大黄酸改善胰岛素敏感性的作用及其机制研究

改善胰岛素敏感性对防治2型糖尿病(T2DM)有重要意义.我国中医药治疗糖尿病效果显著但机制尚不甚清楚,本课题组从改善胰岛素敏感性的角度入手,先后通过药物筛选、细胞和动物实验3个部分研究中药大黄单体成分--大黄酸对糖尿病的治疗作用,其机制可能与上调机体外周组织的过氧化物酶体增殖物活化受体(PPAR)-慵捌咸烟亲说鞍?GLUT)表达,从而促进外周组织葡萄糖摄取并改善机体胰岛素敏感性有关.本文将分别对这3部分实验内容进行介绍并就大黄酸改善胰岛素敏感性的作用机制展开讨论.     

The synaptically evoked late hyperpolarisation in hippocampal CA1 pyramidal cells is resistant to intracellular EGTA

It has been suggested that the late hyperpolarisation following synaptic activation of hippocampal CA1 pyramidal neurons is activated by calcium influx. This hypothesis was examined using microelectrodes containing EGTA. Intracellular injection of EGTA blocked the afterhyperpolarisation which normally followed cell firing produced by injection of a depolarising current or the ionophoresis of glutamate onto the apical dendrites. In contrast, the hyperpolarisation following synaptic activation was resistant to EGTA. The results suggest that this potential is not dependent on intracellular Ca2+. Other possible mechanisms are discussed.     

PPARβ/δ attenuates palmitate-induced endoplasmic reticulum stress and induces autophagic markers in human cardiac cells

Background

Chronic endoplasmic reticulum (ER) stress contributes to the apoptotic cell death in the myocardium, thereby playing a critical role in the development of cardiomyopathy. ER stress has been reported to be induced after high-fat diet feeding in mice and also after saturated fatty acid treatment in vitro. Therefore, since several studies have shown that peroxisome proliferator-activated receptor (PPAR)β/δ inhibits ER stress, the main goal of this study consisted in investigating whether activation of this nuclear receptor was able to prevent lipid-induced ER stress in cardiac cells.

Methods and results

Wild-type and transgenic mice with reduced PPARβ/δ expression were fed a standard diet or a high-fat diet for two months. For in vitro studies, a cardiomyocyte cell line of human origin, AC16, was treated with palmitate and the PPARβ/δ agonist GW501516. Our results demonstrate that palmitate induced ER stress in AC16 cells, a fact which was prevented after PPARβ/δ activation with GW501516. Interestingly, the effect of GW501516 on ER stress occurred in an AMPK-independent manner. The most striking result of this study is that GW501516 treatment also upregulated the protein levels of beclin 1 and LC3II, two well-known markers of autophagy. In accordance with this, feeding on a high-fat diet or suppression of PPARβ/δ in knockout mice induced ER stress in the heart. Moreover, PPARβ/δ knockout mice also displayed a reduction in autophagic markers.

Conclusion

Our data indicate that PPARβ/δ activation might be useful to prevent the harmful effects of ER stress induced by saturated fatty acids in the heart by inducing autophagy.     

Ethnopharmacological in vitro studies on Austria's folk medicine—An unexplored lore in vitro anti-inflammatory activities of 71 Austrian traditional herbal drugs

Ethnopharmacological relevance

In Austria, like in most Western countries, knowledge about traditional medicinal plants is becoming scarce. Searching the literature concerning Austria's ethnomedicine reveals its scant scientific exploration.Aiming to substantiate the potential of medicinal plants traditionally used in Austria, 63 plant species or genera with claimed anti-inflammatory properties listed in the VOLKSMED database were assessed for their in vitro anti-inflammatory activity.

Material and methods

71 herbal drugs from 63 plant species or genera were extracted using solvents of varying polarities and subsequently depleted from the bulk constituents, chlorophylls and tannins to avoid possible interferences with the assays. The obtained 257 extracts were assessed for their in vitro anti-inflammatory activity. The expression of the inflammatory mediators E-selectin and interleukin-8 (IL-8), induced by the inflammatory stimuli tumor necrosis factor alpha (TNF-α) and the bacterial product lipopolysaccharide (LPS) was measured in endothelial cells. The potential of the extracts to activate the nuclear factors PPARα and PPARγ and to inhibit TNF-α-induced activation of the nuclear factor-kappa B (NF-κB) in HEK293 cells was determined by luciferase reporter gene assays.

