The aim of this study was to investigate the protective effects of Mangiferin (MG) on glucolipotoxicity-induced pancreatic beta-cell injury. In vivo administration of MG significantly reduced the level of blood glucose in high-fat diet (HFD)-fed mice. MG treatment inhibited beta-cell apoptosis in HFD-treated mice. In vitro, MG protected INS-1 cells against apoptosis and impairment of insulin secretion following High glucose/Palmitic acid (HG/PA) treatment. MG treatment enhanced autophagy flux which was blocked by HG/PA treatment. Inhibition of autophagosome formation by 3-Methyladenine or blockade of autolysosome by Chloroquine reversed the protective effects of MG on INS-1 cells. MG treatment increased AMPK phosphorylation and reduced mTOR activation in INS-1 cells. Administration of the AMPK blocker abrogated MG-induced autophagy, and similar results were observed in INS-1 cells after cotreatment with MG and mTOR activator. In conclusion, MG ameliorated pancreatic beta-cell injury induced by glucolipotoxicity through modulation of autophagy via the AMPK-mTOR pathway.

Background: Adipose fibrosis is a major factor of adipose dysfunction, which causes metabolic dysfunction during obesity, but its molecular mechanisms are poorly understood. This study investigated the role and potential mechanisms of mTORC1 in obesity-induced adipose fibrosis. Methods: ob/ob mice were injected with rapamycin or the same volume of normal saline. The level of fibrosis in epididymal adipose tissue (EAT) was detected by observing aberrant deposition of extracellular matrix. Expression of fibrotic related genes was analysed using RNA-seq. 3T3-L1 preadipocytes were treated with cobalt chloride (CoCl2) and TGF-β1 to induce preadipocyte fibrosis. The fibrosis-related gene expression and protein levels were determined by RT-PCR, WB, and immunofluorescence in two types of fibrotic preadipocytes with or without rapamycin. Results: Compared with vehicle treatment, EAT fibrosis-related aberrant deposition of extracellular matrix proteins and fibrotic gene expression were reduced in ob/ob mice treated with rapamycin. Both CoCl2-induced hypoxia and TGF-β1 successfully promoted adipocyte fibrosis, and the upregulated fibrosis-related genes expression was inhibited after the mTORC1 pathway was inhibited by rapamycin. Conclusion: Inhibition of the mTORC1 pathway ameliorates adipose fibrosis by suppressing fibrosis-related genes in hypoxia- and TGF-β-induced fibrotic preadipocytes.

目的 探讨叶氏益肾降糖方对早期脾肾阴虚夹瘀型糖尿病肾脏病患者肾功能及氧化应激的影响.方法 选取本院2021年5月～2022年4月就诊的107例早期脾肾阴虚夹瘀型糖尿病肾脏病患者,随机分为两组,对照组52例,中药组55例.对照组采用常规西医治疗,中药组采用常规西医联合叶氏益肾降糖方治疗,治疗24周.治疗后对两组的尿白蛋白/肌酐比值(urinary albumin-to-creatinine ratio,UACR)、肾小球滤过率估算(estimated glomeruar filtration rate,eGFR)、治疗有效率、超氧化物歧化酶(su-peroxide dismutase,SOD)水平进行比较.结果 经治疗后,中药组的治疗总有效率高于对照组(P＜0.05);中药组的UACR低于对照组(P＜0.05);中药组eGFR、SOD水平高于对照组(P＜0.05),差异具有统计学意义.结论 叶氏益肾降糖方能够减少早期脾肾阴虚夹瘀型糖尿病肾脏病的尿微量白蛋白,升高SOD水平,保护肾功能,其作用机制可能是通过改善氧化应激反应而发挥作用.

糖尿病周围神经病变(DPN)是糖尿病的主要慢性并发症之一,具有患病率高、致残率高的特点.中医外治法可改善DPN患者症状,提高神经传导速度.通过分析近年来中医外治法治疗DPN的文献,从中医外治法理论基础、针刺疗法、艾灸疗法、温针灸疗法、足浴疗法等阐述中医外治法治疗DPN的进展,为中医外治法治疗DPN的临床应用及深入研究提供依据.

