RESUMEN
As one of the most significant pathological changes of diabetic nephropathy (DN), tubulointerstitial fibrosis (TIF) had a close relationship with tubulointerstitial inflammation (TI), and the occurrence of TI could have resulted from the disrupted tight junctions (TJs) of renal tubular epithelial cells (RTECs). Studies have demonstrated that sodium butyrate (NaB), a typical short chain fatty acid (SCFA), played an important regulatory role in intestinal TJs and inflammation. In this study, our in vivo and in vitro results showed that accompanied by TI, renal tubular TJs were gradually disrupted in the process of DN-related TIF. In HG and LPS co-cultured HK-2 cells and db/db mice, NaB treatment regained the TJs of RTECs via the sphingosine 1-phosphate receptor-1 (S1PR1)/AMPK signaling pathway, relieving inflammation. Small interfering RNA of S1PR1, S1PR1 antagonist W146 and agonist SEW2871, and AMPK agonist AICAR were all used to further confirm the essential role of the S1PR1/AMPK signaling pathway in NaB's TJ protection in RTECs in vitro. Finally, NaB administration not only improved the renal function and TIF, but also relieved the TI of db/db mice. These findings suggested that the use of NaB might be a potential adjuvant treatment strategy for DN-associated TIF, and this protective effect was linked to the TJ modulation of RTECs via the S1PR1/AMPK signaling pathway, leading to the improvement of TI.
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Diabetes Mellitus , Nefropatías Diabéticas , Ratones , Animales , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Ácido Butírico/farmacología , Ácido Butírico/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Uniones Estrechas/metabolismo , Células Epiteliales/metabolismo , Fibrosis , Diabetes Mellitus/metabolismoRESUMEN
To explore the role of PDE4D in diabetic nephropathy (DN) and investigate whether resveratrol protects against DN via inhibiting PDE4D. Diabetic db/db mouse and glomerular mesangial cell line (GMCs) were used to investigate the role of PDE4D and the protective effect of resveratrol on renal fibrosis under high glucose (HG) environment. Resveratrol alleviated the progress of DN via inhibiting mitochondrial fragmentation and restoring the expression of PDE4D, PKA, phosphorylated Drp1-Ser637 and Drp1 in kidney of db/db mice. In HG-exposed GMCs, resveratrol treatment decreased the expression of PDE4D, increased PKA level, and inhibited Drp1-mediated mitochondrial fission. In contrast, PDE4D over-expression blunted the inhibitory effects of resveratrol on Drp1 expression and mitochondrial fission. Moreover, PKA inhibitor H89 blunted the effects of resveratrol on phosphorylated Drp1-Ser637 expression and mitochondrial fission in HG-treated GMCs. Inhibition of mitochondrial fission with Drp1 inhibitor Mdivi-1 alleviated mitochondrial dysfunction in GMCs under HG. These findings indicate PDE4D plays an important role in the process of DN. Resveratrol attenuates the development of DN by preventing mitochondrial fission through inhibiting PDE4D, which regulates the expression of phosphorylated Drp1-Ser637 directly.
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Diabetes Mellitus Experimental , Nefropatías Diabéticas , Ratones , Animales , Nefropatías Diabéticas/tratamiento farmacológico , Resveratrol/farmacología , Dinámicas Mitocondriales , Diabetes Mellitus Experimental/metabolismo , Células Mesangiales/metabolismoRESUMEN
The development of diabetic nephropathy (DN) could be promoted by the occurrence of tubulointerstitial fibrosis (TIF), which has a close relationship with mitochondrial dysfunction of renal tubular epithelial cells (RTECs). As a key regulator of metabolic homeostasis, Yin Yang 1 (YY1) plays an important role not only in regulating the fibrosis process but also in maintaining the mitochondrial function of pancreatic ß-cells. However, it was not clear whether YY1 participated in maintaining mitochondrial function of RTECs in early DN-associated TIF. In this study, we dynamically detected mitochondrial functions and protein expression of YY1 in db/db mice and high glucose (HG)-cultured HK-2 cells. Our results showed that comparing with the occurrence of TIF, the emergence of mitochondrial dysfunction of RTECs was an earlier even, besides the up-regulated and nuclear translocated YY1. Correlation analysis showed YY1 expressions were negatively associated with PGC-1α in vitro and in vivo. Further mechanism research demonstrated the formation of mTOR-YY1 heterodimer induced by HG up-regulated YY1, the nuclear translocation of which inactivated PGC-1α by binding to the PGC-1α promoter. Overexpression of YY1 induced mitochondrial dysfunctions in normal glucose-cultured HK-2 cells and 8-weeks-old db/m mice. While, dysfunctional mitochondria induced by HG could be improved by knockdown of YY1. Finally, downregulation of YY1 could retard the progression of TIF by preventing mitochondrial functions, resulting in the improvement of epithelial-mesenchymal transition (EMT) in early DN. These findings suggested that YY1 was a novel regulator of mitochondrial function of RTECs and contributed to the occurrence of early DN-associated TIF.
