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.

LAMC2 regulates proliferation, migration, and invasion mediated by the Pl3K/AKT/mTOR pathway in oral

Background:Oral squamous cell carcinoma(OSCC)is a common malignant tumor.Recently,Laminin Gamma 2(LAMC2)has been shown to be abnormally expressed in OSCC;however,how LAMC2 signaling contributes to the occurrence and development of OSCC and the role of autophagy in OSCC has not been fully explored.This study aimed to analyze the role and mechanism of LAMC2 signaling in OSCC and the involvement of autophagy in OSCC.Methods:To explore the mechanism by which LAMC2 is highly expressed in OSCC,we used small interfering RNA(siRNA)to knock down LAMC2 to further observe the changes in the signaling pathway.Furthermore,we used cell proliferation assays,Transwell invasion assays,and wound-healing assays to observe the changes in OSCC proliferation,invasion,and metastasis.RFP-LC3 was used to detect the level of autophagy intensity.A cell line-derived xenograft(CDX)model was used to detect the effect of LAMC2 on tumor growth in vivo.Results:This study found that the level of autophagy was correlated with the biological behavior of OSCC.The downregulation of LAMC2 activated autophagy and inhibited OSCC proliferation,invasion,and metastasis via inhibiting the PI3K/AKT/mTOR pathway.Moreover,autophagy has a dual effect on OSCC,and the synergistic downregulation of LAMC2 and autophagy can inhibit OSCC metastasis,invasion,and proliferation via the PI3K/AKT/mTOR pathway.Conclusions:LAMC2 interacts with autophagy to regulate OSCC metastasis,invasion,and proliferation via the PI3K/AKT/mTOR pathway.LAMC2 down-regulation can synergistically modulate autophagy to inhibit OSCC migration,invasion,and proliferation.

基于TRPM2/Akt/GSK3β通路探讨24-乙酰泽泻醇A保护脑缺血再灌注损伤脑微血管内皮细胞的机制

目的:探讨24-乙酰泽泻醇A(24A)改善小鼠脑缺血再灌注后脑微血管内皮细胞(BMECs)损伤的作用机制。方法:将45只雄性10周龄C57BL/6小鼠按随机数字表法分为假手术组、模型组、24A组,每组15只。模型组、24A组采用20 min双侧颈总动脉结扎后再灌注的方法构建小鼠脑缺血再灌注损伤(CI/RI)模型,假手术组小鼠只分离颈总动脉和迷走神经,不结扎。24 A组采用24 A灌胃,剂量为30 mg/(kg·d);假手术组和模型组则给予等体积的生理盐水进行灌胃,3组均灌胃1次/d,连续干预7 d。分别采用神经功能缺损评分(mNSS)评估小鼠神经功能受损程度;采用新物体识别测试(NORT)和水迷宫(MWM)评价小鼠认知功能;采用免疫荧光法检测内皮细胞CD31表达;采用Western blot法检测脑组织闭锁小带蛋白1(ZO-1)、闭合蛋白(Occludin)、紧密连接蛋白-5(Claudin-5)、磷酸化核因子κB(p-NF-κB)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、瞬时受体电位离子通道-2(TRPM2)、磷酸化苏氨酸蛋白激酶(p-AKT)、磷酸化糖原合成酶激酶-3(p-GSK3β)蛋白表达水平。结果:①神经功能:与假手术组比较,模型组mNSS在第1、3、5、7天均明显升高(P<0.05);与模型组比较,24A组第3、7天mNSS明显降低(P<0.05)。②认知功能:与假手术组比较,模型组NORT 1、24 h识别指数明显降低(P<0.05);与模型组比较,24A组NORT 1、24 h识别指数明显升高(P<0.05)。与假手术组比较,模型组干预后第2~4天逃避潜伏期明显延长,穿越目标平台次数明显减少(P<0.05);与模型组比较,24A组干预后第3、4天逃避潜伏期明显缩短,穿越目标平台次数明显增加(P<0.05)。③内皮细胞CD31、ZO-1、Occludin、Claudin-5蛋白表达:假手术组CD31荧光表达较为密集,呈现红色荧光;与假手术组比较,模型组CD31荧光表达明显降低(P<0.05);与模型组比较,24A组CD31荧光表达明显升高(P<0.05)。与假手术组比较,模型组ZO-1、Occludin、Claudin-5蛋白表达量明显下降(P<0.05);与模型组比较,24A组ZO-1、Occludin、Claudin-5蛋白表达量明显升高(P<0.05)。④p-NF-κB、IL-1β、TNF-α、TRPM2、p-Akt、p-GSK3β蛋白表达:与假手术组比较,模型组p-NF-κB、IL-1β、TNF-α、TRPM2表达明显增加,p-Akt、p-GSK3β表达明显降低(P<0.05);与模型组比较,24A组p-NF-κB、IL-1β、TNF-α、TRPM2蛋白表达水平明显下降,p-Akt、p-GSK3β表达明显增加(P<0.05)。结论:24A可有效改善CI/RI小鼠的神经功能、认知功能,降低炎症反应,改善BMECs损伤,其机制可能与TRPM2/Akt/GSK3β通路的调控有关。

