Neuroprotective and anti-inflammatory effects of a therapy combining agonists of nicotinic α7 and σ1 receptors in a rat model of Parkinson’s disease

To date there is no treatment able to stop or slow down the loss of dopaminergic neurons that characterizes Parkinson’s disease.It was recently observed in a rodent model of Alzheimer’s disease that the interaction between the α7 subtype of nicotinic acetylcholine receptor(α7-nAChR)and sigma-1 receptor(σ1-R)could exert neuroprotective effects through the modulation of neuroinflammation which is one of the key components of the pathophysiology of Parkinson’s disease.In this context,the aim of the present study was to assess the effects of the concomitant administration of N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide(PHA)543613 as an α7-nAChR agonist and 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate(PRE)-084 as aσ1-R agonist in a well-characterized 6-hydroxydopamine rat model of Parkinson’s disease.The animals received either vehicle separately or the dual therapy PHA/PRE once a day until day 14 postlesion.Although no effect was noticed in the amphetamine-induced rotation test,our data has shown that the PHA/PRE treatment induced partial protection of the dopaminergic neurons(15-20%),assessed by the dopamine transporter density in the striatum and immunoreactive tyrosine hydroxylase in the substantia nigra.Furthermore,this dual therapy reduced the degree of glial activation consecutive to the 6-hydroxydopamine lesion,i.e,the 18 kDa translocation protein density and glial fibrillary acidic protein staining in the striatum,and the CD11b and glial fibrillary acidic protein staining in the substantia nigra.Hence,this study reports for the first time that concomitant activation of α7-nAChR andσ1-R can provide a partial recovery of the nigro-striatal dopaminergic neurons through the modulation of microglial activation.The study was approved by the Regional Ethics Committee(CEEA Val de Loire n°19)validated this protocol(Authorization N°00434.02)on May 15,2014.

Targeting α7 nicotinic acetylcholine receptors: a future potential for neuroprotection from traumatic brain injury

Traumatic brain injury （TBI） poses a significant socioeconomic burden in the world. The long lasting consequences in cognitive impairments are often underreported and its mechanisms are unclear. In this perspective, cholinergic dysfunction and thera-peutic strategy targeting this will be reviewed. Novel agents that can target specific subtype of acetylcholine receptors have been developed over the recent years and are at various stages of development, which include AR-R 17779, GTS-21, SSR- 180711A, AR-R17779, and PNU-282987. A detailed review on this topic has been previously published （Shin and Dixon, 2015）.

Therapeutic potential of α7 nicotinic receptor agonists to regulate neuroinflammation in neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer＇s, Parkinson＇s and Huntington＇s diseases, are all character- ized by a component of innate immunity called neuroinflammation. Neuronal loss and neuroinflammation are two phenomena closely linked. Hence, the neuroinflammation is a relevant target for the management of the neurodegenerative diseases given that, to date, there is no treatment to stop neuronal loss. Several studies have investigated the potential effects of activators of alpha 7 nicotinic acetylcholine receptors in animal models of neurodegenerative diseases. These receptors are widely distributed in the central nervous system. After activation, they seem to mediate the cholinergic anti-inflammatory pathway in the brain. This anti-inflammatory pathway, first described in periphery, regulates activation of microglial cells considered as the resident macrophage population of the central nervous system. In this article, we shortly review the agonists of the alpha 7 nicotinic acetylcholine receptors that have been evaluated in vivo and we focused on the selective positive allosteric modulators of these receptors. These compounds represent a key element to enhance receptor activity only in the presence of the endogenous agonist.

