Melatonin alleviates insulin resistance through the PI3K/AKT signaling pathway in ovary granulosa cells of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine gynecological disorder. Insulin resistance (IR) is a major cause of PCOS. Melatonin, a critical endogenous hormone, has beneficial effects on the female reproductive system. This study aims to investigate the molecular effect of melatonin on IR in human ovarian granulosa cells (GCs). Hormone levels of the subjects were determined through clinical examination. The expression levels of insulin receptor substrate (IRS)-1 and glucose transporter (GLUT4) in GCs from PCOS patients and a human granulosa cell line (SVOG) were examined using qRT-PCR and western blot. The IR cell model was established by inducing SVOG cells with palmitic acid (PA). IR was detected in GCs of PCOS patients and SVOG by measuring glucose content and glucose uptake. Cell viability and apoptosis levels were detected by CCK-8 assay and flow cytometry. PI3K/Akt pathway expression in SVOG was assessed by western blot. PCOS patients had higher levels of luteinizing hormone (LH), testosterone, and LH/follicle-stimulating hormone. PA decreased cell viability, promoted apoptosis, and reduced glucose uptake in SVOG cells. IRS-1 and GLUT4 mRNA and protein expression was downregulated, and glucose uptake capacity was reduced in PCOS GCs and SVOG cells. Melatonin significantly upregulated IRS-1 and GLUT4 expression, downregulated p-IRS-1 (Ser307), and improved glucose uptake in PCOS patients' GCs and SVOG cells. PA decreased PI3K and Akt phosphorylation, whereas melatonin increased p-PI3K and p-Akt levels. Melatonin can reduce IR in GCs and PA-induced SVOG cells via the PI3K/Akt signaling pathway, providing more evidence for treating polycystic ovary syndrome.     

象皮生肌膏对肛瘘术后创面修复及PI3K/Akt/mTOR信号通路的影响

摘要 目的：探究象皮生肌膏对肛瘘切除术后大鼠创面的治疗作用及其与PI3K/Akt/mTOR信号通路的相关性。方法：将SD雄性大鼠随机分为假手术组、模型组、象皮生肌膏组、湿润烧伤膏组,共4组,每组10只。4组均采用0号钢丝制造肛瘘模型,造模成功后,除假手术组外,均在麻醉下行肛瘘切除术,创面保持开放,使之形成"开放、渗血、渗液、有脓性分泌物"的感染性肛瘘术后创面,假手术组保留瘘管,不予换药,模型组予以生理盐水冲洗、络合碘消毒后不予换药,治疗组分别予以相应药物进行创面换药,共10天。10天后采用常规面积检测法比较模型组、象皮生肌膏组、湿润烧伤膏组大鼠肛瘘术后创面的创面愈合率及创面肉芽组织覆盖率;HE染色观察各组大鼠肛周组织病理情况;ELISA检测各组大鼠血清中bFGF、EGF、VEGF的表达水平,WB检测比较各组大鼠肛周肉芽组织中PI3K、Akt、mTOR、p70 S6K、p-PI3K（S473）、p-AKT（S473）、p-mTOR（Ser2448）、p-p70 S6K的蛋白表达水平。结果：与模型组比较,治疗第3、7、10天象皮生肌膏组和湿润烧伤膏组的创面愈合率及创面肉芽组织填充率显著升高（P＜0.01）。病理切片显示,假手术组炎性细胞浸润较少,其余各组均可见不同程度的炎性细胞浸润,且创面区可见不同程度炎性修复型肉芽组织增生、胶原纤维新生及血管扩张;其中模型组病理切片显示大量炎性细胞浸润,血管扩张明显,并有明显的血管出血;象皮生肌膏组病理切片显示炎性细胞较少,成纤维细胞成熟且分布整齐,真皮层内胶原纤维丰富且排列整齐,可见血管新生,无明显血管扩张及出血;湿润烧伤膏组病理切片显示少量炎性细胞浸润及血管扩张,真皮层内可见成纤维细胞生成。ELISA检测结果显示,与假手术组比较,模型组血清中bFGF、EGF、VEGF的含量显著降低（P＜0.01）;与模型组比较,象皮生肌膏组血清中bFGF、EGF、VEGF的含量显著升高（P＜0.01）,湿润烧伤膏组大鼠血清中EGF、VEGF的含量显著升高（P＜0.01）。WB检测结果显示,与假手术组比较,模型组肛周组织中p-PI3K、p-Akt 、p-mTOR、p-p70 S6K蛋白表达显著降低（P<0.01）;与模型组比较,象皮生肌膏组肛周组织中p-PI3K、p-Akt 、p-mTOR、p-p70 S6K蛋白表达显著升高（P<0.05）。结论：象皮生肌膏能有效促进肛瘘术后创面修复,减轻肛瘘术后创面炎症反应,促进创面肉芽生长,其促愈机制可能与PI3K/Akt/mTOR信号通路相关蛋白表达有关,其通过上调PI3K/Akt/mTOR信号通路相关蛋白激活PI3K/Akt/mTOR信号通路,进而加快创面修复进程。     