6-姜酚通过Akt/GSK3-β信号通路改善心肌缺血再灌注损伤的研究

目的通过Akt/GSK3-β信号通路研究6-姜酚对心肌缺血再灌注损伤(I/R)的影响。方法(1)选取相同月龄的健康雄性Wistar大鼠32只,依据随机数字表法将大鼠分为对照组1、I/R1组、I/R+2 mg/kg组及I/R+4 mg/kg组各8只,对I/R1组、I/R+2 mg/kg组及I/R+4 mg/kg组制备I/R模型,分别给予I/R+2 mg/kg组及I/R+4 mg/kg组2 mg/kg及4 mg/kg的6-姜酚溶液2 mL灌胃,对照组1及I/R1组大鼠灌注等量蒸馏水。比较各组干预后心肌细胞凋亡水平、半胱氨酸蛋白酶-3(Caspase-3)、血清乳酸脱氢酶(LDH)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、核因子κB(NF-κB)、Akt、p-Akt、GSK3-β及p-GSK3-β表达水平。(2)H9C2心肌细胞分为对照组2、I/R2组、I/R2+50 mmol/L组、I/R2+100 mmol/L组,每组3份。对I/R2组、I/R2+50 mmol/L组、I/R2+100 mmol/L组H9C2心肌细胞建立I/R模型,分别将对照组2、I/R2组、I/R2+50 mmol/L组及I/R2+100 mmol/L组H9C2心肌细胞置于含有0 mmol/L、0 mmol/L、50 mmol/L和100 mmol/L的6-姜酚DMEM培养基中,比较H9C2心肌细胞干预后的凋亡水平、Caspase-3、LDH、Akt、p-Akt、GSK3-β、p-GSK3-β的表达水平。结果(1)I/R+4 mg/kg组的细胞凋亡面积及Caspase-3、LDH、IL-6、TNF-α及NF-κB水平均低于I/R1组(P<0.05),且I/R+4 mg/kg组的细胞凋亡面积及Caspase-3及IL-6水平低于I/R+2 mg/kg组(P<0.05);I/R+4 mg/kg组的p-Akt/Akt及p-GSK3-β/GSK3-β均高于I/R1组及I/R+2 mg/kg组(P<0.05)。(2)I/R2+100 mmol/L组的细胞凋亡率、Caspase-3及LDH均低于I/R2组及I/R2+50 mmol/L组(P<0.05),I/R2+100 mmol/L组的p-Akt/Akt及p-GSK3-β/GSK3-β均高于I/R2组及I/R2+50 mmol/L组(P<0.05)。结论6-姜酚处理后的大鼠心肌细胞I/R程度显著减轻,炎症因子降低,心肌细胞的p-Akt/Akt及p-GSK3-β/GSK3-β信号通路相关蛋白比值升高,考虑6-姜酚通过激活Akt/GSK3-β信号通路达到改善心肌I/R的效果。

SHP2 promotes proliferation of breast cancer cells through regulating Cyclin D1 stabilityviathe PI3K/AKT/GSK3β signaling pathway

Objective:The tyrosine phosphatase SHP2 has a dual role in cancer initiation and progression in a tissue type-dependent manner.Several studies have linked SHP2 to the aggressive behavior of breast cancer cells and poorer outcomes in people with cancer.Nevertheless,the mechanistic details of how SHP2 promotes breast cancer progression remain largely undefined.Methods:The relationship between SHP2 expression and the prognosis of patients with breast cancer was investigated by using the TCGA and GEO databases.The expression of SHP2 in breast cancer tissues was analyzed by immunohistochemistry.CRISPR/Cas9 technology was used to generate SHP2-knockout breast cancer cells.Cell-counting kit-8,colony formation,cell cycle,and EdU incorporation assays,as well as a tumor xenograft model were used to examine the function of SHP2 in breast cancer proliferation.Quantitative RT-PCR,western blotting,immunofluorescence staining,and ubiquitination assays were used to explore the molecular mechanism through which SHP2 regulates breast cancer proliferation.Results:High SHP2 expression is correlated with poor prognosis in patients with breast cancer.SHP2 is required for the proliferation of breast cancer cellsin vitro and tumor growthin vivo through regulation of Cyclin D1 abundance,thereby accelerating cell cycle progression.Notably,SHP2 modulates the ubiquitin–proteasome-dependent degradation of Cyclin D1viathe PI3K/AKT/GSK3βsignaling pathway.SHP2 knockout attenuates the activation of PI3K/AKT signaling and causes the dephosphorylation and resultant activation of GSK3β.GSK3βthen mediates phosphorylation of Cyclin D1 at threonine 286,thereby promoting the translocation of Cyclin D1 from the nucleus to the cytoplasm and facilitating Cyclin D1 degradation through the ubiquitin–proteasome system.Conclusions:Our study uncovered the mechanism through which SHP2 regulates breast cancer proliferation.SHP2 may therefore potentially serve as a therapeutic target for breast cancer.

