Alzheimer’s disease (AD) is a neurodegenerative disease with clinical hallmarks of progressive cognitive impairment. Synergistic effects of the Aβ-Tau cascade reaction are tightly implicated in AD pathology, and microglial NLRP3 inflammasome activation drives neuronal tauopathy. However, the underlying mechanism of how Aβ mediates NLRP3 inflammasome remains unclear. Herein, we determined that oligomeric Aβ (o-Aβ) bound to microglial Kv2.1 and promoted Kv2.1-dependent potassium efflux to activate NLRP3 inflammasome resulting in neuronal tauopathy by using Kv2.1 inhibitor drofenine (Dfe) as a probe. The underlying mechanism has been intensively investigated by assays with Kv2.1 knockdown in vitro (si-Kv2.1) and in vivo (AAV-ePHP-si-Kv2.1). Dfe deprived o-Aβ of its capability to promote microglial NLRP3 inflammasome activation and neuronal Tau hyperphosphorylation by inhibiting the Kv2.1/JNK/NF-κB pathway while improving the cognitive impairment of 5×FAD-AD model mice. Our results have highly addressed that the Kv2.1 channel is required for o-Aβ-driven microglial NLRP3 inflammasome activation and neuronal tauopathy in AD model mice and highlighted that Dfe as a Kv2.1 inhibitor shows potential in the treatment of AD.
In addition to the essential pharmacological effects of opioids, situational cues associated with drug addiction memory are key triggers for drug seeking. CircRNAs, an emerging hotspot regulator in crown genetics, play an important role in central nervous system-related diseases. However, the internal mediating mechanism of circRNAs in the field of drug reward and addiction memory remains unknown. Here, we trained mice on a conditional place preference (CPP) model and collected nucleus accumbens (NAc) tissues from day 1 (T0) and day 8 (T1) for high-throughput RNA sequencing. QRT-PCR analysis revealed that circTmeff-1 was highly expressed in the NAc core but not in the NAc shell, suggesting that it plays a role in addiction memory formation. Meanwhile, the down-regulation of circTmeff-1 by adeno-associated viruses in the NAc core or shell could inhibit the morphine CPP scores. Subsequently, the GO and KEGG analyses indicated that circTmeff-1 might regulate the addiction memory via the MAPK and AMPK pathways. These findings suggest that circTmeff-1 in NAc plays a crucial role in morphine-dependent memory formation.
Histamine is a conserved neuromodulator in mammalian brains and critically involved in many physiological functions. Understanding the precise structure of the histaminergic network is the cornerstone in elucidating its function. Herein, using histidine decarboxylase (HDC)-CreERT2 mice and genetic labeling strategies, we reconstructed a whole-brain three dimensional (3D) structure of histaminergic neurons and their outputs at 0.32 × 0.32 × 2 μm 3 pixel resolution with a cutting-edge fluorescence microoptical sectioning tomography system. We quantified the fluorescence density of all brain areas and found that histaminergic fiber density varied significantly among brain regions. The density of histaminergic fiber was positively correlated with the amount of histamine release induced by optogenetic stimulation or physiological aversive stimulation. Lastly, we reconstructed a fine morphological structure of 60 histaminergic neurons via sparse labeling and uncovered the largely heterogeneous projection pattern of individual histaminergic neurons. Collectively, this study reveals an unprecedented whole-brain quantitative analysis of histaminergic projections at the mesoscopic level, providing a foundation for future functional histaminergic study.
