Mammalian target of rapamycin complex 1 as an inducer of neurotrophic factors in dopaminergic neurons

The defining neuropathological feature of Parkinson＇s disease （PD） is the loss of nigrostriatal dopaminergic （DA） projections. This results in striatal dopamine levels and a biochemical reduction of movement disorders, such as a tremor at rest, rigidity of the limbs, bradykinesia, and postural instability （Kim et al., 2011; Kim et al., 2012; Burke and O＇Malley, 2013; Leem et al., 2014; Namet al., 2014）.

3,6-dichlorobenzo[b]thiophene-2-carboxylic acid alleviates ulcerative colitis by suppressing mammalian target of rapamycin complex 1 activation and regulating intestinal microbiota

BACKGROUND 3,6-dichlorobenzo[b]thiophene-2-carboxylic acid(BT2)is a benzothiophene carboxylate derivative that can suppress the catabolism of branched-chain amino acid(BCAA)-associated mammalian target of rapamycin complex 1(mTORC1)activation.Previous studies have demonstrated the therapeutic effects of BT2 on arthritis,liver cancer,and kidney injury.However,the effects of BT2 on ulcerative colitis(UC)are unknown.AIM To investigate the anti-UC effects of BT2 and the underlying mechanism.METHODS Mouse UC models were created through the administration of 3.5%dextran sodium sulfate(DSS)for 7 d.The mice in the treated groups were administered salazosulfapyridine(300 mg/kg)or BT2(20 mg/kg)orally from day 1 to day 7.At the end of the study,all of the mice were sacrificed,and colon tissues were removed for hematoxylin and eosin staining,immunoblot analyses,and immunohistochemical assays.Cytokine levels were measured by flow cytometry.The contents of BCAAs including valine,leucine,and isoleucine,in mouse serum were detected by liquid chromatography-tandem mass spectrometry,and the abundance of intestinal flora was analyzed by 16S ribosomal DNA sequencing.RESULTS Our results revealed that BT2 significantly ameliorated the inflammatory symptoms and pathological damage induced by DSS in mice.BT2 also reduced the production of the proinflammatory cytokines interleukin 6(IL-6),IL-9,and IL-2 and increased the anti-inflammatory cytokine IL-10 level.In addition,BT2 notably improved BCAA catabolism and suppressed mTORC1 activation and cyclooxygenase-2 expression in the colon tissues of UC mice.Furthermore,highthroughput sequencing revealed that BT2 restored the gut microbial abundance and diversity in mice with colitis.Compared with the DSS group,BT2 treatment increased the ratio of Firmicutes to Bacteroidetes and decreased the abundance of Enterobacteriaceae and Escherichia-Shigella.CONCLUSION Our results indicated that BT2 significantly ameliorated DSS-induced UC and that the latent mechanism involved the suppression of BCAA-associated mTORC1 activation and modulation of the intestinal flora.

MicroRNA-451 from Human Umbilical Cord-Derived Mesenchymal Stem Cell Exosomes Inhibits Alveolar Macrophage Autophagy via Tuberous Sclerosis Complex 1/Mammalian Target of Rapamycin Pathway to Attenuate Burn-Induced Acute Lung Injury in Rats

Objective Our previous studies established that microRNA(miR)-451 from human umbilical cord mesenchymal stem cell-derived exosomes(hUC-MSC-Exos)alleviates acute lung injury(ALI).This study aims to elucidate the mechanisms by which miR-451 in hUC-MSC-Exos reduces ALI by modulating macrophage autophagy.Methods Exosomes were isolated from hUC-MSCs.Severe burn-induced ALI rat models were treated with hUC-MSC-Exos carrying the miR-451 inhibitor.Hematoxylin-eosin staining evaluated inflammatory injury.Enzyme-linked immunosorbnent assay measured lipopolysaccharide(LPS),tumor necrosis factor-α,and interleukin-1βlevels.qRT-PCR detected miR-451 and tuberous sclerosis complex 1(TSC1)expressions.The regulatory role of miR-451 on TSC1 was determined using a dual-luciferase reporter system.Western blotting determined TSC1 and proteins related to the mammalian target of rapamycin(mTOR)pathway and autophagy.Immunofluorescence analysis was conducted to examine exosomes phagocytosis in alveolar macrophages and autophagy level.Results hUC-MSC-Exos with miR-451 inhibitor reduced burn-induced ALI and promoted macrophage autophagy.MiR-451 could be transferred from hUC-MSCs to alveolar macrophages via exosomes and directly targeted TSC1.Inhibiting miR-451 in hUC-MSC-Exos elevated TSC1 expression and inactivated the mTOR pathway in alveolar macrophages.Silencing TSC1 activated mTOR signaling and inhibited autophagy,while TSC1 knockdown reversed the autophagy from the miR-451 inhibitor-induced.Conclusion miR-451 from hUC-MSC exosomes improves ALI by suppressing alveolar macrophage autophagy through modulation of the TSC1/mTOR pathway,providing a potential therapeutic strategy for ALI.

