The phytochemical polydatin ameliorates non‐alcoholic steatohepatitis by restoring lysosomal function and autophagic flux

Impaired autophagic degradation of intracellular lipids is causally linked to the development of non‐alcoholic steatohepatitis (NASH). Pharmacological agents that can restore hepatic autophagic flux could therefore have therapeutic potentials for this increasingly prevalent disease. Herein, we investigated the effects of polydatin, a natural precursor of resveratrol, in a murine nutritional model of NASH and a cell line model of steatosis. Results showed that oral administration of polydatin protected against hepatic lipid accumulation and alleviated inflammation and hepatocyte damage in db/db mice fed methionine‐choline deficient diet. Polydatin also alleviated palmitic acid‐induced lipid accumulation in cultured hepatocytes. In both models, polydatin restored lysosomal function and autophagic flux that were impaired by NASH or steatosis. Mechanistically, polydatin inhibited mTOR signalling and up‐regulated the expression and activity of TFEB, a known master regulator of lysosomal function. In conclusion, polydatin ameliorated NASH through restoring autophagic flux. The polydatin‐regulated autophagy was associated with inhibition of mTOR pathway and restoration of lysosomal function by TFEB. Our study provided affirmative preclinical evidence to inform future clinical trials for examining the potential anti‐NASH effect of polydatin in humans.     

Comparative studies of polydatin and resveratrol on mutual transformation and antioxidative effect in vivo

BackgroundPolydatin and resveratrol are extractives of radix or rhizoma of Polygonum cuspidatum, and as the glycoside forms, it is a natural precursor of resveratrol.PurposeIn this study, we aimed to explore the mutual transformation between polydatin and resveratrol in rats, and to compare the antioxidative effect of them in vivo.Study designIn this study, we analyzed the serum molar concentration of polydatin and resveratrol after oral administration in rats and evaluated the anti-oxidative stress effects of them using a mouse model.MethodsRats were orally administered polydatin or resveratrol and the concentration of them in serum were analyzed by high performance liquid chromatography (HPLC). Their antioxidative effect was compared in mice with oxidative stress cardiomyopathy induced by doxorubicin (DOX).ResultsThe results showed that polydatin and resveratrol could mutually transform in vivo, the molar concentration of polydatin in serum was always averagely 3.35 and 4.28 times as much as resveratrol after oral administration of polydatin and resveratrol at 200 mg/kg, respectively. Both polydatin and resveratrol could significantly decrease the content of malonydialdehyde (MDA), promote the activities of total superoxide dismutase (T-SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in plasma, and increase the content of glutathione (GSH) in myocardial tissue. The effect of polydatin surpassed resveratrol, particularly embodied in increasing the activities of T-SOD and CAT, and the content of GSH.ConclusionIt illustrates that polydatin is the main substance in serum after intragastric administration with polydatin or resveratrol, and the mutual transformation between polydatin and resveratrol keeps balance; they both have the ability of antioxidative stress in vivo, and polydatin has a better effect than resveratrol, which hints that polydatin may be a substitute for resveratrol in antioxidant for clinical use.     

白藜芦醇甙对大鼠心室乳头状肌动作电位的影响及其离子机制(英文)

有研究表明白藜芦醇甙(polydatin)具有抗缺血性心律失常作用,但其电生理学机制尚未明了。本研究旨在应用细胞内记录和全细胞膜片钳方法,探讨白藜芦醇甙对大鼠心室乳头状肌动作电位的影响及其离子机制。结果显示:(1)白藜芦醇甙(50和100μmol/L)可剂量依赖性地缩短正常乳头状肌动作电位复极化50%时间(APD50)和90%时间(APD90)(P<0.01)。白藜芦醇甙对正常乳头状肌静息电位(resting potential,RP)、动作电位幅值(amplitude of action potential,APA)、超射值(overshoot,OS)和0期最大上升速度(Vmax)无影响(P>0.05)。(2)对部分去极化的乳头状肌,白藜芦醇甙(50μmol/L)不但缩短APD50和APD90,而且还降低动作电位OS、APA和Vmax(P<0.05)。(3)ATP敏感钾通道阻断剂格列本脲(10μmol/L)可部分阻断白藜芦醇甙(50μmol/L)的电生理效应。(4)一氧化氮合酶抑制剂L-NAME(1 mmol/L)对白藜芦醇甙的上述效应无影响。(5)白藜芦醇甙(25、50、75、100μmol/L)可浓度依...     