Treponema pallidum membrane protein Tp47 induced autophagy and inhibited cell migration in HMC3 cells via the PI3K/AKT/FOXO1 pathway

The migratory ability of microglia facilitates their rapid transport to a site of injury to kill and remove pathogens. However, the effect of Treponema pallidum membrane proteins on microglia migration remains unclear. The effect of Tp47 on the migration ability and autophagy and related mechanisms were investigated using the human microglial clone 3 cell line. Tp47 inhibited microglia migration, the expression of autophagy-associated protein P62 decreased, the expression of Beclin-1 and LC3-II/LC3-I increased, and the autophagic flux increased in this process. Furthermore, autophagy was significantly inhibited, and microglial cell migration was significantly increased after neutralisation with an anti-Tp47 antibody. In addition, Tp47 significantly inhibited the expression of p-PI3K, p-AKT, and p-mTOR proteins, and the sequential activation of steps in the PI3K/AKT/mTOR pathways effectively prevented Tp47-induced autophagy. Moreover, Tp47 significantly inhibited the expression of p-FOXO1 protein and promoted FOXO1 nuclear translocation. Inhibition of FOXO1 effectively suppressed Tp47-induced activation of autophagy and inhibition of migration. Treponema pallidum membrane protein Tp47-induced autophagy and inhibited cell migration in HMC3 Cells via the PI3K/AKT/FOXO1 pathway. These data will contribute to understanding the mechanism by which T. pallidum escapes immune killing and clearance after invasion into the central nervous system.     

丹参酚酸B通过抑制PI3K/AKT/mTOR通路促进自噬减轻大鼠心肌纤维化的研究

目的:研究丹参酚酸B(SA-B)能否通过抑制PI3K/AKT/mTOR通路促进自噬,从而减轻大鼠心肌纤维化。方法:选用SD大鼠40只,完全随机化分为对照组、模型组、低剂量SA-B治疗组和高剂量SA-B治疗组,采用皮下注射异丙肾上腺素(ISO)构建大鼠心肌纤维化模型。低、高剂量SA-B治疗组在造模同时灌喂丹参酚酸B水溶液,对照组和模型组分别灌胃等体积0.9%生理盐水。测定心重指数(HW/BW)和左心室重指数(LVW/BW);ELISA法测定心肌中Ⅰ型、Ⅲ型胶原水平;Western blot检测自噬相关蛋白PI3K、AKT、p-AKT、mTOR、Beclin1、LC3-Ⅱ水平;大鼠心肌HE染色评估心肌纤维化程度。结果:与对照组比较,模型组中大鼠的心重指数、左心室重指数和心肌中Ⅰ型、Ⅲ型胶原的水平升高(P0.05),HE染色结果提示心肌组织发生明显的纤维化。模型组大鼠心肌细胞中的自噬相关蛋白PI3K、AKT、p-AKT、mTOR表达升高,Beclin1、LC3-Ⅱ表达较对照组明显降低(P0.05)。SA-B组中心重指数、左心室重指数和心肌中Ⅰ型、Ⅲ型胶原的水平明显降低,HE染色未见明显纤维化病灶,其自噬相关蛋白PI3K、AKT、p-AKT、mTOR表达降低,Beclin1、LC3-Ⅱ表达较模型组明显升高(P0.05)。结论:丹参酚酸B能够抑制ISO所致的大鼠心肌纤维化,且具有剂量依耐性,其机制与抑制PI3K/AKT/mTOR传导通路促进细胞自噬密切相关。     