Most α2-AR agonists derived from dexmedetomidine have few structural differences between them and have no selectivity for α2A/2B-AR or Gi/Gs, which can lead to side effects in drugs. To obtain novel and potent α2A-AR agonists, we performed virtual screening for human α2A-AR and α2B-AR to find α2A-AR agonists with higher selectivity. Compound P300–2342 and its three analogs significantly decreased the locomotor activity of mice (p < 0.05). Furthermore, P300–2342 and its three analogs inhibited the binding of [3H] Rauwolscine with IC50 values of 7.72 ± 0.76 and 12.23 ± 0.11 μM, respectively, to α2A-AR and α2B-AR. In α2A-AR-HEK293 cells, P300–2342 decreased forskolin-stimulated cAMP production without increasing cAMP production, which indicated that P300–2342 activated α2A-AR with coupling to the Gαi/o pathway but without Gαs coupling. P300–2342 exhibited no agonist but slight antagonist activities in α2B-AR. Similar results were obtained for the analogs of P300–2342. The docking results showed that P300–2342 formed π-hydrogen bonds with Y394, V114 in α2A-AR, and V93 in α2B-AR. Three analogs of P300–2342 formed several π-hydrogen bonds with V114, Y196, F390 in α2A-AR, and V93 in α2B-AR. We believe that these molecules can serve as leads for the further optimization of α2A-AR agonists with potentially few side effects.
Multiple sclerosis(MS)is a systemic inflammatory illness of the central nervous system that involves demyelinating lesions in the myelin-rich white matter and pathology in the grey matter.Despite signifi-cant advancements in drug research for MS,the dis-ease's complex pathophysiology makes it difficult to treat the progressive forms of the disease.In this study,we identified a natural flavonoid compound icariin(ICA)as a potent effective agent for MS in ameliorating the deterioration of symptoms including the neurological defi-cit score and the body weight in a murine experimental autoimmune encephalomyelitis(EAE)model.These improvements were associated with decreased demyelin-ation in the corpus callosum and neuron loss in the hippo-campus and cortex confirmed by immunohistochemistry analysis.Meanwhile,it was observed that the activation of microglia in cerebral cortex and hippocampus were inhibited followed by the neuroinflammatory cytokines downregulation such as IL-1β,IL-6 and TNF-α after ICA treatment,which was probably attributable to the sup-pression of microglial NLRP3 inflammasome activation.Additionally,molecular docking also revealed the binding force of ICA to NLRP3 inflammasome protein complexes in vitro.Taken together,our findings have demonstrated that ICA,as pleiotropic agent,prevents EAE-induced MS by improving demyelination and neuron loss,which inter-feres with the neuroinflammation via microglial NLRP3 inflammasome activation.
Currently available antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs), generally require weeks to months to produce a therapeutic response, but the mechanism of action underlying the delayed onset of antidepressant-like action remains to be elucidated. The balance between excitatory glutamatergic pyramidal neurons and inhibitory γ-aminobutyric acid (GABA) interneurons, i.e., the excitation:inhibition functional (E:I) balance, in the medial prefrontal cortex (mPFC) is critical in regulating several behaviors and might play an important mediating role in the mechanism of rapid antidepressant-like action reported by several studies. In the present study, the multichannel electrophysiological technique was used to record the firing activities of pyramidal neurons and interneurons and investigate the effects of a single dose of fluoxetine and ketamine (both 10 mg/kg, i.p.) on the E:I functional balance in the rat mPFC after 90 min or 24 h, and the forced swimming test (FST) was used to evaluate the antidepressant-like effects of fluoxetine and ketamine. The present study also explored the effects of chronic treatment with fluoxetine (10 mg/kg, i.g.) for 7 d or 21 d on the E:I functional balance in the mPFC. The present results suggested that a single dose of ketamine could both significantly increase the firing activities of pyramidal neurons and significantly decrease the firing activities of interneurons in the mPFC and exerted significant antidepressant-like action on the FST after 90 min and 24 h, but fluoxetine had no such effects under the same conditions. However, chronic treatment with fluoxetine for 21 d (but not 7 d) could significantly affect the firing activities of pyramidal neurons and interneurons in the mPFC. Taken together, the present results indicated that rapid regulation of the E:I functional balance in the mPFC might be an important common mechanism of rapid-acting antidepressants and the delayed onset of SSRIs might be partly attributed to their inability to rapidly regulate the E:I functional balance in the mPFC. The present study provided a new entry point to the development of rapid-acting antidepressants.