Identification of α7 nicotinic acetylcholine receptor on hippocampal astrocytes cultured in vitro and its role on inflammatory mediator secretion

The present study found expressions of a7 nicotinic acetylcholine receptor on hippocampal slices and hippocampal astrocytes using double immunofluorescence stainings. Expression of glial fibdllary acidic protein in the cultured hippocampal slices and hippocampal astrocytes significantly increased, and levels of macrophage inflammatory protein la, RANTES, interleukin-1β, intedeukin-6, and tumor necrosis factor-α increased in the supernatant of cultured astrocytes following exposure to 200 nM amyloid 13 protein 1-42. Preconditioning of 10 μM nicotine, a nicotinic acetylcholine receptor agonist, could attenuate the influence of amyloid β protein 1-42 in inflammatory mediator secretion of cultured astrocytes. Experimental findings indicated that α7 nicotinic acetylcholine receptor was expressed on the surface of hippocampal astrocytes, and activated a7 nicotinic acetylcholine receptor was shown to inhibit inflammation induced by amyloid β protein 1-42.

Alpha-7 nicotinic acetylcholine receptor agonist treatment in a rat model of Huntington's disease and involvement of heme oxygenase-1

Neuroinflammation is a common element involved in the pathophysiology of neurodegenerative diseases.We recently reported that repeated alpha-7 nicotinic acetylcholine receptor（α7 n ACh R） activations by a potent agonist such as PHA 543613 in quinolinic acid-injured rats exhibited protective effects on neurons.To further investigate the underlying mechanism,we established rat models of early-stage Huntington＇s disease by injection of quinolinic acid into the right striatum and then intraperitoneally injected 12 mg/kg PHA 543613 or sterile water,twice a day during 4 days.Western blot assay results showed that the expression of heme oxygenase-1（HO-1）,the key component of the cholinergic anti-inflammatory pathway,in the right striatum of rat models of Huntington＇s disease subjected to intraperitoneal injection of PHA 543613 for 4 days was significantly increased compared to the control rats receiving intraperitoneal injection of sterile water,and that the increase in HO-1 expression was independent of change in α7 n ACh R expression.These findings suggest that HO-1 expression is unrelated to α7 n ACh R density and the increase in HO-1 expression likely contributes to α7 n ACh R activation-related neuroprotective effect in early-stage Huntington＇s disease.

Nicotinic cholinergic receptors in esophagus:Early alteration during carcinogenesis and prognostic value

AIM: To compare expression of nicotinic cholinergic receptors(CHRNs) in healthy and squamous cell carcinoma-affected esophagus and determine the prognostic value.METHODS: We performed RT-q PCR to measure the expression of CHRNs in 44 esophageal samples from healthy individuals and in matched normal surrounding mucosa, and in tumors from 28 patientsdiagnosed with esophageal squamous cell carcinoma(ESCC). Next, we performed correlation analysis for the detected expression of these receptors with the habits and clinico-pathological characteristics of all study participants. In order to investigate the possible correlations between the expression of the different CHRN subunits in both healthy esophagus and tissues from ESCC patients, correlation matrices were generated. Subsequently, we evaluated whether the detected alterations in expression of the various CHRNs could precede histopathological modifications during the esophageal carcinogenic processes by using receiver operating characteristic curve analysis. Finally, we evaluated the impact of CHRNA5 and CHRNA7 expression on overall survival by using multivariate analysis.RESULTS: CHRNA3, CHRNA5, CHRNA7 and CHRNB4, but not CHRNA1, CHRNA4, CHRNA9 or CHRNA10, were found to be expressed in normal(healthy) esophageal mucosa. In ESCC, CHRNA5 and CHRNA7 were overexpressed as compared with patient-matched surrounding non-tumor mucosa(ESCC-adjacent mucosa; P < 0.0001 and P = 0.0091, respectively). Positive correlations were observed between CHRNA3 and CHRNB4 expression in all samples analyzed. Additionally, CHRNB4 was found to be differentially expressed in the healthy esophagus and the normalappearing ESCC-adjacent mucosa, allowing for distinguishment between these tissues with a sensitivity of 75.86% and a specificity of 78.95%(P = 0.0002). Finally, CHRNA5 expression was identified as an independent prognostic factor in ESCC; patients with high CHRNA5 expression showed an increased overall survival, in comparison with those with low expression. The corresponding age- and tumor stage-adjusted hazard ratio was 0.2684(95%CI: 0.075-0.97, P = 0.0448).CONCLUSION: Expression of CHRNs is homogeneous along healthy esophagus and deregulated in ESCC, suggesting a pathogenic role for these receptors in ESCC development and progression.

Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer＇s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which influence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to persistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in persistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer＇s disease.

Differentiation of the Agonists and Antagonists of the α7 Nicotinic Acetylcholine Receptor

Theα7 nicotinic acetylcholine receptors(nAChRs)are widely expressed in the central and peripheral nervous systems and are important drug targets for the treatment of neurological diseases.However,differentiation of the agonists and antagonists of the nAChR is difficult.In this study we aimed to develop a reliable and efficient computational approach for differentiation of the agonists from the antagonists of the nAChR based on a systematical analysis of 123 ligands(87 agonists,12 partial agonists,and 24 antagonists)binding with the extracellular domain of theα7 n AChR chimera.Our results suggest that the ligand size and ligand binding affinity cannot differentiate the agonists from the antagonists of the nAChR.The ligand efficiency that considers both ligand binding affinity and size for the agonists is overall more left shifted in comparison to the antagonists,but the values of the ligand efficiency still cannot differentiate the agonists from the antagonists unless the values are either relatively high(more than-0.3 kcal mol^-1)or relatively low(less than-0.45 kcal mol^-1).Our results suggest that accurate prediction of the agonist or antagonist of the nAChR is challenging and the ligand innate configuration has to be considered as an extra for differentiation of the agonists from the antagonists of the nAChR.

Activation of α7 nicotinic acetylcholine receptor protects against oxidant stress damage through reducing vascular peroxidase-1 in a JNK signaling-dependent manner in endothelial cells

Aim Alpha7 nicotinic acetylcholine receptor （α7nAChR）, a subtype of nAChR regulating neurotrans- mission in central nervous system, is an essential regulator of cholinergic antiinflammatory pathway in periphery. The present study was to determine the effects of activation of α7nAChR on oxidant stress-induced injury in endo- thelial cells. Methods Cultured human umbilical vein endothelial cells were treated with H202 （400 μmol · L^-1） or H202plus PNU-282987 （ 10 μmol · L^-1 ）. Cell viability and membrane integrity were measured. AnnexinV ＋ PI assay, immunoblotting of bcl-2, bax and cleaved caspase-3, and immunofluorescence of apoptosis inducing factor （AIF） were performed to evaluate apoptosis. Protein expression of vascular peroxidase-1 （ VPO-1 ） and phosphor- JNK were measured by immunoblotting. Results Activation of α7nAChR by a selective agonist PNU-282987 pre-vented H202-indced decrease of cell viability and increase of lactate dehydrogenase release. Activation of α7nAChR markedly reduced cell apoptosis and intracellular oxidative stress level. Moreover, activation of α7nAChR reduced H2 02 -induced VPO-1 protein upregulation and JNK1/2 phosphorylation. The inhibitory effect of α7nAChR activa- tion on VPO-1 was blocked by JNK inhibitor SP600125. In addition, pretreatment of α7nAChR antagonist methyl- lycaconitine blocked the cytoprotective effect of PNU-282987. Conclusion These results provide the first evidence that activation of α7nAChR protects against oxidant stress-induced damage by suppressing VPO-1 in a JNK signa- ling pathway-dependent manner in endothelial cells.

Cholinergic receptor, nicotinic, alpha 7 as a target molecule of Arctic mutant amyloid β

Alzheimer’s disease（AD）is a progressive cognitive disorder that develops predominantly in elderly patients and is characterized by cognitive impairments affecting memory,learning,and attention（Selkoe,2002）.