Lipotoxicity can lead to beta-cell dysfunction and apoptosis because it induces oxidative stress. Recent studies have found that Irisin prevents pancreatic beta-cell dysfunction induced by palmitic acid (PA). However, an association between the protection against oxidative stress conferred by Irisin and beta-cell dysfunction has not been fully elucidated. In this study, we observed that Irisin treatment prevented INS-1 cell apoptosis induced by PA treatment and preserved the insulin-secreting function of INS-1 cells in vitro. These effects probably resulted from the Irisin-induced decrease in intracellular ROS levels triggered by PA treatment. In addition, PA treatment induced oxidative stress partially by inhibiting the activation of thioredoxin 2 (Trx2) through its increase of thioredoxin-interacting protein (Txnip) expression. However, Irisin administration blocked the increase in Txnip expression, which reversed the PA-induced inactivation of Trx2. Irisin also increased the nuclear translocation of Stat3, and the inhibition of Stat3 by siRNAs blocked Irisin-induced Trx2 expression, indicating that both Txnip and Stat3 are involved in Irisin-induced activation of Trx2. Furthermore, blockade of Stat3 by siRNAs led to the decreased gene expression of MafA and Ins and to cessation of glucose-induced insulin secretion that had been enhanced by Irisin. In vivo, HFD treatment led to reduced glucose tolerance and an increase in the level of the oxidative marker malondialdehyde (MDA) compared to that in the control group. However, these effects were ameliorated by Irisin injection due to the inhibition of beta-cell apoptosis and the activation of Trx2, probably through Txnip inhibition and Stat3 activation. In conclusion, our results reveal a possible mechanism for Irisin-induced beta-cell protection, which is mediated through Txnip inhibition and activation of the Stat3-Trx2 pathway.

Objective:To investigate effect and underlying lipid-lowering mechanisms of catalpol in non-alcoholic fatty liver disease(NAFLD).Methods:In vivo model of NAFLD was established with high-fat diet-fed ICR mice for 8 weeks. Low(50 mg/kg), medium(150 mg/kg), and high(300 mg/kg) doses of catalpol were administered, and the body weight, liver weight, hepatic index, and biochemical parameters of the mice were analyzed. Free fatty acid-induced LO2 in human hepatocytes to establish NAFLD cell model. Quantitative realtime PCR reaction to detect fatty acid synthesis-related gene levels. Western blotting assay was adopted to analyze proteins in the endoplasmic reticulum stress(ERS)-mediated protein kinase RNA-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α) signaling pathway. Results:Compared with model mice, body weight [(39.43±1.84)g, (34.01±1.83)g, (32.28±1.11)g vs(42.17±1.37)g, all P<0.001], liver weight [(1.03±0.06)g, (0.79±0.05)g, (0.64±0.04)g vs(1.30±0.13)g, P<0.01 or P<0.001], and liver index [(2.60±0.09)%, (2.32±0.09)%, (1.99±0.11)% vs(3.07±0.30)%, P<0.05 or P<0.001] were reduced in low, medium, and high doses of catapol model. Medium and high doses of catalpol diminished total cholesterol, triglyceride, low density lipoprotein-cholesterol, aspartate aminotransferase, and alanine aminotransferase( P<0.01 or P<0.001), increased high density lipoprotein-cholesterol( P<0.01 or P<0.001). In the cell model, elevated levels of both fatty acid synthesis genes and PERK-eIF2α pathway proteins were attenuated by catalase, and this attenuation was reversed by signaling pathway agonists. Conclusion:The Chinese herb catalpol may play a role in improving NALFD by regulating the ERS-mediated PERK-eIF2α signaling pathway.