Results

In total, extracts from 67 of the 71 assessed herbal drugs revealed anti-inflammatory activity in the applied in vitro test systems. Thereby, 30 could downregulate E-selectin or IL-8 gene expression, 28 were strong activators of PPARα or PPARγ (inducing activation of more than 2-fold at a concentration of 10 µg/mL) and 21 evoked a strong inhibition of NF-κB (inhibition of more than 80% at 10 µg/mL).

Conclusion

Our research supports the efficacy of herbal drugs reported in Austrian folk medicine used for ailments associated with inflammatory processes. Hence, an ethnopharmacological screening approach is a useful tool for the discovery of new drug leads.     

补肺汤对肺纤维化大鼠血清INF-γ IL-4表达水平影响的实验研究

目的：观察补肺汤对肺纤维化大鼠血清IFN-γ、IL-4表达水平的影响,探讨补肺汤对肺纤维化的治疗作用及可能的作用机制。方法：96只SD健康大鼠,随机分为4组（每组各24只）：空白对照组、模型对照组、强的松阳性对照组、补肺汤治疗组。模型对照组和治疗组气管内注射博莱霉素诱导肺纤维化模型,正常对照组在相同条件下给予生理盐水。补肺汤治疗组和强的松阳性对照组分别给予补肺汤流浸膏（2.1g/200g）及强的松片与生理盐水混合制剂（6.3mg/kg）灌胃,其余两组每天给予生理盐水（1mL/100g）灌胃。各组分别于造模后第7、14、28天各处死8只大鼠,取大鼠血清和肺组织。肺组织病理切片HE染色和MASSON染色观察病理变化和肺纤维化分级情况,ELISA法测定血清中INF-γ、IL-4含量的变化。结果：与模型对照组比较,补肺汤治疗组和强的松阳性对照组大鼠各个时期的血清IFN-γ表达明显增高（P〈0.05）;强的松阳性对照组大鼠血清IL-4在各个时期的表达降低明显（P〈0.05）,而补肺汤治疗组在第28天降低明显（P〈0.05）。结论：补肺汤肺纤维化的形成有较好的治疗作用,其机制可能是通过促进血清中IFN-γ的分泌,抑制IL-4的分泌,从而调节了Th1/Th2细胞因子失衡来实现的。     

siRNA沉默PPARα减弱羟喜树碱诱导的人结肠癌细胞凋亡

背景:多药耐药一直是遏制提高结肠癌化疗有效性的瓶颈问题。目的:探讨沉默PPARα能否减弱羟喜树碱诱导的人结肠癌细胞凋亡,从而阐明PPARα在羟喜树碱耐药中的潜在作用。方法:构建针对PPARα基因的siRNA干扰载体,并转染人结肠癌SW1116细胞,经G418筛选出稳定转染的细胞,以RT-PCR和蛋白质印迹法验证干扰效果。经羟喜树碱干预后,以MTT法检测细胞增殖,Annexin V-FITC/PI检测细胞凋亡。以定量RT-PCR检测不同浓度miR-506前体转染SW1116细胞后对PPARα下游调控靶基因mRNA表达的影响。结果:siRNA干扰载体Ⅲ的沉默效果最佳,PPARα mRNA和蛋白表达分别下调了约94.1%±3.4%和70.8%±8.6%。稳定转染siRNA的SW1116细胞的半数抑制浓度(IC_(50))为(332±19)μg/L,亲本SW1116细胞为(152±12)μg/L。在相同浓度羟喜树碱的作用下,亲本SW1116细胞的凋亡率显著高于稳定转染siRNA的SW1116细胞(150μg/L:7.0%±2.5%对2.8%±1.6%;300μg/L:32.4%±7.1%对18.5%±2.7%)。miR-506前体下调HMGCS-2表达,上调FABP-2、CCND1和TGF-β1表达,其中上调CCND1的作用最为明显。结论:成功构建了针对PPARα的siRNA干扰载体;沉默PPARα能增强SW1116细胞对羟喜树碱的抵抗性,可能与下调HMGCS-2表达并上调FABP-2、CCND1和TGF-β1表达有关。     