Humanβ-defensin-1 affects the mammalian target of rapamycin pathway and autophagy in colon cancer cells through long noncoding RNA TCONS_00014506

BACKGROUND Colorectal cancer has a low 5-year survival rate and high mortality.Humanβ-defensin-1(hBD-1)may play an integral function in the innate immune system,contributing to the recognition and destruction of cancer cells.Long non-coding RNAs(lncRNAs)are involved in the process of cell differentiation and growth.AIM To investigate the effect of hBD-1 on the mammalian target of rapamycin(mTOR)pathway and autophagy in human colon cancer SW620 cells.METHODS CCK8 assay was utilized for the detection of cell proliferation and determination of the optimal drug concentration.Colony formation assay was employed to assess the effect of hBD-1 on SW620 cell proliferation.Bioinformatics was used to screen potentially biologically significant lncRNAs related to the mTOR pathway.Additionally,p-mTOR(Ser2448),Beclin1,and LC3II/I expression levels in SW620 cells were assessed through Western blot analysis.RESULTS hBD-1 inhibited the proliferative ability of SW620 cells,as evidenced by the reduction in the colony formation capacity of SW620 cells upon exposure to hBD-1.hBD-1 decreased the expression of p-mTOR(Ser2448)protein and increased the expression of Beclin1 and LC3II/I protein.Furthermore,bioinformatics analysis identified seven lncRNAs(2 upregulated and 5 downregulated)related to the mTOR pathway.The lncRNA TCONS_00014506 was ultimately selected.Following the inhibition of the lncRNA TCONS_00014506,exposure to hBD-1 inhibited p-mTOR(Ser2448)and promoted Beclin1 and LC3II/I protein expression.CONCLUSION hBD-1 inhibits the mTOR pathway and promotes autophagy by upregulating the expression of the lncRNA TCONS_00014506 in SW620 cells.

Production of interleukin-1β related to mammalian target of rapamycin/Toll-like receptor 4 signaling pathway during Aspergillus fumigatus infection of the mouse cornea

AIM：To elucidate the effect of rapamycin on regulating the production of interleukin（IL）-1β in Aspergillus fumigatus（A.fumigatus）-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus keratitis is associated with the mammalian target of rapamycin（mT OR）/Toll-like receptor 4（TLR4） signaling pathway.METHODS：Fungal keratitis mouse models of susceptible C57 BL/6 mice were established using A.fumigatus.The mice were subsequently treated with rapamycin.The protein levels of p-mT OR,TLR4,and IL-1β in normal and infected corneal tissue were measured by Western blot.The TLR4 and IL-1β m RNA levels were determined by real-time polymerase chain reaction（PCR）.RESULTS：In C57 BL/6 mice,rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine,IL-1β.The expression of TLR4,stimulated by A.fumigatus,was reduced as well when the mT OR signaling pathway was suppressed by rapamycin.CONCLUSION：Rapamycin is beneficial for the outcome of fungal keratitis and has an inhibitory effect expression of the inflammatory cytokine IL-1β.The inhibitory effect on IL-1β expression can be associated with the mT OR/TLR4 signaling pathway in A.fumigatus infection in mice.

Role of mammalian target of rapamycin complex 2 in primary and secondary liver cancer

The mammalian target of rapamycin(mTOR)acts in two structurally and functionally distinct protein complexes,mTOR complex 1(mTORC1)and mTOR complex 2(mTORC2).Upon deregulation,activated mTOR signaling is associated with multiple processes involved in tumor growth and metastasis.Compared with mTORC1,much less is known about mTORC2 in cancer,mainly because of the unavailability of a selective inhibitor.However,existing data suggest that mTORC2 with its two distinct subunits Rictor and mSin1 might play a more important role than assumed so far.It is one of the key effectors of the PI3K/AKT/mTOR pathway and stimulates cell growth,cell survival,metabolism,and cytoskeletal organization.It is not only implicated in tumor progression,metastasis,and the tumor microenvironment but also in resistance to therapy.Rictor,the central subunit of mTORC2,was found to be upregulated in different kinds of cancers and is associated with advanced tumor stages and a bad prognosis.Moreover,AKT,the main downstream regulator of mTORC2/Rictor,is one of the most highly activated proteins in cancer.Primary and secondary liver cancer are major problems for current cancer therapy due to the lack of specific medical treatment,emphasizing the need for further therapeutic options.This review,therefore,summarizes the role of mTORC2/Rictor in cancer,with special focus on primary liver cancer but also on liver metastases.

