藏红花素通过PI3K/Akt/GSK-3β通路诱导人乳腺癌细胞凋亡的初步研究

目的:探讨藏红花素对人乳腺癌细胞凋亡的影响及相关调控机制。方法:以人正常乳腺MCF-10A细胞和人乳腺癌MCF-7细胞为受试细胞,分别以不同浓度藏红花素0(空白对照组)、200、400、800 mg/L和LY294002(PI3K特异性抑制剂)15 mg/L干预对数生长期MCF-10A细胞和MCF-7细胞48 h,采用四甲基偶氮唑蓝(MTT)法、克隆形成实验分别检测各组细胞增殖抑制率、细胞克隆能力,Annexin V-FITC染色法检测细胞凋亡水平,运用蛋白免疫印迹法(Western blot)检测磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(protein kinase B,Akt)、磷酸化Akt(p-Akt)、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、磷酸化GSK-3β(p-GSK-3β)、半胱氨酸天门冬氨酸蛋白酶9(cysteine aspartic proteases-9,Caspase-9)、激活型半胱氨酸天门冬氨酸蛋白酶3(cleaved cysteine aspartate protease-3,Cleaved caspase-3)、B淋巴细胞瘤2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(bcl-2 related X protein,Bax)蛋白表达。结果:与空白对照组比较,200、400、800 mg/L藏红花素组和LY294002组MCF-10A细胞增殖抑制率、克隆数目、凋亡率,差异均无统计学意义(P>0.05);400、800 mg/L藏红花素组和LY294002组MCF-7细胞增殖抑制率升高、克隆数目降低、凋亡率升高(P<0.01);p-PI3K、p-Akt、p-GSK-3β、Bcl-2表达下调且Caspase-9、Cleaved Caspase-3、Bax表达上调(P<0.01),磷酸化率p-PI3K/PI3K、p-Akt/Akt、p-GSK-3β/GSK-3β降低(P<0.01),Bax/Bcl-2表达比值升高(P<0.01)。与LY294002组比较,800 mg/L藏红花素组MCF-7细胞增殖抑制率升高、克隆数目降低、凋亡率升高(P<0.05或P<0.01);Caspase-9、Cleaved Caspase-3、Bax表达上调(P<0.05或P<0.01),Bax/Bcl-2比值升高(P<0.01),其他指标两组间比较差异无统计学意义(P>0.05)。结论:藏红花素具有诱导人乳腺癌细胞凋亡的作用,其机制可能与抑制PI3K/Akt/GSK-3β信号通路有关。

中药复方金思维含药血清调控PI3K/AKT/GSK-3β信号通路对拟阿尔茨海默病细胞模型tau蛋白磷酸化的影响

目的探讨中药复方金思维含药血清对拟阿尔茨海默病(Alzheimer disease,AD)细胞模型tau蛋白磷酸化的影响及作用机制。方法采用Aβ25-35诱导人神经母细胞瘤细胞(SH-SY5Y)建立拟AD细胞模型,CCK-8法筛选金思维含药血清、PI3K/AKT/GSK-3β通路抑制剂LY294002最佳作用浓度。将SH-SY5Y细胞分为正常组、模型组、金思维低剂量组、金思维中剂量组、金思维高剂量组以及LY294002组。蛋白免疫印迹法检测金思维含药血清对SH-SY5Y细胞tau蛋白磷酸化以及PI3K/AKT/GSK-3β通路相关蛋白的影响。结果20μmol/L Aβ25-35作用于SH-SY5Y细胞24 h可建立最佳拟AD细胞模型。相对于正常组,模型组P-AKT显著降低(P<0.05),P-tau显著升高(P<0.01)。相对于模型组,金思维低剂量含药血清组PI3K的表达量显著升高(P<0.05),高剂量含药血清组GSK-3β的表达量显著降低(P<0.05),中剂量和高剂量含药血清组P-tau的表达量显著降低(P<0.01、P<0.001)。加入通路抑制剂LY294002后,P-PI3K、PI3K、P-AKT、AKT、GSK-3β、P-tau蛋白的表达量较模型组差异无统计学意义(P>0.05)。结论金思维含药血清可降低拟AD细胞模型tau蛋白磷酸化水平,其机制可能与PI3K/AKT/GSK-3β通路的激活有关。