We have previously shown that phosphodiesterase 4 (PDE4) inhibition protects against neuronal injury in rats following middle cerebral artery occlusion/reperfusion (MCAO/R). However, the effects of PDE4 on brain edema and astrocyte swelling are unknown. In this study, we showed that inhibition of PDE4 by Roflumilast (Roflu) reduced brain edema and brain water content in rats subjected to MCAO/R. Roflu decreased the expression of aquaporin 4 (AQP4), while the levels of phosphorylated protein kinase B (Akt) and forkhead box O3a (FoxO3a) were increased. In addition, Roflu reduced cell volume and the expression of AQP4 in primary astrocytes undergoing oxygen and glucose deprivation/reoxygenation (OGD/R). Consistently, PDE4B knockdown showed similar effects as PDE4 inhibition; and PDE4B overexpression rescued the inhibitory role of PDE4B knockdown on AQP4 expression. We then found that the effects of Roflu on the expression of AQP4 and cell volume were blocked by the Akt inhibitor MK2206. Since neuroinflammation and astrocyte activation are the common events that are observed in stroke, we treated primary astrocytes with interleukin-1β (IL-1β). Astrocytes treated with IL-1β showed decreased AQP4 and phosphorylated Akt and FoxO3a. Roflu significantly reduced AQP4 expression, which was accompanied by increased phosphorylation of Akt and FoxO3a. Furthermore, overexpression of FoxO3a partly reversed the effect of Roflu on AQP4 expression. Our findings suggest that PDE4 inhibition limits ischemia-induced brain edema and astrocyte swelling via the Akt/FoxO3a/AQP4 pathway. PDE4 is a promising target for the intervention of brain edema after cerebral ischemia.
A universal drug delivery system (DDS) with brain-targeted ability is demanded to enhance antiepileptic therapeutic efficacy and reduce side effects in multiple types of epileptic seizures. In this study, we reported a micelle-based DDS possessing the brain-targeted ability and electro-responsive feature for universal delivery of antiepileptic drugs (AEDs). The system is fabricated by ferrocene (Fc)-conjugated D-a-tocopherol polyethylene glycol succinate and amphiphilic block copolymer, which improve the drug encapsulation of different AEDs. Interestingly, the intrinsic nature of TPGS-Fc including transferrin receptor-mediated transcytosis and efflux pump inhibition endows the system with high permeability across the blood-brain barrier. Based on the hydrophobic-hydrophilic transition of Fc, the micelles can respond to epileptiform discharges and thus release the loaded AEDs. Improved antiepileptic efficacy of the micelles has been demonstrated in acute, continuous, and chronic epilepsy models. In summary, we have developed a universal micelle-based DDS for various AEDs delivery, which provides a promising approach to on-demand therapy of different epileptic seizures.
A series of novel conjugates of benzoselenazole or selenazole and CPI-1 were designed, synthesized, and evaluated for inhibitory activities against the botulinum neurotoxin A (BoNT/A) light chain (LC) and BoNT/A in vivo. The results show that these compounds exhibit potent inhibitory activities to the LC with IC50 of 0.5-4.1 mu M. The reaction kinetics and the mass spectra of the reaction products of LC with benzoselenazole- or selenazolecoupled CPI-1 demonstrate that the benzoselenazole group of most inhibitors is coupled to the LC of BoNT/A. These data indicate that the CPI-1 conjugates can inhibit both the active center of BoNT/A LC as well as Cys165, therefore functioning as irreversible bifunctional inhibitors. The detoxification activities in vivo show that one of the benzoselenazole-CPI-1 compounds prolongs the survival time of mice challenged by 2 x LD50 of BoNT/A. This work provides a new strategy to design potent antidotes of BoNT/A.
Major depressive disorder ranks as a major burden of disease worldwide, yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects. The lateral septum (LS) is thought to control of depression, however, the cellular and circuit substrates are largely unknown. Here, we identified a subpopulation of LS GABAergic adenosine A2A receptors (A2AR)-positive neurons mediating depressive symptoms via direct projects to the lateral habenula (LHb) and the dorsomedial hypothalamus (DMH). Activation of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipulation of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive phenotypes. Thus, the optogenetic modulation (stimulation or inhibition) of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH, phenocopied depressive behaviors. Moreover, A2AR are upregulated in the LS in two male mouse models of repeated stress-induced depression. This identification that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant potential of A2AR antagonists, prompting their clinical translation.
