. Therefore, the role of PA in human health needs to be reinforced. Whether PA has a direct benefit on glucose metabolism and insulin sensitivity needs further investigation. OBJECTIVE: The present study aimed to investigate the effect and potential mechanism of action of PA on basal and insulin stimulated glucose uptake in C2C12 myotubes. METHODS: Glucose uptake was determined using a 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. The phosphorylation levels of proteins involved in the insulin and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined by western blotting. RESULTS: We found that PA significantly promoted glucose uptake and GLUT4 translocation to the plasma membrane. PA had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (Tyr632) and protein kinase B (PKB/Akt), but increased phosphorylation of AMPK and Akt substrate of 160 kDa (AS160). Compound C (an AMPK inhibitor) blocked PA-induced AMPK activation and reversed PA-induced GLUT4 translocation, indicating that PA promotes glucose uptake via the AMPK pathway in vitro. Moreover, PA significantly promoted insulin-stimulated glucose uptake in myotubes. Under insulin stimulation, PA did not affect the insulin-dependent pathway, but still activated AMPK. CONCLUSION: PA, an odd-chain SFA, significantly stimulates glucose uptake via the AMPK-AS160 pathway and exhibits an insulin-sensitizing effect in myotubes.
RESUMO
The Tuberous Sclerosis Complex (TSC) protein complex (TSCC), comprising TSC1, TSC2, and TBC1D7, is widely recognised as a key integration hub for cell growth and intracellular stress signals upstream of the mammalian target of rapamycin complex 1 (mTORC1). The TSCC negatively regulates mTORC1 by acting as a GTPase-activating protein (GAP) towards the small GTPase Rheb. Both human TSC1 and TSC2 are important tumour suppressors, and mutations in them underlie the disease tuberous sclerosis. We used single-particle cryo-EM to reveal the organisation and architecture of the complete human TSCC. We show that TSCC forms an elongated scorpion-like structure, consisting of a central "body", with a "pincer" and a "tail" at the respective ends. The "body" is composed of a flexible TSC2 HEAT repeat dimer, along the surface of which runs the TSC1 coiled-coil backbone, breaking the symmetry of the dimer. Each end of the body is structurally distinct, representing the N- and C-termini of TSC1; a "pincer" is formed by the highly flexible N-terminal TSC1 core domains and a barbed "tail" makes up the TSC1 coiled-coil-TBC1D7 junction. The TSC2 GAP domain is found abutting the centre of the body on each side of the dimerisation interface, poised to bind a pair of Rheb molecules at a similar separation to the pair in activated mTORC1. Our architectural dissection reveals the mode of association and topology of the complex, casts light on the recruitment of Rheb to the TSCC, and also hints at functional higher order oligomerisation, which has previously been predicted to be important for Rheb-signalling suppression.
Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Conformação Proteica , Proteína 1 do Complexo Esclerose Tuberosa/química , Proteína 2 do Complexo Esclerose Tuberosa/química , Microscopia Crioeletrônica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes , Relação Estrutura-Atividade , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
Support films are commonly used during cryo-EM specimen preparation to both immobilise the sample and minimise the exposure of particles at the air-water interface. Here we report preparation protocols for carbon and graphene supported single particle electron microscopy samples using a novel 3D-printed sample transfer block to facilitate the direct, wetted, movement of both carbon and graphene supports from the substrate on which they were generated to small volumes (10 µL) of sample. These approaches are simple and inexpensive to implement, minimise hydrophobic contamination of the support films, and are widely applicable to single particle studies. Our approach also allows the direct exchange of the sample buffer on the support film in cases in which it is unsuitable for vitrification, e.g. for samples from centrifugal density gradients that help to preserve sample integrity.