RESUMEN
BACKGROUND: Hepatic lipid accumulation was a major promoter for the further development of non-alcoholic fatty liver disease (NAFLD) in type 2 diabetes (T2DM). mTOR/YY1 signaling pathway regulated many metabolic processes in different organs, and played an important role in hepatic lipid metabolism. Thus, targeting mTOR/YY1 signaling pathway might be a novel therapeutic strategy of T2DM-associated NALFD. PURPOSE: To investigate the effects and the mechanism of quercetin against T2DM-associated NAFLD. STUDY DESIGN AND METHODS: The combine abilities of 24 flavonoid compounds with mTOR were detected by computer virtual screening (VS) and molecular modeling. mTOR/YY1 signaling pathway was examined in the liver of db/db mice, and high glucose (HG) and free fatty acid (FFA) co-cultured HepG2 cells. YY1 overexpression lentivirus vector and mTOR specific inhibitor rapamycin were used to further identify the indispensable role of mTOR/YY1 signaling pathway in quercetin's amelioration effect of hepatic lipid accumulation in vitro. Clinical studies, luciferase assay and chromatin immunoprecipitation (ChIP) assay were all carried out to investigate the potential mechanisms by which quercetin exerted its amelioration effect of hepatic lipid accumulation. RESULTS: Quercetin had the strongest ability to combine with mTOR and could competitively occupy its binding pocked. Along with the alleviated hepatic injury by quercetin, mTOR/YY1 signaling pathway was down-regulated in vivo and in vitro. However, the alleviation effect of quercetin against hepatic lipid accumulation was inhibited by YY1 overexpression in vitro. Mechanistically, the down-regulated nuclear YY1 induced by quercetin directly bound to CYP7A1 promoter and activated its transcription, resulting in the restoration of cholesterol homeostasis via the conversion of cholesterol-to-bile acids (BAs). CONCLUSION: The hepatoprotective effect of quercetin on T2DM-associated NAFLD was linked to the restoration of cholesterol homeostasis by the conversion of cholesterol-to-BAs via down-regulating mTOR/YY1 signaling pathway, leading to the increased CYP7A1 activity.
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Diabetes Mellitus Tipo 2 , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Quercetina/farmacología , Quercetina/uso terapéutico , Ácidos y Sales Biliares/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Hígado/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Colesterol/metabolismo , Metabolismo de los Lípidos , Colesterol 7-alfa-Hidroxilasa/metabolismoRESUMEN
The development of diabetic nephropathy (DN) could be promoted by the occurrence of tubulointerstitial fibrosis (TIF), which had a closely relationship with mitochondrial dysfunction of renal tubular epithelial cells (RTECs). As a key regulator of metabolic homeostasis, Yin Yang 1 (YY1) played an important role not only in regulating fibrosis process, but also in maintaining mitochondrial function of pancreatic ß cells. However, it was not clear whether YY1 participated in maintaining mitochondrial function of RTECs in early DN-associated TIF. In this study, we dynamically detected mitochondrial functions and protein expression of YY1 in db/db mice and high glucose (HG)-cultured HK-2 cells. Our results showed that comparing with the occurrence of TIF, the emergence of mitochondrial dysfunction of RTECs was an earlier even, besides the up-regulated and nuclear translocated YY1. Correlation analysis showed YY1 expressions were negatively associated with PGC-1α in vitro and in vivo. Further mechanism research demonstrated the formation of mTOR-YY1 heterodimer induced by HG upregulated YY1, the nuclear translocation of which inactivated PGC-1α by binding to the PGC-1α promoter. Overexpression of YY1 induced mitochondrial dysfunctions in normal glucose cultured HK-2 cells and 8-week-old db/m mice. While, dysfunctional mitochondria induced by HG could be improved by knockdown of YY1. Finally, downregulation of YY1 could retard the progression of TIF by preventing mitochondrial functions, resulting in the improvement of epithelial-mesenchymal transition (EMT) in early DN. These findings suggested that YY1 was a novel regulator of mitochondrial function of RTECs and contributed to the occurrence of early DN-associated TIF .
