Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

Insensitivity of PI3K/Akt/GSK3 signaling in peripheral blood mononuclear cells of age-related macular degeneration patients

Our recent studies with cultured retinal pigment epithelium cells suggested that overexpression of interleukin 17 receptor C（IL-17RC）,a phenomenon observed in peripheral blood and chorioretinal tissues with age-related macular degeneration（AMD）,was associated with altered activation of phosphatidylinositide 3-kinase（PI3K）,Akt,and glycogen synthase kinase 3（GSK3）.We wondered whether or not altered PI3 K,Akt,and GSK3 activities could be detected in peripheral blood mononuclear cells（PBMC） obtained from AMD patients.In the patients＇ PBMC,absent or reduced serine-phosphorylation of GSK3α or GSK3β was observed,which was accompanied with increased phosphorylation of GSK3 substrates（e.g.CCAAT enhancer binding protein a,insulin receptor substrate 1,and TAU）,indicative of enhanced GSK3 activation.In addition,decreased protein mass of PI3K85α and tyrosinephosphorylation of PI3K50α was present in PBMC of the AMD patients,suggesting impaired PI3 K activation.Moreover,abnormally lowered molecular weight forms of Akt and GSK3 were detected in PBMC of the AMD patients.These data demonstrate that despite the presence of high levels of IL-17 RC,Wnt-3a and vascular endothelial growth factor,the PI3K/Akt/GSK3 signaling pathway is insensitive to these stimuli in PBMC of the AMD patients.Thus,measurement of PI3K/Akt/GSK3 expression and activity in PBMC may serve as a surrogate biomarker for AMD.

Regulatory Effects of Zuogui Pill on Apoptosis of Follicles in Rats Injured by 60Co-γRays Based on PI3K/Akt/m TOR Signaling Pathway

[Objectives]To explore the protective effects of Zuogui Pill on ^(60)Co-γ-ray-induced premature aging of rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.[Methods]Sixty sexually mature female SD rats were irradiated with ^(60)Co-γ-ray(6.0 Gy,LD 40)for 24 h at one time.These rats were randomly divided into model group,Progynova group[0.18(g·kg)/d],Progynova[0.09(g·kg)/d]+Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill high dose[23.625(g·kg)/d)]group,Zuogui Pill medium dose[9.45(g·kg)/d)]group and Zuogui Pill low dose[4.725(g·kg)/d]group.The administration(once a day)lasted 21 d.The rat serum[follicle-stimulating hormone(FSH),luteinizing hormone(LH)and estradiol(E_(2))]were detected by Enzyme-linked immunosorbent assay(ELISA).The morphological changes of ovary were observed by hematoxylin-eosin(HE)staining.The apoptosis rate of granulosa cells was detected by terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL).The protein expression of phosphorylated(p)-PI3K,p-Akt,p-mTOR,B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)in ovarian tissues were detected by Western blot.[Results]Compared with the normal group,the model group showed significant increase in the serum FSH(P<0.01),significant decrease in serum E_(2)(P<0.05),and decrease in the number of early follicles and luteum in the ovary(P<0.01).Besides,the apoptosis rate of granulosa cells increased significantly(P<0.01);the expression of p-PI3K,p-Akt,p-mTOR and Bcl-2 in ovarian tissue decreased significantly,while the expression of Bax increased significantly(P<0.01).Compared with the model group,the number of early follicles in the ovary increased and the apoptosis rate of granulosa cells decreased after intervention in each administration group.In addition,the protein expressions of p-PI3K,p-Akt,p-mTOR and Bcl-2 increased,while the expression of Bax decreased,especially in Progynova+Zuogui Pill high dose group,the differences were statistically significant(P<0.05,P<0.01).[Conclusions]Zuogui Pill may protect the radiation-injured ovary through activating the expression of PI3K/Akt/mTOR protein in ovarian tissue,increasing the amount of Bcl-2 protein and inhibiting the expression of Bax protein.