白藜芦醇甙减轻高糖所致心肌微血管内皮细胞损伤的作用及机制研究

目的:探讨白藜芦醇甙在高糖处理的大鼠心肌微血管内皮细胞损伤中的作用及其可能调控机制。方法:酶消法分离大鼠CMECs,高糖处理CMECs建立细胞损伤模型,实验随机分为6个组:对照组(葡萄糖浓度为5.5 mmol/L)、白藜芦醇甙组、高糖组(葡萄糖浓度为33 mmol/L)、高糖+白藜芦醇甙组、高糖+白藜芦醇甙+3-MA(自噬抑制剂)组和高糖+雷帕霉素(自噬诱导剂)组。白藜芦醇甙组和高糖+白藜芦醇甙组分别加入10μmol/L的白藜芦醇甙孵育24 h,高糖+白藜芦醇甙+3-MA组加入10μmol/L的白藜芦醇甙和10μmmol/L 3-MA孵育24 h,高糖+雷帕霉素组加入100 nmol/L的雷帕霉素孵育24小时。CCK-8实验检测大鼠CMECs增殖;Tunel法检测大鼠CMECs凋亡;FITC-葡聚糖清除实验检测单层CMECs通透性;Western blot检测LC3Ⅱ和p62的表达。结果:与对照组和白藜芦醇甙组相比,高糖组CMECs增殖能力降低(P<0.05),凋亡率显著增加(P<0.05),细胞通透性增加(P<0.05),LC3Ⅱ表达降低(P<0.05),p62的表达增加(P<0.05);与高糖组相比,高糖+白藜芦醇甙组和高糖+雷帕霉素组CMECs增殖能力增加(P<0.05),凋亡率显著降低(P<0.05),细胞通透性降低(P<0.05),LC3Ⅱ表达增加(P<0.05),p62的表达降低(P<0.05);与高糖+白藜芦醇甙组相比,高糖+白藜芦醇甙+3-MA组CMECs增殖能力降低(P<0.05),凋亡率显著增加(P<0.05),细胞通透性增加(P<0.05),LC3Ⅱ表达降低(P<0.05),p62的表达增加(P<0.05)。结论:白藜芦醇甙通过增加自噬减轻高糖处理的大鼠心肌微血管内皮细胞损伤。     

Geraniin inhibits cell growth and promoted autophagy-mediated cell death in the nasopharyngeal cancer C666-1 cells

BackgroundNasopharyngeal carcinoma (NPC) is a rare malignant tumor developing from epithelial linings of nasopharynx, and 10–50 out of 100,000 NPC cases were recorded globally particularly in the Asian countries.MethodologyThe cytotoxicity of geraniin against the NPC C666-1 cells were analyzed using MTT assay. The influences of geraniin on the C666-1 cell viability with the presence of ROS and apoptosis inhibitors were also studied. The expressions of PI3K, Akt, mTOR, and autophagic markers LC3, ATG7, P62/SQSTM1 expressions in the C666-1 cells were studied by western blotting analysis. The ROS production was assayed using DCFH-DA staining. The immunofluorescence assay was performed to detect the NF-κB and β-catenin expressions in the C666-1 cells.ResultsThe cell viability of C666-1 cells were appreciably prevented by the geraniin. The geraniin treatment also inhibited the C666-1 cell growth with the presence of apoptotic inhibitor Z-VAD-FMK. The geraniin-treatment effectively improved the ROS production and inhibited the NF-κB and β-catenin expressions in the C666-1 cells. Geraniin appreciably modulated the PI3K/Akt/mTOR signaling axis and improved the autophagy-mediated cell death via improving the autophagic markers LC3 and ATG7 expressions in the C666-1 cells.ConclusionIn conclusion, our results proved that geraniin inhibits C666-1 cell growth and initiated autophagy-mediated cell death via modulating PI3K/Akt/mTOR cascade and improving LC3 and ATG7 expressions in the C666-1. Geraniin and it could be a hopeful and efficient candidate to treat the human NPC in the future.     

Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells

Abstract

Silibinin mostly has been used as hepatoprotectants, but it has other interesting activities, e.g. anti-cancer, cardial protective and brain-protective activities. A previous study demonstrated that silibinin protected amyloid β (Aβ)-induced mouse cognitive disorder by behavioural pharmacological observation. This study assessed the effect of silibinin on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. Subsequent morphologic observation, flow cytometric analysis and Western blot analysis indicated that treatment with SNP significantly induced apoptosis in PC12 cells. However, silibinin eliminated the apoptotic effect by reactive oxygen species (ROS) generation, especially hydroxyl free radical. Silibinin-induced autophagy through ROS generation when exerting a protective effect and silibinin-induced autophagy also enhanced the ROS generation since 3-methyladenine (3-MA), a specific autophagy inhibitor, decreased the ROS generation and rapamycin, an autophagy inducer, enhanced the ROS generation. Therefore, there exists a positive feedback loop between autophagy and ROS generation. Autophagy prevented SNP-induced apoptosis, since the addition of 3-MA significantly eliminated the protective effect of silibinin. This protective effect was attributed to the generation of ROS and its two downstream Ras/PI3K/NF-κB and Ras/Raf/MEK/ERK pathways. Both prevented PC12 cells from apoptosis. The PI3K/NF-κB pathway induced autophagy to protect PC12 cells, but the Raf/MEK/ERK pathway directly protected PC12 cells bypassing the autophagic effect.     

Polydatin reverses oxidation low lipoprotein (oxLDL)-induced apoptosis of human umbilical vein endothelial cells via regulating the miR-26a-5p/BID axis

Atherosclerosis is a disease in which lipids and inflammatory factors accumulate on the walls of arteries, forming plaques that eventually block the flow of blood. Polydatin was derived from plant knotweed, which could play an important role in inhibiting the progression of atherosclerosis. However, the mechanism by which polydatin regulates the genesis and development of atherosclerosis remains unclear. To detect the function of polydatin in atherosclerosis, the proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs) was detected using 5-ethynyl-2’-deoxyuridine staining, flow cytometry and transwell assays, respectively. In addition, the branch points and capillary length of HUVECs were observed using a tube formation assay, and the lipid accumulation was tested by Oil-red O staining assay. Dual luciferase reporter assays were performed to confirm the association between microRNA (miR)-26a-5p and BH3 interacting domain death agonist (BID) in HUVECs. The data suggested oxidized low-density lipoprotein (oxLDL) notably inhibited the viability of HUVECs in a dose-dependent manner, and polydatin reversed the oxLDL-induced inhibition of HUVECs viability and proliferation. In addition, polydatin inhibited the apoptosis, migration and epithelial mesenchymal transition (EMT) process in oxLDL-treated HUVECs. Polydatin reversed oxLDL-induced lipid accumulation and angiogenesis inhibition in HUVECs. Furthermore, BID was targeted by miR-26a-5p, and polydatin reversed the oxLDL-induced apoptosis of HUVECs via regulating the miR-26a-5p/BID axis. In summary, polydatin reversed the oxLDL-induced apoptosis of HUVECs via regulating the miR-26a-5p/BID axis. Therefore, polydatin could act as a new agent for atherosclerosis treatment.Key words: Atherosclerosis, polydatin, miR-26a-5p, BH3 interacting domain death agonist     

Polydatin Restores Endothelium-Dependent Relaxation in Rat Aorta Rings Impaired by High Glucose: A Novel Insight into the PPARβ-NO Signaling Pathway