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Diabetes Mellitus , Nefropatías Diabéticas , Ratones , Animales , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Fibrosis , Glucosa/farmacología , Glucosa/metabolismo , Transición Epitelial-Mesenquimal , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologíaRESUMEN
Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus, which is characterized in renal tubulointerstitial fibrosis (TIF). The current study was designed to investigate the protective effect of Jujuboside A (Ju A) on TIF in type 2 diabetes (T2DM) mice, and explore its underlying anti-fibrosis mechanism. A mouse T2DM model was established using high fat diet (HFD) feeding combined with intraperitoneal injection of streptozotocin (STZ). Then, diabetic mice were treated with Ju A (10, 20 and 40 mg·kg-1·d-1, i.g.) for 12 weeks. Results showed that administration of Ju A not only down-regulated fasting blood glucose (FBG) levels, but also improved hyperlipidemia and renal function in diabetic mice. Moreover, the reduced ECM accumulation was observed in the renal cortex of Ju A treated diabetic mice, while the TIF progression was also attenuated by Ju A through blocking the epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs). Further mechanism studies showed that Ju A treatment effectively down-regulated the protein expression and subsequent nuclear translocation of Yin Yang 1 (YY1) in the renal cortex of diabetic mice, and reduced the levels of transforming growth factor-ß1 (TGF-ß1) in the serum and renal cortex of Ju A treated mice. According to invitro studies, the up-regulated YY1/TGF-ß1 signaling pathway was restored by Ju A in high glucose (HG) cultured HK-2 cells. Taken together, these findings demonstrated that Ju A can ameliorate the TIF of DN through down-regulating the YY1/TGF-ß1 signaling pathway.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Animales , Glucemia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Fibrosis , Ratones , Saponinas , Transducción de Señal , Estreptozocina , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Podocyte injury following abnormal podocyte autophagy plays an indispensable role in diabetic nephropathy (DN), therefore, restoration of podocyte autophagy is considered as a feasible strategy for the treatment of DN. Here, we investigated the preventive effects of sarsasapogenin (Sar), the main active ingredient in Anemarrhena asphodeloides Bunge, on the podocyte injury in diabetic rats, and tried to illustrate the mechanisms underlying the effects in high glucose (HG, 40 mM)-treated podocytes (MPs). Diabetes model was established in rats with single streptozocin (60 mg· kg-1) intraperitoneal administration. The rats were then treated with Sar (20, 60 mg· kg-1· d-1, i.g.) or a positive control drug insulin (INS) (40 U· kg-1· d-1, i.h.) for 10 weeks. Our results showed that both Sar and insulin precluded the decreases of autophagy-related proteins (ATG5, Beclin1 and LC3B) and podocyte marker proteins (podocin, nephrin and synaptopodin) in the diabetic kidney. Furthermore, network pharmacology was utilized to assess GSK3ß as the potential target involved in the action of Sar on DN and were substantiated by significant changes of GSK3ß signaling in the diabetic kidney. The underlying protection mechanisms of Sar were explored in HG-treated MPs. Sar (20, 40 µM) or insulin (50 mU/L) significantly increased the expression of autophagy- related proteins and podocyte marker proteins in HG-treated MPs. Furthermore, Sar or insulin treatment efficiently regulatedphosphorylation at activation and inhibition sites of GSK3ß. To sum up, this study certifies that Sar meliorates experimental DN through targeting GSK3ß signaling pathway and restoring podocyte autophagy.