化瘀消痞汤对大鼠萎缩性胃炎癌前病变及PI3K/Akt/GSK-3β通路的影响

目的探讨化瘀消痞汤对大鼠萎缩性胃炎癌前病变的作用以及对PI3K/Akt/GSK-3β通路的影响。方法60只SPF级SD雄鼠随机挑选10只为空白组,其余大鼠采用多因素造模法制备萎缩性胃炎癌前病变模型。造模成功后随机分为模型组,化瘀消痞汤高、中、低剂量组(24.8、12.4、6.2 g/kg),叶酸组(2 mg/kg),每组10只,连续给药90 d。造模及给药期间观察大鼠一般状况、记录体质量及3 h进食量;HE染色观察大鼠胃组织形态变化;ELISA法检测大鼠胃组织匀浆液Cyclin D1、c-Myc水平;免疫荧光法检测大鼠胃组织Ki67蛋白表达;Western blot法检测大鼠胃组织PI3K、p-Akt、p-GSK-3β、β-catenin、CDK4蛋白表达。结果与空白组比较,模型组大鼠萎靡倦怠,体质量、3 h进食量降低(P<0.05),胃黏膜明显变薄,腺体数量明显减少且排列紊乱,肠上皮化生明显,胃组织匀浆液Cyclin D1、c-Myc水平升高(P<0.05),胃组织Ki67、PI3K、p-Akt、β-catenin、CDK4蛋白表达升高(P<0.05),p-GSK-3β蛋白表达降低(P<0.05);与模型组比较,各给药组大鼠一般状况有所好转,体质量、3 h进食量增加(P<0.05),胃黏膜修复,腺体数量增多且排列趋于整齐,肠上皮化生减轻,胃组织匀浆液Cyclin D1、c-Myc水平降低,胃组织Ki67、PI3K、p-Akt、β-catenin、CDK4蛋白表达降低,p-GSK-3β蛋白表达升高,以化瘀消痞汤高、中剂量组更明显(P<0.05);与叶酸组比较,化瘀消痞汤高、中剂量组体质量、3 h进食量增加(P<0.05),胃黏膜形态和腺体数量逐渐趋于正常,胃组织匀浆液Cyclin D1、c-Myc水平降低(P<0.05),胃组织Ki67、PI3K、p-Akt、β-catenin、CDK4蛋白表达降低(P<0.05),高剂量组大鼠胃组织p-GSK-3β蛋白表达升高(P<0.05)。结论化瘀消痞汤可抑制萎缩性胃炎癌前病变大鼠胃上皮细胞异常增殖,改善胃黏膜损伤,其机制可能与调控PI3K/Akt/GSK-3β信号通路中关键因子表达有关。

山奈酚调节AKT/GSK-3β/Snail信号通路对鼻咽癌细胞增殖、凋亡和上皮间质转化的影响

目的:探讨山奈酚对鼻咽癌细胞增殖、凋亡和上皮间质转化(EMT)的影响,并探究其作用机制。方法:将CNE-2细胞分为对照组、山奈酚低浓度组、山奈酚中浓度组、山奈酚高浓度组、山奈酚高浓度+SC-79(AKT激活剂)组。CCK-8法和克隆实验测定细胞增殖,流式细胞术检测细胞凋亡,Western blot检测各蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、天冬氨酸蛋白水解酶-3(Caspase-3)、波形蛋白(Vimentin)、上皮钙黏蛋白(E-cadherin)、神经钙黏蛋白(N-cadherin)、蛋白激酶B(AKT)、磷酸化Akt(p-AKT)、糖原合成酶激酶3β(GSK-3β)、磷酸化GSK-3β(p-GSK-3β)、Snail的表达水平。构建鼻咽癌裸鼠模型,分为裸鼠NC组、山奈酚组、山奈酚+SC-79组,测量肿瘤质量与体积,免疫组化法检测移植瘤组织p-AKT、p-GSK-3β、Snail蛋白表达。结果:山奈酚对人鼻咽癌上皮细胞活力无显著影响(P>0.05);鼻咽癌细胞活力随着山奈酚浓度的升高而逐渐降低(P<0.05),选择CNE-2作为后续实验细胞,选择25、50、100μmol/L山奈酚作为后续实验浓度。与对照组相比,山奈酚低、中、高组细胞活力、Bcl-2、N-cadherin、Vimentin、p-AKT/AKT、p-GSK-3β/GSK-3β、Snail水平显著下降,凋亡率、Bax、Caspase-3、E-cadherin上升(P<0.05);与山奈酚高浓度组相比,山奈酚高浓度+SC-79组细胞活力、Bcl-2、N-cadherin、Vimentin、p-AKT/AKT、p-GSK-3β/GSK-3β、Snail水平显著上升,凋亡率、Bax、Caspase-3、E-cadherin下降(P<0.05)。山奈酚能抑制移植瘤质量和体积,降低p-AKT、p-GSK-3β、Snail蛋白表达(P<0.05);SC-79逆转山奈酚对移植瘤的抑制作用,促进AKT/GSK-3β/Snail通路蛋白表达(P<0.05)。结论:山奈酚能抑制鼻咽癌细胞增殖和EMT,促进细胞凋亡,其作用机制可能与抑制AKT/GSK-3β/Snail信号通路有关。

