Naringin Protects against Non-Alcoholic Fatty Liver Disease by Promoting Autophagic Flux and Lipophagy.

The autophagic degradation of lipid droplets, termed lipophagy, is the main mechanism contributing to lipid consumption in hepatocytes. Identifying effective and safe natural compounds that target lipophagy to eliminate excess lipids may be a potential therapeutic strategy for non-alcoholic fatty liver disease (NAFLD). Here the effects of naringin on NAFLD and the underlying mechanisms involved are investigated. Naringin treatment effectively relieves HFD-induced hepatic steatosis in mice and inhibits PA-induced lipid accumulation in hepatocytes. Increased p62 and LC3-II levels are observed with excess lipid support autophagosome accumulation and impaired autophagic flux. Treatment with naringin restores TFEB-mediated lysosomal biogenesis, thereby promoting the fusion of autophagosomes and lysosomes, restoring impaired autophagic flux and further inducing lipophagy. However, the knockdown of TFEB in hepatocytes or the hepatocyte-specific knockout of TFEB in mice abrogates naringin-induced lipophagy, eliminating its therapeutic effect on hepatic steatosis. These results demonstrate that TFEB-mediated lysosomal biogenesis and subsequent lipophagy play essential roles in the ability of naringin to mitigate hepatic steatosis and suggest that naringin is a promising drug for treating NAFLD.

Ajugol enhances TFEB-mediated lysosome biogenesis and lipophagy to alleviate non-alcoholic fatty liver disease.

Lipophagy is the autophagic degradation of lipid droplets. Dysregulated lipophagy has been implicated in the development of non-alcoholic fatty liver disease (NAFLD). Ajugol is an active alkaloid isolated from the root of Rehmannia glutinosa which is commonly used to treat various inflammatory and metabolic diseases. This study aimed to investigate the effect of ajugol on alleviating hepatic steatosis and sought to determine whether its potential mechanism via the key lysosome-mediated process of lipophagy. Our findings showed that ajugol significantly improved high-fat diet-induced hepatic steatosis in mice and inhibited palmitate-induced lipid accumulation in hepatocytes. Further analysis found that hepatic steatosis promoted the expression of LC3-II, an autophagosome marker, but led to autophagic flux blockade due to a lack of lysosomes. Ajugol also enhanced lysosomal biogenesis and promoted the fusion of autophagosome and lysosome to improve impaired autophagic flux and hepatosteatosis. Mechanistically, ajugol inactivated mammalian target of rapamycin and induced nuclear translocation of the transcription factor EB (TFEB), an essential regulator of lysosomal biogenesis. siRNA-mediated knockdown of TFEB significantly abrogated ajugol-induced lysosomal biogenesis as well as autophagosome-lysosome fusion and lipophagy. We conclude that lysosomal deficit is a critical mediator of hepatic steatosis, and ajugol may alleviate NAFLD via promoting the TFEB-mediated autophagy-lysosomal pathway and lipophagy.

Interleukin-10 Protects Schwann Cells against Advanced Glycation End Products-Induced Apoptosis via NF-κB Suppression.

Demyelination resulting from Schwann cell injury is a main pathological feature of diabetic neuropathy, and a key contributor to this process may be inflammation due to advanced glycation end products (AGEs). Therefore, protection by anti-inflammation agents is anticipated. In this study, we showed that interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits apoptosis of Schwann cells induced by AGEs in vitro. We isolated and cultured Schwann cells from rat sciatic nerves. As detected by flow cytometry, apoptosis of Schwann cells markedly increased following incubation with AGEs for 48 h. However, pretreatment with IL-10 inhibited AGE-induced apoptosis. The effect of IL-10 on NF-κB, which is a very important regulator of inflammation, was also evaluated, and results showed high levels of phospho-NF-κB and nuclear localization of NF-κB in cells incubated with AGEs but low levels of phospho-NF-κB and cytoplasmic localization in the cells incubated with IL-10, indicating the activation of NF-κB by AGEs and inhibition of NF-κB by IL-10. Moreover, incubating Schwann cells with an NF-κB inhibitor (caffeic acid phenethyl ester) for 30 min before adding AGEs mimicked IL-10, lowering the amount of reactive oxygen species and activity of caspase-3 and also decreasing apoptosis in Schwann cells. These results indicate that IL-10 may protect Schwann cells against AGE-induced apoptosis by attenuating oxidative stress via the inhibition of activation of NF-κB.

Therapeutic Effect of Glycyrrhizin Arginine Salt on Rat Cholestatic Cirrhosis and its Mechanism.