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Autofagia/efectos de los fármacos , Nefropatías Diabéticas/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Podocitos/efectos de los fármacos , Espirostanos/administración & dosificación , Animales , Autofagia/fisiología , Nefropatías Diabéticas/tratamiento farmacológico , Medicamentos Herbarios Chinos/administración & dosificación , Masculino , Podocitos/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Silent information regulator 1 (Sirt1) is a deacetylase, which plays an important role in the occurrence and development of diabetic nephropathy (DN). Our previous study shows that Yin yang 1 (YY1), a widely expressed zinc finger DNA/RNA-binding transcription factor, is a novel regulator of renal fibrosis in diabetic nephropathy. Since the activity of YY1 is regulated via acetylation and deacetylation modification, this study aimed to explore whether Sirt1-induced deacetylation of YY1 mediated high glucose (HG)-induced renal tubular epithelial-mesenchymal transition (EMT) and renal fibrosis in vivo and in vitro. We first confirmed that Sirt1 expression level was significantly decreased in the kidney of db/db mice and in HG-treated HK-2 cells. Diabetes-induced Sirt1 reduction enhanced the level of YY1 acetylation and renal tubular EMT. Then, we manipulated Sirt1 expression in vivo and in vitro by injecting resveratrol (50 mg·kg-1·d-1. ip) to db/db mice for 2 weeks or application of SRT1720 (2.5 µM) in HG-treated HK-2 cells, we found that activation of Sirt1 reversed the renal tubular EMT and YY1 acetylation induced by HG condition. On the contrary, Sirt1 was knocked down in db/m mice or EX527 (1 µM) was added in HK-2 cells, we found that inhibition of Sirt1 exacerbated renal fibrosis in diabetic mice and enhanced level of YY1 acetylation in HK-2 cells. Furthermore, knockdown of YY1 inhibited the ameliorating effect of resveratrol on renal tubular EMT and renal fibrosis in db/db mice. In conclusion, this study demonstrates that Sirt1 plays an important role in renal tubular EMT of DN through mediating deacetylation of YY1.
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Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/fisiopatología , Sirtuina 1/genética , Factor de Transcripción YY1/metabolismo , Animales , Línea Celular , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/genética , Transición Epitelial-Mesenquimal/genética , Fibrosis , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Masculino , Ratones , Resveratrol/farmacología , Factor de Transcripción YY1/genéticaRESUMEN
The intestinal microenvironment, a potential factor that contributes to the development of non-alcoholic fatty liver disease (NALFD) and type 2 diabetes (T2DM), has a close relationship with intestinal tight junctions (TJs). Here, we show that the disruption of intestinal TJs in the intestines of 16-week-old db/db mice and in high glucose (HG)-cultured Caco-2 cells can both be improved by sodium butyrate (NaB) in a dose-dependent manner in vitro and in vivo. Accompanying the improved intestinal TJs, NaB not only relieved intestine inflammation of db/db mice and HG and LPS co-cultured Caco-2 cells but also restored intestinal Takeda G-protein-coupled (TGR5) expression, resulting in up-regulated serum GLP-1 levels. Subsequently, the GLP-1 analogue Exendin-4 was used to examine the improvement of lipid accumulation in HG and free fatty acid (FFA) co-cultured HepG2 cells. Finally, we used 16-week-old db/db mice to examine the hepatoprotective effects of NaB and its producing strain Clostridium butyricum. Our data showed that NaB and Clostridium butyricum treatment significantly reduced the levels of blood glucose and serum transaminase and markedly reduced T2DM-induced histological alterations of the liver, together with improved liver inflammation and lipid accumulation. These findings suggest that NaB and Clostridium butyricum are a potential adjuvant treatment strategy for T2DM-induced NAFLD; their hepatoprotective effect was linked to the modulation of intestinal TJs, causing the restoration of glucose and lipid metabolism and the improvement of inflammation in hepatocytes.
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Ácido Butírico/farmacología , Intestinos/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Uniones Estrechas/efectos de los fármacos , Animales , Glucemia/metabolismo , Células CACO-2 , Colesterol , Clostridium butyricum , Colon/patología , Citocinas/sangre , Diabetes Mellitus Tipo 2/metabolismo , Exenatida , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Células Hep G2 , Humanos , Hipoglucemiantes/farmacología , Inflamación/metabolismo , Metabolismo de los Lípidos , Hígado/lesiones , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , TriglicéridosRESUMEN
Hyperglycemia/oxidative stress has been implicated in the initiation and progression of diabetic complications while the components of Keap1/Nrf2/ARE signaling are being exploited as therapeutic targets for the treatment/management of these pathologies. Antioxidant agents like drugs, nutraceuticals and pure compounds that target the proteins of this pathway and their downstream genes hold the therapeutic strength to put the progression of this disease at bay. Here, we elucidate how the modulation of Keap1/Nrf2/ARE had been exploited for the treatment/management of end-stage diabetic kidney complication (diabetic nephropathy) by looking into (1) Nrf2 nuclear translocation and phosphorylation by some protein kinases at specific amino acid sequences and (2) Keap1 downregulation/Keap1-Nrf2 protein-protein inhibition (PPI) as potential therapeutic mechanisms exploited by Nrf2 activators for the modulation of diabetic nephropathy biomarkers (Collagen IV, Laminin, TGF-ß1 and Fibronectin) that ultimately lead to the amelioration of this disease progression. Furthermore, we brought to limelight the relationship between diabetic nephropathy and Keap1/Nrf2/ARE and finally elucidate how the modulation of this signaling pathway could be further explored to create novel therapeutic milestones.