The PI3K/AKT/mTOR Signaling Pathway Is Overactivated in Primary Aldosteronism

Background

To date, the available non-invasive remedies for primary aldosteronism are not satisfactory in clinical practice. The phosphoinositide 3-kinase (PI3Ks)/protein kinase B (PKB or AKT)/mammalian target of rapamycin (mTOR) signaling pathway is essential for tumorigenesis and metastasis in many types of human tumors, including renal cancer, adrenal carcinoma and pheochromocytoma. The possibility that this pathway is also necessary for the pathogenesis of primary aldosteronism has not yet been explored. To answer this question, we investigated the activity of the PI3K/AKT/mTOR signaling pathway in normal adrenal glands (NAGs), primary aldosteronism (PA) patients and NCI-H295R cells.

Methodology/Principal Findings

Between January 2005 and December 2011, we retrospectively reviewed the records of 45 patients with PA. We compared clinical characteristics (age, gender and biochemical data) and the expression of phospho-AKT (p-AKT), phospho-mTOR (p-mTOR), phospho-S6 (p-S6) and vascular endothelial growth factor (VEGF) by immunohistochemical staining and western blotting, analyzing 30 aldosterone-producing adenomas (APAs), 15 idiopathic hyperaldosteronism (IHA) tissues and 12 NAGs following nephrectomy for renal tumors (control group). Compared with the control group, most of the PA patients presented with polydipsia, polyuria, resistant hypertension, profound hypokalemia, hyperaldosteronemia and decreased plasma renin activity. Compared with normal zona glomerulosa, the levels of p-AKT, p-mTOR, p-S6 and VEGF were significantly upregulated in APA and IHA. No significant differences were found between APA and IHA in the expression of these proteins. Additionally, positive correlations existed between the plasma aldosterone levels and the expression of p-AKT and p-mTOR. In vitro studies showed that mTOR inhibitor rapamycin could inhibit cell proliferation in NCI-H295R cells in a dose- and time-dependent manner. Furthermore, this inhibitor also decreased aldosterone secretion.

Conclusions

Our data suggest that the PI3K/AKT/mTOR signaling pathway, which was overactivated in APA and IHA compared with normal zona glomerulosa, may mediate aldosterone hypersecretion and participate in the development of PA.     

芒果苷通过PI3K/Akt/mTOR通路抑制缺氧缺血性脑损伤大鼠神经细胞凋亡及炎症反应

目的:探讨芒果苷抑制缺氧缺血性脑损伤大鼠神经细胞凋亡的机制。方法:将144只SD新生大鼠分为空白组、模型对照组、阳性对照组(尼莫地平,0.4 mg·kg~(-1)·d~(-1))、芒果苷低、中、高剂量组(50、100、200 mg·kg~(-1)·d~(-1))。检测脑组织中超氧化物歧化酶(SOD)水平、细胞凋亡率、PI3K/Akt/mTOR通路分子表达量。结果:与空白对照组比较,模型组大鼠脑组织中SOD的含量显著降低、细胞凋亡率显著增加,p-PI3K、p-AKT、p-mTOR的表达量显著减少(P0.05);与模型组比较,芒果苷低、中、高剂量组大鼠脑组织中SOD的含量显著增加,细胞凋亡率均显著减少,p-PI3K、p-AKT、p-mTOR的表达量显著增加且芒果苷剂量越大,上述变化越显著(P0.05)。结论:芒果苷对缺氧缺血性脑损伤大鼠的细胞凋亡及炎症反应具有抑制作用且该抑制作用与抑制PI3K/Akt/mTOR通路有关。     

Down‐regulated lncRNA HOTAIR alleviates polycystic ovaries syndrome in rats by reducing expression of insulin‐like growth factor 1 via microRNA‐130a