Assuntos
Carbono/química , Microscopia Crioeletrônica/métodos , Grafite/química , Manejo de Espécimes/métodos , VitrificaçãoRESUMO
BACKGROUND: Alisol A-24-acetate (AA-24-a) is one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., which possesses multiple biological activities, including a hypoglycemic effect. Whether AA-24-a is a hypoglycemic-active compound of A. orientale (Sam.) Juz. is unclear. The present study aimed to clarify the effect and potential mechanism of action of AA-24-a on glucose uptake in C2C12 myotubes. METHOD: Effects of AA-24-a on glucose uptake and GLUT4 translocation to the plasma membrane were evaluated. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay. Cell membrane proteins were isolated and glucose transporter 4 (GLUT4) protein was detected by western blotting to examine the translocation of GLUT4 to the plasma membrane. To determine the underlying mechanism, the phosphorylation levels of proteins involved in the insulin and 5'-adenosine monophosphate-activated protein kinase (AMPK) pathways were examined using western blotting. Furthermore, specific inhibitors of key enzymes in AMPK signaling pathway were used to examine the role of these kinases in the AA-24-a-induced glucose uptake and GLUT4 translocation. RESULTS: We found that AA-24-a significantly promoted glucose uptake and GLUT4 translocation in C2C12 myotubes. AA-24-a increased the phosphorylation of AMPK, but had no effect on the insulin-dependent pathway involving insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/AKT). In addition, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the AKT substrate of 160 kDa (AS160), two proteins that act downstream of AMPK, was upregulated. Compound C, an AMPK inhibitor, blocked AA-24-a-induced AMPK pathway activation and reversed AA-24-a-induced glucose uptake and GLUT4 translocation to the plasma membrane, indicating that AA-24-a promotes glucose metabolism via the AMPK pathway in vitro. STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, also attenuated AA-24-a-induced glucose uptake and GLUT4 translocation. Moreover, STO-609 weakened AA-24-a-induced phosphorylation of AMPK, p38 MAPK and AS160. CONCLUSIONS: These results indicate that AA-24-a isolated from A. orientale (Sam.) Juz. significantly enhances glucose uptake via the CaMKKß-AMPK-p38 MAPK/AS160 pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Colestenonas/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Alisma/química , Animais , Western Blotting , Linhagem Celular , Camundongos , Fibras Musculares Esqueléticas/enzimologia , Projetos Piloto , Plantas Medicinais/químicaRESUMO
There have been very few reports on protein domains that specifically recognize sulfur. Here we present the crystal structure of the sulfur-binding domain (SBD) from the DNA phosphorothioation (PT)-dependent restriction endonuclease ScoMcrA. SBD contains a hydrophobic surface cavity that is formed by the aromatic ring of Y164, the pyrolidine ring of P165, and the non-polar side chains of four other residues that serve as lid, base, and wall of the cavity. The SBD and PT-DNA undergo conformational changes upon binding. The S187RGRR191 loop inserts into the DNA major groove to make contacts with the bases of the GPSGCC core sequence. Mutating key residues of SBD impairs PT-DNA association. More than 1000 sequenced microbial species from fourteen phyla contain SBD homologs. We show that three of these homologs bind PT-DNA in vitro and restrict PT-DNA gene transfer in vivo. These results show that SBD-like PT-DNA readers exist widely in prokaryotes.
Assuntos
DNA/química , Oligonucleotídeos Fosforotioatos/química , Enxofre/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , DNA/genética , Transferência Genética Horizontal/genética , Filogenia , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Streptococcus/metabolismoRESUMO
Colorectal cancer (CRC) is one of the leading causes of death worldwide. Thus, the development of new therapeutic targets for CRC treatment is urgently needed. SGK1 is involved in various cellular activities, and its dysregulation can result in multiple cancers. However, little is known about its roles and associated molecular mechanisms in CRC. In present study, we found that SGK1 was highly expressed in tumor tissues compared with peri-tumor samples from CRC patients. In vitro experiments revealed that SGK1 overexpression promoted colonic tumor cell proliferation and migration and inhibited cell apoptosis induced by 5-fluorouracil (5-FU), while SGK1 shRNA and inhibitors showed the inverse effects. Using CRC xenograft mice models, we demonstrated that knockdown or therapeutic inhibition of SGK1 repressed tumor cell proliferation and tumor growth. Moreover, SGK1 inhibitors increased p27 expression and promoted p27 nuclear accumulation in colorectal cancer cells, and p27 siRNAs could attenuate the repression of CRC cell proliferation induced by SGK1 inhibitors. Collectively, SGK1 promotes colorectal cancer development via regulation of CRC cell proliferation, migration and survival. Inhibition of SGK1 represents a novel strategy for the treatment of CRC.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas Imediatamente Precoces/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Feminino , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Masculino , Camundongos , Estadiamento de Neoplasias , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mutated in colorectal cancer (MCC) gene is an important colorectal tumor suppressor gene, although few studies have reported the microRNA(s) that could directly target MCC in colorectal cancer. Here, we used microRNA (miRNA) target prediction algorithms, and previously reported microarray data in human colorectal cancer found that only miR-4261 was predicted by all three databases to directly target MCC. Based on specimens from our own cohort of colorectal cancer patients, we further demonstrated that miR-4261 was overexpressed in colorectal cancer. Interestingly, overexpression of miR-4261 could enhance cell proliferation and G1/S phase transition of cell cycle, and promote cell migration in HCT116 and HT29 cells, while inhibition of miR-4261 had opposite effects. Luciferase reporter assay and western blot analysis confirmed MCC as a direct target of miR-4261. MCC small interfering RNA (siRNA) could abolish the suppressive effects of miR-4261 inhibitor on cell proliferation and migration in HCT116 and HT29 cell lines. Finally, we showed that therapeutic intervention with lentivirus-based miR-4261 sponge injection could effectively reduce tumor growth and inhibit cell proliferation in colorectal cancer xenograft. Collectively, our study is the first one to unravel the functional role of miR-4261, and it provides strong evidence that inhibition of miR-4261 through targeting of MCC might exert a therapeutic effect for colorectal cancer.
