Curcumin decreases renal triglyceride accumulation through AMPK–SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats

Diabetic kidney disease has been associated with the presence of lipid deposits. We assumed that curcumin, a polyphenol, would attenuate the tissue dyslipidemic condition through activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and suppression of sterol regulatory element-binding protein (SREBP)-1c in the kidney and would prevent renal progression in experimental type 1 diabetic rats. Diabetes was induced with streptozotocin (STZ) (55 mg/kg) by intraperitoneal injection in male Sprague–Dawley rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic and diabetic treated with curcumin (100 mg/kg/day) by gavage for 8 weeks. We found that curcumin decreased plasma triglyceride and the amount of renal triglyceride significantly. Furthermore, treatment of diabetic rats with curcumin increased the phosphorylation of AMPK and prevented the increased renal expression of SREBP-1c and, as a result, decreased the expression of acetyl CoA carboxylase and fatty acid synthase as well as adipose differentiation-related protein, a marker of cytoplasmic droplets. We also demonstrate that curcumin significantly suppressed the increased expression of transforming growth factor β, vascular endothelial growth factor and extracellular matrix proteins such as type IV collagen and fibronectin. In addition, curcumin treatment increased nephrin expression to near-normal levels in diabetic rats. These results demonstrated that curcumin protects against the development of diabetic nephropathy through the AMPK–SREBP pathway and the reduction of renal triglyceride accumulation which could be a possible mechanism by which curcumin preserves renal function in diabetes.     

Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K–Akt signaling in diabetic rats

Diabetes increases the risk of cardiovascular diseases. Berberine (BBR), an isoquinoline alkaloid used in Chinese medicine, exerts anti-diabetic effect by lowering blood glucose and regulating lipid metabolism. It has been reported that BBR decreases mortality in patients with chronic congestive heart failure. However, the molecular mechanisms of these beneficial effects are incompletely understood. In the present study, we sought to determine whether BBR exerts cardioprotective effect against ischemia/reperfusion (I/R) injury in diabetic rats and the underlying mechanisms. Male Sprague-Dawley rats were injected with low dose streptozotocin and fed with a high-fat diet for 12 weeks to induce diabetes. The diabetic rats were intragastrically administered with saline or BBR (100, 200 and 400 mg/kg/d) starting from week 9 to 12. At the end of week 12, all rats were subjected to 30 min of myocardial ischemia and 3 h of reperfusion. BBR significantly improved the recovery of cardiac systolic/diastolic function and reduced myocardial apoptosis in diabetic rats subjected to myocardial I/R. Furthermore, in cultured neonatal rat cardiomyocytes, BBR (50 μmol/L) reduced hypoxia/reoxygenation-induced myocardial apoptosis, increased Bcl-2/Bax ratio and decreased caspase-3 expression, together with enhanced activation of PI3K–Akt and increased adenosine monophosphate-activated protein kinase (AMPK) and eNOS phosphorylation. Pretreatment with either PI3K/Akt inhibitor wortmannin or AMPK inhibitor Compound C blunted the anti-apoptotic effect of BBR. Our findings demonstrate that BBR exerts anti-apoptotic effect and improves cardiac functional recovery following myocardial I/R via activating AMPK and PI3K–Akt–eNOS signaling in diabetic rats.     

Uric acid induced epithelial−mesenchymal transition of renal tubular cells through PI3K/p-Akt signaling pathway

The phenotypic changes of tubular epithelial cell are hallmark features of renal diseases caused by abnormal uric acid levels. We hereby intend to investigate whether PI3K/p-Akt signaling plays a role in uric-acid induced epithelial−mesenchymal transition process. The normal rat kidney cell line (NRK-52E) was used as a proximal tubular cell model in this study. NRK-52E cells were exposed to different concentrations of uric acid, or PI3K inhibitor LY294002, or both, respectively. The effects of uric acid on cell morphology were examined by phase contrast microscopy, while molecular alternations were assessed by western blot analysis and immunofluorescence staining. We found that uric acid induced visible morphological alterations in NRK-52E cells accompanied by increased expression of α-smooth muscle actin and reduced expression of E-cadherin. Moreover, phosphorylation of Akt protein was obviously increased, whereas Akt level remained stable. Furthermore, the above effects were abolished when PI3K/p-Akt pathway was blocked by the PI3K inhibitor. These findings demonstrated that high uric acid could induce phenotypic transition of cultured renal tubular cells, which was probably via activating PI3K/p-Akt signaling pathway.     