It has been found that long noncoding RNA HOTAIR, microRNA‐130a (miR‐130a) and insulin‐like growth factor 1 (IGF1) expression are associated with ovarian cancer, thus, we hypothesised that the HOTAIR/miR‐130a/IGF1 axis might associate with endocrine disorders and biological behaviours of ovarian granulosa cells in rat models of polycystic ovary syndrome (PCOS). PCOS rat models were established by injection of dehydro‐isoandrosterone, followed by treatment of si‐HOTAIR, oe‐HOTAIR, miR‐130a mimics or miR‐130a inhibitors. Serum hormonal levels were determined to evaluate endocrine conditions. The effect of HOTAIR and miR‐130a on activities of isolated ovarian granulosa cells was assessed, as well as the involvement of IGF1.In the ovarian tissues and granulosa cells of PCOS rat models, highly expressed HOTAIR and IGF1 and poorly expressed miR‐130a were identified. In response to oe‐HOTAIR, serum levels of E₂, T and LH were increased and serum levels of FSH were reduced; the proliferation of granulosa cells was reduced and apoptosis was promoted; notably, expression of miR‐130a was reduced while expression of IGF1 was increased. The treatment of si‐HOTAIR reversed the situation. Furthermore, the binding of HOTAIR to miR‐130a and targeting relationship of miR‐130a and IGF1 were confirmed. LncRNA HOTAIR up‐regulates the expression of IGF1 and aggravates the endocrine disorders and granulosa cell apoptosis through competitive binding to miR‐130a in rat models of PCOS. Based on our finding, we predict that competitive binding of HOTAIR to miR‐130a may act as a novel target for the molecular treatment of PCOS.     

Oxymatrine synergistically enhances antitumor activity of oxaliplatin in colon carcinoma through PI3K/AKT/mTOR pathway

Oxymatrine (OMT), one of the main active components of extracts from the dry roots of Sophora flavescens, has been reported to possess many pharmacological properties including cancer-preventive and anti-cancer effects. The aim of the present study is to explore the efficiency of combination therapy with OMT and oxaliplatin (OXA) and identify the in vitro and in vivo cytotoxicity on colon cancer lines (HT29 and SW480) and mice model. Cells were treated with OMT and/or OXA and subjected to cell viability, colony formation, apoptosis, cell cycle, western blotting, xenograft tumorigenicity assay and immunohistochemistry. The results demonstrated that OMT and OXA inhibited the proliferation of colon cancer cells, and combination therapy of OMT and OXA resulted in a combination index?<?1, indicating a synergistic effect. Co-treatment with OMT and OXA caused G0/G1 phase arrest by upregulating P21, P27 and downregulating cyclin D, and induced apoptosis through decreasing the expression of p-PI3K, p-AKT, p-mTOR, p-p70S6K. In addition, pretreatment with a specific PI3K/AKT activator (IGF-1) significantly neutralized the pro-apoptotic activity of OXA?+?OMT, demonstrating the important role of PI3K/AKT in this process. Moreover, in nude mice model, co-treatment displayed more efficient inhibition of tumor weight and volume on SW480 xenograft mouse model than single-agent treatment with OXA or OMT. Immunohistochemistry analysis suggests the combinations greatly suppressed tumor proliferation, which consistent with our in vitro results. In conclusion, our findings highlight that the combination therapy with OMT and OXA exerted synergistic antitumor effects in colon cancer cells through PI3K/AKT/mTOR pathway and combination treatment with OMT and OXA would be a promising therapeutic strategy for colon carcinoma treatment.     

Cd24a和前列腺素代谢相关酶在PCOS小鼠模型卵巢颗粒细胞中表达水平的实验研究

目的:探讨Cd24a分子和前列腺素代谢相关酶在多囊卵巢综合征(polycystic ovary syndrome, PCOS)小鼠模型卵巢颗粒细胞中的表达水平。方法:取20只雌性C57BL/6小鼠,随机分为对照组(正常小鼠)和实验组(应用脱氢表雄酮建立PCOS小鼠模型),每组各10只。对照组小鼠于颈背部连续皮下注射20天芝麻油溶液(0.1 m L/100 g)。实验组应用脱氢表雄酮(6 mg/100 g)联合芝麻油溶液(0.1 m L/100 g)连续颈背部皮下注射20天。经苏木精-伊红染色观察两组小鼠卵巢组织病理学改变。应用实时荧光定量PCR法对小鼠卵巢颗粒细胞中Cd24a分子和前列腺素代谢相关酶m RNA表达量进行检测。结果:实验组体重显著高于对照组(P0.05);实验组小鼠卵巢呈多囊样改变,卵泡中颗粒细胞数量减少,闭锁卵泡增多,闭锁卵泡直径明显大于对照组;实验组Cd24a分子和前列腺素代谢相关酶m RNA表达量较对照组存在显著差异(P0.05)。结论:PCOS小鼠卵巢颗粒细胞中Cd24a分子和前列腺素代谢相关酶表达量异常,提示Cd24a分子可能与PCOS疾病发生相关。     