Altered Expression of Nicotinic Receptors in Perinatal Life Related to Prenatal Exposure to Toxics—An Overview of the Research Carried Out on This Topic at the “Lino Rossi” Research Center of the Milan University

The article aims to underline the impact of nicotine and pesticides on neuronal α7-nicotinic acetylcholine receptors expression in brainstem regions receiving cholinergic projections, given their fundamental role during the neuronal development. The in-depth histopathological/immunohistochemical examination of the autonomic nervous system performed at the “Lino Rossi” Research Center of the Milan University on a wide group of sudden unexpected fetal and infant deaths, highlighted the frequent hypodevelopment of brainstem structures checking the vital functions associated to altered expression of α7-nicotinic acetylcholine receptors and smoke absorption in pregnancy. A dysregulation of the catecholamine system was also observed in the cerebellar cortex of the same cases. However, in a not negligible percentage of sudden deaths with altered expression of α7-nicotinic receptors, the mothers never smoked but lived in rural areas. Specific analytical procedures showed the presence of agricultural pesticides in cerebral cortex samples of these victims. Therefore, it is possible to believe that the exposition to pesticides during pregnancy can produce the same harmful effects as nicotine on the nicotinic acetylcholine receptors. Moreover, alterations of α7-nicotinic acetylcholine receptors receptor expression were also detected in the lungs of many sudden perinatal death victims, allowing to consider even these findings as possible consequence of maternal exposure to toxic factors.

Nicotinic receptors modulate antitumor therapy response in triple negative breast cancer cells

BACKGROUND Triple negative breast cancer is more aggressive than other breast cancer subtypes and constitutes a public health problem worldwide since it has high morbidity and mortality due to the lack of defined therapeutic targets.Resistance to chemotherapy complicates the course of patients’treatment.Several authors have highlighted the participation of nicotinic acetylcholine receptors(nAChR)in the modulation of conventional chemotherapy treatment in cancers of the airways.However,in breast cancer,less is known about the effect of nAChR activation by nicotine on chemotherapy treatment in smoking patients.AIM To investigate the effect of nicotine on paclitaxel treatment and the signaling pathways involved in human breast MDA-MB-231 tumor cells.METHODS Cells were treated with paclitaxel alone or in combination with nicotine,administered for one or three 48-h cycles.The effect of the addition of nicotine(at a concentration similar to that found in passive smokers’blood)on the treatment with paclitaxel(at a therapeutic concentration)was determined using the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.The signaling mediators involved in this effect were determined using selective inhibitors.We also investigated nAChR expression,and ATP“binding cassette”G2 drug transporter(ABCG2)expression and its modulation by the different treatments with Western blot.The effect of the treatments on apoptosis induction was determined by flow cytometry using annexin-V and 7AAD markers.RESULTS Our results confirmed that treatment with paclitaxel reduced MDA-MB-231 cell viability in a concentration-dependent manner and that the presence of nicotine reversed the cytotoxic effect induced by paclitaxel by involving the expression of functionalα7 andα9 nAChRs in these cells.The action of nicotine on paclitaxel treatment was linked to modulation of the protein kinase C,mitogen-activated protein kinase,extracellular signal-regulated kinase,and NF-κB signaling pathways,and to an up-regulation of ABCG2 protein expression.We also detected that nicotine significantly reduced the increase in cell apoptosis induced by paclitaxel treatment.Moreover,the presence of nicotine reduced the efficacy of paclitaxel treatment administered in three cycles to MDA-MB-231 tumor cells.CONCLUSION Our findings point to nAChRs as responsible for the decrease in the chemotherapeutic effect of paclitaxel in triple negative tumors.Thus,nAChRs should be considered as targets in smoking patients.

Modulation by desensitized nicotinic receptors on metabolism of DA in striatum derived from the hemiparkinsonian model

Objective: The purpose of this study was to investigate the effects of desensitized nicotinic receptors(n ACh Rs) on striatal dopaminergic system in the hemiparkinsonian rats treated with 6-hydroxydopamine(6-OHDA). Methods: We examined the effects of desensitized n ACh Rs on the levels of dopamine(DA) and its metabolites, m RNA expression of dopamine receptor D1,D2 and monoamine oxidase B(MAO-B) in the striatum of 6-OHDA-lesioned rats using high-performance liquid chromatography and reverse transcription-polymerase chain reaction. Results: The results showed that n ACh Rs desensitization following repeated nicotine stimulation could reverse significantly the decrease of striatal DA and its metabolites levels and the increase in DA turnover in lesioned side striatum of hemiparkinsonian rats. Dopamine D1 receptor m RNA expression increased significantly, whereas dopamine D2 receptor m RNA expression remained unchanged in lesioned side striatum of nicotine-treated rats compared to 6-OHDA-lesioned rats when n ACh Rs were desensitized. Meanwhile, nicotine-treated rats displayed a significant decrease in MAO-B m RNA expression in lesioned side striatum compared to 6-OHDA-lesioned rats after n ACh Rs desensitization. Conclusion: These results suggest that n ACh Rs desensitization could promote DA level, upregulate dopamine D1 receptor expression and downregulate MAO-B expression in striatum of hemiparkinsonian rats.