PI3K/AKT信号通路与辐射抵抗机制的研究概况

辐射抵抗的三个主要机制为内在辐射敏感性、肿瘤细胞增殖和乏氧。磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路是肿瘤细胞的存活通路之一,该通路的激活与辐射抵抗密切相关。该文就PI3K/AKT信号通路与辐射抵抗机制作一综述。

Tongxinluo Activates PI3K/AKT Signaling Pathway to Inhibit Endothelial Mesenchymal Transition and Attenuate Myocardial Fibrosis after Ischemia-Reperfusion in Mice

Objective To investigate the potential role of Tongxinluo(TXL)in attenuating myocardial fibrosis after myocardial ischemia-reperfusion injury(MIRI)in mice.Methods A MIRI mouse model was established by left anterior descending coronary artery ligation for 45 min.According to a random number table,66 mice were randomly divided into 6 groups(n=11 per group):the sham group,the model group,the LY-294002 group,the TXL group,the TXL+LY-294002 group and the benazepril(BNPL)group.The day after modeling,TXL and BNPL were administered by gavage.Intraperitoneal injection of LY-294002 was performed twice a week for 4 consecutive weeks.Echocardiography was used to measure cardiac function in mice.Masson staining was used to evaluate the degree of myocardial fibrosis in mice.Qualitative and quantitative analysis of endothelial mesenchymal transition(EndMT)after MIRI was performed by immunohistochemistry,immunofluorescence staining and flow cytometry,respectively.The protein expressions of platelet endothelial cell adhesion molecule-1(CD31),α-smoth muscle actin(α-SMA),phosphatidylinositol-3-kinase(PI3K)and phospho protein kinase B(p-AKT)were assessed using Western blot.Results TXL improved cardiac function in MIRI mice,reduced the degree of myocardial fibrosis,increased the expression of CD31 and inhibited the expression ofα-SMA,thus inhibited the occurrence of EndMT(P<0.05 or P<0.01).TXL significantly increased the protein expressions of PI3K and p-AKT(P<0.05 or P<0.01).There was no significant difference between TXL and BNPL group(P>0.05).In addition,the use of the PI3K/AKT pathway-specific inhibitor LY-294002 to block this pathway and combination with TXL intervention,eliminated the protective effect of TXL,further supporting the protective effect of TXL.Conclusion TXL activated the PI3K/AKT signaling pathway to inhibit EndMT and attenuated myocardial fibrosis after MIRI in mice.

Liposomalα-cyperone targeting bone resorption surfaces suppresses osteoclast differentiation and osteoporosis progression via the PI3K/Akt axis

Osteoporosis is a metabolic dysregulation of bone that occurs mainly in postmenopausal women,and the hyperfunction of osteoclasts is the primary contributor to postmenopausal osteoporosis.However,the development of effective therapeutic drugs and precise delivery systems remains a challenge in the field of anti-absorption therapy.Here,we reported theα-cyperone(α-CYP)for anti-osteoporosis and developed a liposome-based nano-drug delivery system ofα-CYP,that specifically targets the bone resorption interface.Firstly,we found that theα-CYP,one of the major sesquiterpenes of Cyperus rotundus L.,attenuated the progression of osteoporosis in ovariectomized(OVX)mice and down-regulated the expression of phosphorylated proteins of phosphoinositide 3-kinase(PI3K)and protein kinase B(Akt),causing down-regulation of osteoclast-related genes/proteins and curbing osteoclast differentiation.Furthermore,α-CYP reversed the activation of osteoclastic differentiation and enhanced osteoporosis-related proteins expression caused by PI3K/Akt agonist(YS-49).More importantly,we adopted the osteoclastic resorption surface targeting peptide Asp8 and constructed the liposome(lipαC@Asp8)to deliverα-CYP to osteoclasts and confirmed its anti-osteoporosis effect and enhanced osteoclast inhibition by blocking PI3K/Akt axis.In conclusion,this study demonstrated thatα-CYP inhibits osteoclast differentiation and osteoporosis development by silencing PI3K/Akt pathway,and the liposome targeting delivery systems loaded withα-CYP might provide a novel and effective strategy to treat osteoporosis.