萝卜硫素通过下调Cox-2/Akt/GSK3β信号传导抑制人皮肤鳞状细胞癌A431细胞增殖

目的:明确萝卜硫素对人皮肤鳞状细胞癌A431细胞株增殖、凋亡的影响及其对Cox-2/Akt/GSK3β信号传导相关蛋白水平的影响。方法:体外培养皮肤鳞状细胞癌A431细胞株,分为不同剂量(30μM,60μM,120μM)萝卜硫素组、单纯顺铂组及顺铂+萝卜硫素组,采用CCK8法、流式细胞术分别检测A431细胞相对存活率及凋亡率,Western Blot检测了A431细胞中Survivin、Casepase-3、Cox-2、Akt、p-Akt、GSK3β及p-GSK3β蛋白的表达水平。结果:与对照组比较,60μM及120μM萝卜硫素组、单纯顺铂组及顺铂+萝卜硫素组细胞相对存活率显著降低(均P<0.05),细胞凋亡率显著增高(均P<0.05),Survivin、Cox-2、p-Akt、p-GSK3β蛋白表达水平显著降低(均P<0.05),而Caspase-3蛋白表达水平显著升高(P<0.05);120μM萝卜硫素组与顺铂组比较,细胞相对存活率、细胞凋亡率,Survivin、Cox-2、p-Akt、p-GSK3β蛋白表达水平及Caspase-3蛋白表达,无明显统计学差异(P>0.05)。结论:萝卜硫素可通过诱导A431细胞凋亡、抑制细胞增殖发挥抗肿瘤作用,其机制可能与抑制Cox-2/Akt/GSK3β信号通路活化有关。

人参皂苷Rh2调控PI3K/AKT/GSK-3β信号通路诱导人结肠癌细胞凋亡

目的探究人参皂苷Rh2(ginsenoside Rh2,Rh2)诱导人结肠癌细胞SW480凋亡作用机制。方法 CCK-8法检测Rh2对SW480细胞增殖的影响;流式细胞术(Flow cyto Metry,FCM)检测Rh2对SW480细胞凋亡的影响;Hoechst33258染色观察Rh2对SW480细胞凋亡形态学的影响;Western blot检测经Rh2诱导SW480细胞中凋亡相关蛋白Bcl-2、Bax、p53、cleaved caspase-3,PI3K/AKT/GSK-3β信号通路相关蛋白PI3K、AKT、P-AKT、GSK-3β、P-GSK-3β表达量变化;LY294002、Rh2单独及联合诱导SW480细胞后,蛋白PI3K、AKT、P-AKT、GSK-3β、P-GSK-3β表达量变化。结果CCK-8结果显示Rh2呈时间浓度依赖抑制SW480细胞增殖。FCM结果显示细胞早期凋亡率由正常对照组的(0.70±0.09)%增至(11.06±1.04)%(P<0.05)。Hoechst33258结果显示,Rh2诱导SW480细胞48h后呈现典型的凋亡形态学改变。Western blot结果显示,经Rh2诱导的SW480细胞,凋亡相关蛋白Bcl-2表达降低,Bax、p53、激活型胱天蛋白酶3(cleaved caspase-3)蛋白表达增加;PI3K/AKT/GSK-3β信号通路蛋白PI3K、P-AKT、P-GSK-3β表达量与对照组比较明显减少,AKT、GSK-3β表达量无明显变化;LY294002、Rh2和LY294002与Rh2联合诱导SW480细胞后,总AKT蛋白和总GSK-3β蛋白表达量基本一致,LY294002与Rh2联合用药对SW480细胞中PI3K、P-AKT和P-GSK-3β的表达抑制作用较单独用药更加明显。结论Rh2可能是通过抑制PI3K/AKT/GSK-3β通路,激活p53信号通路,激活caspase-3,破坏Bcl-2/Bax比例,诱导结肠癌细胞SW480凋亡。

Neuroprotective Effects of astaxanthin against cerebral ischemia- induced apoptosis via the PI3K/Akt/GSK3βNrf2 signaling pathway

Astaxanthin （ATX） , the most abundant flavonoids in propolis, has been proven to exert neuroprotective property against cerebral ischemia-induced apoptosis. However, the mechanisms by which ATX mediates its thera- peutic effects in vitro are unclear. In the present study, the article explored the underlying mechanisms involved in the protective effects of ATX via the PI3IC/Akt/GSK3β/Nrf2 signaling pathway in SH-SY5Y cells. For study of mechanism, the phosphoinositide 3 kinase （PI3K）-Akt inhibitor LY294002, Glycogen synthase kinase 3β （GSK313） inhibitor LiC1 were used. Pre-treatmentwith ATX for 24h significantly reduced the OGD induced viability loss, apoptotic rate and attenuated OGD-mediated ROS production. In addition, ATX inhibited OGD-induced mito- chondrial membrane potential, decreased Bcl-2/Bax ratio. PI3 IC/Akt/GSK3β/Nrf2 signaling pathway activation in SH-SY5Y was tested by Western blot. Nrf2 expression was increasing by ATX and counteracted by PI3IC/Akt in- hibitor LY294002, GSK3β inhibitor LiC1 in SH-SY5Y. Nrf2 Immunocytochemistry showed Nrf2 nuclear transloca- tion was increasing by ATX and counteracted by LY294002 or LiC1 in SH-SY5Y, respectively. It may be suggested that astaxanthin against cerebral ischemia-induced apoptosis in vitro via a programmed PI3 IC/Akt/GSK3β/Nrf2 sig- naling pathway in vitro.