The Expression of Mammalian Target of Rapamycin in Ishikawa and HEC-1A Cells

The activation of mammalian target of rapamycin （mTOR） signaling pathway in endometrial carcinoma cells Ishikawa and HEC-1A was investigated. The expression of mTOR was detected by confocal fluorescence microscopy in Ishikawa and HEC-1A cells. The mRNA levels of PTEN and mTOR, the downstream substrate S6K1 and 4E-BP1 protein were assayed by RT-PCR and Western blot, respectively. The expression of PTEN in Ishikawa cells was deficient, but intact in HEC-1A cells respectively （P〈0.01）. There was mTOR expression in both Ishikawa and HEC-1A cells and the phosporylated substrate levels in Ishikawa cells were higher than those in HEC-1A cells （P〈0.05）. mTOR signaling pathway is activated in two endometrial carcinoma cell strains and the status of activation is related with PTEN expression of the cells. The activation level of mTOR is higher in PTEN-deficient endometrial carcinoma cells than that in PTEN-intact endometrial carcinoma cells.

WJH 6^(th) Anniversary Special Issues(2): Hepatocellular carcinoma Mammalian target of rapamycin inhibition in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is one of the leading causes of cancer-related death worldwide. It is associated with a poor prognosis and has limited treatment options. Sorafenib, a multi-targeted kinase inhibitor, is the only available systemic agent for treatment of HCC that improves overall survival for patients with advanced stage disease; unfortunately, an effective second-line agent for the treatment of progressive or sorafenib-resistant HCC has yet to be identified. This review focuses on components of the mammalian target of rapamycin(mTOR) pathway, its role in HCC pathogenesis, and dual mTOR inhibition as a therapeutic option with potential efficacy in advanced HCC. There are several important upstream and downstream signals in the mTOR pathway, and alternative tumor-promoting pathways are known to exist beyond mTORC1 inhibition in HCC. This review analyzes the relationships of the upstream and downstream regulators of mTORC1 and mTORC2 signaling; it also provides a comprehensive global picture of the interaction between mTORC1 and mTORC2 which demonstrates the pre-clinical relevance of the mTOR pathway in HCC pathogenesis and progression. Finally, it provides scientific rationale for dual mTORC1 and mTORC2 inhibition in the treatment of HCC. Clinical trials utilizing mTORC1 inhibitors and dual mTOR inhibitors in HCC are discussed as well. The mTOR pathway is comprised of two main components, mTORC1 and mTORC2; each has a unique role in the pathogenesis and progression of HCC. In phase Ⅲ studies, mTORC1 inhibitors demonstrate anti-tumor ac-tivity in advanced HCC, but dual mTOR(mTORC1 and mTORC2) inhibition has greater therapeutic potential in HCC treatment which warrants further clinical investigation.

青藤碱调节AMPK/mTOR/ULK1信号通路对IL-1β诱导的关节软骨细胞自噬和凋亡的影响

目的探讨青藤碱(SN)调节单磷酸腺苷活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)/UNC-51样激酶1(ULK1)信号通路对白介素-1β(IL-1β)诱导的关节软骨细胞自噬和凋亡的影响。方法将关节软骨细胞分为Control组(正常培养)、IL-1β组(10μg/L的IL-1β诱导12 h)、L-SN、M-SN、H-SN组(在IL-1β诱导的基础上添加25、50、100μmol/L的SN)、SN+Compound C组(在H-SN组的基础上添加10μmol/L AMPK抑制剂Compound C)。MTT法、透射电子显微镜(TEM)、流式细胞仪分别检测SN对各组关节软骨细胞增殖、自噬、凋亡的影响;ELISA试剂盒检测各组细胞中COX-2、TNF-α、MMP-3、MMP-13的表达;蛋白印迹实验(WB)检测各组细胞中p-AMPK、AMPK、p-mTOR、mTOR、p-ULK1、ULK1蛋白水平。结果与Control组比较,IL-1β组关节软骨细胞的A 490值、p-AMPK/AMPK、p-ULK1/ULK1蛋白水平降低,自噬空泡数、凋亡率、COX-2、TNF-α、MMP-3、MMP-13、p-mTOR/mTOR蛋白水平升高(P<0.05);与IL-1β组比较,L-SN组、M-SN组、H-SN组A 490值、自噬空泡数、p-AMPK/AMPK、p-ULK1/ULK1蛋白水平升高,凋亡率、COX-2、TNF-α、MMP-3、MMP-13、p-mTOR/mTOR蛋白水平降低(P<0.05);与H-SN组比较,SN+Compound C组A 490值、自噬空泡数、p-AMPK/AMPK、p-ULK1/ULK1蛋白水平降低,凋亡率、COX-2、TNF-α、MMP-3、MMP-13、p-mTOR/mTOR蛋白水平升高(P<0.05)。结论SN可以通过促进IL-1β诱导的关节软骨细胞自噬,抑制细胞凋亡,其机制可能是通过激活AMPK/mTOR/ULK1信号通路实现的。