Downregulation of lncRNA ZFAS1 and upregulation of microRNA-129 repress endocrine disturbance,increase proliferation and inhibit apoptosis of ovarian granulosa cells in polycystic ovarian syndrome by downregulating HMGB1

ObjectiveThe objective was to find the role of long-non-coding RNA zinc finger antisense 1 (lncRNA ZFAS1)/microRNA (miR)-129/high-mobility group box protein 1 (HMGB1) axis in polycystic ovary syndrome (PCOS).MethodsOvarian granulosa cells from non-PCOS patients and PCOS patients were collected, and HMGB1, miR-129 and lncRNA ZFAS1 expression were detected. Ovarian granulosa cells were transfected with si-ZFAS1 or miR-129 mimics to verify their roles in P4 and E2 secretion, and the biological functions of ovarian granulosa cells.ResultsLncRNA ZFAS1 and HMGB1 were elevated, while miR-129 was down-regulated in ovarian granulosa cells of PCOS patients. Down-regulated lncRNA ZFAS1 or overexpressed miR-129 could decrease HMGB1 expression, increase P4 and E2 secretion, promote proliferation activity while inhibit apoptosis of ovarian granulosa cells in PCOS.ConclusionLncRNA ZFAS1 could bind to miR-129 to promote HMGB1 expression, thereby affecting the endocrine disturbance, proliferation and apoptosis of ovarian granulosa cells in PCOS.     

Dehydroepiandrosterone stimulates inflammation and impairs ovarian functions of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in child-bearing-age women. It is characterized by ovulation dysfunction, polycystic ovaries, and hyperandrogenism. Inflammation is likely to be a crucial contributor to the pathogenesis of PCOS. However, the association between the inflammatory cytokines and the development of PCOS has not been reported. To explore the relationship between the inflammatory cytokines and PCOS, alterations of serum proteins in dehydroepiandrosterone (DHEA)-induced PCOS rats were screened by protein array, and the concentration of IFN-γ was further measured by using enzyme-linked immunosorbent assay (ELISA). DHEA-induced PCOS rats had a decreased level of IFN-γ compared with the control rats, which was restored partly in flutamide (an androgen receptor antagonist)-treated rats. Moreover, the level of IFN-γ in serum of patients with PCOS was also lower than that in healthy women. Using the ovarian granulosa cells (KGN), we demonstrated that DHEA downregulated the expression and secretion of IFN-γ in dose- and time-dependent manners, which could be restored to some extent by treating with flutamide. Furthermore, flutamide ameliorated the inhibitory effect on cell proliferation and promotive effect on cell apoptosis by DHEA. The results also revealed that IFN-γ promoted the proliferation but inhibited the apoptosis of KGN cells, which was suppressed by DHEA via activating the downstream PI3K/AKT signaling pathway. Taken together, these results showed that DHEA inhibited the proliferation and promoted the apoptosis of ovarian granulosa cells through downregulating the expression of IFN-γ which could be restored by flutamide, and IFN-γ may serve as a potential inflammatory biomarker for PCOS detection.     