Melatonin improves synapse development by PI3K/Akt signaling in a mouse model of autism spectrum disorder

Autism spectrum disorders are a group of neurodevelopmental disorders involving more than 1100 genes,including Ctnnd2 as a candidate gene.Ctnnd2knockout mice,serving as an animal model of autis m,have been demonstrated to exhibit decreased density of dendritic spines.The role of melatonin,as a neuro hormone capable of effectively alleviating social interaction deficits and regulating the development of dendritic spines,in Ctnnd2 deletion-induced nerve injury remains unclea r.In the present study,we discove red that the deletion of exon 2 of the Ctnnd2 gene was linked to social interaction deficits,spine loss,impaired inhibitory neurons,and suppressed phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt) signal pathway in the prefrontal cortex.Our findings demonstrated that the long-term oral administration of melatonin for 28 days effectively alleviated the aforementioned abnormalities in Ctnnd2 gene-knockout mice.Furthermore,the administration of melatonin in the prefro ntal cortex was found to improve synaptic function and activate the PI3K/Akt signal pathway in this region.The pharmacological blockade of the PI3K/Akt signal pathway with a PI3K/Akt inhibitor,wo rtmannin,and melatonin receptor antagonists,luzindole and 4-phenyl-2-propionamidotetralin,prevented the melatonin-induced enhancement of GABAergic synaptic function.These findings suggest that melatonin treatment can ameliorate GABAe rgic synaptic function by activating the PI3K/Akt signal pathway,which may contribute to the improvement of dendritic spine abnormalities in autism spectrum disorders.

罗布麻总黄酮通过PI3K/Akt/GSK3β抑制H_2O_2诱导的EA.hy926凋亡机制研究

心血管疾病严重危害人类健康,其致病机制主要是氧化应激诱导的内皮细胞凋亡,保护内皮细胞免受氧化应激损伤是防治心血管疾病的关键。为了探讨罗布麻总黄酮(total flavonoids of Folium Apocyni Veneti,TFF)抑制过氧化氢(hydrogen peroxide,H_2O_2)诱导的EA.hy926细胞凋亡的机制,采用不同浓度TFF处理EA.hy926细胞,一定浓度的H_2O_2造模,通过MTT法、Hoechst33342/PI荧光双染、流式细胞术检测各组的细胞凋亡情况,并用Western-blot和荧光定量PCR检测相关蛋白及m RNA的表达水平。研究发现,H_2O_2能明显诱导EA.hy926细胞凋亡,而TFF可降低H_2O_2引起的内皮细胞凋亡,表现为凋亡率降低、线粒体膜电位(mitochondrial membrane potential,MMP)增加、裂解的半胱天冬酶-3,-9(cleaved caspase-3,-9)表达减少、髓细胞白血病因子-1(myeloid cell leukemia-1,Mcl-1)表达增加、磷脂酰肌醇-3-激酶/蛋白激酶B/糖原合成酶激酶-3β(phosphatidylinositol-3-kinase/protein kinase B/glycogen synthase kinase-3β,PI3K/Akt/GSK3β)通路中Akt、GSK3β蛋白的磷酸化水平升高。上述结果表明,TFF主要是通过激活PI3K/Akt/GSK3β通路,调节Mcl-1的表达,保护MMP,进而抑制caspase蛋白的剪切活化,抑制H_2O_2诱导的EA.hy926凋亡。