下调Ppp1r17对小鼠饮酒相关行为及AKT/GSK-3β/CREB信号通路磷酸化的影响

目的:观察下调蛋白磷酸酶1调节因子亚基17(Ppp1r17)对小鼠饮酒相关行为的作用,并分析其对蛋白激酶B(protein kinase B,PKB/AKT)/糖原合成酶激酶3β(glycogen synthase kinase-3β,GSK-3β)/cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)通路磷酸化的影响。方法:取40只雄性C57BL/6J小鼠随机分为4组(n=10):control组;shPpp1r17①组;shPpp1r17②组和shPpp1r17③组。给予AAV-shPpp1r173周后检测其在海马组织中的mRNA和蛋白表达水平。取20只雄性C57BL/6J小鼠随机分为control组和shPpp1r17组(n=10)。注射AAV-shPpp1r173周后进行旷场实验、条件性位置偏好实验和翻正反射实验,并检测AAV-shPpp1r17定位及蛋白表达情况。取20只雄性C57BL/6J小鼠分为4组(n=5):shNC+Water组、shNC+EtOH组、shPpp1r17+Water组和shPpp1r17+EtOH组,其中EtOH组小鼠稳定自主饮用9%酒精30 d。Western blot检测Ppp1r17、p-AKT、AKT、p-GSK-3β、GSK-3β、p-CREB和CREB蛋白表达情况。结果:(1)实时荧光定量PCR结果显示下调序列shPpp1r17①为最佳Ppp1r17下调序列。(2)行为学结果显示,shPpp1r17组小鼠以增强运动能力和减少焦虑样情绪为特征,Ppp1r17下调可以增加饮酒CPP分数,并降低小鼠对酒精的敏感性。(3)免疫荧光结果显示,shPpp1r17可以在海马脑区特异性表达。(4)Western blot结果显示,在慢性酒精暴露后,Ppp1r17蛋白表达、p-AKT/AKT、p-GSK-3β/GSK-3β和p-CREB/CREB的比值均显著增加。而在海马敲减Ppp1r17后,Ppp1r17蛋白表达显著降低,并且增强AKT/GSK-3β/CREB通路的活性。结论:Ppp1r17下调后可以增强酒精对小鼠的奖赏效应、增强小鼠的运动能力并降低小鼠对酒精的敏感性,其机制可能与激活AKT/GSK-3β/CREB信号通路有关。

Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway

BACKGROUND Development of end-stage renal disease is predominantly attributed to diabetic nephropathy(DN).Previous studies have indicated that myricetin possesses the potential to mitigate the pathological alterations observed in renal tissue.Never-theless,the precise molecular mechanism through which myricetin influences the progression of DN remains uncertain.AIM To investigate the effects of myricetin on DN and explore its potential therapeutic mechanism.METHODS Db/db mice were administered myricetin intragastrically on a daily basis at doses of 50 mg/kg or 100 mg/kg for a duration of 12 wk.Subsequently,blood and urine indexes were assessed,along with examination of renal tissue pathology.Kidney morphology and fibrosis were evaluated using various staining techniques including hematoxylin and eosin,periodic acid–Schiff,Masson’s trichrome,and Sirius-red.Additionally,high-glucose culturing was conducted on the RAW 264.7 cell line,treated with 25 mM myricetin or co-administered with the PI3K/Akt inhibitor LY294002 for a period of 24 h.In both in vivo and in vitro settings,quantification of inflammation factor levels was conducted using western blotting,real-time qPCR and ELISA.RESULTS In db/db mice,administration of myricetin led to a mitigating effect on DN-induced renal dysfunction and fibrosis.Notably,we observed a significant reduction in expressions of the kidney injury markers kidney injury molecule-1 and neutrophil gelatinase associated lipocalin,along with a decrease in expressions of inflammatory cytokine-related factors.Furthermore,myricetin treatment effectively inhibited the up-regulation of tumor necrosis factor-alpha,interleukin-6,and interluekin-1βinduced by high glucose in RAW 264.7 cells.Additionally,myricetin modulated the M1-type polarization of the RAW 264.7 cells.Molecular docking and bioinformatic analyses revealed Akt as the target of myricetin.The protective effect of myricetin was nullified upon blocking the polarization of RAW 264.7 via inhibition of PI3K/Akt activation using LY294002.CONCLUSION This study demonstrated that myricetin effectively mitigates kidney injury in DN mice through the regulation of macrophage polarization via the PI3K/Akt signaling pathway.

Hypoglycemic mechanism of Tegillarca granosa polysaccharides on type 2 diabetic mice by altering gut microbiota and regulating the PI3K-akt signaling pathwaye

Type 2 diabetes mellitus(T2DM)is a complex metabolic disease threatening human health.We investigated the effects of Tegillarca granosa polysaccharide(TGP)and determined its potential mechanisms in a mouse model of T2DM established through a high-fat diet and streptozotocin.TGP(5.1×10^(3) Da)was composed of mannose,glucosamine,rhamnose,glucuronic acid,galactosamine,glucose,galactose,xylose,and fucose.It could significantly alleviate weight loss,reduce fasting blood glucose levels,reverse dyslipidemia,reduce liver damage from oxidative stress,and improve insulin sensitivity.RT-PCR and Western blotting indicated that TGP could activate the phosphatidylinositol-3-kinase/protein kinase B signaling pathway to regulate disorders in glucolipid metabolism and improve insulin resistance.TGP increased the abundance of Allobaculum,Akkermansia,and Bifidobacterium,restored the microbiota abundance in the intestinal tracts of mice with T2DM,and promoted short-chain fatty acid production.This study provides new insights into the antidiabetic effects of TGP and highlights its potential as a natural hypoglycemic nutraceutical.