Heat shock protein 90 protects rat mesenchymal stem cells against hypoxia and serum deprivation-induced apoptosis via the PI3K/Akt and ERK1/2 pathways

Mesenchymal stem cell （MSC） transplantation has shown a therapeutic potential to repair the ischemic and infracted myocardium, but the effects are limited by the apoptosis and loss of donor cells in host cardiac microenvironment. The aim of this study is to explore the cytoprotection of heat shock protein 90 （Hsp90） against hypoxia and serum deprivation-induced apoptosis and the possible mechanisms in rat MSCs. Cell viability was determined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Apoptosis was assessed by Hoechst 33258 nuclear staining and flow cytometric analysis with annexin V/PI staining. The gene expression of Toll-like receptor-4 （TLR-4） and V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 （ErbB2） was detected by real-time poly- merase chain reaction （PCR）. The protein levels of cleaved caspase-3, Bcl-2, Bcl-xL, Bax, totaI-ERK, phospho-ERK, totaI-Akt, phospho-Akt, and Hsp90 were detected by Western blot. The production of nitric oxide was measured by spectrophotometric assay. Hsp90 improves MSC viability and protects MSCs against apoptosis induced by serum deprivation and hypoxia. The protective role of Hsp90 not only elevates Bcl-2/Bax and Bcl-xL/Bax expression and attenuates cleaved caspase-3 expression via down-regulating membrane TLR-4 and ErbB2 receptors and then ac- tivating their downstream PI3K/Akt and ERK1/2 pathways, but also enhances the paracrine effect of MSCs. These findings demonstrated a novel and effective treatment strategy against MSC apoptosis in cell transplantation.

Epidermal growth factor upregulates Skp2/Cks1 and p27^(kip1) in human extrahepatic cholangiocarcinoma cells

AIM:To evaluate the expression status of S-phase kinase-associated protein 2(Skp2)/cyclin-dependent kinases regulatory subunit 1(Cks1)and p27kip1,and assess the prognostic significance of Skp2/Cks1 expression with p27kip1in patients with extrahepatic cholangiocarcinoma.METHODS:Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December1994 to March 2008 were enrolled.Immunohistochemical staining for Skp2,Cks1,p27kip1,and Ki67,along with other relevant molecular biologic experiments,were performed.RESULTS:By Cox regression analyses,advanced age(>65 years),advanced AJCC tumor stage,poorly differentiated histology,and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma.Exogenous epidermal growth factor(EGF,especially 0.1-10 ng/mL)significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1in SNU-1196,SNU-1079,and SNU-245 cells.The protein levels of Skp2/Cks1(from nuclear lysates)and p27kip1(from cytosolic lysate)were also significantly increased in these cells.There were significant reductions in the protein levels of Skp2/Cks1and p27kip1(from nuclear lysate)after the treatment of LY294002.By chromatin immunoprecipitation assay,we found that E2F1 transcription factor directly binds to the promoter site of Skp2.CONCLUSION:Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma.EGF upregulates the mRNA and protein levels of Skp2/Cks1and p27kip1via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.

PI 3K/Akt/mTOR信号通路介导艾灸改善大鼠卵巢早衰的研究

目的:探讨艾灸对雷公藤多苷片诱导的卵巢早衰大鼠卵巢的保护作用及机制。方法:雌性SD大鼠随机分为空白组、模型组、艾灸组,每组15只。除空白组外,模型组和艾灸组均采用雷公藤多苷片(40mg/kg)灌胃,每日1次,连续6周;灌胃第4周给予艾灸组悬灸"关元"及双侧"三阴交"10min,每日1次,治疗3周。HE染色观察卵巢组织形态结构;ELISA法检测血清中雌二醇(E2)、促黄体生成素(LH)、促卵泡生成素(FSH)等激素水平,白介素-6(IL-6)、IL-1β等炎性因子含量;Western blot法测定卵巢组织中磷脂酰肌醇3-激酶(PI 3K)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)含量及磷酸化水平。结果:与空白组相比,模型组卵巢内卵泡减少,卵泡的粒细胞层次明显减少,黄体减少,闭锁卵泡明显增多,血清中E2显著降低(P<0.01),LH、FSH、IL-6、IL-1β含量均显著增加(P<0.01),卵巢组织中p-PI 3K、p-Akt和p-mTOR蛋白表达显著增加(P<0.01);与模型组相比,艾灸组卵巢组织病理损伤改善,艾灸能够显著降低血清中LH(P<0.05)、FSH(P<0.01)、IL-6(P<0.01)、IL-1β(P<0.01)含量,增加E2含量(P<0.05),降低卵巢组织磷酸化PI 3K、Akt、mTOR的表达水平(P<0.01)。结论:艾灸能够改善雷公藤多苷片诱导的大鼠卵巢早衰,其机制可能与抑制PI 3K/Akt/mTOR信号通路磷酸化相关。