To investigate the therapeutic effect of glycyrrhizin arginine salt on rat cholestatic cirrhosis, we subjected male Sprague Dawley rats to common bile duct ligation for 14 days and treated them with distilled water (model group), arginine, or a low or high dose of glycyrrhizin arginine salt by gavage. A sham-operated group was used as a control group. Treatment with glycyrrhizin arginine salt substantially improved animal growth rates, reduced the ratio of liver weight to body weight and decreased total bilirubin, aspartate aminotransferase, 8-isoprostane and malondialdehyde compared with the values measured in the model group. The progress of liver fibrosis, as detected by hematoxylin and eosin and Masson's trichrome staining, was slower in the glycyrrhizin arginine salt groups than in the model group or the arginine group. Reductions of bile salt pool size, hepatic hydroxyproline content and fibrosis score were also seen in the glycyrrhizin arginine salt groups compared with the model group. Furthermore, glycyrrhizin arginine salt significantly reduced the expression of transforming growth factor [Formula: see text]1 (TGF-[Formula: see text]1), [Formula: see text]-smooth muscle actin, tumor necrosis factor-[Formula: see text] and matrix metalloproteinases 2 and 9. Glycyrrhizin arginine salt also inhibited the expression of [Formula: see text]-SMA and matrix metalloproteinases 2 and 9 in response to TGF-[Formula: see text]1 in LX-2 cells and primary rat hepatic stellate cells and mitigated the cytotoxicity induced by rat bile in HepG2 cells and primary rat hepatocytes.

Inhibition of Miro1 disturbs mitophagy and pancreatic ß-cell function interfering insulin release via IRS-Akt-Foxo1 in diabetes.

Mitochondrial function is essential to meet metabolic demand of pancreatic beta cells respond to high nutrient stress. Mitophagy is an essential component to normal pancreatic ß-cell function and has been associated with ß-cell failure in Type 2 diabetes (T2D). Our previous studies have indicated that mitochondrial Rho (Miro) GTPase-mediated mitochondrial dysfunction under high nutrient stress leads to NOD-like receptor 3 (NLRP3)-dependent proinflammatory responses and subsequent insulin resistance. However, the in vivo mechanism by which Miro1 underlies mitophagy has not been identified. Here we show firstly that the expression of Miro is reduced in human T2D and mouse db/db islets and in INS-1 cell line exposed to high glucose and palmitate. ß-cell specific ablation of Miro1 (Miro1f/f: Rip-cre mice, or (IKO) under high nutrient stress promotes the development of hyperglycemia. ß-cells from IKO mice display an inhibition of mitophagy under oxidative stress and induces mitochondrial dysfunction. Dysfunctional mitophagy in IKO mice is represented by damaged islet beta cell mitochondrial and secretory capacity, unbalanced downstream MKK-JNK signalling without affecting the levels of MEK, ERK or p38 activation and subsequently, impaired insulin secretion signaling via inhibition IRS-AKT-Foxo1 pathway, leading to worsening glucose tolerance in these mice. Thus, these data suggest that Miro1 may be responsible for mitophagy deficiency and ß-cell dysfunction in T2D and that strategies target Miro1 in vivo may provide a therapeutic target to enhance ß-cell mitochondrial quality and insulin secretion to ameliorate complications associated with T2D.

TRB3 mediates advanced glycation end product-induced apoptosis of pancreatic ß-cells through the protein kinase C ß pathway.

Advanced glycation end products (AGEs), which accumulate in the body during the development of diabetes, may be one of the factors leading to pancreatic ß-cell failure and reduced ß-cell mass. However, the mechanisms responsible for AGE­induced apoptosis remain unclear. This study identified the role and mechanisms of action of tribbles homolog 3 (TRB3) in AGE-induced ß-cell oxidative damage and apoptosis. Rat insulinoma cells (INS-1) were treated with 200 µg/ml AGEs for 48 h, and cell apoptosis was then detected by TUNEL staining and flow cytometry. The level of intracellular reactive oxygen species (ROS) was measured by a fluorescence assay. The expression levels of receptor of AGEs (RAGE), TRB3, protein kinase C ß2 (PKCß2) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) were evaluated by RT-qPCR and western blot analysis. siRNA was used to knockdown TRB3 expression through lipofection, followed by an analysis of the effects of TRB3 on PKCß2 and NOX4. Furthermore, the PKCß2-specific inhibitor, LY333531, was used to analyze the effects of PKCß2 on ROS levels and apoptosis. We found that AGEs induced the apoptosis of INS-1 cells and upregulated RAGE and TRB3 expression. AGEs also increased ROS levels in ß-cells. Following the knockdown of TRB3, the AGE-induced apoptosis and intracellular ROS levels were significantly decreased, suggesting that TRB3 mediated AGE-induced apoptosis. Further experiments demonstrated that the knockdown of TRB3 decreased the PKCß2 and NOX4 expression levels. When TRB3 was knocked down, the cells expressed decreased levels of PKCß2 and NOX4. The PKCß2­specific inhibitor, LY333531, also reduced AGE-induced apoptosis and intracellular ROS levels. Taken together, our data suggest that TRB3 mediates AGE-induced oxidative injury in ß-cells through the PKCß2 pathway.