Spi1 regulates the microglial/macrophage inflammatory response via the PI3K/AKT/mTOR signaling pathway after intracerebral hemorrhage

Preclinical and clinical studies have shown that microglia and macrophages participate in a multiphasic brain damage repair process following intracerebral hemorrhage.The E26 transformation-specific sequence-related transcription factor Spi1 regulates microglial/macrophage commitment and maturation.However,the effect of Spi1 on intracerebral hemorrhage remains unclear.In this study,we found that Spi1 may regulate recovery from the neuroinflammation and neurofunctional damage caused by intracerebral hemorrhage by modulating the microglial/macrophage transcriptome.We showed that high Spi1expression in microglia/macrophages after intracerebral hemorrhage is associated with the activation of many pathways that promote phagocytosis,glycolysis,and autophagy,as well as debris clearance and sustained remyelination.Notably,microglia with higher levels of Soil expression were chara cterized by activation of pathways associated with a variety of hemorrhage-related cellular processes,such as complement activation,angiogenesis,and coagulation.In conclusion,our results suggest that Spi1 plays a vital role in the microglial/macrophage inflammatory response following intracerebral hemorrhage.This new insight into the regulation of Spi1 and its target genes may advance our understanding of neuroinflammation in intracerebral hemorrhage and provide therapeutic targets for patients with intracerebral hemorrhage.

Downregulation of Serum PTEN Expression in Mercury-Exposed Population and PI3K/AKT Pathway-Induced Inflammation

Objective This study investigated the impact of occupational mercury(Hg) exposure on human gene transcription and expression, and its potential biological mechanisms.Methods Differentially expressed genes related to Hg exposure were identified and validated using gene expression microarray analysis and extended validation. Hg-exposed cell models and PTEN lowexpression models were established in vitro using 293T cells. PTEN gene expression was assessed using qRT-PCR, and Western blotting was used to measure PTEN, AKT, and PI3K protein levels. IL-6 expression was determined by ELISA.Results Combined findings from gene expression microarray analysis, bioinformatics, and population expansion validation indicated significant downregulation of the PTEN gene in the high-concentration Hg exposure group. In the Hg-exposed cell model(25 and 10 μmol/L), a significant decrease in PTEN expression was observed, accompanied by a significant increase in PI3K, AKT, and IL-6 expression.Similarly, a low-expression cell model demonstrated that PTEN gene knockdown led to a significant decrease in PTEN protein expression and a substantial increase in PI3K, AKT, and IL-6 levels.Conclusion This is the first study to report that Hg exposure downregulates the PTEN gene, activates the PI3K/AKT regulatory pathway, and increases the expression of inflammatory factors, ultimately resulting in kidney inflammation.

和枢消积方通过调节AsTP3正反馈AKT/GSK-3β/mTOR信号通路对人肝癌细胞增殖及凋亡的影响

目的:探讨和枢消积方通过调节三氧化二砷反式激活蛋白3(AsTP3)对人肝癌细胞SMMC-7721增殖、凋亡的作用机制。方法:体外培养SMMC-7721肝癌细胞,采用CCK8法筛选出和枢消积方最适浓度,联合CCK8法与平板克隆形成实验检测细胞增殖,细胞划痕实验及Transwell小室实验检测细胞迁移、侵袭能力,流式细胞实验检测细胞凋亡,qRT-PCR检测细胞AsTP3 mRNA水平,Western Blot检测细胞中AsTP3、AKT、GSK-3β、mTOR蛋白相对表达量及磷酸化水平。结果:与对照组比较,和枢消积方能抑制SMMC-7721细胞增殖、迁移、侵袭及克隆能力,促进SMMC-7721细胞凋亡(P<0.05);和枢消积方可下调SMMC-7721细胞内AsTP3、P-AKT、P-GSK-3β、P-mTOR、Bcl2的mRNA表达及蛋白水平并上调Bax蛋白的表达。结论:和枢消积方能抑制SMMC-7721细胞增殖,促进其凋亡,其机制可能与下调AsTP3表达从而抑制AKT/GSK-3β/mTOR信号通路有关。

MicroRNA (let-7b-5p)-targeted DARS2 regulates lung adenocarcinoma growth by PI3K/AKT signaling pathway