Elucidation of the hepatoprotective effect and mechanism of Melastoma dodecandrum Lour. based on network pharmacology and experimental validation

Objective:To systematically explore the effect and mechanism of melastomatis dodecandri herba(Melastoma dodecandrum Lour.)in the treatment of hepatitis based on network pharmacology.Method:We evaluated the hepatoprotective effects of M.dodecandrum in concanavalin A(Con A)-induced hepatitis in mice by assessing survival rate,histological analysis,serum transaminases,and related cytokines.Then the mechanism of action was predicted by a network pharmacology-based strategy.Based on the results,we measured the hepatic expression of related genes at mRNA level and proteins related to the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)and nuclear factorkappa B(NF-кB)pathways.Results:Our study results clearly demonstrated that M.dodecandrum pretreatment significantly alleviated liver injury.This was demonstrated by an increase in survival rate,decreased severity of liver damage,and reduced serum transaminase levels compared with those in the Con A group.Moreover,M.dodecandrum significantly reduced the serum levels of tumor necrosis factor-a,interleukin-6,and interferon-g and increased the liver levels of superoxide dismutase,which indicated that M.dodecandrum exhibits anti-inflammatory and antioxidant activities.On the basis of network pharmacology,50 nodes were selected as major hubs based on their topological importance.Pathway enrichment analyses indicated that the putative targets of M.dodecandrum mostly participate in various pathways associated with the anti-inflammation response,which implies the underlying mechanism by which M.dodecandrum acts on hepatitis.Real-time fluorescent quantitative PCR analysis showed that M.dodecandrum downregulates the mRNA expression of interleukin-6,Toll-like receptor 7,interleukin-1 receptor-associated kinase-4,NF-кB and tumor necrosis factor-a in liver tissues.Western blotting showed that M.dodecandrum pretreatment protected against inflammation through activating the PI3K-Akt pathway by upregulating phosphorylated Akt(p-Akt)expression and suppressing NF-кB activation by inhibiting the phosphorylation of IKK,IkBa,and p65.Conclusion:The present work demonstrated the hepatoprotective effects of M.dodecandrum by regulating the PI3K/Akt and NF-кB pathways in Con A-induced mice,which provide insights into the treatment of hepatitis using M.dodecandrum.

Novel nervous and multi-system regenerative therapeutic strategies for diabetes mellitus with mTOR

Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.

磷脂酰肌醇3-激酶/蛋白激酶B磷酸化是胰岛素受体后信号转导通路控制PC12细胞凋亡的机制

目的:研究胰岛素抵抗1-甲基-4苯基砒啶(MPP+)诱导的PC12细胞凋亡的信号转导途径中,磷脂酰肌醇3-激酶/蛋白激酶B(PI3-K/PKB)的活力变化。方法:应用Wortmannin(PI3-K抑制剂),比较用药前后细胞生存率的变化;应用Western印迹分析检测此间PKB及其磷酸化水平的变化。结果:Wortmannin预处理组细胞生存率较之胰岛素干预组明显下降;PKB的特异性磷酸化程度(Ser473磷酸化程度/激酶蛋白量)与细胞生存率的变化有关。结论:胰岛素主要通过调节PI3-K活性后再促进PKB的磷酸化,从而促进PKB的激活,并导致生物学效应的变化,但是尚不能排除其他机制的参与。