lncRNA NEAT1通过Klotho/mTOR轴调控慢性脑低灌注大鼠的认知障碍

目的 探讨长链非编码RNA (lncRNA)核富集丰度转录物1 (NEAT1)对慢性脑低灌注(CCH)大鼠认知障碍的调控作用和潜在机制。方法双侧颈总动脉闭塞术(BCCAO)诱导CCH模型大鼠。将大鼠分为5组(每组n=10):Ⅰ组(假手术组);Ⅱ组(BCCAO手术组);Ⅲ组[BCCAO后于海马区注射0.3 mg·kg^(-1)·w^(-1)的沉默lncRNA NEAT1的短发夹RNA重组质粒(shNEAT1),持续12 w];Ⅳ组[BCCAO后于海马区每周注射0.3 mg·kg^(-1)·w^(-1)的shNEAT1和50 mg·kg^(-1)·w^(-1)的沉默克洛托蛋白(Klotho)的短发夹RNA重组质粒(shKlotho),持续12 w];V组[BCCAO后于海马区注射0.3 mg·kg^(-1)·w^(-1)的shNEAT1和25mg·kg^(-1)哺乳动物雷帕霉素靶蛋白(mTOR)的抑制剂(AZD-8055),持续12w]。行Morris水迷宫实验记录逃避潜伏期。实时荧光定量PCR (qRT-PCR)测lncRNA NEAT1的表达。Western blot法检测Klotho、mTOR、磷酸化的mTOR (p-mTOR)、神经核抗原(NeuN)、多聚腺苷酸核糖聚合酶(PARP)、半胱天冬酶-3(caspase-3)、切割模式的半胱天冬酶-3(cleaved-caspase-3)的表达。免疫荧光化学检测海马区NeuN阳性细胞数。结果 与Ⅰ组比,Ⅱ组大鼠的逃避潜伏期延长,lncRNA NEAT1、PARP、cleaved-caspase-3的表达均上调,NeuN、Klotho和p-mTOR的表达均下调,NeuN阳性的细胞数减少(~均P<0.05)。与Ⅱ组比,Ⅲ组大鼠的逃避潜伏期缩短,且海马组织中lncRNA NEAT1、PARP、cleaved-caspase-3的表达均下调,NeuN、Klotho和p-mTOR的表达均上调,NeuN阳性的细胞数增加(~均P><0.05)。与Ⅲ组比,Ⅳ组和Ⅴ组中大鼠的逃避潜伏期延长,且海马组织中PARP、cleaved-caspase-3的表达均上调,NeuN、Klotho和p-mTOR的表达均下调,NeuN阳性细胞数都减少(~均P <0.05)。结论 lncRNA NEAT1通过负调控Klotho/mTOR轴促进CCH大鼠的认知障碍,为研究CCH的有效治疗靶点提供理论依据。

Neuroprotective effects of rapamycin on spinal cord injury in rats by increasing autophagy and Akt signaling

Rapamycin treatment has been shown to increase autophagy activity and activate Akt phosphorylation, suppressing apoptosis in several models of ischemia reperfusion injury. However, little has been studied on the neuroprotective effects on spinal cord injury by activating Akt phosphorylation. We hypothesized that both effects of rapamycin, the increased autophagy activity and Akt signaling, would contribute to its neuroprotective properties. In this study, a compressive spinal cord injury model of rat was created by an aneurysm clip with a 30 g closing force. Rat models were intraperitoneally injected with rapamycin 1 mg/kg, followed by autophagy inhibitor 3-methyladenine 2.5 mg/kg and Akt inhibitor IV 1 μg/kg. Western blot assay, immunofluorescence staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay were used to observe the expression of neuronal autophagy molecule Beclin 1, apoptosis-related molecules Bcl-2, Bax, cytochrome c, casp ase-3 and Akt signaling. Our results demonstrated that rapamycin inhibited the expression of mTOR in injured spinal cord tissue and up-regulated the expression of Beclin 1 and phosphorylated-Akt. Rapamycin prevented the decrease of bcl-2 expression in injured spinal cord tissue, reduced Bax, cytochrome c and caspase-3 expression levels and reduced the number of apoptotic neurons in injured spinal cord tissue 24 hours after spinal cord injury. 3-Methyladenine and Akt inhibitor IV intervention suppressed the expression of Beclin-1 and phosphorylated-Akt in injured spinal cord tissue and reduced the protective effect of rapamycin on apoptotic neurons. The above results indicate that the neuroprotective effect of rapamycin on spinal cord injury rats can be achieved by activating autophagy and the Akt signaling pathway.