GSK-3ß mediates dexamethasone-induced pancreatic ß cell apoptosis.

AIMS: Glucocorticoids, such as dexamethasone, are widely used anti-inflammatory drugs. Their use is frequently associated with the development of steroid- associated diabetes. Pancreatic ß-cell dysfunction has been suggested to be one of the main causes of steroid-associated diabetes. However, the mechanism is not fully understood. Glycogen synthase kinase-3ß (GSK-3ß) is a multifunctional serine/threonine kinase and plays an important role in energy metabolism, cell growth and apoptosis. Therefore, the contribution of GSK-3ß in dexamethasone-induced pancreatic ß-cell apoptosis was determined in the present study. MAIN METHODS: The effect of dexamethasone treatment on rat pancreatic ß-cell line (INS-1) apoptosis (determined by TUNEL and Flow Cytometry), generation of reactive oxidative stress (ROS), and the phosphorylation status of GSK-3ß was determined. The inhibitory effect of GSK-3ß inhibitor-lithium chloride (LiCl) on dexamethasone-induced ß-cell apoptosis was also evaluated. KEY FINDINGS: Dexamethasone (0.1 µM) treatment induced INS-1 apoptosis, which was associated with increased GSK-3ß activation and increased NOX4-derived ROS generation. Pretreatment of INS-1 with LiCl inhibited dexamethasone induced ROS generation and INS-1 apoptosis. SIGNIFICANCE: This study provides a new mechanism of Dex induced pancreatic ß cell apoptosis and may serve as a new therapeutic option for treating GC induced diabetes.

TRB3 is involved in free fatty acid-induced INS-1-derived cell apoptosis via the protein kinase C δ pathway.

Chronic exposure to free fatty acids (FFAs) may induce ß cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger ß cell apoptosis is still unclear. Tribbles homolog 3 (TRB3) is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 ß cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC) δ was enhanced by upregulation of TRB3. Preventing PKCδ nuclear translocation and PKCδ selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 ß cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.

Evaluation of islets derived from human fetal pancreatic progenitor cells in diabetes treatment.

INTRODUCTION: With the shortage of donor organs for islet transplantation, insulin-producing cells have been generated from different types of stem cell. Human fetal pancreatic stem cells have a better self-renewal capacity than adult stem cells and can readily differentiate into pancreatic endocrine cells, making them a potential source for islets in diabetes treatment. In the present study, the functions of pancreatic islets derived from human fetal pancreatic progenitor cells were evaluated in vitro and in vivo. METHODS: Human pancreatic progenitor cells isolated from the fetal pancreas were expanded and differentiated into islet endocrine cells in culture. Markers for endocrine and exocrine functions as well as those for alpha and beta cells were analyzed by immunofluorescent staining and enzyme-linked immunosorbent assay (ELISA). To evaluate the functions of these islets in vivo, the islet-like structures were transplanted into renal capsules of diabetic nude mice. Immunohistochemical staining for human C-peptide and human mitochondrion antigen was applied to confirm the human origin and the survival of grafted islets. RESULTS: Human fetal pancreatic progenitor cells were able to expand in medium containing basic fibroblast growth factor (bFGF) and leukemia inhibitor factor (LIF), and to differentiate into pancreatic endocrine cells with high efficiency upon the actions of glucagon-like peptide-1 and activin-A. The differentiated cells expressed insulin, glucagon, glucose transporter-1 (GLUT1), GLUT2 and voltage-dependent calcium channel (VDCC), and were able to aggregate into islet-like structures containing alpha and beta cells upon suspension. These structures expressed and released a higher level of insulin than adhesion cultured cells, and helped to maintain normoglycemia in diabetic nude mice after transplantation. CONCLUSIONS: Human fetal pancreatic progenitor cells have good capacity for generating insulin producing cells and provide a promising potential source for diabetes treatment.

[The research applications of db/db mouse].