Upregulation of GAD65 mRNA in the medulla of the rat model of metabolic syndrome

Metabolic syndrome is characterized by obesity, elevated blood pressure (BP), insulin resistance, and hypercholesterolemia. Recently an animal model of this disorder has been proposed in rats selectively bred based on their performance on a treadmill-running task. Accordingly, low capacity runner (LCR) rats exhibited all of the diagnostic criteria for metabolic syndrome, including elevated BP, as compared to their high capacity runner (HCR) counterparts [U. Wisløff, S.M. Najjar, O. Ellingsen, P.M. Haram, S. Swoap, Q. Al-Share, M. Fernstrom, K. Rezaei, S.J. Lee, L.G. Koch, S.L. Britton, Cardiovascular risk factors emerge after artificial selection for low aerobic capacity, Science 307 (2005) 418–420]. Previous studies have highlighted the importance of GABAergic neurotransmission in the medullary cardiovascular-regulatory areas in the central control of BP. Thus, we hypothesized a dysregulation in GABAergic transmission in the medullary cardiovascular-regulatory nuclei of LCR rats. To begin testing this hypothesis we carried out experiments examining expression of the GABA synthetic enzymes, GAD65 and GAD67, mRNAs in the two rat strains via radioactive in situ hybridization. Our results showed GAD65 and GAD67 mRNAs were widely expressed throughout the brainstem; quantification revealed increased GAD65 mRNA expression in LCR animals in the caudal nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (VLM) as compared to HCR rats. Conversely, no differences in the expression of GAD67 were detected in these regions. These data are consistent with the notion of altered GABAergic neurotransmission in the NTS and VLM in metabolic syndrome, and point to the importance of these regions in cardiovascular regulation.     

Mendelian susceptibility to mycobacterial diseases: state of the art

BackgroundMendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to infections caused by intracellular pathogens, such as mycobacteria, due to impaired IFN-γ immunity. To date, 18 different genes associated with MSMD have been reported.ObjectivesThis review describes recent discoveries, a 2020–2021 update, in MSMD through the introduction of three novel genetic disorders, namely, AR IFN-γ, T-bet, and ZNFX1 complete deficiency, as well as molecular mechanisms underlying multifocal osteomyelitis in patients with this condition.SourcesPubMed databases were searched for reports of MSMD since January 2020. Relevant articles and their references were screened.ContentThe review covers a general overview, known genes, classifications, symptoms, and treatments for MSMD. MSMD is classified into two groups: isolated MSMD and syndromic MSMD. Among the 18 genes responsible, 13 cause isolated MSMD, which is characterized by selective predisposition to one or more mycobacterial and related infections, and 8 cause syndromic MSMD, which involves the combination of the mycobacterial disease infectious phenotype with additional clinical phenotypes. Among the three genetic etiologies described herein, AR IFN-γ deficiency is classified as isolated MSMD, whereas AR T-bet and ZNFX1 deficiency are classified as syndromic MSMD. Multifocal osteomyelitis is a representative symptom of MSMD, and a high frequency of multifocal osteomyelitis is reported in MSMD patients due to impaired IFN-γ responses, such as with AD IFN-γR1, AD IFN-γR2, or AD STAT1 deficiency. Impaired inhibition of osteoclast differentiation and bone resorption owing to a poor response to IFN-γ has been shown to be in association with multifocal osteomyelitis in MSMD.ImplicationsOver the past decade, genetic dissection by next-generation sequencing techniques has contributed to the understanding of the molecular bases of human immunity to mycobacteria. However, genetic etiologies are lacking for half of MSMD cases. Further studies will be needed to elucidate the pathogenesis of MSMD.