TMEM158 promotes pancreatic cancer aggressiveness by activation of TGFβ1 and PI3K/AKT signaling pathway

Pancreatic cancer (PC) is one of the most deadly digestive cancers world-wide, with a dismal five-year survival rate of <8%. Upregulation of transmembrane protein 158 (TMEM158) is known to facilitate the progression of several carcinomas. However, little is known concerning the potential roles of TMEM158 in PC. Herein, we first found that TMEM158 was significantly upregulated in PC samples as well as PC cell lines. The overexpression of TMEM158 was significantly correlated with advanced clinicopathologic features (including tumor size, TNM stage, and blood vessel invasion) and poorer prognosis of patients with PC in clinic. Evidenced based on a series of loss- and gain-of-function assays uncovered that TMEM158 enhanced PC cell proliferation, migration, and invasion by stimulating the progression of cell cycle, epithelial–mesenchymal transition, and MMP-2/9 production. Furthermore, mechanism-related investigations disclosed that activation of TGFβ1 and PI3K/AKT signal might be responsible for TMEM158-triggered PC aggressiveness. Collectively, TMEM158 was upregulated in PC and promoted PC cell proliferation, migration, and invasion through the activation of TGFβ1 and PI3K/AKT signaling pathways, highlighting its potential as a tumor promoter and a therapeutic target for PC.     

Dapagliflozin alleviates cardiac fibrosis through suppressing EndMT and fibroblast activation via AMPKα/TGF-β/Smad signalling in type 2 diabetic rats

Diabetic cardiomyopathy (DCM) is one of the leading causes of heart failure in patients with diabetes mellitus, with limited effective treatments. The cardioprotective effects of sodium-glucose cotransporter 2(SGLT2) inhibitors have been supported by amounts of clinical trials, which largely fills the gap. However, the underlying mechanism still needs to be further explored, especially in terms of its protection against cardiac fibrosis, a crucial pathophysiological process during the development of DCM. Besides, endothelial-to-mesenchymal transition (EndMT) has been reported to play a pivotal role in fibroblast multiplication and cardiac fibrosis. This study aimed to evaluate the effect of SGLT2 inhibitor dapagliflozin (DAPA) on DCM especially for cardiac fibrosis and explore the underlying mechanism. In vivo, the model of type 2 diabetic rats was built with high-fat feeding and streptozotocin injection. Untreated diabetic rats showed cardiac dysfunction, increased myocardial fibrosis and EndMT, which was attenuated after treatment with DAPA and metformin. In vitro, HUVECs and primary cardiac fibroblasts were treated with DAPA and exposed to high glucose (HG). HG-induced EndMT in HUVECs and collagen secretion of fibroblasts were markedly inhibited by DAPA. Up-regulation of TGF-β/Smad signalling and activity inhibition of AMPKα were also reversed by DAPA treatment. Then, AMPKα siRNA and compound C abrogated the anti-EndMT effects of DAPA in HUVECs. From above all, our study implied that DAPA can protect against DCM and myocardial fibrosis through suppressing fibroblast activation and EndMT via AMPKα-mediated inhibition of TGF-β/Smad signalling.     