Polydatin, a natural component from Polygonum Cuspidatum, has important therapeutic effects on metabolic syndrome. A novel therapeutic strategy using polydatin to improve vascular function has recently been proposed to treat diabetes-related cardiovascular complications. However, the biological role and molecular basis of polydatin’s action on vascular endothelial cells (VECs)-mediated vasodilatation under diabetes-related hyperglycemia condition remain elusive. The present study aimed to assess the contribution of polydatin in restoring endothelium-dependent relaxation and to determine the details of its underlying mechanism. By measuring endothelium-dependent relaxation, we found that acetylcholine-induced vasodilation was impaired by elevated glucose (55 mmol/L); however, polydatin (1, 3, 10 μmol/L) could restore the relaxation in a dose-dependent manner. Polydatin could also improve the histological damage to endothelial cells in the thoracic aorta. Polydatin’s effects were mediated via promoting the expression of endothelial NO synthase (eNOS), enhancing eNOS activity and decreasing the inducible NOS (iNOS) level, finally resulting in a beneficial increase in NO release, which probably, at least in part, through activation of the PPARβ signaling pathway. The results provided a novel insight into polydatin action, via PPARβ-NO signaling pathways, in restoring endothelial function in high glucose conditions. The results also indicated the potential utility of polydatin to treat diabetes related cardiovascular diseases.     

Guttiferone K induces autophagy and sensitizes cancer cells to nutrient stress-induced cell death

BackgroundMedicinal plants have long been an excellent source of pharmaceutical agents. Autophagy, a catabolic degradation process through lysosomes, plays an important role in tumorigenesis and cancer therapy.PurposeThrough a screen designed to identify autophagic regulators from a library of natural compounds, we found that Guttiferone K (GUTK) can activate autophagy in several cancer cell lines. The objective of this study is to investigate the mechanism by which GUTK sensitizes cancer cells to cell death in nutrient starvation condition.MethodsCell death analysis was performed by propidium iodide staining with flow cytometry or Annexin V-FITC/PI staining assay. DCFH-DA staining was used for intracellular ROS measurement. Protein levels were analyzed by western blot analysis. Cell viability was measured by MTT assay.ResultsExposure to GUTK was observed to markedly induce GFP-LC3 puncta formation and activate the accumulation of LC3-II and the degradation of p62 in HeLa cells, suggesting that GUTK is an autophagy inducer. Importantly, hydroxychloroquine, an autophagy inhibitor, was found to significantly prevent GUTK-induced cell death in nutrient starvation conditions, suggesting that the cell death observed is largely dependent on autophagy. We further provide evidence that GUTK inhibits Akt phosphorylation, thereby inhibiting the mTOR pathway in cancer cells during nutrient starvation. In addition, GUTK causes the accumulation of reactive oxygen species (ROS) and the phosphorylation of JNK in EBSS, which may mediate both autophagy and apoptosis.ConclusionThese data indicate that GUTK sensitizes cancer cells to nutrient stress-induced cell death though Akt/mTOR dependent autophagy pathway.     

Polydatin prevents angiotensin II-induced cardiac hypertrophy and myocardial superoxide generation

Our studies and others recently demonstrate that polydatin, a resveratrol glucoside, has antioxidative and cardioprotective effects. This study aims to investigate the direct effects of polydatin on Ang II-induced cardiac hypertrophy to explore the potential role of polydatin in cardioprotection. Our results showed that in primary cultured cardiomyocytes, polydatin blocked Ang II-induced cardiac hypertrophy in a dose-dependent manner, which were associated with reduction in the cell surface area and [³H]leucine incorporation, as well as attenuation of the mRNA expressions of atrial natriuretic factor and β-myosin heavy chain. Furthermore, polydatin prevented rat cardiac hypertrophy induced by Ang II infusion, as assessed by heart weight-to-body weight ratio, cross-sectional area of cardiomyocyte, and gene expression of hypertrophic markers. Further investigation demonstrated that polydatin attenuated the Ang II-induced increase in the reactive oxygen species levels and NADPH oxidase activity in vivo and in vitro. Polydatin also blocked the Ang II-stimulated increases of Nox4 and Nox2 expression in cultured cardiomyocytes and the hearts of Ang II-infused rats. Our results indicate that polydatin has the potential to protect against Ang II-mediated cardiac hypertrophy through suppression of NADPH oxidase activity and superoxide production. These observations may shed new light on the understanding of the cardioprotective effect of polydatin.     