PURPOSE:To investigate the role of thioredoxin 2 (Trx2) inhibition induced by intracellular methylglyoxal (MGO) in pancreatic beta-cell mitochondrial dysfunction and apoptosis.METHODS:Rat pancreatic beta-cell line INS-1 cells were treated with Glo1 siRNAs or exogenous MGO to increase intracellular MGO. AGEs formation was detected by ELISA and mitochondrial ROS was detected by probe MitoSOX. Transmission electron microscopy (TEM) analysis and ATP content were measured to evaluate mitochondrial function. Trx2 expression was manipulated by overexpression with recombinant Trx2 lentivirus or knockdown with Trx2 siRNAs, and effects on apoptosis and insulin secretion were measured by flow cytometry and ELISA, respectively.RESULTS:The increase of intracellular MGO by Glo1 blockage or MGO treatment led to advanced glycation end products (AGEs) overproduction, mitochondrial ROS increase, and insulin secretion paralysis. These were probably due to MGO-induced inhibition of mitochondrial Trx2. Trx2 inhibition by blockage of either Glo1 or Trx2 impaired mitochondrial integrity, inhibited cytochrome C oxidases subunit 1 and 4 (Cox1 and Cox4) expression and further reduced ATP generation, and all of these might lead to insulin paralysis; whereas Trx2 overexpression partially reversed MGO-induced oxidative stress, attenuated insulin secretion by preventing mitochondrial damage. Trx2 overexpression also retarded MGO-induced apoptosis of INS-1 cell through inhibiting ASK1 activation and downregulation of the ASK1-p38 MAPK pathway.CONCLUSIONS:Our results reveal a possible mechanism for beta-cell oxidative damage upon intracellular MGO-induced Trx2 inactivation and mitochondrial dysfunction and apoptosis.

Non-alcoholic fatty liver disease (NAFLD) has become the leading cause of liver disease in children, with evidence that the maternal diet and the early life nutritional environment are potential risk for such disease. This study was aimed to investigate the effects of maternal high-fat diet (HFD) on the occurrence of NAFLD in offspring rats and the underlying mechanisms. In this study, the incidence of NAFLD was compared in F1 offspring rats between the maternal HFD group and standard chow (SC) group. In addition, the expression levels of inflammatory cytokines in the placenta, in the umbilical cord blood, and in the livers of neonate offsprings were compared between two groups. HepG2 cells were treated with recombinant IL6 (rIL6) to assess stearoyl-CoA desaturase 1 (SCD1) expression and lipid synthesis in an inflammatory condition. Lipid accumulation was assayed in both SCD1 overexpression and interference HepG2 cells as well as in neonatal rats. Our results showed that HFD exposure before and throughout the pregnancy induced the elevated hepatic TG content of F1 neonates. The levels of inflammatory cytokines in the placenta, umbilical cord blood, and the livers of HFD F1 neonates were significantly higher than those of the SC group. In addition, rIL6 treatment led to TG accumulation accompanied by the upregulation of SCD1 in HepG2 cell lines. Overexpression of SCD1 led to the accumulation of TG contents in HepG2 cells, whereas Scd1 knockdown attenuated the effects of rIL6 treatment. Overexpression of SCD1 in F1 neonatal rats led to hepatic lipid accumulation. Our study indicated that maternal HFD led to intrauterine inflammation, which subsequently caused transgenerationally abnormal hepatic lipid metabolism of F1 neonates. This modulation might be mediated by upregulating SCD1 expression in hepatic cells.

Type 2 diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose and/or high serum free fatty acids. Chronic hyperlipidemia causes the dysfunction of pancreatic beta cells, which is aggravated in the presence of hyperglycemia (glucolipotoxicity). Long noncoding RNAs (lncRNAs) have been suggested to play key roles in type 1 diabetes mellitus development. However, their roles in glucolipotoxicity-induced beta cell dysfunction are not fully understood. In the present study, we identified the differentially expressed lncRNAs in INS-1 cells exposed to high glucose and palmitate (HG/PA). Among the dysregulated lncRNAs, NONRATT003679.2 (low expression in glucolipotoxicity-treated beta cells (LEGLTBC)) was involved in glucolipotoxicity-evoked rat islet beta cell damage. LEGLTBC functioned as a molecular sponge of miR-34a in INS-1 cells. Additionally, SIRT1 was identified as a target of miR-34a and LEGLTBC promoted SIRT1 expression by sponging miR-34a. The upregulation of LEGLTBC attenuated HG/PA-induced INS-1 cell injury through the promotion of SIRT1-mediated suppression of ROS accumulation and apoptosis. This is the first study to comprehensively identify the lncRNA expression profiling of HG/PA-treated INS-1 beta cells and to demonstrate that LEGLTBC functions as a competing endogenous RNA and regulates miR-34a/SIRT1-mediated oxidative stress and apoptosis in INS-1 cells undergoing glucolipotoxicity.