RESUMO
Colorectal cancer (CRC) is one of the most leading causes of cancer-related death worldwide. The serum and glucocorticoid inducible kinase SGK1 is highly expressed and involved in several tumors. GSK650394, a SGK1 inhibitor, has been proved to be effective in impeding tumor growth in vitro. In this study, we developed a novel analog of GSK650394, and evaluated its effects on CRC cells and tumor growth both in vitro and in vivo. HCT116 cells were treated with a concentration gradient of new developed compounds and cholecystokinin octapeptide (CCK-8) assay was used to calculate the IC50 value of every analog. Cell proliferation analysis was estimated from EdU staining and flow cytometry in vitro, and immunohistochemistry of Ki67 and PCNA in vivo. Cell migration analysis was examined using the transwell assay. In vivo tumor growth was determined in athymic nude mice by injecting the HCT116 cells in the subcutaneous tissue, followed by the injection of QGY-5-114-A. We found that new developed GSK650394 analog QGY-5-114-A has lower IC50 value, and treatment with QGY-5-114-A significantly inhibited CRC cell proliferation and migration in vitro. Besides that, colonic tumor growth was also dramatically restricted by QGY-5-114-A in vivo. In conclusion, pharmacological treatment with QGY-5-114-A impedes CRC tumor cell proliferation, migration and tumor growth.
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BACKGROUND: Treatment with angiotensin receptor blockers (ARBs) is successful in mitigating IgA nephropathy (IgAN), independent of blood pressure changes, but the therapeutic role of ARB in advanced IgAN with impaired renal function is to be ascertained. The present study was performed to investigate the effect of losartan on advanced IgAN induced by staphylococcal enterotoxin B (SEB) combined with 5/6 nephrectomy in rats. METHODS: Fifty-four male SD rats were randomly divided into three group: Rats in the model group were treated with SEB plus 5/6 nephrectomy, and those in the losartan group were gavaged with losartan (33.3 mg kg(-1 )d(-1)) besides the treatment with SEB plus 5/6 nephrectomy. The urine and blood biochemical changes of rats were tested. IgA, IgG, IgM and C3 depositions were studied dynamically with immunofluorescence. The renal tissue structures were observed under light microscopy. The expressions of TGF-ß1, FN, alpha-SMA and FGF-1 in rat renal tissues were determined with immunohistochemical methods and real-time PCR. RESULTS: At 12 weeks, rats with SEB treatment plus 5/6 nephrectomy showed gradually increased urinary red blood cell (URBC) with a gradual elevation of the 24 h urinary protein, serum BUN and Scr, but losartan treatment lowered the levels of 24 h urinary protein, serum BUN and Scr. A large number of IgA depositions in the mesangial area, glomerulosclerosis and tubulointerstitial fibrosis were found in the model group, and the losartan group showed relieved injury. The expressions of TGF-ß1, FN, alpha-SMA and FGF-1 were significantly elevated in the model. Losartan lessened their expressions. CONCLUSION: Losartan treatment can delay the progression of advanced IgA nephropathy with impaired renal function.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Glomerulonefrite por IGA/tratamento farmacológico , Testes de Função Renal , Losartan/uso terapêutico , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Biomarcadores/sangue , Biomarcadores/urina , Avaliação Pré-Clínica de Medicamentos , Enterotoxinas , Glomerulonefrite por IGA/sangue , Glomerulonefrite por IGA/urina , Testes de Função Renal/métodos , Losartan/farmacologia , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
Tubulointerstitial fibrosis (TIF) is the final common pathway in the end-stage renal disease. Epithelial-to-mesenchymal transition (EMT) is considered a major contributor to the TIF by increasing the number of myofibroblasts. Curcumin, a polyphenolic compound derived from rhizomes of Curcuma, has been shown to possess potent anti-fibrotic properties but the mechanism remains elusive. We found that curcumin inhibited the EMT as assessed by reduced expression of α-SMA and PAI-1, and increased E-cadherin in TGF-ß1 treated proximal tubular epithelial cell HK-2 cells. Both of the conventional TGF-ß1/Smad pathway and non-Smad pathway were investigated. Curcumin reduced TGF-ß receptor type I (TßR-I) and TGF-ß receptor type II (TßR II), but had no effect on phosphorylation of Smad2 and Smad3. On the other hand, in non-Smad pathway curcumin reduced TGF-ß1-induced ERK phosphorylation and PPARγ phosphorylation, and promoted nuclear translocation of PPARγ. Further, the effect of curcumin on α-SMA, PAI-1, E-cadherin, TßR I and TßR II were reversed by ERK inhibitor U0126 or PPARγ inhibitor BADGE, or PPARγ shRNA. Blocking PPARγ signaling pathway by inhibitor BADGE or shRNA had no effect on the phosphorylation of ERK whereas the suppression of ERK signaling pathway inhibited the phosphorylation of PPARγ. We conclude that curcumin counteracted TGF-ß1-induced EMT in renal tubular epithelial cells via ERK-dependent and then PPARγ-dependent pathway.