Protective Effects of Polydatin from Polygonum cuspidatum against Carbon Tetrachloride-Induced Liver Injury in Mice

Polydatin is one of main compounds in Polygonum cuspidatum, a plant with both medicinal and nutritional value. The possible hepatoprotective effects of polydatin on acute liver injury mice induced by carbon tetrachloride (CCl₄) and the mechanisms involved were investigated. Intraperitoneal injection of CCl₄ (50 µl/kg) resulted in a significant increase in the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and hepatic malondialdehyde (MDA), also a marked enhancement in the expression of hepatic tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and nuclearfactor-kappa B (NF-κB). On the other hand, decreased glutathione (GSH) content and activities of glutathione transferase (GST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were observed following CCl₄ exposure. Nevertheless, all of these phenotypes were evidently reversed by preadministration of polydatin for 5 continuous days. The mRNA and protein expression levels of hepatic growth factor-beta1 (TGF-β₁) were enhanced further by polydatin. These results suggest that polydatin protects mice against CCl₄-induced liver injury through antioxidant stress and antiinflammatory effects. Polydatin may be an effective hepatoprotective agent and a promising candidate for the treatment of oxidative stress- and inflammation-related diseases.     

(-)-Epicatechin gallate accumulates in foamy macrophages in human atherosclerotic aorta: implication in the anti-atherosclerotic actions of tea catechins

The localization and target sites of tea catechins underlying their biological activity including anti-atherosclerotic activity have not yet been fully understood. To identify the target sites of catechins in vivo, we have developed a novel monoclonal antibody (mAb5A3) specific for (−)-epicatechin-3-gallate (ECg), one of the major tea catechins. The immunoreactive materials with mAb5A3 were detected in the human atherosclerotic lesions but not in the normal aorta, and were specifically localized in the macrophage-derived foam cells. In vitro experiments using macrophage-like cell lines also showed the significant accumulation of ECg in the cells. We also demonstrated that ECg could suppress the gene expression of a scavenger receptor CD36, a key molecule for foam cell formation, in macrophage cells. These results, for the first time, showed the target site of a tea component ECg in the aorta and might provide a mechanism for the anti-atherosclerotic actions of the catechins.     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

miR-155靶向PTEN-PI3KAKT通路促进鼻咽癌细胞增殖并抑制其凋亡的实验研究

摘要 目的：研究miR-155靶向PTEN-PI3KAKT通路促进鼻咽癌细胞增殖并抑制其凋亡的作用机制。方法：选取105只成年健康SD雄性大鼠作为研究对象,将其以随机抽签法等分成观察组、对照组、试验组,每组35只。所有大鼠通过诱癌剂二甲硝基哌嗪腋部皮下注射以及促癌剂佛波醇酯皮下注射进行鼻咽癌大鼠模型的建立。观察组大鼠予以miR-155抑制剂干预,试验组予以PI3K抑制剂干预,对照组不予以任何干预。以实时荧光定量PCR法检测miR-155相对表达量,采用免疫组化法检测PTEN、PI3K、P-AKT表达情况,通过Western blot法检测Bcl-2、Bax、Ezrin蛋白表达水平。比较三组大鼠上述相关指标水平,并作相关性分析。结果：试验组、观察组、对照组大鼠的miR-155相对表达量分别为（34.88±1.32）、（29.72±1.23）、（35.01±1.34）,观察组显著低于试验组和对照组,差异明显（F=23.105,P=0.000）。试验组、观察组、对照组大鼠PTEN表达率逐渐升高,而PI3K、P-AKT表达率逐渐降低（均P＜0.05）。经Pearson相关性分析可得：鼻咽癌大鼠miR-155相对表达量与PTEN表达率呈正相关关系,而与PI3K、P-AKT表达率呈负相关关系（均P＜0.05）。试验组、观察组、对照组Bcl-2、Ezrin蛋白表达水平呈逐渐升高趋势,而Bax蛋白表达水平呈逐渐减低趋势,且经单因素方差分析发现：各组间对比差异显著（均P＜0.05）。结论：miR-155可能是通过靶向作用于PTEN-PI3KAKT信号通路,继而发挥促进鼻咽癌细胞增殖以及抑制鼻咽癌细胞凋亡的作用。临床工作中可能将其作为鼻咽癌治疗的新靶点。     

