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.

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.

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.

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.

Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats

AIM:To determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt,also known as PKB) pathway. METHODS:Rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane.RESULTS:After Wistar rats were fed with high-fat diet for 16 wk,their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7,P < 0.01),deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%,P < 0.05),and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%,P < 0.05). In addition,the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced,as was the expression of the anti-apoptotic protein Bcl-2,while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt,instead of high-fat diet,a similar pattern of hepatocellular fat accumulation,mitochondrial impairment,and change in the levels of PI3K,Akt,Bcl-2 was observed. CONCLUSION:High-fat diet appears to inhibit the PI3K/Akt signaling pathway,which may lead to hepa-tocellular injury through activation of the mitochondrial membrane pathway of apoptosis.

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.

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.

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.

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。

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.