The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.
The preference for social novelty is crucial to the social life of humans and rodents. However, the neural mechanisms underlying social novelty preference are poorly understood. Here, we found that chronic social defeat stress (CSDS) reduced the preference for social novelty in mice by impairing the response of CaMKIIα + neurons in the CA3 region of dorsal hippocampus (dCA3) during approach to an unfamiliar mouse. The deficits of social novelty preference in CSDS-treated mice were reversed by activating the output from dCA3 to the GABAergic neurons in the lateral septum (LS). The activation of GABAergic projection from LS recruited a circuit that inhibited the Foxb1 + neurons in the parvafox nucleus (PFN), which drove social avoidance by projecting to the lateral periaqueductal gray (lPAG). These results suggest that a previously unidentified circuit of dCA3 CaMKIIα+ →LS GABA+ →PFN Foxb1+ →lPAG mediates the deficits of social novelty preference induced by CSDS.
OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro-vascular endothelial cells(CMEC)after oxygen-glucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemia-reperfusion model,and were divided into normal,model,low(10 μmol·L-1),medium(20 μmol·L-1)and high(40 μmol·L-1)ICA group,and high ICA+ inhibitor group(40 μmol·L-1+20 nmol·L-1).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col-lect ICA-related targets,the GeneCards data-base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar-get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft-ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec-tive effect at 10-160 μmol·L-1;the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P＜0.01,P＜0.01);and the results of tube-for-mation assay showed that each group of ICA could significantly promote the generation of branches(P＜0.01)and the capillary length exten-sion(P＜0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar-gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich-ment analysis found 53 results,involving AGE-RAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu-lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra-tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha-nism of action of ICA against myocardial isch-emia-reperfusion injury may be related to the reg-ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.
目的 阐明不同成瘾性物质(甲基苯丙胺、可卡因和吗啡)引起的腹侧被盖区多巴胺神经元及其投射脑区伏隔核内多巴胺递质的实时动力学变化.方法 将携带多巴胺神经递质荧光探针和携带酪氨酸羟化酶钙启动子钙指示剂蛋白病毒分别注射在小鼠伏隔核和腹侧被盖区,每个脑区各28只.小鼠分为生理盐水组、甲基苯丙胺(1 mg·kg-1,ip)组、可卡因(10 mg·kg-1,ip)组和吗啡(10 mg·kg-1,sc)组,给药后,立即采用光纤记录系统监测2h内多巴胺神经递质和多巴胺能神经元的实时动力学变化,包括多巴胺神经递质荧光变化曲线下面积(AUC),曲线极值及达极值的潜伏时间,多巴胺能神经元钙离子信号变化AUC,曲线最高值或最低值及达极值的潜伏时间和作用持续时间.结果 ip给予甲基苯丙胺(1 mg·kg-1)和可卡因(10 mg·kg-1)均会引起多巴胺能神经元的Ca2+信号快速而持续地下降,而sc给予吗啡(10 mg·kg-1)引起多巴胺能神经元的Ca2+信号缓慢而持续地上升,甲基苯丙胺给药后的多巴胺能神经元Ca2+信号变化曲线极值和AUC显著低于可卡因(P＜0.05);甲基苯丙胺对腹侧被盖区的多巴胺能神经元的持续作用时间显著长于可卡因(P＜0.05),而吗啡对腹侧被盖区的多巴胺能神经元持续作用时间显著长于甲基苯丙胺和可卡因(P＜0.01,P＜0.05).上述物质引起多巴胺神经递质表现不同,与生理盐水组相比,吗啡、甲基苯丙胺和可卡因均显著诱导小鼠伏隔核内多巴胺显著升高,但也显示了不同的动力学特征:甲基苯丙胺给药后导致的伏隔核脑区多巴胺变化AUC显著高于可卡因和吗啡(P＜0.01,P＜0.05);甲基苯丙胺和可卡因给药后,多巴胺变化达到极值所需时间显著早于吗啡(P＜0.01).结论 不同成瘾性物质对腹侧被盖区多巴胺能神经元的初始作用显著不同,但均可显著升高伏隔核内多巴胺神经递质,并存在动力学变化的显著差异,提示不同种类物质导致成瘾的神经机制很可能存在不同.