METTL14通过PI3K/AKT/GSK3β/β-catenin信号通路调控巨噬细胞分化对宫颈癌细胞凋亡和增殖的影响

目的探讨METTL14通过调控巨噬细胞分化抑制宫颈癌病理性发展及相关机制。方法检测宫颈癌病变样本METTL14 m RNA和蛋白,以及IL-6、iNOS、Arg-1和CD206表达变化。PMA诱导THP-1细胞转化为巨噬细胞,慢病毒过表达或抑制METTL14表达,检测IL-6、iNO、Arg-1和CD206表达变化以及PI3K/AKT/GSK3β/β-catenin信号通路相关蛋白表达情况。随后加入PI3K/AKT/GSK3β/β-catenin信号通路激动剂和抑制剂,检测过表达或抑制METTL14后,巨噬细胞IL-6、iNO、Arg-1和CD206表达变化,并取其上清制成条件培养基,孵育Hela细胞,检测细胞凋亡和增殖情况。结果1)宫颈癌病变组织中METTL14 mRNA和蛋白表达降低(P<0.05),巨噬细胞M1型标志物IL-6和iNOS表达明显降低(P<0.05),而M2型标志物Arg-1和CD206表达明显升高(P<0.05)。2)巨噬细胞过表达METTL14后,IL-6和iNOS表达明显升高(P<0.05),而Arg-1和CD206表达明显降低(P<0.05),M1/M2比例升高;抑制METTL14表达后,M1型标志物降低(P<0.05),M2型标志物升高(P<0.05),M1/M2比例降低。3)巨噬细胞中转染OE-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被抑制(P<0.05);加入PI3K/AKT激动剂后,M1型标志物降低而M2型标记物升高(P<0.05),M1/M2比例降低;OE-METTL14可逆转此趋势。Sh-METTL14慢病毒组PI3K/AKT/GSK3β/β-catenin信号通路被激活(P<0.05),加入PI3K/AKT抑制剂后,M1型标志物升高而M2型标记物降低(P<0.05),M1/M2比例升高;Sh-METTL14可逆转此趋势。4)取转染OE-METTL14慢病毒后的巨噬细胞上清培养Hela细胞,可见细胞凋亡明显增多(P<0.05),增殖明显减少(P<0.05)。Sh-METTL14组的Hela细胞则表现出细胞凋亡减少(P<0.05),增殖增多(P<0.05)。结论METTL14通过PI3K/AKT/GSK3β/β-catenin信号通路调控巨噬细胞分化可能有促进宫颈癌细胞凋亡,抑制增殖的作用。

罗布麻总黄酮通过PI3K/AKT/GSK3β抑制H2O2诱导的EA.hy926凋亡机制研究

目的研究罗布麻总黄酮（TFF）对H2O2诱导的人脐静脉融合细胞EA.hy926凋亡的保护作用,并探讨其可能的作用机制.方法MTT法检测细胞存活率、凋亡检测试剂盒测定细胞凋亡率、Hoechst33342/PI荧光双染观察细胞形态变化,JC1染色检测细胞线粒体膜电位的变化;Westernblot检测凋亡相关蛋白Mcl-1、cleavedcaspase-3、cleavedcaspase9及给药组与加抑制剂组AKT、GSK3β蛋白表达和磷酸化水平;荧光定量PCR检测各组GSK3β、Mcl-1、cleavedcaspase-3、cleavedcaspase9mRNA的表达.结果与对照组相比,模型组细胞存活率降低、凋亡率升高、线粒体膜电位降低,与模型组相比,TFF各剂量组（9mg·L^-1、18mg·L^-1、36mg·L^-1）细胞存活率明显增加（P〈0.05）,线粒体膜电位升高（P〈0.05）;Westernblot结果,与模型组相比,TFF各剂量组的Mcl-1蛋白表达升高,而cleavedcaspase-3、cleavedcaspase-9蛋白表达量降低（P〈0.05）,与正常组相比,单独给药TFF各剂量组使通路中的AKT、GSK3β磷酸化水平增高,而加PI3K抑制剂LY294002后,与给药组相比AKT及GSK3β磷酸化水平均降低（P〈0.05）;荧光定量PCR结果,与模型组相比,随着给药剂量的增加Mcl-1的mRNA表达逐渐增高,而cleavedcaspase-3、cleavedcaspase-9、GSK3β的mRNA表达降低.结论罗布麻总黄酮对H2O2诱导的EA.hy926细胞损伤有明显的保护作用,其主要作用机制可能是通过激活PI3K/AKT/GSK3β通路,增加AKT、GSK3β的磷酸化,减少Bcl-2家族抗凋亡蛋白Mcl-1的泛素化降解,进而抑制基于caspase家族的线粒体凋亡,发挥细胞保护作用.

COX-2活化PI3K/Akt信号通路促结直肠癌肝转移的实验研究

目的:探讨COX-2促进结直肠癌肝转移效果及其机制,从而为结直肠癌肝转移防治提供依据。方法:建立COX-2低表达和高表达CT26结肠癌细胞株,通过注射0.2 m L的对数生长期细胞浓度为1×10~6个/mL的正常CT26结肠癌细胞悬液(A组,n=20)、COX-2高表达的CT26结肠癌细胞悬液(B组,n=20)以及COX-2低表达的结肠癌细胞悬液(C组,n=20)至脾脏制作小鼠结肠癌肝转移模型,检测比较三组小鼠移植瘤COX-2、PI3K、p-Akt表达、移植瘤体积、肝脏肿瘤大小和数量以及生存期,并分析结直肠癌肝转移小鼠COX-2、PI3K、p-Akt表达的相互关系以及三者与其移植瘤体积、肝脏肿瘤大小和数量以及生存期的关系。结果:与A组比较,B组和C组移植瘤COX-2、PI3K、p-Akt蛋白检测阳性率均升高,移植瘤体积降低,肝脏肿瘤大小和数量降低,生存期延长(P<0.05)。与B组比较,C组移植瘤COX-2、PI3K、p-Akt蛋白检测阳性率均升高,移植瘤体积降低,肝脏肿瘤大小和数量降低,生存期延长(P<0.05)。Spearman相关分析结果显示,结直肠癌肝转移小鼠移植瘤COX-2蛋白表达与其PI3K、p-Akt蛋白表达呈正相关(P<0.05),且其移植瘤COX-2、PI3K、p-Akt与其移植瘤体积、肝脏肿瘤大小和数量均呈正相关(P<0.05),与其生存期均呈负相关(P<0.05)。结论:COX-2可促结直肠癌肝转移和影响其预后,在此过程中可能涉及其活化PI3K/Akt信号通路作用。