miR-506与PI3K/AKT通路在自发性高血压大鼠心脏重构中的作用

目的:探讨mi R-506和PI3K/AKT信号通路在自发性高血压大鼠心脏重构中的作用。方法:将12只雄性自发性高血压大鼠(Spontaneous Hypertension Rat, SHR)随机分为2组,每组6只。分别为SHR模型组和治疗组(卡托普利,30 mg·kg~(-1)),6只健康WKY大鼠作为空白对照组。SHR模型组和空白对照组灌胃等体积生理盐水,连续给药8周,采用尾动脉测压法测定给药前后各组大鼠血压,采用qRT-PCR法检测各组大鼠心肌miR-506表达量,并检测大鼠心肌组织中SOD和GPx mRNA表达水平,免疫印迹检测大鼠心肌中p-PI3K和p-AKT的蛋白表达量。结果:SHR模型组血压为(184.79±3.35)mmHg,与空白对照组比较显著升高(P0.05),治疗组血压为(133.57±1.43)mm Hg,与SHR模型组相比均显著降低(P0.05)。SHR模型组大鼠心肌中mi R-506、SOD、GPx的RNA相对表达量分别为(0.36±0.05)、(0.27±0.04)和(0.32±0.02),与空白对照组比较显著降低(P0.05),而p-PI3K、p-AKT蛋白水平显著降低(P0.05),与SHR模型组比较,治疗组大鼠心肌中mi R-506以及SOD、GPx的RNA水平显著升高(P0.05),p-PI3K、p-AKT蛋白水平显著升高(P0.05)。结论:在卡托普利治疗高血压的过程中,mi R-506可能通过抑制PI3K/AKT信号通路提高机体的抗氧化能力促进SHR心脏重塑。     

RHCG基因在非小细胞肺癌中的表达及对细胞增殖的影响

为了探讨Rh type C glycoprotein (RHCG)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞增殖的影响及可能的作用机制,本研究使用荧光定量PCR法检测12对NSCLC及癌旁组织样本中RHCG mRNA的表达水平及pcDNA3.1-RHCG质粒对A549细胞RHCG m RNA的表达;采用CCK-8法检测细胞增殖能力;运用PI染色法检测细胞周期;使用免疫印迹法检p-PI3K、PI3K、p-AKT以及AKT蛋白表达水平。本研究发现,与癌旁组织比较,NSCLC中RHCG m RNA表达水平明显降低。RHCG过表达能抑制NSCLC细胞系A549细胞增殖能力。此外,RHCG过表达使A549细胞周期G1/S期转化发生阻滞。本研究还发现,RHCG过表达可下调A549细胞p-PI3K/PI3K和p-AKT/AKT水平。本研究表明,RHCG抑制NSCLC细胞增殖的作用与其抑制PI3K/AKT信号通路有关。     

Dexmedetomidine alleviates hepatic ischaemia-reperfusion injury via the PI3K/AKT/Nrf2-NLRP3 pathway

Hepatic ischaemia-reperfusion (I/R) injury constitutes a tough difficulty in liver surgery. Dexmedetomidine (Dex) plays a protective role in I/R injury. This study investigated protective mechanism of Dex in hepatic I/R injury. The human hepatocyte line L02 received hypoxia/reoxygenation (H/R) treatment to stimulate cell model of hepatic I/R. The levels of pyroptosis proteins and inflammatory factors were detected. Functional rescue experiments were performed to confirm the effects of miR-494 and JUND on hepatic I/R injury. The levels of JUND, PI3K/p-PI3K, AKT/p-AKT, Nrf2, and NLRP3 activation were detected. The rat model of hepatic I/R injury was established to confirm the effect of Dex in vivo. Dex reduced pyroptosis and inflammation in H/R cells. Dex increased miR-494 expression, and miR-494 targeted JUND. miR-494 inhibition or JUND upregulation reversed the protective effect of Dex. Dex repressed NLRP3 inflammasome by activating the PI3K/AKT/Nrf2 pathway. In vivo experiments confirmed the protective effect of Dex on hepatic I/R injury. Overall, Dex repressed NLRP3 inflammasome and alleviated hepatic I/R injury via the miR-494/JUND/PI3K/AKT/Nrf2 axis.     

Autophagy participates in the protection role of 1,25-dihydroxyvitamin D3 in acute myocardial infarction via PI3K/AKT/mTOR pathway

Vitamin D deficiency is associated with acute myocardial infarction (AMI); thus we aimed to explore improvement effects of 1,25-dihydroxyvitamin D3 (VD3) on the AMI and its potential mechanism. AMI models were constructed using male C57/BL6J mice and randomly treated with normal saline or VD3, using sham rats as control. Heart functions, myocardial damage, apoptosis, and inflammation were evaluated. Cardiomyocytes isolated from 3-day-old suckling mice were used for in vitro verification. After VD3 treatment, AMI-induced cardiac dysfunction was reversed with better cardiac function parameters. VD3 treatment reduced inflammatory cell infiltration and myocardial infarction area accompanied by the reduction of inflammatory factors and myocardial infarction markers compared with the AMI group. VD3 treatment obviously alleviated AMI-induced myocardial apoptosis, along with Bcl-2 upregulation and downregulation of caspase-3, caspase-9, and Bax. Both in vivo and in vitro experiments revealed that VD3 enhanced the expression of LC3II and Beclin-1 and decreased soluble p62. Furthermore, VD3 enhanced the AMI-caused inhibition of PI3K, p-AKT, and p-mTOR expression, which was conversely reversed by the addition of 3-methyladenine in vitro. The study highlights the improvement effects of VD3 on cardiac functions. We proposed a potential mechanism that VD3 protects against myocardial damage, inflammation, and apoptosis by promoting autophagy through PI3K/AKT/mTOR pathway.     