目的 建立苯并[a]芘(BaP)恶性转化人支气管上皮细胞T-16HBE-C1(THBEc1)的裸小鼠原位肺癌模型,评估血清人源神经纤维网蛋白2(NRP2)对该模型的诊断价值.方法 12只雄性ICR小鼠随机分为3组,右肺分别一次性注射无菌生理盐水20,30和40 μL,观察14d内小鼠死亡数,以确定小鼠肺内注射安全体积.16只雄性BALB/c-nu裸小鼠随机分为4组:对照组[皮下和肺内同时一次性接种人支气管上皮16HBE(HBE)细胞,皮下1×106,肺内2×105]、皮下荷瘤模型组(皮下一次性接种THBEc1细胞1×106)、原位肺癌模型组(肺内一次性接种THBEc1细胞2×105或5×105).接种后每7d监测小鼠体重和皮下肿瘤体积;HE染色检测肺肿瘤和皮下肿瘤组织病理特征,计算成瘤率;酶联免疫吸附实验(ELISA)检测裸小鼠血清和胸水中人源NRP2水平;采用受试者工作特征(ROC)曲线确定血清人源NRP2对THBEc1细胞导致的裸小鼠原位肺癌模型的诊断价值.结果 肺内注射40 μL无菌生理盐水可致2/4小鼠死亡,20和30 μL组未见小鼠死亡,但整体表现欠佳,故后续实验注射体积选择20 μL.对照组裸小鼠均未在接种部位检出肿瘤,小鼠体重持续增长;皮下荷瘤模型组祼小鼠全部成瘤,体重显著低于对照组(P＜0.05).接种细胞后第10天,原位肺癌模型5×105细胞组裸小鼠全部成瘤(4/4);第14天,原位肺癌模型2×105细胞组裸小鼠3/4成瘤,且2个剂量组小鼠体重均低于对照组(P＜0.01,P＜0.05),肿瘤组织均呈鳞状细胞癌特征.ELISA结果显示,对照组裸小鼠血清人源NRP2水平均显著低于皮下荷瘤模型(4只)和原位肺癌模型(7只)裸小鼠(P＜0.05).原位肺癌模型组(4只)裸小鼠胸水中均可检出高水平的人源NRP2.ROC曲线显示,血清人源NRP2水平能有效区分THBEc1细胞在裸小鼠肺内是否成瘤,最佳截断值为123.00 ng·L-1.结论 通过肺内注射接种THBEc1细胞建立了裸小鼠原位肺癌模型,血清人源NRP2可用于该模型诊断.