Eukaryotic elongation factor-1α 2 knockdown inhibits hepatocarcinogenesis by suppressing PI3K/Akt/NF-κB signaling

AIM: To assess the impact of eukaryotic elongation factor 1 alpha 2 (eEF1A2) on hepatocellular carcinoma (HCC) cell proliferation, apoptosis, migration and invasion, and determine the underlying mechanisms.METHODS: eEF1A2 levels were detected in 62 HCC tissue samples and paired pericarcinomatous specimens, and the human HCC cell lines SK-HEP-1, HepG2 and BEF-7402, by real-time PCR and immunohistochemistry. Experimental groups included eEF1A2 silencing in BEL-7402 cells with lentivirus eEF1A2-shRNA (KD group) and eEF1A2 overexpression in SK-HEP-1 cells with eEF1A2 plasmid (OE group). Non-transfected cells (control group) and lentivirus-based empty vector transfected cells (NC group) were considered control groups. Cell proliferation (MTT and colony formation assays), apoptosis (Annexin V-APC assay), cell cycle (DNA ploidy assay), and migration and invasion (Transwell assays) were assessed. Protein levels of PI3K/Akt/NF-&#x003ba;B signaling effectors were evaluated by Western blot.RESULTS: eEF1A2 mRNA and protein levels were significantly higher in HCC cancer tissue samples than in paired pericarcinomatous and normal specimens. SK-HEP-1 cells showed lower eEF1A2 mRNA levels; HepG2 and BEL-7402 cells showed higher eEF1A2 mRNA levels, with BEL-7402 cells displaying the highest amount. Efficient eEF1A2 silencing resulted in reduced cell proliferation, migration and invasion, increased apoptosis, and induced cell cycle arrest. The PI3K/Akt/NF-&#x003ba;B signaling pathway was notably inhibited. Inversely, eEF1A2 overexpression resulted in promoted cell proliferation, migration and invasion.CONCLUSION: eEF1A2, highly expressed in HCC, is a potential oncogene. Its silencing significantly decreases HCC tumorigenesis, likely by inhibiting PI3K/Akt/NF-&#x003ba;B signaling.

Niuhuang(Bovis Calculus)-Shexiang(Moschus)combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explore its anti-HCC mechanism.Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro.SMMC-7721 was divided into the BC-Moschus group and the control group,and different doses(rude drug dosage 0.625,1.25,2.5,and 5 mg/m L)of BC-Moschus extract were used for the intervention.The proliferation ability of HCC cells was detected using the Cell Counting Kit-8(CCK-8)assay,and the migration ability was detected by a wound healing assay.A subcutaneous xenograft model was prepared using nude mice with human HCC.Specific pathogen-free-grade BALB/c nude mice(5-week-old)were randomly divided into the following groups(n=6 per group):control(0.9%physiological saline 0.2 m L/d),BC-Moschus[BC 45.5 mg/(kg·d)+Moschus 13 mg/(kg·d)],and cisplatin(DDP,intraperitoneal injection5 mg/kg per week)groups.All groups were administered for 14 d.The volume and mass of the subcutaneous xenografts in nude mice were observed.The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway,apoptosis-associated factor p70 S6 Kinase(S6K),Bax,Bcl-2,caspase-3,and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR(RT-qPCR)and Western blot.Terminal Deoxynucleotidy Transferase-Mediated d UTP NickEnd Labeling(TUNEL)was used for quantitative analysis of apoptotic cells.Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone,and the inhibition effect was dose-and time-dependent(P<0.01).The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration(P<0.01).In the subcutaneous xenograft model of nude mice with human HCC,we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group(P<0.01).The levels of the PI3K/AKT/m TOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased(P<0.01).The expression level of the anti-apoptotic gene Bcl-2 was downregulated(P<0.05),and the expression of the pro-apoptotic gene Baxand apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated(P<0.01).The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC(P<0.01).Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice.The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway,regulation of apoptosis-related protein caspase-3,caspase-9,Bcl-2,and Bax expression,and promotion of apoptosis.

