The obstruction of post-insulin receptor signaling is the main mechanism of insulin-resistant diabetes. Progestin and adipoQ receptor 3 (PAQR3), a key regulator of inflammation and metabolism, can ...negatively regulate the PI3K/AKT signaling pathway. Here, we report that gentiopicroside (GPS), the main bioactive secoiridoid glycoside of Gentiana manshurica Kitagawa, decreased lipid synthesis and increased glucose utilization in palmitic acid (PA) treated HepG2 cells. Additionally, GPS improved glycolipid metabolism in streptozotocin (STZ) treated high-fat diet (HFD)-induced diabetic mice. Our findings revealed that GPS promoted the activation of the PI3K/AKT axis by facilitating DNA-binding protein 2 (DDB2)-mediated PAQR3 ubiquitinated degradation. Moreover, results of surface plasmon resonance (SPR), microscale thermophoresis (MST) and thermal shift assay (TSA) indicated that GPS directly binds to PAQR3. Results of molecular docking and cellular thermal shift assay (CETSA) revealed that GPS directly bound to the amino acids of the PAQR3 NH2-terminus including Leu40, Asp42, Glu69, Tyr125 and Ser129, and spatially inhibited the interaction between PAQR3 and the PI3K catalytic subunit (P110α) to restore the PI3K/AKT signaling pathway. In summary, our study identified GPS, which inhibits PAQR3 expression and directly targets PAQR3 to restore insulin signaling pathway, as a potential drug candidate for the treatment of diabetes.
Gentiopicroside (GPS) decreased progestin and adipoQ receptor 3 (PAQR3) expression by promoting DNA-binding protein 2 (DDB2)-mediated PAQR3 ubiquitinated degradation. Importantly, GPS bound directly to PAQR3 and spatially inhibited the combination between PAQR3 and P110α to restore PI3K/AKT signaling. Display omitted
Renal tubulointerstitial fibrosis (TIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells, is the typical pathological alteration in diabetic nephropathy. ...Gentiopicroside (GPS), a natural compound with anti-inflammatory activity, has been demonstrated to alleviate glomerulosclerosis, whereas whether GPS inhibits TIF
via
regulating inflammation remains unclear. In this study, diabetic db/db mice and high glucose (HG)-stimulated renal tubular epithelial cells (NRK-52E) were applied to explore the effects and mechanisms of GPS on TIF. The results
in vivo
showed that GPS effectively improves glycolipid metabolism disorder, renal dysfunction, and TIF. In particular, GPS treatment reversed the abnormal expressions of EMT marker proteins including elevated α-smooth muscle actin and vimentin and decreased E-cadherin in the kidney of db/db mice. Moreover, GPS treatment also inhibited protein expressions of angiotensinⅡ type 1 receptor (AT1R) and CK2α and the activation of the NF-κB pathway. Importantly, the aforementioned effects of GPS acted
in vivo
were further observed
in vitro
in HG-stimulated NRK-52E cells, which were independent of its effects on glucose and lipid-lowering activity but were reversed by AT1R over-expression. Together, our results indicate that GPS that directly inhibits the CK2/NF-κB inflammatory signaling pathway
via
AT1R may also contribute to the amelioration of TIF in diabetes.
The multiple roles of TGR5 in the regulation of glucose metabolism, inflammation, and oxidative stress have drawn attention as therapeutic candidates for diabetes-related kidney disease. However, ...diabetes induces downregulation of renal TGR5 protein expression, and the regulatory mechanisms have not been clarified. Here, we identify that Smurf1, an E3 ubiquitin ligase, is a critical interactor of TGR5 and mediates the ubiquitination and proteasomal degradation of TGR5 under high glucose stimulation in glomerular mesangial cells. Genetic deficiency of Smurf1 restores TGR5 protein expression and attenuates renal injuries in diabetic mice. Mechanistically, Smurf1 interacts with the TGR5 ICL2 region by its HECT domain and induces K11/K48-linked polyubiquitination of TGR5 at K306 residue. Moreover, restoration of TGR5 protects db/db mice from diabetic nephropathy. These observations elucidate the critical role of Smurf1 in regulating TGR5 stability, suggesting that pharmacological targeting of the interaction between Smurf1 and TGR5 could serve as a promising therapeutic strategy against diabetic nephropathy.