The db/db mice are perfect animal models of type 2 diabetes which have been widely used. The phenotypes of severe obesity, hyperphagia, polydipsia, and polyuria are due to a spontaneous mutation of the leptin receptor (Lepr). The course of the disease is markedly influenced by genetic background, which is more serious in the C57BLKS/J background. And there are many other spontaneous mutations in different sites of Lepr, which produce a series of animal models of obesity, including db(3J)/ db(3J) mice, db(5j)/db(5J) mice, db(pas)/db(pas) mice, Zucker fa/fa rats, and Koletsky fa(k)/fa(k) rats, etc. These rodents appear similar hyperphagia and severe obesity, but different levels of blood glucose, kidney damage and reproductive ability, providing profuse material to investigate the complex function of Lepr. Here we review the history of the discovery of the leptin signaling pathway, the abnormal phenotypes of db/db mice in metabolic, reproductive, immune, etc. Discuss their research applications, reproductive strategy, genotyping guideline, the phenotypic diversity of those animal models with Lepr spontaneous mutation and their mutation patterns, respectively.

[Significance of apolipoprotein A1 as biomarker for early diagnosis and classification of bladder urothelial carcinoma].

OBJECTIVE: To investigate the significance of apolipoprotein (Apo)-A1 in urine as a biomarker for early diagnosis and classification of bladder urothelial carcinoma (BUC). METHODS: Urine samples were divided into four groups: normal control group, benign bladder disease group, low-grade malignant BUC group, and high-grade malignant BUC group. Apo-A1, which showed significantly different expression among the four groups, was selected according to the two-dimensional electrophoresis (2-DE) images of the four groups, and enzyme-linked immunosorbent assay (ELISA) was used to quantify Apo-A1 in the four groups. A receiver operating characteristic (ROC) curve was generated, and the optimal operating points on the ROC curve were found to determine the critical concentrations of Apo-A1 for early diagnosis of BUC and differentiation of low-grade and high-grade malignant BUC. The results were verified clinically, and the specificity and sensitivity were calculated. RESULTS: The 2-DE images showed that that the level of Apo-A1 increased from the normal control grouP to high-grade malignant BUC group. The ELISA showed that there was no significant difference in Apo-A1 level between the normal control grouP and benign bladder disease group, but the Apo-A1 level was significantly higher in the BUC groups than in the normal control grouP and benign bladder disease grouP (P < 0.01); the high-grade BUC grouP had a significantly higher Apo-A1 level than the low-grade BUC grouP (P < 0.01). The BUC patients and those without BUC could be differentiated with an Apo-A1 concentration of 18.22 ng/ml, while the low-grade and high-grade malignant BUC could be differentiated with an Apo-A1 concentration of 29.86 ng/ml. When used as a biomarker, Apo-A1 had a sensitivity of 91.6% (98/107) and a specificity of 85.7% (42/49) for diagnosis of BUC and had a sensitivity of 83.7% (41/49) and a specificity of 89.7% (52/58) for BUC classification. CONCLUSION: Apo-A1 may be a biomarker for early diagnosis and classification of BUC and shows promise for clinical application.

[Three-dimensional mini-type permanent magnetic resonance imaging device and medical imaging of mice].

We developed a three-dimensional mini-type permanent magnetic resonance imaging (MRI) device in our lab. The purposes of this study were (1) for further development of MRI technologies, (2) for support of broadening practices of animal test modeling in medical research, and (3) for training more specialists from colleges or universities in the field of MRI. This paper describes the research and development at our lab(s), especially stressing on the design of the main magnet, the gradient coil and the radio frequency coil. In addition, the specific methodologies used in our lab(s) and the related data are emphasized. The 3D MRI technologies have met the needs of using small animals, super thin sections of live animal body and high imaging resolutions. MRI images of mice head and abdominal have been obtained successfully by using the imager that we developed. The imaging results and analyses have also been discussed.

Involvement of the Ca2+-responsive transactivator in high glucose-induced ß-cell apoptosis.

The calcium-regulated transcription coactivator, Ca(2)(+)-responsive transactivator (CREST) was expressed in pancreatic ß-cells. Moreover, CREST expression became significantly increased in pancreatic islets isolated from hyperglycemic Goto-Kakizaki rats compared with normoglycemic Wistar controls. In addition, culture of ß-cells in the presence of high glucose concentrations also increased CREST expression in vitro. To further investigate the role of this transactivator in the regulation of ß-cell function, we established a stable ß-cell line with inducible CREST expression. Hence, CREST overexpression mimicked the glucotoxic effects on insulin secretion and cell growth in ß-cells. Moreover, high glucose-induced apoptosis was aggravated by upregulation of the transactivator but inhibited when CREST expression was partially silenced by siRNA technology. Further investigation found that upregulation of Bax and downregulation of Bcl2 was indeed induced by its expression, especially under high glucose conditions. In addition, as two causing factors leading to ß-cell apoptosis under diabetic conditions, endoplasmic reticulum stress and high free fatty acid, mimicked the high glucose effects on CREST upregulation and generation of apoptosis in ß-cells, and these effects were specifically offset by the siRNA knockdown of CREST. These results indicated that CREST is implicated in ß-cell apoptosis induced by culture in high glucose and hence that CREST may become a potential pharmacological target for the prevention and treatment of type 2 diabetes mellitus.