目的 探讨IL-6对HepG2细胞硬脂酰辅酶A去饱和酶1(SCD1)基因的影响及其SCD1基因表达的改变对细胞脂质合成的影响.方法 应用rIL-6刺激HepG2细胞,检测细胞内脂质合成以及细胞SCD1基因水平变化.分别构建人SCD1真核表达质粒和小干扰(small interfering)RNA,转染HepG2细胞,观察改变SCD1水平后HepG2细胞脂代谢相关基因表达的变化以及细胞内脂质合成的变化.结果 rIL-6刺激HepG2细胞甘油三酯水平显著高于对照组(P<0.05);与空质粒组比,转染真核表达质粒细胞SCD1蛋白表达增加,细胞脂质合成相关基因SREBP1c和FASN相对水平增加1.35倍和1.27倍,脂质氧化代谢相关基因PPARα和ASCL3水平降低12.3％和13.5％;细胞甘油三酯水平显著升高(P<0.01),而转染small interfering RNA,再用rIL-6刺激HepG2细胞SCD1蛋白表达显著降低,细胞甘油三酯水平也显著降低(P<0.05),脂质合成相关基因SREBP1c和FASN水平显著下降了32.3％和51.9％,脂质分解相关基因PPARα相对水平也下降了80％(P<0.05),而ASCL3水平无显著变化(P=0.832).结论 rIL-6通过上调HepG2细胞SCD1基因表达,引起细胞内脂质合成增加,导致甘油三酯含量增多.

Objective To explore the role of the pyrin domain-containing 3 ( NLRP3) inflammasome in advanced glycation end products ( AGEs )-induced mice pancreatic β-cell damage. Methods AGEs were administered intraperitoneally for 6 weeks in NLRP3 knockout mice or C57BL/6J mice. Intraperitoneal glucose tolerance test and insulin releasing test were performed. Pancreatic sections were stained with haematoxylin and eosin, or with F4/80 and NLRP3 antibodies. Insulin and pancreatic tissue monocyte chemotactic protein 1 ( MCP-1) as well as interleukin-1β( IL-1β) levels were measured with ELISA kits. Expression of MCP-1 protein was determined by western blot. MIN6 cells and mouse peritoneal macrophages cells were treated with AGEs and different interventions (antioxidant NAC, adenovirus NLRP3 shRNA or NLRP3 knockout). Reactive oxygen species production, NLRP3 mRNA expression, IL-1β secretion, caspase 1 activity, apoptosis and glucose stimulated insulin release were determined. Results Injection of AGEs induced an abnormal response to glucose, enhanced the insulitis score, and increased the levels of pancreatic tissue MCP-1 and IL-1β, as well as raised the expression of NLRP3 and F4/80 in pancreatic islet. Remarkably, co-localization of NLRP3 and macrophage marker F4/80 was observed in islet. The damages were improved in NLRP3 knockout mice. After incubation with AGEs, reactive oxygen species production and cell apoptosis was enhanced, NLRP3 inflammasome activated, with glucose-stimulated insulin release impaired in MIN6 cells. NAC treatment alliviated the above damages, but NLRP3 gene silencing had no effect on ROS level, apoptosis, and insulin secretion. Finally NAC treatment and NLRP3 gene knockout inhibited activation of NLRP3 inflammasome induced by AGEs in mouse peritoneal macrophages cells. Conclusion NLRP3 knockout ameliorates the islet β-cell damage induced by AGEs. These effects were associated with AGEs-induced islets macrophage infiltrating by up-regulation of MCP-1 expression, and AGEs-induced activation of NLRP3 inflammasome in macrophage through ROS pathway, which results in the release of active IL-1βand leads to the lesions of β-cell.