Assuntos
Curcumina/farmacologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Túbulos Renais Proximais/citologia , PPAR gama/metabolismo , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Butadienos/farmacologia , Caderinas/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: This study investigated the effects of losartan intervention on the expressions of hypoxia-inducible factor-1α (HIF-1α), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in renal fibrosis in rats with 5/6 nephrectomy. METHODS: Sprague Dawley rats were randomly divided into three groups. Rats in the losartan group were gavaged with losartan (33.3 mg/kg/day) from 1 week after 5/6 nephrectomy, and those in the sham group and the model group only received an equal volume of saline solution by gavage. Rats were sacrificed at the ends of the 4, 8 and 12 weeks, respectively. Urinary N-acetyl-glucosaminidase (NAG), 24-h urinary protein, serum cystatin C, blood urea nitrogen (BUN), and serum creatinine (Scr) levels were assessed. Kidney tissues were observed under light and electron microscope. The expressions of HIF-1α, transforming growth factor-ß1 (TGF-ß1), MMP-9, and TIMP-1 were determined by immunohistochemistry and Western blotting. RESULTS: Twenty-four hour urinary protein, urinary NAG, serum cystatin C, BUN, and Scr levels in the model group were significantly higher than those in the sham group (p < 0.05), but losartan treatment improved these changes. The apparent glomerular sclerosis and tubulointerstitial fibrosis were also found in the model group, which were ameliorated by losartan. The expressions of HIF-1α, TGF-ß1, MMP-9, and TIMP-1 were elevated and MMp-9/TIMP-1 ratio was lowered in the model group (p < 0.05), but losartan increased the expression of MMP-9 and MMp-9/TIMP-1 ratio (p < 0.05) and lessened the expressions of HIF-1α, TGF-ß1, and TIMP-1 (p < 0.05). CONCLUSION: Losartan may ameliorate renal fibrosis partly by down-regulating HIF-1α and up-regulating MMP-9/TIMP-1 in rats with 5/6 nephrectomy.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/patologia , Losartan/farmacologia , Losartan/uso terapêutico , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Nefrectomia/métodos , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-1/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Fibrose/tratamento farmacológico , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: To investigate the renal microvascular injury in chronic aristolochic acid nephropathy (AAN) and the protective effects of Cozaar. METHODS: Male Sprague-Dawley rats were randomized into three groups. Rats in the model group received Caulis Aristolochiae manshuriensis (CAM) decoction by gavage (10 mL/kg/day); those in the Cozaar group were gavaged with CAM and Cozaar (33.3 mg/kg/day); and those in the control group only received an equal daily volume of saline solution by gavage. Kidney tissues were observed under a light and electron microscope. CD34, caspase-3, and bone morphogenetic protein-7 (BMP-7) were determined by immunohistochemistry, and expressions of angiopoietin (Ang) 1 and 2, Tie-2, BMP-7, and vascular endothelial growth factor (VEGF) mRNA were monitored via real-time polymerase chain reaction (PCR). RESULTS: (1) The kidney tissue injury in the chronic AAN model group was apparent, compared to the normal structure in the normal control group, and the Cozaar group showed relieved injury. (2) The expression of caspase-3 in the model group was elevated, while expressions of BMP-7 and CD34 were decreased (p < 0.05). Cozaar lessened caspase-3 expression (p < 0.05) and promoted BMP-7 and CD34 expressions (p < 0.05). (3) Real-time PCR demonstrated a downregulation of Ang-1, Tie-2, BMP-7, and VEGF mRNA (p < 0.05) and an upregulation of Ang-2 mRNA (p < 0.01) in the renocortex, while Cozaar upregulated the expression of Ang-1, Tie-2, BMP-7, and VEGF mRNA (p < 0.05). CONCLUSION: Renal microvascular injury was observed in chronic AAN, which was hypothetically correlated with a lack in the expressions of Ang-1, BMP-7, Tie-2, and VEGF and an excess in caspase-3 and Ang-2. Cozaar can significantly ameliorate the renal microvascular injury and protect renal function.