Synthesis of 2-[^(18)F]fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine as a radioligand for imaging nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors(nChRs) are involved in the various pharmacological effects or disease states.In order to study the central nChRs by PET or SPECT,some radioligands have been investigated.In this paper,the procedure for synthesis of 2-[^18F] fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine(2-[^18F0-A-85380),a potential PET ligand for in vivo imaging nicotinic acetylcholine receptor was described.2-[^18F]-A-85380 was prepared from the precursor,2-nitro-3-[(1-(tert-butoxycarbonyl)-2-(S0-azetidinyl)methoxy] pyridine(4),which was synthesized with commercial (S)-2-zaetid-inecarboxylic acid as starting material.The whole procedure for radiosynthesis and purification was executed in about 1h and 45-55% of the added fluorine-18 was found in the purified 2-[^18F]-A-85380,with specific activity of 1.0-2.2×10^11 Bq/umol.

Cloning and Sequence of Nicotinic Acetylcholine Receptor α Subunit from Chilo suppressalis

Nicotinic acetylcholine receptors (nAChRs) play a significant role in excitatory synaptic transmission in insects and are the target for chloronicotinyl and nereistoxin insecticides.In recent years,Chilo suppressalis,an economically important pest of rice,developed high resistance against monosultap,a nereistoxin insecticide acting on nAChR.In order to reveal the hypothesized target insensitive mechanism,studies on the molecular property of nAChR from Chilo suppressalis are required.In this study,the full length cDNA of nAChR α subunit from this pest was cloned by RT-PCR.Sequence analysis shows that it is a novel nAChR α subunit,which was named as Cs α 1(Genbank accession No.AF418987).It contains 1?997?bp nucleotides and involves an open reading frame (ORF) encoding a mature protein of 509 amino acids excluding a signal peptide of 24 amino acids.The deduced amino acid sequence was 52%-94% identical to the reported insect nAChR genes.

电针预处理对切口痛大鼠中脑导水管周围灰质5-HT_(7)受体表达的影响

目的:建立足趾部切口痛大鼠模型,探讨重复电针预处理对切口痛大鼠镇痛效果及其对中脑导水管周围灰质(periaqueductal gray,PAG)5-HT_(7)受体(5-HT_(7)R)表达的影响。方法:40只成年雄性SD大鼠按随机数字表法均等分为对照组(Control,Con组)、切口痛模型组(Incision pain,IP组)、正常+电针预处理组(Control+Electroacupuncture,Con+EA组)、模型+电针预处理组(Incision Pain+Electroacupuncture,IP+EA组)。IP组和IP+EA组大鼠右足趾部行疼痛造模,且造模前,Con+EA组和IP+EA组大鼠行右侧“足三里”穴和“环跳”穴电针刺激(2/10 Hz疏密波,刺激强度数值为1档,每日1次30 min),连续5天。于第1次电针预处理前2 h(T1)、术前2 h(T2)、术后4 h(T3)、术后24 h(T4)测定大鼠机械刺激缩足反射阈值(mechanical withdrawal threshold,MWT)和热缩足潜伏期(thermal withdrawal latency,TWL);酶联免疫吸附法检测脑脊液中5-HT浓度;免疫组化和免疫荧光方法分别检测大鼠PAG中c-Fos和5-HT_(7)R蛋白表达情况。结果:与Con组比较,IP组大鼠T3、T4时间点MWT和TWL均明显降低,脑脊液中5-HT浓度增加,PAG中c-Fos蛋白和5-HT_(7)R表达明显上调(P<0.05);Con+EA组和IP+EA组脑脊液中5-HT含量和PAG中5-HT_(7)R表达均上升(P<0.05)。与IP组相比,IP+EA组大鼠T3、T4时间点的MWT和TWL显著升高,PAG中c-Fos蛋白表达减少,5-HT_(7)R蛋白表达增加(P<0.05)。结论:电针预处理可能通过上调PAG中5-HT_(7)R蛋白表达发挥镇痛作用。