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•Renal TGR5 is downregulated via ubiquitin-proteasome pathway under HG stimulation•Restoration of TGR5 attenuates renal injuries in diabetic mice•Smurf1 is a negative regulator of TGR5 through its ubiquitination activity•Smurf1 interacts with TGR5 ICL2 region by its HECT domain
TGR5 has drawn attention as a therapeutic candidate for diabetic nephropathy. However, diabetes induces downregulation of renal TGR5 expression. Lin et al. show that TGR5 undergoes ubiquitination and degradation induced by Smurf1 in diabetic nephropathy. They find that genetic deficiency of Smurf1 and restoration of TGR5 attenuate renal injuries in diabetic mice.
Diabetic nephropathy (DN) is quickly becoming the largest cause of end-stage renal disease (ESRD) in diabetic patients, as well as a major source of morbidity and mortality. Our previous studies ...indicated that the activation of Nrf2/ARE pathway via Connexin43 (Cx43) considerably contribute to the prevention of oxidative stress in the procession of DN. Fraxin (Fr), the main active glycoside of Fraxinus rhynchophylla Hance, has been demonstrated to possess many potential pharmacological activities. Whereas, whether Fr could alleviate renal fibrosis through regulating Cx43 and consequently facilitating the activation of Nrf2/ARE pathway needs further investigation. The
results showed that: 1) Fr increased the expression of antioxidant enzymes including SOD1 and HO-1 to inhibit high glucose (HG)-induced fibronectin (FN) and inflammatory cell adhesion molecule (ICAM-1) overexpression; 2) Fr exerted antioxidant effect through activating the Nrf2/ARE pathway; 3) Fr significantly up-regulated the expression of Cx43 in HG-induced glomerular mesangial cells (GMCs), while the knock down of Cx43 largely impaired the activation of Nrf2/ARE pathway induced by Fr; 4) Fr promoted the activation of Nrf2/ARE pathway via regulating the interaction between Cx43 and AKT. Moreover, in accordance with the results
, elevated levels of Cx43, phosphorylated-AKT, Nrf2 and downstream antioxidant enzymes related to Nrf2 were observed in the kidneys of Fr-treated group compared with model group. Importantly, Fr significantly improved renal dysfunction pathological changes of renal fibrosis in diabetic db/db mice. Collectively, Fr could increase the Cx43-AKT-Nrf2/ARE pathway activation to postpone the diabetic renal fibrosis and the up-regulation of Cx43 is probably a novel mechanism in this process.
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•Gentiopicroside reversed the downregulation of TGR5 and the upregulation of ECM and inflammatory factors in HG-induced GMCs.•Gentiopicroside inhibited the activation of the NF-κB ...signalling pathway by preventing the phosphorylation of IκBα.•The depletion of TGR5 blocked the inhibition of the NF-κB signalling pathway by GPS.•Gentiopicroside enhanced the interaction between β-arrestin2 and IκBα via TGR5 activation.
Our previous studies indicated that the G-protein-coupled bile acid receptor, Gpbar1 (TGR5), inhibits inflammation by inhibiting the NF-κB signalling pathway, eventually attenuating diabetic nephropathy (DN). Gentiopicroside (GPS), the main active secoiridoid glycoside of Gentiana manshurica Kitagawa, has been demonstrated to inhibit inflammation in various diseases via inhibiting the inflammatory signalling pathways. However, whether GPS inhibits the NF-κB signalling pathway by activating TGR5 and regulates the pathological progression of diabetic renal fibrosis requires further investigation. In this study, we found that GPS significantly reversed the downregulation of TGR5 and inhibited the overproduction of fibronectin (FN), transforming growth factor β1 (TGF-β1), intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in glomerular mesangial cells (GMCs) exposed to high glucose (HG). Additionally, GPS prevented the phosphorylation and degradation of IκBα, and subsequently inhibited the activation of the NF-κB signalling pathway. Further investigation found that GPS enhanced the stabilization of IκBα by promoting the interaction of β-arrestin2 with IκBα via TGR5 activation, which contributed to the inhibition of NF-κB signalling pathway. Importantly, the depletion of TGR5 blocked the inhibition of the NF-κB signalling pathway and reversed the downregulation of FN, ICAM-1, VCAM-1 and TGF-β1 by GPS in HG-induced GMCs. Moreover, GPS increased the TGR5 protein levels and promoted the interaction between IκBα and β-arrestin2, thereby inhibiting the reduction of IκBα and blocked NF-κB p65 nuclear translocation in the kidneys of STZ-induced diabetic mice. Collectively, these data suggested that GPS regulates the TGR5-β-arrestin2-NF-κB signalling pathway to prevent inflammation in the kidneys of diabetic mice, and ultimately ameliorates the pathological progression of diabetic renal fibrosis.