Assuntos
Ácidos Aristolóquicos/efeitos adversos , Nefropatias/induzido quimicamente , Nefropatias/prevenção & controle , Rim/irrigação sanguínea , Losartan/uso terapêutico , Microvasos/efeitos dos fármacos , Doenças Vasculares/complicações , Doenças Vasculares/prevenção & controle , Animais , Doença Crônica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Left ventricular hypertrophy (LVH) and peripheral vascular disease (PVD) are highly prevalent among patients undergoing maintenance haemodialysis (MHD) and contribute to most cardiovascular mortalities in this population. Ankle-brachial index (ABI) has been recently demonstrated to be a predictor for all-cause and cardiovascular mortality in MHD Patients. The main objective of the present study is to see whether ABI is correlated with LVH in MHD patients. METHODS: One hundred and sixteen MHD patients selected from our dialysis unit were enrolled in this study. Colour Doppler ultrasonic examinations were performed at their hearts, both lower extremities and non-fistula upper extremities to determine the morphological and haemodynamic changes of their hearts and the systolic pressures of both their posterior tibial arteries, dorsalis pedis arteries and non-fistula brachial arteries. ABI was calculated on the basis of systolic pressures of the lower extremities and non-fistula upper extremities, and the correlation between ABI and LVH was analysed. RESULTS: Seventy-four (63.8%) MHD patients presented with LVH and related haemodynamic changes of varying degrees. There was a significant difference in ABI between the LVH group and the non-LVH group (0.96 +/- 0.15 vs 1.25 +/- 0.12, P < 0.001). Bivariate analysis showed that left ventricular mass index (LVMI) was negatively correlated with ABI, serum Alb and Hb (r = -0.482, -0.329, -0.247, P < 0.01), positively correlated with hypertension, serum calcium, serum phosphorus and calcium-phosphorus product. (r = 0.235, 0.168, 0.231,0.282, P < 0.05), and ABI was reversely correlated with hypertension, serum calcium, serum phosphorus and calcium-phosphorus product. (r = -0.195, -0.405, -0.271, -0.384, -0.461, P < 0.05), positively correlated with serum albumin (r = 0.338, P < 0.001). Multiple linear regression demonstrated ABI was independently associated with LVMI in MHD patients (beta = -103.522, P = 0.000). CONCLUSION: There is high incidence of LVH and decreased ABI in MHD patients. A decrease in ABI to some extent reflects the degree of LVH in this population.
Assuntos
Determinação da Pressão Arterial/métodos , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Diálise Renal/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Tornozelo , Braço , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) results from a combination of environmental and genetic factors. Secreted protein acidic and rich in cysteine (SPARC) can be expressed by many different cell types and is associated with development, remodelling, cell turnover and tissue repair. The analysis of SPARC would help evaluate the effect of the unique matricellular glycoprotein on renal disease progression in ADPKD. METHODS: The concentration of SPARC was measured with an enzyme-linked immunosorbent assay (ELISA); distribution and expression levels were measured with in situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and western blot assays. Apoptosis was assessed by morphological observation and fluorescence-activated cell sorting (FACS) apoptosis index (AI) analysis. Cell cycle phase was examined by FACS analysis. Cell proliferation was studied using bromodeoxyuridine (BrdU) incorporation ELISA. RESULTS: The SPARC level in the renal cyst fluid of patients with ADPKD was greater than that in patients with simple renal cyst (SRC), and also greater than that found in the plasma and urine of patients with either ADPKD or SRC and normal subjects. SPARC mRNA and protein levels in polycystic renal tissue were greater than that in normal renal tissue. Additionally, SPARC could inhibit cyst-lining epithelial cell proliferation, bring about cell cycle arrest in the G0/G1 phase and induce apoptosis in vitro. SPARC treatment resulted in decreased mRNA levels of PCNA (proliferating cell nuclear antigen), MCM2 (minichromosome maintenance protein 2), ClnD1 and Bcl-2, but an increased mRNA level of p21(Waf1) in cyst-lining epithelial cells. CONCLUSION: Our findings suggest that the increased SPARC expression in ADPKD renal tissue may provide negative feedback in ADPKD patients.