Background:The aberrant intraellular expression of a mitochondrial aspartyl tRNA synthetase 2(DARS2)has been reported in human cancers.Nevertheless its critical role and detailed mechanism in lung adenocarcinoma(LUAD)remain unexplored.Methods:Initially,The Cancer Genome Atlas(TCGA)based Gene Expression Profiling Interactive Analysis(GEPIA)database (http:/gepia.cancer-pku.cn/)was used to analyze the prognostic relevance of DARS2 expression in LUAD.Further,cell counting kit(CCK)8,immunostaining,and transwell invasion assays in LUAD cell lines in vitro,as well as DARS2 silence on LUAD by tumorigenicity experiments in wivo in nude mice,were performed.Besides,we analyzed the expression levels of p-PI3K(phosphorylated Phosphotylinosital3 kinase),PI3K,AKT(Protein Kinase B),p-AKT(phosphorylated Protein Kinase B),PCNA(proliferating cell nudear antigen),cleaved-caspase 3,E cadherin,and N-cadherin proteins using the Westem blot analysis.Results:LUAD tissues showed higher DARS2 expression compared to normal tissues.Upregulation of DARS2 could be related to Tumor-Node-Metastasis(TNM)stage,high lymph node metastasis,and inferior prognosis.DARS2 silence decreased the proliferation,migration,and invasion abilities of LUAD cells.In addition,the DARS2 downregulation decreased the PCNA and N-cadherin expression and increased cleaved:caspase 3 and E cadherin expressions in LUAD cells,coupled with the inactivation of the PI3K/AKT signaling pathway.Moreover,DARS2 silence impaired the tumonigenicity of LUAD in vivo.Interestingly,let:7b-5p could recognize DARS2 through a complementary sequence.Mechanistically,the increased let 7b 5p expression attenuated the promo oncogenic action of DARS2 during LUAD progression,which were inversely correlated to each other in the LUAD tssues Conclusion:In summary,let 7b-5p,downregulated DARS2 expression,regulating the progression of LUAD cells by the PI3K/AKT signaling pathway.

PI3K/AKT/GSK-3β信号通路在高尿酸血症大鼠尿酸、血糖及脂质代谢中的作用机制

目的 研究PI3K/AKT/GSK-3β信号通路在高尿酸血症大鼠尿酸、血糖及脂质代谢中的作用机制。方法 取40只SD大鼠随机分为4组,其中2组为未建模SD大鼠,分别为模型对照组(C组)、LY294002组(L组),剩余2组为建模成功的SD大鼠,分为高尿酸血症组(H组)和高尿酸血症加LY294002干预组(HL组)。C组大鼠经口给予纯水(10 mL/kg体重)和腹腔注射0.2%二甲基亚砜溶液(6 mL/kg体重);L组大鼠给予相同剂量的纯水和腹腔注射LY294002溶液(6 mL/kg体重);H组大鼠经口给予腺嘌呤(100 mg/kg体重)和乙胺丁醇(250 mg/kg体重);HL组大鼠在给予相同剂量的腺嘌呤和乙胺丁醇的同时腹腔注射LY294002溶液(6 mL/kg体重)。各组持续干预15 d,每3天从眼眶静脉丛采集血样,测定血尿酸(Uric Acid, UA)、肌酐(Serum Creatinine, Scr)、尿素氮(Blood Urea Nitrogen, BUN)、血糖(Glucose, GLU)、甘油三酯(Triglyceride, TG)、胆固醇(Total Cholesterol, TC)。第15天麻醉后采集各组大鼠肾脏组织,采用荧光定量PCR法和Western blotting法检测大鼠肾脏组织中PI3K/AKT/GSK-3β的表达水平。结果 4组C组、L组、H组和HL组大鼠实验前血清尿酸、肌酐、尿素氮、血糖、甘油三酯和胆固醇水平差异无统计学意义(P<0.05)。H组和HL组大鼠UA、Scr、BUN水平在实验后均高于C组和L组,与C组第6天比较,H组和HL组UA水平降低,第9天至第15天UA水平升高,差异有统计学意义(P<0.05)。L组、H组和HL组GLU水平较C组升高,其中L组在第12天,GLU水平高于C组,H组和HL组在实验第6天,GLU水平高于L组,差异有统计学意义(P均<0.05)。H组和HL组TC、TG水平较H组和L组升高,其中HL组在实验第9天,TG水平高于H组,差异有统计学意义(P<0.05)。荧光定量PCR检测结果显示,与C组比较,H组和HL组PI3K、AKT、GSK-3β增大,L组AKT减小,差异有统计学意义(P均<0.05)。与L组比较,H组和HL组PI3K、AKTGSK-3β增大,差异有统计学意义(P均<0.05)。与H组比较,HL组PI3K、AKT和GSK-3β增大,差异有统计学意义(P均<0.05)。Western-Blot结果显示,与C组比较,H组和HL组PI3K、AKT、GSK-3β增大,差异有统计学意义(P均<0.05)。与L组比较,H组和HL组PI3K、AKTGSK-3β增大,差异有统计学意义(P均<0.05)。与H组比较,HL组PI3K、AKT和GSK-3β增大,差异有统计学意义(P均<0.05)。结论 PI3K/AKT/GSK-3β信号通路与高尿酸血症之间存在联系,提示该通路对治疗和预防高尿酸血症具有重要意义。

Alleviatory effect of isoquercetin on benign prostatic hyperplasia via IGF-1/PI3K/Akt/mTOR pathway