Involvement of dynamin-related protein 1 in free fatty acid-induced INS-1-derived cell apoptosis.

Elevated extracellular free fatty acids (FFAs) can induce pancreatic beta cell apoptosis, thereby contributing to the pathogenesis of type 2 diabetes mellitus (T2D). Mitochondrial dysfunction has been implicated in FFA-induced beta cell apoptosis. However, molecular mechanisms linking mitochondrial dysfunction and FFA-induced beta cell apoptosis are not clear. Dynamin-related protein 1 (DRP-1) is a mitochondrial fission modulator. In this study, we investigated its role in FFA-induced INS-1 beta cell apoptosis. DRP-1 protein was promptly induced in INS-1 cells and rat islets after stimulation by FFAs, and this DRP-1 upregulation was accompanied by increased INS-1 cell apoptosis. Induction of DRP-1 expression significantly promoted FFA-induced apoptosis in DRP-1 WT (DRP-1 wild type) inducible INS-1-derived cell line, but not in DRP-1K38A (a dominant negative mutant of DRP-1) inducible INS-1-derived cell line. To validate these in vitro results, we transplanted DRP-1 WT or DRP-1 K38A cells into renal capsules of streptozotocin (STZ)-treated diabetic mice to study the apoptosis in xenografts. Consistent with the in vitro results, the over-expression of DRP-1 led to aggravated INS-1-derived cell apoptosis triggered by FFAs. In contrast, dominant-negative suppression of DRP-1 function as represented by DRP-1 K38A significantly prevented FFA-induced apoptosis in xenografts. It was further demonstrated that mitochondrial membrane potential decreased, while cytochrome c release, caspase-3 activation, and generation of reactive oxygen species (ROS) were enhanced by the induction of DRP-1WT, but prevented by DRP-1 K38A in INS-1-derived cells under FFA stimulation. These results indicated that DRP-1 mediates FFA-induced INS-1-derived cell apoptosis, suggesting that suppression of DRP-1 is a potentially useful therapeutic strategy for protecting against beta cell loss that leads to type 2 diabetes.

[Preventive effects of Salvia miltiorrhiza on multiple organ edema in the rats of limb ischemia/reperfusion].

OBJECTIVE: To investigate the preventive effects of Salvia miltiorrhiza (SM) on multiple organ edema in the rats which suffered from hind limb ischemia/reperfusion( LI/R). METHODS: Twenty four Wistar rats were randomly divided into 3 groups (n = 8): control group (C group), ischemia/reperfusion group (I/R group ), Salvia miltiorrhiza group (SM group). Referring to Tourniquet method, the model rats which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made. Thirty minutes before reperfusion, SM was given to the rats in SM group by tail vein injection at the dose of 5 mL/kg. Accurately weighed one gram of heart, liver, kidney, lung, brain, intestine and skeletal muscle from every animals, weigh these specimens after baking (60 degrees C, 55 hours), calculated the ratio of wet and dry (Wet/Dry,W/D). The levels of interleukin-1 (IL-1) ,interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in plasma and the contents of Superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The morphologic changes of skeletal muscle were observed with microscope. RESULTS: It was found that after suffering from ischemia/reperfusion, the W/D of every specimens increased in different degree (P < 0.05, P < 0.01). In plasma, the values of SOD decreased but MDA increased obviously (P < 0.05, P < 0.01). The levels of IL-1, IL-6 ,TNF-alpha-a in plasma increased (P < 0.05, P <0.01). After LI/R, infiltration of inflammatory cells, broaden interstitial around muscle fiber and disordered arrangement of muscle fibers could be seen under microscope. However, Compared with LI/R group, W/D and levels of serum inflammatory factors in SM group were all lower, the values of SOD in plasma increased but MDA in plasma failed down. Pathological changes in skeletal muscle were improved. CONCLUSION: Limb ischemia/reperfusion can lead to multiple organ edema, Salvia miltiorrhiza can prevent the edema in some degree by anti-oxidation and anti-inflammation.