Anti-tumor effect of carrimycin on oral squamous cell carcinoma cells in vitro and in vivo

Purpose: Carrimycin is a newly synthesized macrolide antibiotic with good antibacterial effect. Exploratory experiments found its function in regulating cell physiology, proliferation and immunity, suggesting its potential anti-tumor capacity. The aim of this study is to investigate the anti-tumor effect of carrimycin against human oral squamous cell carcinoma cells in vitro and in vivo.Methods: Human oral squamous cell carcinoma cells (HN30/HN6/Cal27/HB96 cell lines) were treated with gradient concentration of carrimycin. Cell proliferation, colony formation and migration ability were analyzed. Cell cycle and apoptosis were assessed by flow cytometry. The effect of carrimycin on OSCC in vivo was investigated in tumor xenograft models. Immunohistochemistry, western blot assay and TUNEL assays of tissue samples from xenografts were performed. The key proteins in PI3K/AKT/mTOR pathway and MAPK pathway were examined by western blot.Results: As the concentration of carrimycin increased, the proliferation, colony formation and migration ability of OSCC cells were inhibited. After treating with carrimycin, cell cycle was arrested in G0/G1 phase and cell apoptosis was promoted. The tumor growth of xenografts was significantly suppressed. Furthermore, the expression of p-PI3K, p-AKT, p-mTOR, p-S6K, p-4EBP1, p-ERK and p-p38 were down-regulated in vitro and in vivo.Conclusions: Carrimycin can inhibit the biological activities of OSCC cells in vitro and in vivo, and regulate the PI3K/AKT/mTOR and MAPK pathways.     

Activation of the PI3K/mTOR Pathway following PARP Inhibition in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive malignancy with limited treatment options. We previously found that PARP is overexpressed in SCLC and that targeting PARP reduces cell line and tumor growth in preclinical models. However, SCLC cell lines with PI3K/mTOR pathway activation were relatively less sensitive to PARP inhibition. In this study, we investigated the proteomic changes in PI3K/mTOR and other pathways that occur following PAPR inhibition and/or knockdown in vitro and in vivo. Using reverse-phase protein array, we found the proteins most significantly upregulated following treatment with the PARP inhibitors olaparib and rucaparib were in the PI3K/mTOR pathway (p-mTOR, p-AKT, and pS6) (p≤0.02). Furthermore, amongst the most significantly down-regulated proteins were LKB1 and its targets AMPK and TSC, which negatively regulate the PI3K pathway (p≤0.042). Following PARP knockdown in cell lines, phosphorylated mTOR, AKT and S6 were elevated and LKB1 signaling was diminished. Global ATP concentrations increased following PARP inhibition (p≤0.02) leading us to hypothesize that the observed increased PI3K/mTOR pathway activation following PARP inhibition results from decreased ATP usage and a subsequent decrease in stress response signaling via LKB1. Based on these results, we then investigated whether co-targeting with a PARP and PI3K inhibitor (BKM-120) would work better than either single agent alone. A majority of SCLC cell lines were sensitive to BKM-120 at clinically achievable doses, and cMYC expression was the strongest biomarker of response. At clinically achievable doses of talazoparib (the most potent PARP inhibitor in SCLC clinical testing) and BKM-120, an additive effect was observed in vitro. When tested in two SCLC animal models, a greater than additive interaction was seen (p≤0.008). The data presented here suggest that combining PARP and PI3K inhibitors enhances the effect of either agent alone in preclinical models of SCLC, warranting further investigation of such combinations in SCLC patients.     