Nimbolide ameliorates the streptozotocin-induced diabetic retinopathy in rats through the inhibition of TLR4/NF-κB signaling pathway

BackgroundDiabetic retinopathy (DR) is a common problem in the diabetic patients due to the high blood glucose level. DR affects more number of diabetic patients worldwide with irreversible vision loss.ObjectiveThe current investigation was focused to reveal the therapeutic actions of nimbolide against the streptozotocin (STZ)-provoked DR in rats through inhibition of TLR4/NF-κB pathway.MethodologyDR was provoked to the rats through administering a single dose of STZ (60 mg/kg) intraperitoneally. The DR rats were then supplemented with the 50 mg/kg of nimbolide for 60 days. The bodyweight and blood glucose level was measured using standard methods. The lipid profiles (cholesterol, TG, LDL, and HDL), inflammatory markers, and antioxidants level was detected using respective kits. The level of MCP-1, VEGF, and MMP-9 was quantified using kits. The morphometric analysis of retinal tissues were done. The mRNA expressions of target genes were studied using RT-PCR assay.ResultsNimbolide treatment effective decreased the food intake and blood glucose, and improved the bodyweight of STZ-provoked animals. The levels of pro-inflammatory mediators, cholesterol, TG, LDL, and HDL, MCP-1, VEGF, and MMP-9 was remarkably suppressed by the nimbolide treatment. Nimbolide also improved the antioxidants, retinal thickness and cell numbers. The TLR4/NF-κB pathway was appreciably inhibited by the nimbolide.ConclusionOverall, our findings demonstrated that the nimbolide attenuated the STZ-provoked DR in rats through inhibiting the TLR4/NF-κB pathway.     

ERO1α promotes testosterone secretion in hCG-stimulated mouse Leydig cells via activation of the PI3K/AKT/mTOR signaling pathway

ER oxidoreduclin 1α (ERO1α) is an oxidase, participating in formation of secretory and membrane proteins. However, the other physiological functions ERO1α is not well known. We found that ERO1α is high in the Leydig cells of the testis. Therefore, the purposes of the current study are to explore the role of ERO1α and the possible mechanisms in regulating cell proliferation, apoptosis, and testosterone secretion of Leydig cells. ERO1α was mainly localized in Leydig cells in the adult mice testes by immunofluorescence staining. Western blot analysis showed that ERO1α was higher in Leydig cells than that in the seminiferous tubules. The effect of ERO1α on cell proliferation, apoptosis, and testosterone secretion was detected by transducing ERO1α overexpression and knockdown lentiviruses into cultured primary Leydig cells (PLCs) together with hCG exposure. Flow cytometry analysis showed that ERO1α promoted cell proliferation by increasing cell distribution at the S phase and decreasing that at the G0/G1 phase. Western bolt analysis showed that ERO1α increased CDK2 and CDK6 expression. Cell apoptosis determination found that ERO1α inhibited PLC apoptosis. Western bolt analysis showed that ERO1α increased the ratio of BCL-2/BAX, and decreased BAD and Caspase-3 expression. Enzyme-linked immunosorbent assay analysis demonstrated that ERO1α enhanced testosterone secretion. Western bolt analysis found that ERO1α increased StAR, 3β-HSD, and CYP17A1 expression. Furthermore, ERO1α could activate the PI3K/AKT/mTOR signaling pathway. In summary, these results suggest that ERO1α might play proliferation promotion and antiapoptotic roles and enhance testosterone secretion in PLC, at least partly, via activation of the PI3K/AKT/mTOR signaling pathway.     

Methylene blue offers neuroprotection after intracerebral hemorrhage in rats through the PI3K/Akt/GSK3β signaling pathway

Inflammation and apoptosis are two key factors contributing to secondary brain injury after intracerebral hemorrhage (ICH). In the present study, we explored the neuroprotective role of methylene blue (MB) in ICH rats and studied the potential mechanisms involved. Rats were subjected to local injection of collagenase IV in the striatum or sham surgery. We observed that MB treatment could exert a neuroprotective effect on ICH by promoting neurological scores, decreasing the brain water content, alleviating brain–blood barrier disruption, and improving the histological damages in the perihematomal areas. Furthermore, we demonstrated that the various mechanisms underlying MB’s neuroprotective effects linked to inhibited apoptosis and inhibited neuroinflammation. In addition, wortmannin, a selective inhibitor of phosphoinositide 3-kinase (PI3K), could reverse the antiapoptotic and anti-inflammatory effects of MB, which suggested that the PI3K–Akt pathway played an important role. In conclusion, these data suggested that MB could inhibit apoptosis and ameliorate neuroinflammation after ICH, and its neuroprotective effects might be exerted via the activation of the PI3K/Akt/GSK3β pathway.     