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Elementos de Respuesta Antioxidante/fisiología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de los fármacos , Elementos de Respuesta Antioxidante/genética , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Oxidación-ReducciónRESUMEN
BACKGROUND: To investigate the correlation between the level of circulating vitamin D and the development of colorectal cancer (CRC) and to clarify the effect and mechanism of vitamin D on the development of CRC. METHODS: Serum samples from 63 patients with CRC (CRC group) and 61 healthy volunteers (normal group) were collected. Azoxymethane + dextran sodium sulfate-induced CRC mouse model and dietary models with different doses of vitamin D were established to verify whether vitamin D supplementation could reverse the occurrence and development of CRC at the overall animal level. Intestinal barrier integrity and microbial defense response were evaluated by detection of intestinal flora and expression of related genes. RESULTS: In the clinical serum samples, compared with the normal group, the level of 25 (OH) D3 in the CRC group was relatively low (P < 0.01), which was consistent with the clinical situation in mice. Vitamin D deficiency aggravated the deterioration of enteritis and intestinal cancer in CRC mice, whereas the overall condition of CRC mice improved after vitamin D supplementation. Vitamin D has a significant regulatory effect on the homeostasis of the intestinal flora, particularly in the regulation of intestinal probiotics, Akkermansia muciniphila-mediated colon barrier integrity. CONCLUSIONS: Vitamin D deficiency is closely related to the high incidence of CRC, and vitamin D supplementation can inhibit the occurrence and development of CRC. Vitamin D plays a role in the reversal of CRC mainly through the regulation of intestinal flora, especially the regulation of A. muciniphila-mediated colon barrier integrity.
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Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/microbiología , Neoplasias Colorrectales/prevención & control , Microbioma Gastrointestinal/efectos de los fármacos , Deficiencia de Vitamina D/complicaciones , Vitamina D/administración & dosificación , Vitamina D/farmacología , Akkermansia , Animales , Suplementos Dietéticos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , VerrucomicrobiaRESUMEN
Multiphase pathological processes involve in Type 2 diabetes (T2DM)-induced nonalcoholic fatty liver disease (NAFLD). However, the therapies are quite limited. In the present study, the hepatoprotective effects and underlying mechanisms of quercetin in T2DM-induced NAFLD were investigated. T2DM-induced NAFLD and quercetin treatment models were established in vivo and in vitro. The results revealed that quercetin alleviated serum transaminase levels and markedly reduced T2DM-induced histological alterations of livers. Additionally, quercetin restored superoxide dismutase, catalase, and glutathione content in livers. Not only that, quercetin markedly attenuated T2DM-induced production of interleukin 1 beta, interleukin 6, and TNF-α. Accompanied by the restoration of the increased serum total bile acid (p = .0001) and the decreased liver total bile acid (p = .0005), quercetin could reduce lipid accumulation in the liver of db/db mice. Further mechanism studies showed that farnesoid X receptor 1/Takeda G-protein-coupled receptor 5 signaling pathways was involved in quercetin regulation of lipid metabolism in T2DM-induced NAFLD. In high D-glucose and free fatty acid cocultured HepG2 cells model, quercetin eliminated lipid droplets and restored the upregulated total cholesterol and triglyceride levels. Similar to the findings in mice, quercetin could also activate farnesoid X receptor 1/Takeda G-protein-coupled receptor 5 signaling pathway. These findings suggested that quercetin might be a potentially effective drug for the treatment of T2DM-induced NAFLD.