We evaluated the effect of isoquercetin(quercetin-O-3-glucoside-quercetin,IQ)as a functional component of Abeliophyllum disistichum Nakai ethanol extract(ADLE)on prostate cell proliferation and apoptosis and its effects on the IGF-1/PI3K/Akt/mTOR pathway in benign prostatic hyperplasia(BPH).Metabolites in ADLE were analyzed using UHPLC-qTOF-MS and HPLC.IQ was orally administered(1 or 10 mg/kg)to a testosterone propionate-induced BPH rat model,and its effects on the prostate weight were evaluated.The effect of IQ on androgen receptor(AR)signaling was analyzed in LNCaP cells.Whether IGF-1 and IQ affect the IGF-1/PI3K/Akt/mTOR pathway in BPH-1 cells was also examined.The metabolites in ADLE were identified and quantified,which confirmed that ADLE contained abundant IQ(20.88 mg/g).IQ significantly reduced the prostate size in a concentration-dependent manner in a BPH rat model,and significantly decreased the expression of AR signaling factors in the rat prostate tissue and LNCaP cells in a concentration-dependent manner.IQ also inhibited the PI3K/AKT/mTOR pathway activated by IGF-1 treatment in BPH-1 cells.In BPH-1 cells,IQ led to G0/G1 arrest and suppressed the expression of proliferation factors while inducing apoptosis.Thus,IQ shows potential for use as a pharmaceutical and nutraceutical for BPH.

槲皮素基于PI3K/AKT/GSK-3β/β-Catenin通路改善Dox诱导小鼠心肌损伤的机制

目的:研究槲皮素(Que)改善多柔比星(Dox)所致小鼠心肌损伤作用机制。方法:将50只小鼠随机分为正常组(Control)、模型组(Dox)、槲皮素低、中、高剂量组(Que-L、Que-M、Que-H),除Control外腹腔注射Dox建立心肌损伤模型。心脏超声及血流动力学评价小鼠心脏功能,ELISA法检测小鼠血清中肌酸激酶同工酶(CK-MB)与乳酸脱氢酶(LDH)含量,HE染色、Tunel染色、WGA染色分别观察小鼠心肌组织病理变化、心肌细胞凋亡、心肌细胞横截面积变化;免疫荧光检测心肌组织中p-GSK-3β表达,Western blot法检测小鼠心肌组织PI3K/AKT/GSK-3β通路与凋亡蛋白表达。结果:与Control组比较,Dox组小鼠左室射血分数(LVEF)与左室短轴缩短率(LVFS)极显著下降(P<0.01),左心室舒张末期内径(LVEDd)与左心室收缩末期内径(LVEDs)极显著增加(P<0.01),血清中CK-MB与LDH含量极显著增加(P<0.01),心肌细胞肿胀且排列紊乱;心肌组织中PI3K、p-AKT、p-GSK-3β、β-catenin表达极显著降低(P<0.01),Bax/Bcl-2、Cleaved Caspase-3表达极显著增加(P<0.01)。与Dox组比较,Que各给药组小鼠心肌损伤均有不同程度改善,LVEF与LVFS极显著升高(P<0.01),(LVEDd)与(LVEDs)极显著降低(P<0.01),血清中CK-MB、LDH含量极显著减少(P<0.01),心脏功能增强;心肌细胞肿胀、凋亡、纤维增生减轻,心肌组织中PI3K、p-AKT、p-GSK-3β、β-catenin蛋白表达极显著升高(P<0.01),PI3K/AKT/GSK-3β/β-catenin被激活,凋亡蛋白Bax/Bcl-2、Cleaved Caspase-3表达极显著降低(P<0.01),其中Que-H组治疗效果最佳。结论:Que具有一定的心肌保护作用,可通过激活PI3K/AKT/GSK-3β/β-catenin通路改善Dox引起的心肌损伤与凋亡。

miR-152-3p通过AKT/GSK-3β/Nrf2信号通路促进脑出血大鼠神经元铁死亡

目的:探讨miR-152-3p通过AKT/GSK-3β/Nrf2信号通路促进脑出血(ICH)大鼠神经元铁死亡的机制。方法:使用大鼠神经元细胞为研究对象,采用氧合血红蛋白(oxyHb)刺激神经元细胞构建ICH体外细胞模型,分别将miR-152-3p inhibitor和(或)PIK3CA抑制剂分别处理细胞,采用流式细胞术观察细胞凋亡情况,RT-qPCR、Western blot检测相关基因和蛋白表达情况;并用细胞免疫荧光观察Nrf2蛋白的入核率;用双荧光素酶靶标实验验证miR-152-3p与PIK3CA的关系。结果:将miR-152-3p inhibitor转染的细胞构建ICH细胞模型后,细胞凋亡率明显降低(P<0.01);SLC7A11、GPx4、PIK3CA、Nrf2蛋白表达水平以及p-AKT/AKT比率显著升高而GSK-3β蛋白表达水平显著降低(P<0.01);同时,Nrf2蛋白入核量也显著升高(P<0.01);而此作用在使用PIK3CA抑制剂干预后,miR-152-3p inhibitor的改善效果消失;荧光素酶靶标实验证实,miR-152-3p可靶向调控PIK3CA。结论:miR-152-3p通过AKT/GSK-3β/Nrf2信号通路促进ICH大鼠神经元铁死亡。