We investigated whether maternal over-nutrition during pregnancy and lactation affects the offspring’s lipid metabolism at weaning by assessing liver lipid metabolic gene expressions and analysing its mechanisms on the development of metabolic abnormalities. Female Sprague–Dawley rats were fed with standard chow diet (CON) or high-fat diet (HFD) for 8 weeks, and then continued feeding during gestation and lactation. The offspring whose dams were fed with HFD had a lower birth weight but an increased body weight with impaired glucose tolerance, higher serum cholesterol, and hepatic steatosis at weaning. Microarray analyses showed that there were 120 genes differently expressed between the two groups. We further verified the results by qRT-PCR. Significant increase of the lipogenesis ( Me1 , Scd1 ) gene expression was found in HFD ( P <0.05), and up-regulated expression of genes ( PPAR-α , Cpt1α , Ehhadh ) involved in β-oxidation was also observed ( P <0.05), but the Acsl3 gene was down-regulated ( P <0.05). Maternal over-nutrition could not only primarily induce lipogenesis, but also promote lipolysis through an oxidation pathway as compensation, eventually leading to an increased body weight, impaired glucose tolerance, elevated serum cholesterol and hepatic steatosis at weaning. This finding may provide some evidence for a healthy maternal diet in order to reduce the risk of metabolic diseases in the early life of the offspring.

This study aimed to assess the impact of perinatal high-fat (HF) diet in female Sprague-Dawley rats (F0) on glucose metabolism and islet function in their early life of second-generation of offspring (F2).

An increased intracellular methylglyoxal (MGO) under hyperglycemia led to pancreatic beta cell death. However, its mechanism in which way with MGO induced beta cell death remains unknown. We investigated both high glucose and MGO treatment significantly inclined intracellular MGO concentration and inhibited cell viability in vitro. MGO treatment also triggered intracellular advanced glycation end products (AGEs) formation, declined mitochondrial membrane potential (MMP), increased oxidative stress and the expression of ER stress mediators Grp78/Bip and p-PERK; activated mitochondrial apoptotic pathway, which could mimic by Glo1 knockdown. Aminoguanidine (AG), a MGO scavenger, however, prevented AGEs formation and MGO-induced cell death by inhibiting oxidative stress and ER stress. Furthermore, both antioxidant N-acetylcysteine (NAC) and ER stress inhibitor 4-phenylbutyrate (4-PBA) could attenuate MGO-induced cell death through ameliorating ER stress. MGO treatment down-regulated Ire1α, a key ER stress mediator, increased JNK phosphorylation and activated mitochondrial apoptosis; down-regulated Bcl-2 expression which could be attenuated by the JNK inhibitor SP600125 and further inhibited cytochrome c leakage from mitochondria and blocked the conversion of pro caspase 3 into cleaved caspase 3, all these might contribute to the inhibition of INS-1 cell apoptosis. Ire1α down-regulation by Ire1α siRNAs mimicked MGO-induced cytotoxicity by activating the JNK phosphorylation and mitochondrial apoptotic pathway. In summary, we demonstrated that increased intracellular MGO induced cytotoxicity in INS-1 cells primarily by activating oxidative stress and further triggering mitochondrial apoptotic pathway, and ER stress-mediated Ire1α-JNK pathway. These findings may have implication on new mechanism of glucotoxicity-mediated pancreatic beta-cell dysfunction.

Objective To study the impact of maternal high-fat diet during pregnancy and lactation on hepatic steatosis in the early life of offspring rats and its possible mechanism. Methods Female Sprague-Dawley rats were fed either a high fat diet (HF) or control (C) diet for 8 weeks before mating and throughout gestation and ifrst 3 weeks of lactation. The expressions of hepatic fatty acid catabolism related genes, including peroxisome proliferator-activated receptor alpha (PPARα), acyl-CoA syn-thease long-chain family member3 (ACSL3), carnitine palmitoyltransferase-1α(CPT-1α) and 3-hydroxyacyl CoA dehydrogenase (Ehhadh) were determined in offspring liver tissue. The liver pathology was examined in offspring rats at 3 weeks of age. Results Pathohistological ifndings at 3 weeks of age showed that there were diffuse vacuolar degeneration in cytoplasm of hepatocytes and spot necrosis in hepatic lobular in the HF offspring liver. The mRNA expressions of PPARαand Ehhadh genes were markedly increased in the HF offspring as compared to the control group (P<0.05). The mRNA expression of CPT-1αgene was also higher in the HF offspring than that in control group (P=0.19). The level of ACSL3 gene expression, however, was markedly decreased (P<0.05). Conclusions Maternal high fat diet during pregnancy and lactation could result in an increased expression of genes related to hepatic fatty acidβ-oxidation, including PPARα, CPT1αand Ehhadh, but the liver steatosis cannot be reversed in the early life of offspring.