低氧诱导因子-1的转录活性调控及其信号传导

低氧诱导因子 1(hypoxia induciblefactor 1,HIF 1)是氧平衡调控相关的转录因子 .依赖HIF 1的基因表达调控系统广泛影响葡萄糖代谢、细胞增殖、凋亡和血管发生 ,与机体低氧适应、胚胎发育、各种缺血性疾病及肿瘤相关 .HIF 1自身活性调节是低氧应答基因表达调控的中心环节 .调控主要发生在源于Ras的两条信号途径 :Ras/Raf/MEK介导的HIF 1反式激活功能调控 ,PI(3)K/Akt依赖的HIF 1alpha蛋白稳定性调控 .这两个信号传导途径分别独立又协调地调控着HIF

Transcriptomic landscape regulated by the 14 types of bone morphogenetic proteins(BMPs)in lineage commitment and differentiation of mesenchymal stem cells(MSCs)

Mesenchymal stem cells(MSCs)are ubiquitously-existing multipotent progenitors that can self-renew and differentiate into multiple lineages including osteocytes,chondrocytes,adipocytes,tenocytes and myocytes.MSCs represent one of the most commonly-used adult progenitors and serve as excellent progenitor cell models for investigating lineagespecific differentiation regulated by various cellular signaling pathways,such as bone morphogenetic proteins(BMPs).As members of TGFb superfamily,BMPs play diverse and important roles in development and adult tissues.At least 14 BMPs have been identified in mammals.Different BMPs exert distinct but overlapping biological functions.Through a comprehensive analysis of 14 BMPs in MSCs,we demonstrated that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of MSCs.Nonetheless,a global mechanistic view of BMP signaling in regulating the proliferation and differentiation of MSCs remains to be fully elucidated.Here,we conducted a comprehensive transcriptomic profiling in the MSCs stimulated by 14 types of BMPs.Hierarchical clustering analysis classifies 14 BMPs into three subclusters:an osteo/chondrogenic/adipogenic cluster,a tenogenic cluster,and BMP3 cluster.We also demonstrate that six BMPs(e.g.,BMP2,BMP3,BMP4,BMP7,BMP8,and BMP9)can induce ISmads effectively,while BMP2,BMP3,BMP4,BMP7,and BMP11 up-regulate Smad-independent MAP kinase pathway.Furthermore,we show that many BMPs can upregulate the expression of the signal mediators of Wnt,Notch and PI3K/AKT/mTOR pathways.While the reported transcriptomic changes need to be further validated,our expression profiling represents the first-of-its-kind to interrogate a comprehensive transcriptomic landscape regulated by the 14 types of BMPs in MSCs.

头部浅低温联合胰岛素样生长因子-1对大鼠全脑缺血再灌注损伤的影响

目的 探讨头部浅低温联合胰岛素样生长因子-1（IGF-1）对大鼠全脑缺血再灌注损伤的影响，研究PI3K/Akt信号通路在脑缺血再灌注损伤中的作用机制。方法 健康雄性SD大鼠60只，体质量250~280 g，随机分为五组（n=12）：假手术组（S组），全脑缺血再灌注组（C组），浅低温处理组（H组），IGF-1处理组（I组），浅低温联合IGF-1处理组（HI组）。C组、H组、I组和HI组应用改良Pulsinelli四血管阻断法制备大鼠全脑缺血再灌注模型，缺血时间15 min；H组和HI组进行鼻咽温降温将海马温度降至（33.0±0.5）℃；I组和HI组在再灌注后1 h经尾静脉缓慢推注IGF-1 5 μg。缺血再灌注8 h后每组取6只大鼠用免疫组化法测定海马CA1区pAkt、pFoxO3a、Bcl-2和Bax蛋白表达情况；每组另6只大鼠用Western blot方法检测海马pAkt和总Akt蛋白表达情况。结果 与S组比较，C组pAkt、pFoxO3a、Bcl-2蛋白及Bax蛋白表达均增加；与C组比较，H组、I组和HI组pAkt、pFoxO3a及Bcl-2蛋白表达均增加，Bax蛋白表达均减少；HI组Bax表达较H组和I组显著减少，而pAkt、 pFoxO3a和Bcl-2蛋白表达显著增加（P〈0.05）。Western blot：与S组比较, C组pAkt水平升高；与C组比较，H组、I组和HI组pAkt蛋白表达均增加，HI组pAkt蛋白表达显著高于其他组（P〈0.05）；各组总Akt蛋白表达比较差异无统计学意义（P〉0.05）。结论 头部浅低温联合IGF-1更能减轻大鼠全脑缺血再灌注损伤，其机制可能是通过上调Akt磷酸化，进一步磷酸化下游底物，从而抑制神经元凋亡发挥脑保护作用。