Oxidative stress (OS) is the main cause leading to diabetic renal fibrosis. Recently, Fyn was paid much attention on OS and emerged as a pivotal player in acute kidney injury, while whether Fyn ...regulates oxidative stress in chronic diabetes nephropathy (DN) has not been clarified yet. The purpose of this study was to identify the role of Fyn in DN and elucidated its regulatory mechanism.
The db/db mice and littermate control C57BKS/J mice were injected by tail vein with Fyn interfering adenovirus or Fyn overexpressing adenovirus to investigate the role of Fyn in vivo. Primary glomerular mesangial cells (GMCs) were used for in vitro studies.
Fyn was up-regulated in high glucose (HG)-induced GMCs and kidneys of diabetic mice. Additionally, Fyn knockdown reduced the level of OS in HG-induced GMCs and kidneys of diabetic mice, thereby ameliorating diabetic renal fibrosis. While overexpression of Fyn significantly increased the level of OS in GMCs and kidney tissues, resulting in renal damage. Moreover, Fyn deficiency exerted antioxidant effects by activating the Sirt1/Foxo3a pathway. Mechanistically, Fyn facilitated the combination of c-Cbl and Sirt1 by phosphorylating c-Cbl at Tyr731, which triggered K48-linked polyubiquitination of Sirt1 at Lys377 and Lys513 by c-Cbl and promoted Sirt1 degradation, impairing the antioxidant effects of Foxo3a.
Fyn deficiency promoted Foxo3a nuclear transcription via reducing the ubiquitination of Sirt1 by c-Cbl, thereby alleviating renal oxidative damage in diabetic mice. These results identified Fyn as a potential therapeutic target against DN.
•This study reported the role of Fyn in diabetic nephropathy (DN).•We proposed Fyn as a potential therapeutic target against DN.•Fyn deficiency exerted antioxidant effects by activating the Sirt1/Foxo3a pathway.•Fyn facilitated the bond of c-Cbl and Sirt1 by phosphorylation at Tyr731 of c-Cbl.•c-Cbl promoted K48-linked polyubiquitination at Lys377 and Lys513 of Sirt1.
Chicken is one of the highest selling meats worldwide. Different cooking methods produce various flavor compounds in cooked chicken, which affect consumers’ food choices. In this study, the volatile ...flavor components of Gushi chicken breast after stewing and air frying were detected using gas chromatography‐ion mobility chromatography (GC‐IMS) and principal component analysis (PCA), and the relative odor activity value (ROAV) was determined using PCA. A total of 43 volatile substances were identified. 3‐(methylthio)propionaldehyde‐M and benzene acetaldehyde can be used as characteristic flavor markers in the air‐fried chicken breast.ethyl acetate, (E)‐2‐octenal‐D, and 2‐ethyl‐1‐hexanol‐D can be used as characteristic flavor markers in the stewed chicken breast. Because these five substances have the most obvious contrast in the fingerprint. The following part describes the specific substances that have mainly contributed to the flavor system in the volatile system obtained by different cooking methods. Finally, the main flavor compounds of the stewed chicken breast were 3‐methylbutanal‐D, octanal‐D, (E)‐2‐nonenal, and other 11 types of compounds (ROAV ≥1). Nine types of compounds (ROAV ≥1), including 3‐methylbutanal‐D, (E)‐2‐nonenal‐M octanal‐M, and hexanal‐M, were the main compounds to characterize the air‐fried chicken breast.
Practical applications
GC‐IMS technology combined with the principal component analysis of ROAV can be used to rapidly identify the main flavor substances and the flavor substances having an important role in modifying the overall taste of the sample. It plays a considerably important role in the aroma recombination of substances and can play a role in food processing.
First, 3‐(methylthio)propionaldehyde‐M and benzene acetaldehyde only appeared in the air‐fried chicken breast, and ethyl acetate, (E)‐2‐octenal‐D, and 2‐ethyl‐1‐hexanol‐D only appeared in the stewed chicken breast. Second, the main flavour compounds of the stewed chicken breast were 3‐methylbutanal‐D, octanal‐D, (E)‐2‐nonenal, and other 11 types of compounds (ROAV ≥1). Finally, nine types of compounds (ROAV ≥1), including 3‐methylbutanal‐D, (E)‐2‐nonenal‐M octanal‐M, and hexanal‐M, were the main compounds to characterise the air‐fried chicken breast.