目的 研究T-2毒素的抗癌活性,并探讨其作用机制.方法 人食管癌细胞EC109和EC1、人胃癌细胞MGC-803、人肺癌细胞H460及人正常胃黏膜细胞GES-1,各细胞分别分为细胞对照组(二甲亚砜,终浓度＜0.01%)和T-2毒素0.375～100 nmol·L-1组,处理细胞24,48和72h后,采用MTT法检测细胞存活率.EC1细胞分为细胞对照组和T-2毒素5和10 nmol·L-1组,实时无标记动态细胞分析技术(RTCA)进一步分析EC1细胞增殖和迁移;EC1细胞分为细胞对照组和T-2毒素5,10和20 nmol·L-1组,T-2毒素处理48h后,流式细胞术检测细胞凋亡和细胞周期及活性氧(ROS)水平和线粒体膜电位(MMP),Western印迹法检测细胞色素c、多聚ADP核糖聚合酶(PARP)、剪切形式PARP、P21、γ-H2AX、P53、Bax和活化胱天蛋白酶3/7蛋白表达.结果 T-2毒素作用EC109,EC1,MGC-803和H460 4种人癌细胞72 h,IC50值分别为6.34,5.54,6.94和5.96 nmol·L-1,T-2毒素对正常胃黏膜细胞GES-1的IC50 为16.4 nmol·L-1.在0～25 nmol·L-1 浓度范围内,T-2毒素对EC1细胞增殖具有明显抑制作用,且呈浓度和时间依赖性(24 h,r=0.9204;48 h,r=0.9745;72 h,r=0.9772),此外T-2毒素亦可强烈抑制EC1细胞迁移;与细胞对照组相比,T-2毒素5,10和20 nmol·L-1组EC1细胞凋亡率(P＜0.01)、G2/M期细胞比例(P＜0.01)、ROS含量(P＜0.05,P＜0.01)、低MMP细胞比例(P＜0.01)、细胞质中细胞色素c含量(P＜0.01)、剪切形式PARP含量(P＜0.01)、P21(P＜0.01)和γ-H2AX、P53、Bax及活化胱天蛋白酶3/7含量(P＜0.05,P＜0.01)均显著升高.结论 T-2毒素通过诱导细胞凋亡和周期阻滞抑制癌细胞生长,其机制与激活线粒体凋亡通路和上调周期抑制因子P21表达有关.
目的 探索苯并[a]芘(BaP)恶性转化人支气管上皮细胞T-16HBE-C1(THBEc1)中微RNA(miRNA)调控叉头框蛋白A1(FOXA1)表达上调的机制,并筛选和验证FOXA1的潜在靶基因.方法 利用TargetScan,ENCORI数据库和THBEc1细胞与非转化细胞16HBE间miRNA的二代测序(NGS)结果,综合预测靶向调控FOXA1的miRNA,并通过实时荧光定量PCR(RT-qPCR)进一步筛选预测的miRNA.采用miRNA模拟物(mimics)转染THBEc1细胞,Western印迹法测定FOXA1蛋白表达水平,对靶向调控FOXA1的miRNA进行鉴定.利用hTF,JASPAR和ENCODE数据库结合FOXA1敲除细胞THBEc1-ΔFOXA1-c34和对照细胞THBEc1-ctrl间mRNA的NGS结果,综合预测FOXA1的潜在靶基因.利用RT-qPCR进一步对预测的靶基因[跨膜蛋白98(TMEM98)、IKAROS家族锌指2(IKZF2)、异柠檬酸脱氢酶1(IDH1)、肿瘤坏死因子受体相关因子5(TRAF5)、骨形态发生蛋白2型受体(BMPR2)、热休克蛋白B1(HSPB1)、Runt相关转录因子2(RUNX2)、golgin A7家族成员B(GOLGA7B)和维甲酸相关孤核受体A(RORA)]进行筛选.通过转染过表达质粒构建稳定表达FOXA1的细胞模型THBEc1-ΔFOXA1-c34-oe,即FOXA1功能回补,并利用RT-qPCR和Western印迹法验证FOXA1的潜在靶基因.结果 TargetScan和ENCORI数据库分别预测到213和145个可能靶向调控FOXA1的miRNA.NGS共发现351个miRNA在THBEc1细胞中表达下调(差异倍数＜0.5,且错误发现率＜0.05),其中hsa-miR-584-5p(miR-584-5p),hsa-miR-142-5p(miR-142-5p)和hsa-miR-211-5p(miR-211-5p)在上述2个数据库中均被预测可靶向调控FOXA1.RT-qPCR结果证实,THBEc1细胞中miR-584-5p,miR-142-5p和miR-211-5p表达水平显著低于16HBE细胞(P＜0.01).