Melatonin attenuates cisplatin-induced HepG2 cell death via the regulation of mTOR and ERCC1 expressions

AIM:To elucidate the effects of melatonin on cisplatininduced hepatocellular carcinoma(HepG2) cell death and to identify potential cross-talk pathways.METHODS:Hepatocellular carcinoma HepG2 cells were treated with melatonin and/or cisplatin for 24 to 48 h.Cell viability and the 50% cytotoxic concentration(CC50) were calculated by MTT assays.The effects and intracellular events induced by the selected concentrations of melatonin(1 mmol/L) and cisplatin(20 μmol/L) were investigated.Cell death and survival detection were primarily evaluated using a fluorescence microscope to assess 4',6 diamideno-2-phenylindol DNA staining and acridine orange lysosome staining and then further analyzed with immunocytochemistry using an anti-LC3 antibody.The potential molecularresponses mediated by melatonin against cisplatin after the combined treatment were investigated by reverse transcription-polymerase chains reaction and Western blot analyses of the genes and proteins associated with cell survival and death.A cell cycle analysis was performed using a flow cytometry assay.RESULTS:Melatonin had a concentration-dependent effect on HepG2 cell viability.At 1 mmol/L,melatonin significantly increased the cell viability percentage and decreased reactive oxygen species production due to cisplatin.Melatonin reduced cisplatin-induced cell death,decreasing phosphorylated p53 apoptotic protein,cleaved caspase 3 and Bax levels but increasing anti-apoptotic Bcl-2 gene and protein expression.When combined with cisplatin,melatonin induced S phase(DNA synthesis) cell cycle arrest and promoted autophagic events in HepG2 cells.Melatonin also had a concentration-dependent effect on Beclin-1 and its autophagic regulator mammalian target of rapamycin(mTOR) as well as the DNA excision repair cross complementary 1(ERCC1) protein.The expression levels of these proteins were altered in HepG2 cells during cisplatin or melatonin treatment alone.In the combination treatment,melatonin reversed the effects of cisplatin by suppressing the over-expression of mTOR and ERCC 1 and enhancing the expression levels of Beclin-1 and microtubule-associated protein-light chain3-Ⅱ,leading to intracellular autophagosome progression.CONCLUSION:Melatonin attenuated cisplatin-induced cell death in HepG2 cells via a counter-balance between the roles of apoptotic- and autophagy-related proteins.

Neuroprotection by dipeptidyl-peptidase-4 inhibitors and glucagonlike peptide-1 analogs via the modulation of AKT-signaling pathway in Alzheimer’s disease

Alzheimer’s disease(AD)is the most common reason for progressive dementia in the elderly.It has been shown that disorders of the mammalian/mechanistic target of rapamycin(mTOR)signaling pathways are related to the AD.On the other hand,diabetes mellitus(DM)is a risk factor for the cognitive dysfunction.The pathogenesis of the neuronal impairment caused by diabetic hyperglycemia is intricate,which contains neuro-inflammation and/or neurodegeneration and dementia.Glucagon-like peptide-1(GLP1)is interesting as a possible link between metabolism and brain impairment.Modulation of GLP1 activity can influence amyloid-beta peptide aggregation via the phosphoinositide-3 kinase/AKT/mTOR signaling pathway in AD.The GLP1 receptor agonists have been shown to have favorable actions on the brain such as the improvement of neurological deficit.They might also exert a beneficial effect with refining learning and memory on the cognitive impairment induced by diabetes.Recent experimental and clinical evidence indicates that dipeptidyl-peptidase-4(DPP4)inhibitors,being currently used for DM therapy,may also be effective for AD treatment.The DPP-4 inhibitors have demonstrated neuroprotection and cognitive improvements in animal models.Although further studies for mTOR,GLP1,and DPP4 signaling pathways in humans would be intensively required,they seem to be a promising approach for innovative AD-treatments.We would like to review the characteristics of AD pathogenesis,the key roles of mTOR in AD and the preventive and/or therapeutic suggestions of directing the mTOR signaling pathway.