Neuroprotective effects of linarin through activation of the PI3K/Akt pathway in amyloid-β-induced neuronal cell death

Linarin, a natural occurring flavanol glycoside derived from Mentha arvensis and Buddleja davidii is known to have anti-acetylcholinesterase effects. The present study intended to explore the neuroprotective effects of linarin against Aβ(25-35)-induced neurotoxicity with cultured rat pheochromocytoma cells (PC12 cells) and the possible mechanisms involved. For this purpose, PC12 cells were cultured and exposed to 30 μM Aβ(25-35) in the absence or presence of linarin (0.1, 1.0 and 10 μM). In addition, the potential contribution of the PI3K/Akt neuroprotective pathway in linarin-mediated protection against Aβ(25-35)-induced neurotoxicity was also investigated. The results showed that linarin dose-dependently increased cell viability and reduced the number of apoptotic cells as measured by MTT assay, Annexin-V/PI staining, JC-1 staining and caspase-3 activity assay. Linarin could also inhibit acetylcholinesterase activity induced by Aβ(25-35) in PC12 cells. Further study revealed that linarin induced the phosphorylation of Akt dose-dependently. Treatment of PC12 cells with the PI3K inhibitor LY294002 attenuated the protective effects of linarin. Furthermore, linarin also stimulated phosphorylation of glycogen synthase kinase-3β (GSK-3β), a downstream target of PI3K/Akt. Moreover, the expression of the anti-apoptotic protein Bcl-2 was also increased by linarin treatment. These results suggest that linarin prevents Aβ(25-35)-induced neurotoxicity through the activation of PI3K/Akt, which subsequently inhibits GSK-3β and up-regulates Bcl-2. These findings raise the possibility that linarin may be a potent therapeutic compound against Alzheimer's disease acting through both acetylcholinesterase inhibition and neuroprotection.     

Induction of primitive streak and mesendoderm formation in monolayer hESC culture by activation of TGF-β signaling pathway by Activin B

Human embryonic stem cells (hESCs) have the ability to differentiate into all human cells, however controlling the differentiation has always been a challenge. In the present study we have investigated the direct differentiation of hESCs on MEFs by using TGF-β signaling pathway activators Activin A and Activin B. Activation of the TGF-β pathway with Activin B in low serum highly induced primitive streak and mesendoderm formation after 24 h, which included up-regulation of SOX 17 and BRACHYURY protein and gene expression. Continuous stimulation with Activin B in 2% serum further induced mesendoderm formation by increased gene expression of Brachyury, SOX17, MEOX and FOX at the same time we found down-regulation of neuroectodermal marker genes. Further, by stimulating the mesodermal cells by BMP-2 we succeeded to induce mesenchymal like cells with high expression of mesenchymal markers including; MEOX, FOX, RUNX2, COL1 and OSTEOPONTIN. In conclusion we have directed the differentiation of hESCs as monolayer to primitive streak like cells with Activin B and further into pure mesoderm and mesenchymal like cells by BMP-2.     

RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERK signaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1–10 ng/ml) of TNF-α and decreased in high concentration (50–100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.     

Extracts of black and brown rice powders improve hepatic lipid accumulation via the activation of PPARα in obese and diabetic model mice

Rice powder extract (RPE) from black and brown rice (Oryza sativa L. indica) improves hepatic lipid accumulation in obese and diabetic model mice via peroxisomal fatty acid oxidation. RPE showed PPARα agonistic activity which did not differ between black and brown RPE despite a higher anthocyanin content in black RPE.     

Obesity reduces bone density associated with activation of PPARγ and suppression of Wnt/β-catenin in rapidly growing male rats

Background

It is well established that excessive consumption of a high fat diet (HFD) results in obesity; however, the consequences of obesity on postnatal skeletal development have not been well studied.