转染miR-584-5p模拟物或miR-211-5p模拟物均可显著下调THBEc1细胞FOXA1蛋白表达水平(P＜0.01),而转染miR-142-5p模拟物对THBEc1细胞FOXA1蛋白表达水平无明显影响.THBEc1-ΔFOXA1-c34和THBEc1-ctrl细胞间的20个差异表达基因(差异倍数＜0.5或＞2,且错误发现率＜0.05)被hTF,JASPAR和ENCODE数据库均预测为FOXA1 的潜在靶基因.RT-qPCR结果显示,在THBEc1-ΔFOXA1-c34中,TMEM98,IKZF2,IDH1,TRAF5,BMPR2,HSPB1和RUNX2 mRNA表达水平显著下调(P＜0.05,P＜0.01),GOLGA7B和RORA mRNA表达水平显著上调(P＜0.05,P＜0.01);在THBEc1-ΔFOXA1-c34-oe中,IDH1和HSPB1的mRNA表达水平显著上调(P＜0.01),余7个基因mRNA表达水平未见改变.Western印迹结果显示,THBEc1-ΔFOXA1-c34细胞中IDH1和HSPB1表达水平较THBEc1-ctrl均显著下调(P＜0.01);而恢复FOXA1表达的THBEc1-ΔFOXA1-c34-oe细胞中IDH1和HSPB1表达水平较对照细胞THBEc1-ΔFOXA1-c34-ctrl均显著上调(P＜0.01).结论 BaP恶性转化细胞THBEc1中miR-584-5p/miR-211-5p/FOXA1轴参与IDH1和HSPB1转录调控.
目的 探讨利拉鲁肽(liraglutide)抗CXC趋化因子配体16(CXCL16)诱导的人足细胞脂质沉积的作用及机制.方法 ① 葡萄糖40 mmol·L-1刺激人足细胞24 h,棕榈酸250 μmol·L-1刺激人足细胞6h,Western印迹法检测足细胞CXCL16蛋白表达水平.②重组CXCL16 100 μg·L-1刺激足细胞0,6,12和24 h,Western印迹法检测足细胞裂隙素蛋白表达水平.③足细胞分为细胞对照组、CXCL16 100 μg·L-1组、CXCL16+利拉鲁肽10,50和100 nmol·L-1组及CXCL16+辛伐他汀100 nmol·L-1组,加药2h后加重组CXCL16 100 μg·L-1继续培养24 h,油红O染色检测足细胞脂滴面积.④足细胞分为细胞对照组、CXCL16 100 μg·L-1 组、CXCL16+利拉鲁肽100 nmol·L-1 组和CXCL16+辛伐他汀100 nmol·L-1 组,加药2h后加CXCL16 100 μg·L-1继续培养24 h,鬼笔环肽染色检测足细胞肌动蛋白应力纤维百分比,ELISA检验细胞培养液中肿瘤坏死因子α(TNF-α)、转化生长因子β(TGF-β)和白细胞介素1β(IL-1β)蛋白浓度,实时荧光定量PCR检测TNF-α,TGF-β和IL-1βmRNA表达水平;Western印迹法检测足细胞裂隙素、胆固醇调节元件结合蛋白1(SREBP1)、SREBP2和脂肪分化相关蛋白(ADRP)表达水平.结果 ①与细胞对照组相比,高糖和棕榈酸刺激均可引起人足细胞CXCL16表达水平显著升高(P＜0.01).②与细胞对照组相比,CXCL16刺激可引起足细胞裂隙素表达显著下调(P＜0.01).③与细胞对照组相比,CXCL16刺激可引起足细胞脂滴面积显著升高(P＜0.01),辛伐他汀和不同浓度利拉鲁肽均可显著缓解这一变化(P＜0.01).④与细胞对照组相比,重组CXCL16可引起足细胞肌动蛋白应力纤维百分比显著下降(P＜0.01),培养液中TNF-α,TGF-β和L-1β蛋白浓度显著升高(P＜0.01),足细胞TNF-α,TGF-β和IL-1β mRNA水平显著升高(P＜0.05,P＜0.01),裂隙素表达显著下调(P＜0.01),SREBP1,SREBP2和ADRP蛋白表达水平显著增高(P＜0.01);与CXCL16组比较,利拉鲁肽和辛伐他汀可以显著恢复足细胞肌动蛋白应力纤维百分比(P＜0.01),下调培养液中TNF-α,TGF-β和IL-1β蛋白浓度(P＜0.01),抑制足细胞TNF-α,TGF-β和IL-1βmRNA水平(P＜0.05,P＜0.01),上调裂隙素蛋白表达水平(P＜0.01),下调SREBP1,SREBP2和ADRP蛋白表达水平(P＜0.01).结论 利拉鲁肽可通过抑制CXCL16诱导的脂质沉积减轻足细胞损伤,并缓解脂质沉积引起的炎症激活,这可能与利拉鲁肽能够抑制SREBP和ADRP表达有关.