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Antioxidantes/uso terapéutico , Diabetes Mellitus Tipo 2/complicaciones , Inflamación/tratamiento farmacológico , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Quercetina/uso terapéutico , Animales , Antioxidantes/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Masculino , Ratones , Quercetina/farmacología , Transducción de SeñalRESUMEN
BACKGROUND: Renal fibrosis promotes the development of diabetic nephropathy (DN). A growing number of studies have reported that Yin Yang 1 (YY1), which is involved in cellular proliferation and differentiation, plays a crucial role in the pathogenesis of many diseases, such as pulmonary fibrosis, hepatic steatosis and cancer. METHODS: We detected the expression of YY1 under various glucose concentration and time gradient conditions. Rapamycin was used to verify the mTORC1/p70S6K/YY1 signaling pathway in HK-2 cells. We used db/db mice to examine the connection between renal fibrosis and YY1. A luciferase assay and chromatin immunoprecipitation (ChIP) assay were used to identify whether YY1 directly regulated α-SMA by binding to the α-SMA promoter. RNA silencing and overexpression were performed by using a YY1 expression/knockdown plasmid to investigate the function of YY1 in renal fibrosis of DN. RESULTS: YY1 expression and subsequent nuclear translocation were upregulated in a glucose- and time-dependent manner via the mTORC1/p70S6K signaling pathway in HK-2 cells. YY1 expression and nuclear translocation was significantly upregulated in db/db mice. Furthermore, YY1 upregulated α-SMA expression and activity in high-glucose-cultured HK-2 cells. Overexpression of YY1 promoted renal fibrosis in db/m mice mainly by upregulating α-SMA expression and inducing epithelial-mesenchymal transition (EMT) in vitro and in vivo. Finally, downregulation of YY1 reversed renal fibrosis by improving EMT in vivo and in vitro. CONCLUSIONS: These results reveal that upregulation of YY1 plays a critical role in HG-induced deregulation of EMT-associated protein expression, which finally results in renal fibrosis of DN. Therefore, decreasing YY1 expression might represent a new therapeutic target for diabetic nephropathy-induced renal fibrosis.
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Nefropatías Diabéticas/tratamiento farmacológico , Factor de Transcripción YY1/efectos de los fármacos , Actinas/metabolismo , Animales , Línea Celular , Nefropatías Diabéticas/complicaciones , Nefropatías Diabéticas/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Fibrosis , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Glucosa/farmacología , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Transcripción YY1/biosíntesis , Factor de Transcripción YY1/genéticaRESUMEN
One of the earliest critical secondary complications of diabetes is the opacification of the eye lens - a condition strictly associated with diabetic cataract. The study presented here was designed to investigate the effect of Ginkgo biloba extract (GbE), rutin and quercetin on streptozotocin (STZ) induced diabetic cataract (DC) rats. Ten weeks after administration of GbE, rutin and quercetin, the opacity of diabetic rats' lenses was graded under a slit lamp. Then, the levels of malondialdehyde (MDA), reduced glutathione (GSH), advanced glycosylation end products (AGEs), and the activities of aldose reductase (AR) were estimated. The DC-induced rats produced less GSH, higher levels of MDA and AGEs as well as elevated AR activity when compared to the normal group. Administration of GbE, rutin and quercetin remarkably inhibited the AR activity, stimulated the production of glutathione, and decreased the levels of MDA and AGEs in the lenses of DC-induced rats, which eventually delayed the progression of lens opacification in diabetic rats to various degrees. Our results revealed that quercetin had the highest significant (P<0.05) potential to delay the progression of STZ-induced diabetic cataract when compared with rutin and GbE. The mechanism dictating this interesting prowess of quercetin might be attributed to its AR inhibitory strength, anti-lipid peroxidation potential and anti-AGEs activity.