Hyperglycemia-induced renal epithelial-to-mesenchymal transition (EMT) is a key pathological factor in diabetic renal tubulointerstitial fibrosis (RIF). Our previous studies have shown that connexin ...43 (Cx43) activation attenuated the development of diabetic renal fibrosis. However, whether Cx43 regulates the EMT of renal tubular epithelial cells (TECs) and the pathological process of RIF under the diabetic conditions remains to be elucidated. In the present study, we identified that Cx43 protein expression was down-regulated in the kidney tissues of db/db mice as well as in high glucose (HG)-induced NRK-52E cells. Overexpression of Cx43 improved renal function in db/db spontaneous diabetic model mice, increased SIRT1 levels, decreased hypoxia-inducible factor (HIF)-1α expression, and reduced production of EMT markers and extracellular matrix (ECM) components. Additionally, Cx43 overexpression inhibited the EMT process and reduced the expression of ECM components such as fibronectin (FN), Collagen I, and Collagen IV in HG-induced NRK-52E cells, whereas Cx43 deficiency had the opposite effects. Mechanistically, Cx43 in a carboxyl-terminal signal transduction-dependent manner could up-regulate SIRT1 expression and enhance SIRT1-dependent deacetylation of HIF-1α to reduce HIF-1α activity, which eventually ameliorated renal EMT and diabetic RIF. Our study indicates the essential role of Cx43 in regulating renal EMT and diabetic RIF via regulating the SIRT1-HIF-1α signaling pathway and provides an experimental basis for Cx43 as a potential target for diabetic nephropathy (DN).
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Renal chronic inflammation is an important hallmark of diabetic renal fibrosis. Casein kinase 2 interacting protein 1 (CKIP-1) performs a nephroprotective role in the pathogenesis of ...diabetic nephropathy (DN), which is dramatically decreased in diabetic kidneys. However, whether CKIP-1 regulates inflammation to ameliorate renal fibrosis remains unclear and it is interesting to clarify the degradation mechanism of CKIP-1. Here, we identified CKIP-1 expression was down-regulated in diabetic kidneys and knockout (KO) of CKIP-1 increased c-Jun expression and extra cellular matrix (ECM) in kidneys of normal mice, and knockout (KO) of CKIP-1 further exacerbated renal inflammatory fibrosis in diabetic mice. Moreover, the activated Src kinase interacted with CKIP-1 at Lys252 and increased K48 linked polyubiquitination and proteasome degradation of CKIP-1 in HG induced GMCs and diabetic kidneys. Mechanistically, Src facilitating the binding of c-Cbl with CKIP-1 by promoting the phosphorylation of c-Cbl, thereby increasing Cbl-mediated ubiquitination of CKIP-1 to down-regulate CKIP-1 protein expression. Thus, our study highlighted the anti-inflammation role of CKIP-1 and clarified the mechanism of CKIP-1 degradation in DN.
Renal oxidative stress (OSS) is the leading cause of diabetic nephropathy (DN). The silent information regulator 1/forkhead boxo3a (Sirt1/Foxo3a) pathway plays an essential role in regulating the ...antioxidant enzyme system. In this study, we aimed to investigate the mechanism of connexin32 (Cx32) on the antioxidant enzyme system in DN.
In this study, Cx32 overexpression significantly reduced reactive oxygen species generation and effectively inhibited the excessive production of extracellular matrix such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in high-glucose (HG)-induced glomerular mesangial cells. In addition, Cx32 overexpression reversed the downregulation of Sirt1, and promoted the nuclear transcription of Foxo3a, subsequently activating the antioxidant enzymes including catalase and manganese superoxide dismutase (MnSOD), however, Cx32 knockdown showed the opposite effects. A further mechanism study showed that Cx32 promoted the autoubiquitination and degradation of Smad ubiquitylation regulatory factor-1 (Smurf1), thereby reducing the ubiquitination of Sirt1 at Lys
and the degradation of Sirt1. Moreover, the
results showed that adenovirus-mediated Cx32 overexpression activated the Sirt1/Foxo3a pathway, and inhibited OSS in the kidney tissues, eventually improving the renal function and glomerulosclerosis in diabetic mice.
This study highlighted the antioxidant role of Cx32-Sirt1-Foxo3a axis to alleviate DN, which is a new mechanism of Cx32 alleviating DN.
Cx32 alleviated DN
activating the Sirt1/Foxo3a antioxidant pathway. The specific mechanism was that Cx32 upregulated the Sirt1 expression through reducing the ubiquitination of Lys
of Sirt1 by inhibiting Smurf1.
39, 241-261.
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