P2X7R过表达的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达观察

目的观察嘌呤能受体P2X配体门控离子通道7的配体(P2X7R)过表达白血病细胞诱导分化的巨噬细胞单钠尿酸盐(MSU)晶体诱导痛风炎症反应过程中NOD样受体家族3(NLRP3)蛋白、IL-1β、TNF-α表达情况。方法取人单核细胞白血病细胞系THP-1,并随机分为过表达组、空白组、模型组、对照组;过表达组和空白组分别转染P2X7R过表达质粒、空白载体质粒,转染5 d,将过表达组、空白组、模型组THP-1细胞用100 ng/mL的PMA刺激3 h后分化为巨噬细胞,另将MSU晶体用氢氧化钠溶解配制成浓度为100μg/mL的MSU乳糜状悬液加入培养液中孵育6 h;对照组正常培养。分别采用RT-PCR法和Western blot法测算巨噬细胞P2X7R mRNA、蛋白,ELISA法检测巨噬细胞上清液IL-1β、TNF-α,Western blot法测算巨噬细胞NOD样受体家族3(NLRP3)蛋白。结果与对照组比较,过表达组、空白组、模型组P2X7R mRNA和蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05);与模型组、空白组比较,过表达组P2X7R mRNA、蛋白相对表达量升高,细胞上清液IL-1β、TNF-α水平升高,细胞NLRP3蛋白相对表达量升高(P均<0.05)。结论P2X7R过表达白血病细胞诱导分化的巨噬细胞MSU晶体诱导痛风炎症反应过程中IL-1β、TNF-α、NLRP3表达增加,IL-1β、TNF-α水平升高可能通过激活NLRP3蛋白来实现。

非剥脱点阵激光联合侧柏叶酊对斑秃小鼠IL-7/IL-7Rα信号通路和Tregs细胞亚群的影响

目的:研究1565 nm非剥脱点阵激光联合侧柏叶酊(Platycladus orientalis tincture,POT)对斑秃(Alopecia areata,AA)小鼠治疗作用以及对白细胞介素7(Interleukin 7,IL-7)/白细胞介素7受体α(Interleukin-7 receptorα,IL-7Rα)信号通路和调节性T细胞(Regulatory T cells,Tregs)亚群的影响。方法:将50只成年雄性C3H/HeJ小鼠随机分为对照组(C组),模型组[M组,环磷酰胺(Cyclophosphamide,CTX)诱导AA模型],M+1565 nm组(1565 nm非剥脱点阵激光治疗AA),M+POT组(POT治疗AA)、M+1565 nm+POT组(1565 nm非剥脱点阵激光联合POT治疗AA),每组10只。流式细胞术检测C组和M组皮损组织中Tregs细胞亚群的比例和所有组血液中单个核细胞中Tregs细胞亚群的比例。Western blot法检测各组小鼠皮损组织中IL-7和IL-7Rα的表达。结果:与C组比,M组皮肤组织IL-7和IL-7Rα的表达均明显增加,而且Tregs细胞比例明显减少(P<0.05)。与M组比,M+1565 nm组和M+POT组IL-7的表达均降低(P<0.05)。与M组比,M+1565 nm+POT组IL-7和IL-7Rα的表达均降低,且Tregs细胞比例都显著增加(P<0.05)。结论:1565 nm非剥脱点阵激光联合POT治疗可以抑制斑秃小鼠IL-7/IL-7Rα信号并减少Tregs细胞的比例。