Methodology and Principal Findings

Total enteral nutrition (TEN) was used to feed postnatal day 27 male rats intragastrically with a high 45% fat diet (HFD) for four weeks to induce obesity. Fat mass was increased compared to rats fed TEN diets containing 25% fat (medium fat diet, MFD) or a chow diet (low fat diet, LFD) fed ad libitum with matched body weight gains. Serum leptin and total non-esterified fatty acids (NEFA) were elevated in HFD rats, which also had reduced bone mass compared to LFD-fed animals. This was accompanied by decreases in bone formation, but increases in the bone resorption. Bone marrow adiposity and expression of adipogenic genes, PPARγ and aP2 were increased, whereas osteoblastogenic markers osteocalcin and Runx2 were decreased, in bone in HFD rats compared to LFD controls. The diversion of stromal cell differentiation in response to HFD stemmed from down-regulation of the key canonical Wnt signaling molecule β-catenin protein and reciprocal up-regulation of nuclear PPARγ expression in bone. In a set of in vitro studies using pluripotent ST2 bone marrow mesenchymal stromal cells treated with serum from rats on the different diets or using the free fatty acid composition of NEFA quantified in rat serum from HFD-fed animals by GC-MS, we were able to recapitulate our in vivo findings.

Conclusions/Significance

These observations strongly suggest that increased NEFA in serum from rats made obese by HFD-feeding impaired bone formation due to stimulation of bone marrow adipogenesis. These effects of obesity on bone in early life may result in impaired attainment of peak bone mass and therefore increase the prevalence of osteoporosis later on in life.     

Over-expression of PDGFR-β promotes PDGF-induced proliferation, migration, and angiogenesis of EPCs through PI3K/Akt signaling pathway

The proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) play critical roles in postnatal neovascularization and re-endothelialization following vascular injury. Here we evaluated whether the over-expression of platelet-derived growth factor receptor-β (PDGFR-β) can enhance the PDGF-BB-stimulated biological functions of EPCs through the PDGFR-β/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We first confirmed the expression of endogenous PDGFR-β and its plasma membrane localization in spleen-derived EPCs. We then demonstrated that the PDGFR-β over-expression in EPCs enhanced the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. Using AG1295 (a PDGFR kinase inhibitor), LY294002 (a PI3K inhibitor), and sc-221226 (an Akt inhibitor), we further showed that the PI3K/Akt signaling pathway participates in the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. In addition, the PI3K/Akt signaling pathway is required for PDGFR-β over-expression to enhance these PDGF-BB-induced phenotypes.     

Lung injury after ischemia-reperfusion of small intestine in rats involves apoptosis of type II alveolar epithelial cells mediated by TNF-α and activation of Bid pathway

Although ischemia-reperfusion (I/R) of small intestine is known to induce lung cell apoptosis, there is little information on intracellular and extracellular molecular mechanisms. Here, we investigated the mechanisms of apoptosis including the expression of Fas, Fas ligand (FasL), Bid, Bax, Bcl-2, cytochrome c, and activated caspase-3 in the rat lung at various time-points (0–24 h) of reperfusion after 1-h ischemia of small intestine. As assessed by TUNEL, the number of apoptotic epithelial cells, which were subsequently identified as type II alveolar epithelial cells by electron microscopy and immunohistochemical double-staining, increased at 3 h of reperfusion in the lung. However, intravenous injections of anti-TNF-α antibody decreased the number of TUNEL-positive cells, indicating involvement of tumor necrosis factor-α (TNF-α) in the induction of lung cell apoptosis. Western blotting and/or immunohistochemistry revealed a marked up-regulation of Fas, FasL, Bid, Bax, cytochrome c and activated caspase-3 and down-regulation of Bcl-2 in lung epithelial and stromal cells at 3 h of reperfusion. Our results indicate that I/R of small intestine results in apoptosis of rat alveolar type II cells through a series of events including systemic TNF-α, activation of two apoptotic signaling pathways and mitochondrial translocation of Bid.