肝细胞DEP结构域蛋白5/哺乳动物雷帕霉素靶蛋白复合物1信号轴在非酒精性脂肪肝形成中的作用

目的建立肝细胞Dishevelled/Egl-10/pleckstrin(DEP)结构域蛋白5(DEPDC5)基因(Depdc5)肝细胞特异性敲除小鼠高脂喂养模型,探讨DEPDC5/哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)信号轴对非酒精性脂肪肝的调控。方法构建肝细胞特异性敲除Depdc5^(flox/flox)模型;Alb-Cre小鼠(LKO),Depdc5^(flox/flox)小鼠(Loxp)作为对照。32只2~3月龄雄性小鼠随机分为高脂LKO组、高脂Loxp对照组、高脂+雷帕霉素LKO组及高脂+雷帕霉素Loxp对照组,每组8只。检测肝脏血清生物化学指标、脂质含量、蛋白、mRNA及病理切片,采用GraphPad Prism 8软件进行统计学分析。结果高脂喂养导致LoxP小鼠肝脏脂肪变性,LKO小鼠肝脏脂肪变性减轻但合并出现肝损伤;雷帕霉素抑制了Depdc5敲除引起的mTORC1通路激活,显著改善Loxp小鼠肝脏脂肪变性,并改善LKO小鼠的肝损伤。结论Depdc5基因敲除能够保护高脂喂养小鼠肝脏脂肪变性,雷帕霉素可以改善DEPDC5缺失诱发的肝损伤。

前癃通胶囊介导miR-216a-5p/TPT1/mTORC1通路调控良性前列腺增生的实验研究

目的通过细胞实验探讨前癃通胶囊(qian long tong capsule,QLTC)能否通过调控miR-216a-5p/肿瘤蛋白翻译控制1/哺乳动物雷帕霉素靶蛋白复合物1(miR-216a-5p/tumor protein translationally controlled 1/mammalian target of rapamycin complex 1,miR-216a-5p/TPT1/mTORC1)信号通路抑制良性前列腺增生(benign prostatic hyperplasia,BPH)。方法将25只大鼠随机分为对照组(等体积生理盐水),QLTC低(56.25 mg/mL)、中(112.50 mg/mL)、高(225.00 mg/mL)剂量组,LBSC组(168.75 mg/mL),每组5只。每组灌胃1 mL/次,2次/d,连续5 d。各组大鼠麻醉后制备含药血清。根据实验目的不同,将CP-H022细胞分5步做实验处理,每部分实验进行独立分组。将miR-216a-5p过表达和沉默表达,及TPT1过表达进行对照研究;RT-qPCR法检测正常和BPH模型CP-H022细胞内miR-216a-5p表达量,并观察不同浓度QLTC处理的BPH细胞中miR-216a-5p表达量的差异;细胞集落形成实验检测细胞增殖能力;CCK-8法检测BPH模型细胞增殖;RT-qPCR法检测miR-216a-5p、TPT1 mRNA表达水平;流式细胞术检测细胞凋亡;生信分析、双荧光素酶实验验证miR-216a-5p与TPT1的靶向关系;过表达TPT1后,Western blot法检测BPH细胞中TPT1/mTORC1信号通路相关分子表达情况。结果与对照组1比较,模型组1的CP-H022细胞内miR-216a-5p表达量下调(P<0.05);不同浓度的QLTC均能上调miR-216a-5p表达量(P<0.05);根据本实验结果,本研究将选用QLTC(高剂量)组CP-H022细胞进行后续实验。与模型组2比较,QLTC组2细胞增殖减少、凋亡增加(P<0.05),B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)表达降低(P<0.05),Bcl-2关联X蛋白单克隆抗体(monoclonal antibody to Bcl-2 associated X protein,Bax)、cleaved Caspase-3表达升高(P<0.05)。敲低miR-216a-5p后,与模型组4比较,QLTC组4细胞增殖增强、凋亡减少(P<0.05),Bcl-2表达升高(P<0.05),Bax、cleaved Caspase-3表达降低(P<0.05)。与mimic-NC组比较,miR-216a-5p mimic组TPT1表达量降低(P<0.05);QLTC处理后,细胞TPT1、p-mTORC1表达均降低(P<0.05);过表达TPT1后BPH细胞增殖功能增强(P<0.05),凋亡减少(P<0.05),Bcl-2表达升高(P<0.05),Bax、cleaved Caspase-3表达下降(P<0.05)。结论QLTC可通过介导miR-216a-5p下调TPT1/mTORC1通路,进而抑制BPH。

Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling

Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.