LncRNA MEG3 contributes to adenosine-induced cytotoxicity in hepatoma HepG2 cells by downregulated ILF3 and autophagy inhibition via regulation PI3K-AKT-mTOR and beclin-1 signaling pathway

Adenosine is a promising cytotoxic reagent for tumors, long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been indicated to play critical roles in tumorigenesis, ILF3 has been recognized as a MEG3-binding protein, however, the roles of adenosine and MEG3 on hepatoma are still ambiguous. To clarify the effects of MEG3 on the adenosine-induced cytotoxicity in hepatoma, MEG3 and ILF3 lentivirus were transduced into human hepatoma HepG2 cells to stimulate overexpression of MEG3 (OE MEG3) and overexpression of ILF3 (OE ILF3), furthermore, ILF3 small interfering RNA (siRNA) was also applied to downregulate the expression of ILF3. In this study, autophagy was markedly inhibited by low concentration of adenosine, which present by not only inhibited transformation from LC3-I to LC3-II and autophagosomes formation, but also the elevation of mTOR and reduction of beclin-1 proteins. Furthermore, low concentration of adenosine also exerted marked cytotoxicity representing induced cell apoptosis together with reductions of cell viability and migration, which were also markedly enhanced by OE MEG3. Novelly and excitingly, adenosine markedly stimulated MEG3 expression, OE MEG3 markedly decreased the ILF3 expression in HepG2 cells, and the adenosine-induced autophagy inhibition, together with the ratio of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR were also boosted by OE MEG3. More interestingly, OE ILF3 increased autophagy, whereas downregulated ILF3, especially in the case of adenosine, led to marked autophagy inhibition by decreasing beclin-1. The present study demonstrates autophagy inhibition is involved in the adenosine-induced cytotoxicity in HepG2 cells, the cytotoxicity can be synergized by OE MEG3 via downregulated ILF3 to activate PI3K/Akt/mTOR and inactivate the beclin-1 signaling pathway. In conclusion, MEG3 and inhibition of autophagy might be potential targets for augmenting adenosine-induced cytotoxicity in hepatoma.     

优思悦对多囊卵巢综合征模型大鼠卵巢功能的影响及其机制研究

摘要 目的：探讨优思悦对硫酸脱氢表雄酮（dehydroepiandrosterone,DHEA）诱导的多囊卵巢综合征模型大鼠体内性激素：睾酮( testosterone,T)、雌二醇( estradiol,E2 )、促黄体生成素( luteinizing hormone,LH)及卵泡刺激素( follicle－stimulating hormone,FSH)及PI3K /AKT信号通路的的影响。方法：将60只雌性SD大鼠随机均分为3组,包括空白组、模型组及治疗组。空白组每日颈部皮下注射0.2 mL大豆油,其余各组每日颈部皮下注射DHEA 60 mg/kg+0.2 mL大豆油,持续注射35 d。造模第21 d时,每日上午制备大鼠阴道涂片,将其放置在显微镜下观察,根据细胞形态判定大鼠的动情周期,无动情周期规律视为造模成功。造模第36 d时,连续4 w对大鼠进行灌胃,每天一次。检测大鼠阴道分泌物的细胞形态、大鼠卵巢中卵泡发育的情况。放射免疫法测定大鼠血清中T、E2、LH、FSH的含量;RT-qPCR法检测大鼠卵巢组织中PI3K /AKT信号通路相关因子（IＲS-1、AKT-2、CSK-3β、GLUT-4 mRNA和PTEN mRNA）的表达变化。结果：空白组大鼠的动情周期有规律性,而模型组大鼠的动情周期失去规律性,且模型组大鼠卵巢中的卵泡呈囊状扩张,血清中T、LH水平明显升高（P<0.05）,同时卵巢组织中的IRS-1、AKT-2、CSK-3β、GLUT-4 mRNA表达减少（P<0.05）,PTEN mRNA表达增多（P<0.05）。与模型组比较,优思悦可改善PCOS模型大鼠动情周期,降低血清T、LH水平（P<0.05）,改善卵泡发育,减少囊状扩张卵泡的形成,上调IRS-1、AKT-2、CSK-3β、GLUT-4 mRNA表达量,下调PTEN mRNA 表达量。结论：优思悦可有效治疗DHEA诱导的PCOS模型大鼠,与卵巢中的PI3K /AKT信号通路相关因子的基因表达相关。