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Catarata/tratamiento farmacológico , Complicaciones de la Diabetes/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Aldehído Reductasa/efectos de los fármacos , Animales , Catarata/etiología , Catarata/prevención & control , Diabetes Mellitus Experimental , Ginkgo biloba , Cristalino/patología , Extractos Vegetales/uso terapéutico , Polímeros/metabolismo , Quercetina/uso terapéutico , Ratas , Rutina/uso terapéuticoRESUMEN
The aim of this study is to investigate effects and potential mechanisms of sarsasapogenin (Sar), an active component purified from Rhizoma Anemarrhenae, on high glucose-induced amyloid-beta (Aß) peptide overproduction in HT-22 cells. HT-22 cells were divided into normal glucose; high glucose (HG); HG co-treated with low, middle, and high concentration of Sar (1, 5, 25 µmol/L); and peroxisome proliferator-activated receptor γ (PPARγ) agonist (10 µmol/L pioglitazone). After treatment for 24 h, protein expression of Aß and ß-site Aß precursor protein cleaving enzyme 1 (BACE1) and activated PPARγ level were determined by Western blot; Aß42 levels were also measured by using both immunofluorescence and ELISA methods. BACE1 activity and mRNA level were assessed by fluorospectrophotometry and quantitative PCR, respectively. Cell viability was assayed with a CCK-8 kit. Elevated Aß expression and Aß42 level were found in HG-treated HT-22 cells, accompanied by increased BACE1 protein and mRNA levels as well as enzymatic activity, which was markedly attenuated by three concentrations of Sar and pioglitazone. Moreover, HG reduced nuclear PPARγ levels, which was reversed by middle and high concentrations of Sar as well as pioglitazone. PPARγ antagonist GW9662 (20 µmol/L) pretreatment reversed the effect of Sar on BACE1 protein expression in HG-cultured HT-22 cells. Additionally, Sar suppressed HG-induced decreases in cell viability of HT-22 cells. High glucose can induce an increase in Aß levels and a decrease in cell viability in HT-22 cells, while co-treatment with Sar improves these results, which is mediated likely through activation of PPARγ and subsequent downregulation of BACE1.
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Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Medicamentos Herbarios Chinos/farmacología , Glucosa/toxicidad , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/biosíntesis , Espirostanos/farmacología , Animales , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , RatonesRESUMEN
Hydrophilic interaction chromatography (HILIC) is an emerging separation mode of liquid chromatography (LC). Using highly hydrophilic stationary phases capable of retaining polar/ionic metabolites, and accompany with high organic content mobile phase that offer readily compatibility with mass spectrometry (MS) has made HILIC an attractive complementary tool to the widely used reverse-phase (RP) chromatographic separations in metabolomic studies. The combination of HILIC and RPLC coupled with an MS detector expands the number of detected analytes and provides more comprehensive metabolite coverage than use of only RP chromatography. This review describes the recent applications of HILIC-MS/MS in metabolomic studies, ranging from amino acids, lipids, nucleotides, organic acids, pharmaceuticals, and metabolites of specific nature. The biological systems investigated include microbials, cultured cell line, plants, herbal medicine, urine, and serum as well as tissues from animals and humans. Owing to its unique capability to measure more-polar biomolecules, the HILIC separation technique would no doubt enhance the comprehensiveness of metabolite detection, and add significant value for metabolomic investigations. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:574-600, 2016.
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Cromatografía Liquida , Interacciones Hidrofóbicas e Hidrofílicas , Metabolómica , Animales , Cromatografía de Fase Inversa , Humanos , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Recently, extract of Ginkgo biloba leaves (GbE) have become widely known phytomedicines and have shown various pharmacological activities, including improvement of blood circulation, protection of oxidative cell damage, prevention of Alzheimer's disease, treatment of cardiovascular disease and diabetes complications. This study was designed to investigate the effects of an ethanolic GbE on renal fibrosis in diabetic nephropathy (DN) and to clarify the possible mechanism by which GbE prevents renal fibrosis. STUDY DESIGN: We investigated the protective effects of GbE on renal fibrosis in STZ-induced diabetic rats. Rats were randomized into six groups termed normal control, diabetes mellitus, low dose of GbE (50 mg/kg/d), intermediate dose of GbE (100 mg/kg/d), high dose of GbE (200 mg/kg/d) and rapamycin (1 mg/kg/d). METHODS: After 12 weeks, the rats were sacrificed and then fasting blood glucose (FBG), creatinine (Cr), blood urea nitrogen (BUN), urine protein, relative kidney weight, glycogen and collagen accumulation, and collagen IV and laminin expression were measured by different methods. The amounts of E-cadherin, α-SMA and snail, as well as the phosphorylation of Akt, mTOR and p70S6K in the renal cortex of rats, were examined by western blotting. RESULTS: Compared with diabetic rats, the levels of Cr, BUN, urine protein, relative kidney weight, accumulation of glycogen and collagen, and expression of collagen IV and laminin in the renal cortex were all decreased in GbE treated rats. In addition, GbE reduced the expression of E-cadherin, α-SMA, snail and the phosphorylation of Akt, mTOR and p70S6K in diabetic renal cortex. CONCLUSION: GbE can prevent renal fibrosis in rats with diabetic nephropathy, which is most likely to be associated with its abilities to inhibit the Akt/mTOR signaling pathway.