大黄糖络丸通过AMPK/mTOR/ULK1通路调控糖尿病肾病小鼠足细胞自噬的作用机制研究

目的:探究大黄糖络丸(DHT)基于腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白/unc-51样激酶1(AMPK/mTOR/ULK1)信号通路对糖尿病肾病(diabetic nephropathy,DN)小鼠的干预作用。方法:40只造模成功的C57BL/KSJ-db/db(以下简称db/db)小鼠随机分为模型组,达格列净组(1.5 mg·kg^(-1)·d^(-1)),DHT高、中、低剂量组(3.6、1.8、0.9 g·kg^(-1)·d^(-1)),每组8只;另取10只C57BL/KSJ-db/dm(以下简称db/m)小鼠为正常组,正常组和模型组给予生理盐水,治疗组小鼠分别给予相应药物,连续给药10周,1次/d。于给药0、4、8、10周固定时间,禁食不禁水12 h,取尾静脉血检测空腹血糖(FBG);于给药0、5、10周末收集尿液检测尿中白蛋白、肌酐含量,计算尿白蛋白肌酐比值(ACR);给药10周后,检测各组小鼠24 h尿总蛋白,血肌酐(Scr),尿素氮(BUN)含量;蛋白免疫印迹法检测肾脏组织p-AMPK、p-mTOR及p-ULK1蛋白的表达水平,以及自噬关键分子酵母Atg6同系物1(Beclin-1)、微管相关蛋白1轻链3(LC3)、P62蛋白的表达水平;免疫组化法检测肾脏组织足细胞裂孔膜蛋白(Nephrin、Podocin)的表达水平;采用光学显微镜和透射电镜观察肾脏组织病理形态学变化。结果:与模型组比较,达格列净组和DHT组小鼠FBG、ACR、24 h尿总蛋白均降低,Scr、BUN无统计学差异;肾组织中p-AMPK、p-ULK1表达水平升高,p-mTOR表达水平降低及LC3Ⅱ/LC3Ⅰ、Beclin-1表达水平升高,P62表达水平降低(P<0.01,P<0.05);肾小球的足细胞裂孔膜蛋白Nephrin、Podocin表达水平升高(P<0.01,P<0.05);肾脏病理损害减轻;透射电镜显示自噬小体、自噬溶酶体数量增加。结论:DHT可能通过调控AMPK/mTOR/ULK1信号通路,增强足细胞自噬,保护肾小球,延缓DN发展进程。

橄榄苦苷调节PRAS40/mTORC1信号通路对噪声性耳聋大鼠耳蜗组织损伤的影响

目的研究橄榄苦苷调节脯氨酸蛋白激酶底物蛋白(PRAS40)/哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)信号通路对噪声性耳聋(NIHL)大鼠耳蜗组织损伤的影响。方法总共选取48只大鼠,随机选出12只大鼠作为对照组,剩余大鼠采用白噪声来构建NIHL大鼠模型。随后将NIHL模型大鼠随机分为模型组、橄榄苦苷组、橄榄苦苷+NV-5138组,每组12只。各组给予相应干预7 d。检测大鼠的听性脑干反应(ABR)阈值;HE染色检测大鼠耳蜗组织病理损伤;原位末端标记法染色观察大鼠耳蜗组织细胞凋亡情况;共聚焦显微镜观察耳蜗组织基底膜毛细胞序列变化;蛋白印迹法检测大鼠耳蜗组织磷酸化PRAS40(p-PRAS40)、PRAS40、磷酸化mTORC1(p-mTORC 1)、mTORC1、B淋巴细胞瘤-2相关X蛋白(Bax)蛋白表达。结果与对照组比较,模型组耳蜗组织螺旋神经节细胞数目明显减少,形态异常,基底膜毛细胞结构损坏严重,未见明显的界限,细胞排列不整齐,ABR阈值、耳蜗组织凋亡细胞数目、p-mTORC1/mTORC1蛋白比值、Bax蛋白表达显著升高,p-PRAS40/PRAS40蛋白比值显著降低(P<0.05)。与模型组比较,橄榄苦苷组耳蜗组织螺旋神经节细胞数目增加,且形态有所恢复,基底膜毛细胞损伤减轻,细胞排列逐渐恢复正常,界限逐渐清晰,ABR阈值、耳蜗组织凋亡细胞数目、p-mTORC1/mTORC1蛋白比值、Bax蛋白表达显著降低,p-PRAS40/PRAS40蛋白比值显著升高(P<0.05)。NV-5138干预后减弱了橄榄苦苷对NIHL大鼠耳蜗组织病理损伤、基底膜毛细胞序列和上述指标的改善作用(P<0.05)。结论橄榄苦苷可能通过调控PRAS40/mTORC1信号通路,促进PRAS40磷酸化,抑制mTORC1磷酸化,从而减轻NIHL大鼠耳蜗组织损伤。