Acalypha australis L.extract inhibits B16 melanoma cell metastasis through PI3K/AKT signaling pathway

Background:Melanoma is a deadly skin tumor resulting from the malignant transformation of melanocytes.It is highly malignant and invasive,with the highest mortality rate among skin cancers.Acalypha australis L.(AAL),a plant with dual medicinal and culinary purposes,is commonly regarded as an edible wild vegetable in southern China.Additionally,AAL has a long history of medicinal use in China,often employed for its hemostatic,anti-diarrheal,and anti-inflammatory properties.Modern pharmacology has demonstrated that AAL possesses functions such as weight loss,antimicrobial activity,antiviral effects,and treatment for ulcerative colitis.However,there is currently no research available regarding its effectiveness and mechanisms of action on melanoma.Methods:In this investigation,we used methyl thiazolyl tetrazolium assay to detect cell viability,transwell assay to detect cell migration and invasion ability,and Western blot assay to detect relevant signaling pathways.Results:The present study reveals that 2 mg/mL AAL effectively suppresses the metastasis of B16 cells,while simultaneously triggering the expression of key apoptosis-related proteins,including Bcl-2,Bax,and cleaved caspased 3.Subsequent investigations demonstrate that AAL exerts this inhibitory effect via the PI3K/AKT signal transduction pathway,as evidenced by the observed deficits in Ras,AKT,p-AKT,and PI3K expression levels.Conclusion:These findings indicated that AAL could be a valuable therapeutic option for reducing the metastatic potential of B16 melanoma cells.

辣椒素纳米胶囊通过抑制PI3K/Akt/GSK-3β途径改善心肌纤维化的研究

目的探讨辣椒素纳米颗粒(Capsaicin/Nanoparticles,Cap/NPs)胶囊对大鼠心肌梗死后心肌纤维化及磷酸肌醇3激酶/蛋白激酶B/糖原合成酶激酶3β(Phosphatidylinositol Kinase 3-Kinase/Protein Kinase B/Glycogen Synthase Kinase 3β,PI3K/Akt/GSK-3β)信号通路的影响。方法于2022年1月—2023年6月自贵州医科大学动物中心购进40只雄性健康Sprague-Dawley(SD)大鼠,在适应性喂养7 d后,按随机数表法分为4组(正常对照组、假手术组、模型组、干预组),每组10只。正常对照组、假手术组、模型组予以生理盐水,干预组予以Cap/NPs胶囊口服,1次/周,连续4周。对模型组与干预组行手术结扎,构建心肌梗死模型,假手术组则开展相同手术操作不予以结扎处理,对照组为正常大鼠。术后4周,观察各组心肌纤维化情况,检测PI3K/Akt/GSK-3β通路蛋白在大鼠心肌组织中表达情况。结果模型组血清激活素A(Activin A,ACT-A)水平为(2.33±0.28)pg/mg、心房钠尿肽(Atrial Natriuretic Peptide,ANP)水平为(2.21±0.57)pg/mg、脑钠肽(Brain Natriuretic Peptide,BNP)水平为(212.04±17.36)ng/L、基质金属蛋白酶-9(Matrix Metalloproteinases,MMP-9)水平为(221.65±23.34)ng/L、胶原纤维蛋白Ⅲ水平为(67.62±1.85)ng/mL,各指标均明显高于干预组,差异有统计学意义(P均<0.05)。模型组大鼠心肌组织PI3K/Akt/GSK-3β蛋白表达水平明显低于干预组,差异有统计学意义(P<0.05)。结论大鼠心肌梗死模型建立后予以Cap/NPs胶囊干预,能够有效抑制心肌纤维化发展,减轻大鼠心肌损伤,其机制与PI3K、Akt、GSK-3β蛋白活性调控以及PI3K/Akt/GSK-3β信号通路传导抑制有关。

Downregulation of LncRNAH19 and MiR-675 Promotes Migration and Invasion of Human Hepatocellular Carcinoma Cells through AKT/GSK-3β/Cdc25A Signaling Pathway