目的 观察母代高脂饮食对F2代大鼠的不良代谢影响.方法 3周龄SD雌性大鼠随机分为高脂饮食组(F0-HF组)和对照组(F0-CON组),至11周龄时交配怀孕产仔(F1代),母鼠按原饲养要求喂养至哺乳期结束.F1代大鼠断乳后均予普通饲料喂养至11周龄时再次交配产仔(F2-HCC组和F2-CON组),两组F2代大鼠均普通饲料喂养至3周龄,观察其糖脂代谢相关指标变化.结果 F2-HCC组大鼠出生、1周龄时体质量明显高于对照组大鼠,差异有统计学意义(P均＜0.05);F2-HCC组大鼠3周龄时空腹血糖和糖耐量曲线下面积均高于对照组,血清总胆固醇和肝脏三酰甘油水平均高于对照组,差异有统计学意义(P均＜0.05);1日龄及3周龄时F2-HCC组大鼠表现出不同程度的脂肪肝,对照组大鼠肝脏镜下结构均正常.结论 母代高脂饮食可引起F2代大鼠体质量增加、肝脏脂肪变性及糖耐量受损,后代发生代谢性疾病的危险性增加.

目的 探讨细胞内基质金属蛋白酶2(MMP-2)基因过表达对大鼠胰岛β细胞株INS-1细胞功能的影响. 方法 采用基因重组技术将大鼠MMP-2 cDNA插入真核表达载体pcDNA 3.1(+)构建大鼠MMP-2真核表达质粒,培养INS-1细胞.随机分为正常对照组,空质粒转染组及MMP-2质粒转染组.脂质体Lipofectmine 2000转染INS-1细胞,观察INS-1细胞内MMP-2基因和蛋白表达量变化,MMP-2酶活性变化,以及INS-1细胞凋亡情况与胰岛素分泌功能的变化. 结果 与正常对照组、空质粒转染组比较,MMP-2质粒转染组MMP-2 mRNA表达增加(P均＜0.05),蛋白表达水平和酶活性上调(P均＜0.05); MMP-2质粒转染组细胞凋亡率(56.07±3.68)％高于正常对照组(33.70±6.53)％及空质粒转染组(38.02±5.60)％(P＜0.05),而IRI(1.30±0.27)低于正常对照组(3.37±0.76)与空质粒转染组(2.90±0.84) (P＜0.05). 结论 细胞内MMP-2基因的过表达可引起胰岛β细胞凋亡增加,胰岛素分泌功能下降.

OBJECTIVE:To investigate the role of matrix metalloproteinase 2 (MMP2) in pancreatic beta cell injury induced by oxidative stress.METHODS:Rat pancreatic beta cell line INS-1 cells were treated with advanced glycation end-products (AGE) to induce intracellular oxidative stress. Intracellular MMP2 expression and activity were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and zymography, respectively. MMP2 expression and activity were manipulated by over-expression with recombinant MMP2 plasmids or knockdown with either MMP2 specific siRNA or inhibitors, and effects on apoptosis and insulin-secretion were measured by flow cytometry and ELISA.RESULTS:AGE treatment induced intracellular oxidative stress in INS-1 cells, as indicated by elevated ROS levels, apoptotic cell death, and suppressed insulin secretion. This was accompanied by increased MMP2 expression and activity. However, Antioxidant N-acetylcysteine (NAC) treatment inhibited MMP2 expression and activity, and partially reversed cell apoptosis and insulin secretion dysfunction induced by AGE. Forced expression of MMP2 mimicked the effects of AGE treatment while inhibition of MMP2 either by a specific MMP2 inhibitor or MMP2 siRNA protected oxidative stress induced by AGE.CONCLUSION:MMP2 expression and intracellular activity are increased by oxidative stress, contributing to cellular dysfunction and apoptosis in INS-1 cells after AGE challenge.