iNOS和mTORC1在胎膜早破合并绒毛膜羊膜炎中的表达及其与巨噬细胞极化的关系

目的探讨诱导型一氧化氮合酶(iNOS)、哺乳动物雷帕霉素靶蛋白C1(mTORC1)在胎膜早破(PROM)合并绒毛膜羊膜炎(CA)中的表达及其与巨噬细胞极化的关系,以期为临床早期诊断及干预提供依据。方法选取徐州医科大学附属医院于2022年10月—2023年10月收治的PROM孕产妇100例为研究组,均为剖宫产结束分娩。根据胎膜破裂的时间分为足月胎膜早破组(TPROM组)50例和未足月胎膜早破组(PPROM组)50例,根据胎膜病理结果是否存在CA,将2组分为4个亚组,分别为足月胎膜早破合并绒毛膜羊膜炎组(TPROM-CA+组)、未足月胎膜早破合并绒毛膜羊膜炎组(PPROM-CA+组)、足月胎膜早破未合并绒毛膜羊膜炎组(TPROM-CA-组)以及未足月胎膜早破未合并绒毛膜羊膜炎组(PPROM-CA-组)。对照组为正常妊娠足月剖宫产分娩的孕产妇30例。采用ELISA法检测各组孕妇血清中mTORC1的表达水平;所有胎膜均送病理检查,采用免疫组化法检测各组胎膜组织中iNOS、mTORC1的表达情况。结果①TPROM组CA的发生率为42%(21/50),PPROM组为56%(28/50),对照组为0,3组比较,差异有统计学意义(P<0.05);②血清中mTORC1的表达水平在TPROM-CA+组和PPROM-CA+组明显高于其余3组(P<0.05);TPROM-CA+组与PPROM-CA+组比较,差异无统计学意义(P>0.05);ROC曲线分析显示,PROM孕妇血清中mTORC1的表达水平对PROM合并CA有诊断价值;③免疫组化结果显示TPROM-CA+组及PPROM-CA+组胎膜组织中iNOS、mTORC1的表达水平明显高于其余3组(P<0.05);2因子在TPROM-CA+组与PPROM-CA+组比较,差异无统计学意义(P>0.05)。iNOS在CAⅠ期中的表达水平高于CAⅡ期和CAⅢ期,mTORC1则相反,差异有统计学意义(P<0.05)。结论血清中mTORC1水平升高可能对胎膜早破合并绒毛膜羊膜炎有预测价值;iNOS和mTORC1在PROM合并CA中的表达水平上调,提示iNOS和mTORC1可能通过诱导巨噬细胞极化参与CA的发生发展。

失代偿期肝硬化并发自发性细菌性腹膜炎患者危险因素和PBMC CD36/mTORC1信号通路变化研究

目的探讨失代偿期肝硬化患者并发自发性细菌性腹膜炎(SBP)的危险因素,分析患者外周血单个核细胞(PBMC)分化抗原(CD)36/哺乳动物雷帕霉素靶蛋白1(mTORC1)信号通路水平变化。方法2020年7月~2023年12月我院诊治的失代偿期肝硬化患者82例,其中并发SBP者43例。取腹水培养,进行细菌鉴定,采用PCR法检测PBMC CD36/mTORC1 mRNA水平。应用多因素Logistic回归分析影响失代偿期肝硬化患者并发SBP的危险因素。结果在本组43例SBP患者中,分离出病原菌8株(9.8%),其中科氏葡萄球菌和溶血葡萄球菌各1株,大肠埃希菌2株,肺炎克雷伯菌2株和阴沟杆菌2株;SBP患者既往SBP发生史、血清总胆红素、血清白蛋白、INR、MELD评分及PBMC CD36和mTORC1 mRNA水平分别为51.2%、(45.7±5.2)μmol/L、(21.7±3.1)g/L、(1.5±0.5)、(24.9±7.5)、(3.2±0.8)和(2.4±0.7),与肝硬化组【分别为18.0%、(12.3±1.4)μmol/L、(35.3±5.4)g/L、(1.2±0.3)、(12.8±3.7)、(1.4±0.5)和(1.1±0.4)】比,差异显著(P<0.05);多因素Logistic回归分析结果显示,SBP发生史、血清总胆红素、ALB、MELD评分及PBMC CD36和mTORC1水平为影响失代偿期肝硬化并发SBP的独立危险因素(P<0.05)。结论失代偿期肝硬化并发SBP患者PBMC CD36/mTORC1信号通路表达上调,其在SBP发生过程中的作用还有待于进一步研究。