Summary： LncRNAH19 has been implicated as having both oncogenic and tumor suppression properties in cancer. LncRNAH19 transcripts also serve as a precursor for miR-675. However, it is unknown whether LncRNAH19 and miR-675 are involved in the migration and invasion of hepatocellular carcinoma （HCC） cells. The purpose of this study was to investigate the effect and mechanism of LncRNAH19 and miR-675 on migration and invasion of HCC cells. The migration and invasion of HCC cells were measured by Transwell migration and invasion assays after transfection of HCC cells with miR-675 inhibitors and LncRNAH19siRNA. The levels of LncRNAH19 and miR-675 were detected by quantitative reverse transcriptase real-time polymerase chain reaction （qRT-PCR）, and the protein expression of AKT, GSK-3[3 and Cdc25A by Western blotting analysis. The expression levels of LncRNAHI9 and miR-675 were higher in MHCC-97H cells than in L02, Huh-7 and HepG2 cells. Transwell migration assay revealed that the miR-675 inhibitor and LncRNAH19siRNA could significantly increase the migration of HCC cells （P〈0.01） as compared with the control group. Transwell invasion assay demonstrated that the miR-675 inhibitor and LncRNAH19siRNA could significantly increase the invasion of HCC cells （P〈0.01） as compared with the control group. Western blotting analy- sis showed that the expression levels of AKT and Cdc25A were significantly increased （P〈0.05）, and the expression level of GSK-313 was significantly decreased （P〈0.05） after treatment with miR-675 inhibitors and LncRNAH19siRNA as compared with the control group. These findings suggested that inhibition of LncRNAH 19 and miR-675 expression can promote migration and invasion of HCC cells via AKT/GSK-3[3/Cdc25A signaling pathway.

Liqi Huoxue dripping pill protects against myocardial ischemia-reperfusion injury via the PI3K/Akt/GSK-3β signaling pathway in rats

Background:Liqi Huoxue dripping pill(LQHXDP),a traditional Chinese drug for coronary heart disease,has a protective effect on the heart of rats with myocardial ischemia-reperfusion injury(MIRI)in previous studies;however,its mechanism of action remains unclear.The purpose of this study was to investigate the protective mechanism of LQHXDP on MIRI in rats and its relationship with the PI3K/Akt signaling pathway.Methods:In this study,Sprague-Dawley rats were pre-infused with LQHXDP(175 mg/kg/d)for 10 days.PI3K inhibitor LY294002(0.3 mg/kg)was intravenously injected 15 minutes before ischemia.The rat model of MIRI was established by ligating the left anterior descending coronary artery.Subsequently,cardiac hemodynamics,serum myocardial injury markers,inflammatory factors,myocardial infarct size,antioxidant indexes,myocardial histopathology,and phosphorylation levels of key proteins of PI3K/Akt signaling pathway were assessed in rats.Results:LQHXDP was found to improve cardiac hemodynamic indexes,reduce serum creatine kinase MB isoenzyme activity and cardiac troponin and heart-type fatty acid binding protein levels,lower serum interleukin-1 beta,interleukin-6 and tumour necrosis factorαlevels,reduce the myocardial infarct size and enhance the antioxidant capacity of myocardial tissue in MIRI rats.Pathological analysis revealed that LQHXDP attenuated the extent of myocardial injury and protected mitochondria from damage in MIRI rats.Immunoblot analysis revealed that LQHXDP increased the expression levels of p-Akt and p-GSK-3βin MIRI rat cardiomyocytes.PI3K inhibitor LY294002 could impair these effects of LQHXDP.Conclusion:LQHXDP attenuated myocardial injury,attenuated oxidative stress injury and reduced inflammatory response in MIRI rats,and its protective effects were mediated by activating of PI3K/Akt/GSK-3βsignaling pathway.

Ezetimibe Protects Endothelial Cells against Oxidative Stress through Akt/GSK-3β Pathway

Ezetimibe was reported to pharmacologically defend against oxidative stress. This study was designed to investigate whether ezetimibe can protect against the oxidative stress induced by oxidized low-density lipoprotein （oxLDL） in vitro and the underlying mechanism. Human umbilical vein endothelial cells （HUVECs） were pretreated with ezetimibe and then exposed to oxLDL for 24 h. TUNEL assay and detection for the protein levels of cleaved caspase-3, Bcl-xl and Bcl-2 were employed to assess the oxLDL-induced endothelial apoptosis. Intracellular reactive oxygen species （ROS） generation was evaluated by measuring dichlorofluorescein （DCF） fluorescence. The activities of endothelial antioxidant enzymes [superoxide dismutase （SOD） and catalase] were tested via an enzymatic assay. The mitochondrial membrane potential （MMP） was monitored by flow cytometry using JC-I staining. Phosphorylation levels of glycogen synthase kinase-3β （p-GSK-3β） and Akt （p-Akt）, as well as total GSK-3β and Akt were determined by Western blotting. The results showed that ezetimibe treatment inhibited HUVECs apoptosis, intracellular ROS production, and enhanced antioxidant enzyme activities elicited by oxLDL. HUVECs exposed to oxLDL alone had reduced mitochondrial function, while ezetimibe pre-intervention could significantly rescue the MMP. Furthermore, the protein levels of p-GSK-3β and p-Akt in ezetimibe-pretreated HUVECs were markedly increased as compared with those in oxLDL-induced HUVECs. However, no significant effect on total GSK- 3β and Akt was found in ezetimibe-pretreated HUVECs. Taken together, it was concluded that ezetimibe protects against oxLDL-induced oxidative stress through restoring the MMP, which may be mediated by Akt-dependent GSK-3β phosphorylation.