芝麻素通过抑制AKT/GSK3β/NFATc1信号通路减轻2型糖尿病大鼠骨质疏松症

目的:探究芝麻素对2型糖尿病性骨质疏松症(T2DOP)的治疗作用及其可能机制。方法:将健康雄性SD大鼠分为对照组、模型组、低剂量芝麻素组(L-Ses组)和高剂量芝麻素组(H-Ses组),每组10只。模型组、L-Ses组和H-Ses组大鼠给予高脂高糖饲料喂饲4周,并一次性腹腔注射链脲佐菌素(STZ,剂量35 mg/kg)溶液建立2型糖尿病性骨质疏松症大鼠模型。造模成功后,L-Ses组和H-Ses组大鼠分别按照10 mg/kg和40 mg/kg的剂量给予芝麻素灌胃处理,每日1次,持续药物处理8周。对照组和模型组大鼠同时间段给予等量的生理盐水灌胃处理。分别检测大鼠血清中空腹血糖值(Fasting plasma glucose,FPG)、空腹胰岛素(Fasting insulin,FINS)、糖化血红蛋白(Glycosylated hemoglobin,HbA1c)、血清总胆固醇(Serum total cholesterol,TC)、甘油三酯(Triglyceride,TG)、低密度脂蛋白胆固醇(Low-density lipoprotein cholesterol,LDL-C)、高密度脂蛋白胆固醇(High-density lipoprotein cholesterol,HDL-C)、超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化氢酶(Catalase,CAT)、谷胱甘肽(Glutathione,GSH)和丙二醛(Malondialdehyde,MDA)的水平。采用苏木素-伊红(Hematoxylin-eosin staining,HE)染色检测大鼠股骨组织形态。μCT扫描仪分析大鼠股骨干骺端三维结构以及骨密度(Bone mineral density,BMD)、骨小梁数(Trabecular number,Tb.N)和骨小梁厚度(Trabecular thickness,Tb.Th)参数,Western blot检测骨形成指标骨碱性磷酸酶(Bone alkaline phosphate,BALP)、骨钙素(Osteocalcin,OCN)、成骨相关基因2(Runt-related transcription factor 2,Runx2),以及磷脂酰肌醇-3激酶(Phosphoinositide 3 kinase,PI3K)/蛋白激酶B(Protein kinase B,Akt)/糖原合成酶激酶3β(Glycogen synthase kinase 3β,GSK3β)/活化T细胞核因子1(Nuclear factor of activated T cells c1,NFATc1)信号通路相关蛋白的表达。结果:与对照组比较,模型组大鼠血清FPG、FINS、HBA1c、TC、TG、LDL-C和MDA水平均升高(P<0.05),HDL-C、SOD、CAT和GSH水平均降低(P<0.05);股骨组织中骨小梁断裂状态较多,排列紊乱,骨质严重流失,BMD、Tb.N和Tb.Th均降低(P<0.05),股骨组织中BALP、OCN和Runx2蛋白表达水平均降低(P<0.05),AKT和NFATc1蛋白磷酸化水平均升高(P<0.05),GSK3β蛋白磷酸化水平降低(P<0.05)。与模型组比较,L-Ses组和H-Ses组大鼠血清中FPG、FINS、HBA1c、TC、TG、LDL-C和MDA水平均降低(P<0.05),HDL-C、SOD、CAT和GSH水平均升高(P<0.05);股骨组织中骨小梁断裂状态较少,排列、形态结构和骨质流失均呈现较大改善,BMD、Tb.N和Tb.Th均升高(P<0.05),股骨组织中BALP、OCN和Runx2蛋白表达水平均升高(P<0.05),AKT和NFATc1蛋白磷酸化水平均降低(P<0.05),GSK3β蛋白磷酸化水平升高(P<0.05)。与L-Ses组比较,H-Ses组大鼠血清中FPG、FINS、HBA1c、TC、TG、LDL-C、SOD、CAT、GSH和MDA水平均降低(P<0.05),HDL-C水平升高(P<0.05);大鼠股骨组织和形态稍有缓解,BMD、Tb.N和Tb.Th均升高(P<0.05),股骨组织中BALP、OCN和Runx2蛋白表达水平均升高(P<0.05),AKT和NFATc1蛋白磷酸化水平均降低(P<0.05),GSK3β蛋白磷酸化水平升高(P<0.05)。结论:芝麻素可能通过抑制AKT/GSK3β/NFATc1信号通路,促进骨形成,进而缓解T2DOP大鼠的骨质疏松症进展。

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.