Apelin protects against cardiomyocyte apoptosis induced by glucose deprivation

Background Apoptosis is a major cause of ischemic heart dysfunction. Apelin, the endogenous ligand for the G-protein-coupled APJ receptor, has been reported to exert cardioprotective effects during myocardial injury. The aim of this study was to investigate the effects of apelin on apoptosis of rat cardiomyocytes induced by glucose deprivation （GD） and study the related signaling pathway. Methods Apelin and APJ mRNA expression were determined by RT-PCR in neonatal rat cardiomyocytes during different durations of GD. Cardiomyocyte apoptosis was detected by annexin V-FITC/propidium iodide （PI） staining after GD for 12 hours with or without apelin-13 （10 and 100 nmol/L） pretreatment. Protein levels of Akt and the mammalian target of rapamycin （mTOR） as well as cell apoptosis were detected in the presence or absence of LY294002 （a phosphatidylinositol 3-kinases （PI3K） inhibitor） or rapamycin （a mTOR inhibitor）. Results Apelin mRNA expression was up-regulated when cardiomyocytes were exposed to GD for 6, 12, 18, and 24 hours compared with the base level （P 〉0.05, P 〈0.01, P 〈0.01, P 〈0.01）. However, when cardiomyocytes were exposed to GD for up to 36 hours, apelin mRNA expression was 17% lower than the base level （P〈0.05）. APJ mRNA expression paralleled that of apelin. Apelin-13 pretreatment at 100 nmol/L significantly inhibited GD-induced cardiomyocyte apoptosis （P 〈0.05） and increased Akt and mTOR phosphorylation （P 〈0.01, P 〈0.01）. At the same time apelin-13 （100 nmol/L） up-regulated Bcl-2 protein expression and down-regulated Bax and cleaved caspase-3 expression （P 〈0.01, P 〈0.05, P 〈0.05）. The anti-apoptotic effect of apelin-13 was blocked by LY294002 （P 〈0.01） but not by rapamycin. Conclusions The endogenous apelin-APJ system is compensatorily up-regulated and ultimately down-regulated following sustained myocardial ischemia. Apelin protects against ischemic cardiomyocyte apoptosis via activation of the PI3K/Akt pathway.

淫羊藿素对多囊卵巢综合征颗粒细胞凋亡和自噬的影响

目的基于磷脂酰肌醇-3激酶(Phospoinositide 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)通路探讨淫羊藿素对多囊卵巢综合征颗粒细胞凋亡和自噬的影响。方法分离多囊卵巢综合征大鼠卵巢颗粒细胞,分成对照组、低、中、高剂量实验组(15,30,60μmol·L^(-1)淫羊藿素)和抑制剂组(60μmol·L^(-1)淫羊藿素和PI3K/Akt通路抑制剂LY294002处理)。以蛋白质印迹法检测PI3K/Akt通路关键蛋白、凋亡相关蛋白B细胞淋巴瘤/白血病-2(B cell lymphoma/lewkmia-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)和自噬相关蛋白Beclin1、LC3Ⅱ/LC3Ⅰ表达变化,以细胞计数法-8(CCK-8)实验测定细胞增殖活性,以流式细胞术测定细胞凋亡。结果对照组、低、中、高剂量实验组和抑制剂组细胞增殖活性(OD值)分别为0.41±0.05、0.63±0.06、0.75±0.08、0.88±0.06、0.69±0.06,LC3Ⅱ/LC3Ⅰ表达量分别为1.76±0.15、1.45±0.11、1.12±0.07、1.05±0.09、1.32±0.13,细胞凋亡率分别为(42.65±6.32)%,(37.01±3.24)%,(26.47±2.11)%,(19.84±1.40)%,(35.12±0.30)%,p-PI3K表达量分别为0.35±0.05,0.48±0.05,0.68±0.07,0.89±0.09,0.41±0.06,PI3K表达量分别为0.94±0.11,0.94±0.09,0.92±0.11,0.87±0.12,0.90±0.09,以上指标,对照组与低、中、高剂量实验组比较,差异均有统计学意义(均P<0.05);低、中、高剂量实验组间比较,差异均有统计学意义(均P<0.05);高剂量实验组与抑制剂组比较,差异均有统计学意义(P<0.05)。结论淫羊藿素通过激活PI3K/Akt通路抑制多囊卵巢综合征颗粒细胞凋亡和自噬。