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Nefropatías Diabéticas/tratamiento farmacológico , Ginkgo biloba/química , Enfermedades Renales/prevención & control , Extractos Vegetales/farmacología , Transducción de Señal , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Fibrosis/prevención & control , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Fitoterapia , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The mesangial cell (MC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN), but the compounds interacting with cell are still unknown, which may be potential bioactive components. Based on this, the determination of GBE in MC lysates was proposed by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in this study. The MC was cultured with normal or high glucose with GBE for 4, 8, 12, 16, 24 and 48h. The harvested cell was extracted with 40% acetic acid in water and further analyzed by LC-MS/MS. All the validation data including linearity, intra-day and inter-day precision, limit of detection and quantification, matrix effect, and stability were within the required limits. The validated method was successfully applied to quantify 11 compounds of GBE in cell lysates. The results showed that high glucose prolonged the peak time of all observed 11 compounds and peak concentrations of bilobalide, ginkgolide C, ginkgolide B, quercetin, luteolin, kaempferol, isorhamnetin and genkwanin in cell lysates, which hinted that these compounds may be the potential bioactive components of GBE with preventive effect against DN.
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Cromatografía Líquida de Alta Presión/métodos , Ginkgo biloba/química , Células Mesangiales/metabolismo , Extractos Vegetales/análisis , Extractos Vegetales/metabolismo , Espectrometría de Masas en Tándem/métodos , Técnicas de Cultivo de Célula , Nefropatías Diabéticas , Glucosa/metabolismo , Humanos , Reproducibilidad de los ResultadosRESUMEN
This study was designed to investigate the effects of mangiferin on renal fibrosis, osteopontin production, and inflammation in the kidney of diabetic rats. Diabetes was induced through the single administration of streptozotocin (55 mg/kg, i.p.). Diabetic rats were treated with mangiferin (15, 30, and 60 mg/kg/day, i.g.) for 9 weeks. The kidney was fixed in 10% formalin for glomerulus fibrosis examination using Masson trichrome staining. Kidney and blood were obtained for assays of the associated biochemical parameters. Chronic mangiferin treatment prevented renal glomerulus fibrosis evidenced by decreases in Mason-stained positive area of glomeruli, protein expression of type IV collagen, and α-smooth muscle actin in the kidney of diabetic rats, in comparison with decreases in mRNA and protein expression of osteopontin as well as protein expression of cyclooxygenase 2 and NF-кB p65 subunit in the renal cortex of diabetic rats. Moreover, mangiferin reduced the levels of interleukin 1ß in both the serum and the kidney of diabetic rats. Our findings demonstrate that mangiferin prevents the renal glomerulus fibrosis of diabetic rats, which is realized through the suppression of osteopontin overproduction and inflammation likely via inactivation of NF-кB.
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Diabetes Mellitus Experimental/fisiopatología , Glomérulos Renales/efectos de los fármacos , Osteopontina/metabolismo , Xantonas/farmacología , Actinas/metabolismo , Animales , Glucemia/análisis , Peso Corporal/efectos de los fármacos , Colágeno Tipo IV/metabolismo , Ciclooxigenasa 2/metabolismo , Regulación hacia Abajo , Fibrosis , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/patología , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Estreptozocina , Factor de Transcripción ReIA/metabolismoRESUMEN
A rapid and useful approach for screening potential bioactive components in Ginkgo biloba extract (GBE) with preventive effect against diabetic nephropathy (DN) was developed using mesangial cells extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Mesangial cells were first divided into two groups according to their treatments with high glucose or high glucose plus GBE. After incubation for 4, 8, 12, 16, 24 and 48 h, the cells were harvested and extracted with 40% acetic acid in water before LC-MS/MS analysis. Then, 19 compounds and five metabolites were found to selectively combine with mesangial cells. Notably, compounds including quercetin and rutin were identified or tentatively characterized according to the results of retention time and MS spectra, which is highly consistent with our previous reports that quercetin and rutin are potent protective agents against glomerulosclerosis in DN. Therefore, all these results indicate that target cell extraction coupled with LC-MS/MS analysis can be successfully applied for predicting the bioactive components in GBE with preventive effect against DN.