槐杞黄对高糖诱导HK-2细胞损伤的保护效应及机制

目的探讨槐杞黄是否通过调控AKT/GSK3β信号通路抑制高糖诱导的人肾小管上皮细胞(human kidney-2,HK-2)氧化应激和凋亡。方法将HK-2细胞分为正常对照组、甘露醇组、高糖模型组、槐杞黄组和卡托普利组。采用CCK-8法检测HK-2细胞存活率,活性氧(reactive oxygen species,ROS)检测试剂盒检测ROS含量,脂质过氧化丙二醛(malondialdehyde,MDA)试剂盒检测MDA含量,超氧化物歧化酶(superoxide dismutase,SOD)活性检测试剂盒检测SOD含量,RT-qPCR检测肾损伤分子(kidney injury molecule-1,KIM-1)和中性粒细胞相关载脂蛋白(neutrophil gelatinase-associated lipocalin,NGAL)mRNA表达水平,Western blot检测凋亡相关蛋白(Bax、Bcl-2、cleaved-Caspase-3、Caspase-3)和AKT/GSK3β通路蛋白(p-AKT、AKT、p-GSK-3β、GSK-3β)表达水平。结果与正常对照组比较,高糖模型组HK-2细胞存活率明显降低(P<0.001),KIM-1和NGAL mRNA表达水平明显上升(P<0.001),ROS生成增加(P<0.001),MDA含量显著升高(P<0.001),SOD活性显著下降(P<0.001),Bcl-2蛋白水平显著下调(P<0.001),Bax和cleaved-Caspase-3的蛋白水平显著上调(P<0.001),p-AKT蛋白水平显著下调(P<0.001),p-GSK-3β蛋白水平显著上调(P<0.001)。与高糖模型组比较,槐杞黄组和卡托普利组HK-2细胞存活率显著增加(P<0.001),KIM-1和NGAL mRNA表达水平明显下降(P<0.001),ROS生成减少(P<0.001),MDA含量显著降低(P<0.001),SOD活性显著上升(P<0.001),Bcl-2的蛋白水平显著上调(P<0.01),Bax和cleaved-Caspase-3的蛋白水平显著下调(P<0.01),p-AKT的蛋白水平显著上调(P<0.01),p-GSK-3β的蛋白水平显著下调(P<0.01)。槐杞黄组与卡托普利组各指标差异无统计学意义。结论槐杞黄对高糖诱导的HK-2细胞氧化应激和凋亡具有抑制作用,其作用机制可能与调控AKT/GSK3β信号通路有关。

Macrophage-derived exosomal miR-342-3p promotes the progression of renal cell carcinoma through the NEDD4L/CEP55 axis

Due to its difficulty in early diagnosis and lack of sensitivity to chemotherapy and radiotherapy,renal cell carcinoma(RCC)remains to be a frequent cause of cancer-related death.Here,we probed into new targets for its early diagnosis and treatment for RCC.microRNA(miRNA)data of M2-EVs and RCC were searched on the Gene Expression Omnibus database,followed by the prediction of the potential downstream target.Expression of target genes was measured via RT-qPCR and Western blot,respectively.M2 macrophage was obtained viaflow cytometry with M2-EVs extracted.The binding ability of miR-342-3p to NEDD4L and to CEP55 ubiquitination was studied with their roles in the physical abilities of RCC cells assayed.Subcutaneous tumor-bearing mouse models and lung metastasis models were prepared to observe in vivo role of target genes.M2-EVs induced RCC growth and metastasis.miR-342-3p showed high expression in both M2-EVs and RCC cells.M2-EVs carrying miR-342-3p promoted RCC cell abilities to proliferate,invade and migrate.In RCC cells,M2-EV-derived miR-342-3p could specifically bind to NEDD4L and consequently elevate CEP55 protein expression via suppressing NEDD4L,thereby exerting tumor-promoting effects.CEP55 could be degraded by ubiquitination under the function of NEDD4L,and miR-342-3p delivered by M2-EVs facilitated the RCC occurrence and development by activating the PI3K/AKT/mTOR signaling pathway.In conclusion,M2-EVs promote RCC growth and metastasis by delivering miR-342-3p to suppress NEDD4L and subsequently inhibit CEP55 ubiquitination and degradation via activation of the PI3K/AKT/mTOR signaling pathway,strongly driving the proliferative,migratory and invasive of RCC cells.

基于PI3K/AKT/GSK3β通路探讨解毒通络调肝方对2型糖尿病认知障碍大鼠的影响

55只SD大鼠随机分为对照组、模型组、解毒通络调肝方组以及盐酸二甲双胍组,其中45只采用高糖高脂联合腹腔注射链脲佐菌素的方式诱导2型糖尿病模型,给药4周后麻醉处死取样。Morris水迷宫试验法测试大鼠空间学习能力,血生化检测空腹血糖、空腹血清胰岛素,HE染色法观察SD大鼠海马组织病理变化,免疫组织化学法检测BDNF蛋白表达,Western blot法检测CREB、GLUT4、PI3K、AKT、GSK3β蛋白表达。结果提示:与对照组相比,模型组空腹血糖、空腹血清胰岛素显著升高(P<0.01),水迷宫试验相关指标提示大鼠空间学习能力降低(P<0.01),BDNF蛋白阳性表达明显降低(P<0.01),CREB、GLUT4、PI3K、AKT蛋白表达也均呈现出明显降低(P<0.001,P<0.01),GSK3β蛋白表达升高(P<0.001);与模型组相比,给药组大鼠空腹血糖、空腹血清胰岛素均明显下降(P<0.01),水迷宫试验相关指标提示大鼠空间学习能力提升(P<0.01),BNDF阳性表达增加(P<0.01),CREB、GLUT4、PI3K、AKT蛋白表达均显著增加(P<0.001,P<0.01,P<0.05),GSK3β蛋白表达下降(P<0.001)。结果表明,解毒通络调肝方能够提高2型糖尿病认知障碍大鼠的认知能力,其作用机制可能是通过调控海马组织中BDNF、CREB、GLUT4蛋白表达,影响PI3K/AKT/GSK-3β信号通路实现的。