Effects of epoxyeicosatrienoic acids on levels of eNOS phosphorylation and relevant signaling transduction pathways involved

Endothelial nitric oxide synthase (eNOS) is a key enzyme responsible for the regu-lation of vascular homeostasis. Many humor factors and mechanical forces can affect eNOS ac-tivity via phosphorylation modification but the mechanisms involved vary with stimuli applied. We have demonstrated that cytochrome P450 (CYP) epoxygenase-dependent metabolites of ara-chidonic acid, epoxyeicosatrienoic acids (EETs), can robustly up-regulate eNOS expression and its activity, however the relevant signaling pathways responsible for activity regulation are not well known. In this study, we explored the role of PI3 kinase (PI3K)/protein kinase B (Akt) sig-naling pathway in eNOS expression and its phosphorylation in response to EETs via direct addi-tion of EETs into cultured bovine aorta endothelial cells (BAECs) and recombinant adeno- asso-ciated virus-mediated transfection of CYP epoxygenase genes CYPF87V and CYP2C11 to pro-duce endogenous EETs followed by co-treatment with PI3K or Akt inhibitor. Results show that both exogenous and endogenous EETs could remarkably enhance eNOS expression and its phosphorylation at Ser1179 and Thr497 residues; PI3K inhibitor LY294002 could inhibit EETs-induced increase in eNOS-Ser(P)1179 but had no effect on the change of eNOS-Thr(P)497, while Akt inhibitor could attenuate the increase in phosphor-eNOS at both residues; both of the two inhibitors could block EETs-enhanced eNOS expression. These results lead to conclusions: (i) EETs-mediated regulation of eNOS activity may be related with the changes of phosphoryla-tion level at eNOS-Ser1179 via PI3K/Akt and eNOS-Thr497 via Akt; (ii) PI3K/Akt signaling pathway is involved in the up-regulation of eNOS expression by EETs.

Targeting the complement system in pancreatic cancer drug resistance:a novel therapeutic approach

Pancreatic cancer is ranked as the fourth leading cause of cancer-related mortality and is predicted to become the second leading cause of cancer-related death by 2030.The cause of this high mortality rate is due to pancreatic ductal adenocarcinoma’s rapid progression and metastasis,and development of drug resistance.Today,cancer immunotherapy is becoming a strong candidate to not only treat various cancers but also to combat against chemoresistance.Studies have suggested that complement system pathways play an important role in cancer progression and chemoresistance,especially in pancreatic cancer.A recent report also suggested that several signaling pathways play an important role in causing chemoresistance in pancreatic cancer,major ones including nuclear factor kappa B,signal transducer and activator of transcription 3,c-mesenchymal-epithelial transition factor,and phosphoinositide-3-kinase/protein kinase B.In addition,it has also been proven that the complement system has a very active role in establishing the tumor microenvironment,which would aid in promoting tumorigenesis,progression,metastasis,and recurrence.Interestingly,it has been shown that the downstream products of the complement system directly upregulate inflammatory mediators,which in turn activate these chemo-resistant pathways.Therefore,targeting complement pathways could be an innovative approach to combat against pancreatic cancer drugs resistance.In this review,we have discussed the role of complement system pathways in pancreatic cancer drug resistance and a special focus on the complement as a therapeutic target in pancreatic cancer.