心肌梗死是冠状动脉急性闭塞引起的严重而持久的缺血性心肌坏死,传统药物治疗靶向性较差,到达损伤区域的药物剂量不足且停留时间偏短.近年来研究显示,纳米药物实现了对病变组织的选择性靶向输送,在心肌梗死的靶向治疗中,借助其独特的理化性质,纳米载体可携带炎症因子受体、炎症信号通路抑制剂、抗氧化剂、治疗基因、特异性抗体和生长因子等到达心肌组织释放药物.纳米药物通过中和炎症因子、抑制炎症信号通路和炎症基因表达、调节巨噬细胞表型等对抗单核巨噬细胞及中性粒细胞介导的炎症损伤;通过抗氧化剂消除过氧化物的方式对抗氧化应激性损伤;通过生长因子促进心肌组织的再生和修复;通过水凝胶修复心脏电传导功能并促进心脏间充质干细胞的增殖分化.研究发现,纳米药物可保护缺血心肌组织,改善心脏功能.本文对纳米药物在心肌梗死治疗中的应用研究进展进行综述.
目的 研究大麻二酚(CBD)对口腔颌面部炎性痛的镇痛作用及其中枢机制.方法 采用上唇右侧sc给1%福尔马林溶液(37%～40%甲醛)诱发口腔颌面部炎性痛模型.①成年雄性C57BL/6J小鼠随机分为正常对照组、模型组和模型+CBD(0.01,0.03和0.06 mg)组(上唇右侧sc给药),给药5 min后,上唇右侧sc给予1%福尔马林溶液.②成年雄性C57BL/6J小鼠随机分为正常对照组、模型组和模型+CBD 0.06 mg组,给药方式及部位同分组①.观察记录分组①小鼠一相痛(0～6 min)和二相痛(15～45 min)小鼠抓脸时间;行为实验结束45 min后(相当于注射福尔马林90 min后),分组①中每组随机选取4只,采用免疫荧光方法检测小鼠三叉神经脊束核尾侧亚核(Sp5C)和前扣带回(ACC)脑区c-Fos蛋白表达.采用内源性大麻素探针结合光纤记录方法检测分组②中小鼠二相痛时期小鼠中央导水管周围灰质(PAG)、基底外侧杏仁核(BLA)和ACC脑区内源性大麻素水平.结果 ①与正常对照组相比,模型组小鼠一相痛和二相痛抓脸时间显著延长(P＜0.01);与模型组相比,模型+CBD 0.06 mg组二相痛时期小鼠的抓脸时间显著缩短(P＜0.01),一相痛时期的抓脸时间无明显变化.免疫荧光结果显示,与正常对照组相比,模型组小鼠Sp5C和ACC的c-Fos阳性神经元数量显著升高(P＜0.01);与模型组相比,模型+CBD 0.06 mg组上述2个脑区的c-Fos蛋白表达水平显著降低(P＜0.01).②探针结合光纤记录结果显示,与模型组相比,模型+CBD 0.06 mg BLA的内源性大麻素水平显著升高(P＜0.05);但与正常对照组相比,模型组无显著变化.结论 在口腔颌面部炎性痛中,sc给予CBD可通过降低口面部疼痛上行传导通路中Sp5C和ACC脑区c-Fos蛋白表达,上调疼痛下行抑制通路内源性大麻素系统功能,从而发挥镇痛作用.