A Potential Role for the Inhibition of PI3K Signaling in Glioblastoma Therapy

Glioblastoma multiforme (GBM) is the most common primary brain tumor and among the most difficult to treat malignancies per se. In almost 90% of all GBM alterations in the PI3K/Akt/mTOR have been found, making this survival cascade a promising therapeutic target, particular for combination therapy that combines an apoptosis sensitizer, such as a pharmacological inhibitor of PI3K, with an apoptosis inducer, such as radio- or chemotherapy. However, while in vitro data focusing mainly on established cell lines has appeared rather promising, this has not translated well to a clinical setting. In this study, we analyze the effects of the dual kinase inhibitor PI-103, which blocks PI3K and mTOR activity, on three matched pairs of GBM stem cells/differentiated cells. While blocking PI3K-mediated signaling has a profound effect on cellular proliferation, in contrast to data presented on two GBM cell lines (A172 and U87) PI-103 actually counteracts the effect of chemotherapy. While we found no indications for a potential role of the PI3K signaling cascade in differentiation, we saw a clear and strong contribution to cellular motility and, by extension, invasion. While blocking PI3K-mediated signaling concurrently with application of chemotherapy does not appear to be a valid treatment option, pharmacological inhibitors, such as PI-103, nevertheless have an important place in future therapeutic approaches.     

Inhibition of phosphatidylinositide 3-kinase impairs the benzyl isothiocyanate-induced accumulation of autophagic molecules and Nrf2 in human colon cancer cells

The regulating role of phosphatidylinositide 3-kinase (PI3K) in benzyl isothiocyanate (BITC)-induced Nrf2 activation, contributing to the inducible expression of cytoprotective genes, was investigated. BITC significantly enhanced the accumulation of Nrf2 as well as autophagic molecules in human colorectal cancer HCT-116 cells. Experiments using a PI3K-specific inhibitor suggested that PI3K plays the key role in the non-canonical Nrf2 activation by BITC.     

Bone marrow‐derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion‐induced lung injury

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM‐MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion‐induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM‐MSCs. Seventy mice were pre‐treated with BM‐MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro‐vascular endothelial cells (HPMVECs) were pre‐conditioned with BM‐MSCs by oxygen‐glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI‐treated mice, administration of BM‐MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD‐treated HPMVECs, co‐culture with BM‐MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM‐MSCs decreased the level of PI3K class I and p‐Akt while the expression of PI3K class III was increased. Finally, BM‐MSCs‐induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM‐MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM‐MSCs and will help to develop new cell‐based therapeutic strategies in lung injury.     

Oxyresveratrol activates parallel apoptotic and autophagic cell death pathways in neuroblastoma cells

BackgroundDrug resistance from apoptosis is a challenging issue with different cancer types, and there is an interest in identifying other means of inducing cytotoxicity. Here, treatment of neuroblastoma cells with oxyresveratrol (OXYRES), a natural antioxidant, led to dose-dependent cell death and increased autophagic flux along with activation of caspase-dependent apoptosis.MethodsFor cell viability, we performed the CCK-8 assay. Protein expression changes were with Western blot and immunocytochemistry. Silencing of proteins was with siRNA. The readouts for cell cycle, mitochondria membrane potential, caspase-3, autophagy and apoptosis were performed with flow cytometry.ResultsPhosphorylation of p38 MAPK increased with OXYRES treatment and inhibition of p38 reduced autophagy and cell death from OXYRES. In contrast, PI3K/AKT/mTOR signaling decreased in the target cells with OXYRES and inhibition of PI3K or mTOR enhanced OXYRES-mediated cytotoxicity with increased levels of autophagy. Modulation of either of the apoptosis and autophagy flux pathways affected the extent of cell death by OXYRES, but did not affect the indicators of these pathways with respect to each other. Both pathways were independent of ROS generation or p53 activation.ConclusionOXYRES led to cell death from autophagy, which was independent of apoptosis induction. The OXYRES effects were due to changes in the activity levels of p38 MAPK and PI3K/AKT/mTOR.General significanceWith two independent and parallel pathways for cytotoxicity induction in target cells, this study puts forward a potential utility for OXYRES or the pathways it represents as novel means of inducing cell death in neuroblastoma cells.