表皮生长因子蛛毒素受体7次跨膜结构域1通过血管内皮生长因子信号通路调控胃癌迁移的机制研究

目的探究表皮生长因子蛛毒素受体7次跨膜结构域1(ADGRL4)通过血管内皮生长因子(VEGF)信号通路调控胃癌迁移的机制。方法将正常胃黏膜上皮细胞GES⁃1、胃癌细胞SGC⁃7901、HGC⁃27、Hs⁃746T传代培养后,检测ADGRL4表达量。将胃癌SGC⁃7901细胞分为对照组、过表达组、沉默组。对照组转染空载体,过表达组转染ADGRL4上调质粒,沉默组转染ADGRL4沉默质粒。分析并比较3组胃癌细胞侵袭、迁移数及VEGF信号通路蛋白表达量。结果与正常胃黏膜上皮细胞GES⁃1比较,胃癌细胞SGC⁃7901、HGC⁃27、Hs⁃746T中ADGRL4表达量均上升(P<0.05);与胃癌细胞HGC⁃27、Hs⁃746T比较,胃癌细胞SGC⁃7901中ADGRL4表达量较高(P<0.05),故选择SGC⁃7901进行后续实验。与对照组比较,过表达组ADGRL4表达量上升,沉默组ADGRL4表达量下降(P<0.05);与过表达组比较,沉默组ADGRL4表达量下降(P<0.05),说明ADGRL4转染成功。与对照组比较,过表达组细胞侵袭数、迁移数、基质金属蛋白酶(MMP)⁃2、MMP⁃9、VEGF、血管内皮生长因子受体⁃2(VEGFR⁃2)表达量上升,E⁃钙黏蛋白(E⁃cadherin)表达量下降,沉默组细胞侵袭数、迁移数、MMP⁃2、MMP⁃9、VEGF、VEGFR⁃2表达量下降,E⁃cadherin表达量上升(P<0.05);与过表达组比较,沉默组细胞侵袭数、迁移数、MMP⁃2、MMP⁃9、VEGF、VEGFR⁃2表达量下降,E⁃cadherin表达量上升(P<0.05)。结论胃癌细胞经下调ADGRL4干预后,侵袭、迁移数减少,迁移、侵袭相关蛋白表达量得到调节,其机制可能与VEGF通路受到抑制有关。

射干制剂对失眠大鼠自主活动及TLR7/MyD88信号通路的影响

目的 探研射干制剂对PCPA致失眠大鼠自主活动、脑电波以及脑组织TLR7、MyD88基因表达的影响。方法 随机将70只健康SD大鼠分为空白对照组、模型对照组、射干制剂低剂量组(26 mg·kg^(-1))、射干制剂中剂量组(52 mg·kg^(-1))、射干制剂高剂量组(104 mg·kg^(-1))、朱砂安神丸组(1.62 g·kg^(-1))、地西泮片组(0.9 mg·kg^(-1))。荧光定量PCR检测各组TLR7、My D88基因的相对表达量;采用Etho Vision大小鼠行为学系统记录大鼠自主活动情况;通过无线遥测系统记录各组睡眠时脑电图。结果 与空白组对照比较,模型对照组脑组织TLR7、MyD88相对表达量显著升高(P<0.01);同模型对照组相比,射干制剂低剂量组、射干制剂中剂量组TLR7、MyD88的mRNA表达量均降低(P<0.05),射干制剂高剂量组TLR7、MyD88 mRNA表达量显著降低(P<0.01);与空白对照组相比,PCPA造模后大鼠运动量明显增加,不同剂量射干制剂组、朱砂安神丸组及地西泮片组运动有不同程度的减少,射干制剂高剂量组运动减少较为明显;给药120 min后,与空白对照组相比,模型对照组脑电波中的δ波百分比降低(P<0.05);与模型对照组相比,射干低中剂量组、朱砂安神丸组、地西泮片组δ波百分比均升高(P<0.05)。结论 射干制剂可有效抑制大鼠兴奋性,增加脑电图δ波百分比,改善睡眠,这些作用可能是通过调节脑组织TLR7和MyD88基因的表达来实现。