δ-Opioid receptor as a potential therapeutic target for ischemic stroke

Ischemic stroke is a global epidemic condition due to an inadequate supply of blood and oxygen to a specific area of brain either by arterial blockage or by narrowing of blood vessels.Despite having advancement in the use of thrombolytic and clot removal medicine,significant numbers of stroke patients are still left out without option for treatment.In this review,we summarize recent research work on the activation ofδ-opioid receptor as a strategy for treating ischemic stroke-caused neuronal injury.Moreover,as activation ofδ-opioid receptor by a non-peptidicδ-opioid receptor agonist also modulates the expression,maturation and processing of amyloid precursor protein andβ-secretase activity,the potential role of these effects on ischemic stroke caused dementia or Alzheimer’s disease are also discussed.

Expression of mu-opioid receptors in human chronic inflamed knee joint synovium tissue

Objective: To examine the changes of mu-opioid receptors (MORs) expression in human chronic inflamed knee joint synovium tissue. Methods:Knee joint synovium tissues were taken from 21 patients with chronic arthritis (inflamed group) and 6 fresh bodies with normal knee joints (control group). And the expression of MORs was detected by using immunohistochemistry. flow cytometry(FCM) and reverse-transcription polymerase chain reaction (RT-PCR). Results: The expression of MORs in the inflamed group was significantly higher than that in the normal group by using the 3 techniques(P<0. 05). Conclusion: Chronic inflammation enhances the up-regulation of MORs in human knee joint synovium tissue.

Molecular characterization and functional expression of opioid receptor-libe_1 receptor

The opioid receptor-libel receptor (ORL), an orphan receptor whose human and murine complementary DNAs,has been characterized recently. ORL transcripts are particularly abundant in the central nervous system. We demonstrated that ORL expressed in human neuroblastoma SK-N-SH and SH-SY5Y cell lines by radioligand binding assay, reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis in the present study. Stimulation with ORL1 specific agonist, nociceptin/orphanin Fo, increased [34S]GTPrγS binding to SK-N-SH cell membranes (EC50 = 14 ±0.45 nM), and attenuated forskolin-stimulated accumulation of cellular cAMP (EC50= 0.80 ±0.45 nM, indicative that activation of ORL1 activates G proteins and inhibits adenylyl cyclase. Activation of ORL1 receptor was also accessed using CHO:hORL1 cell line by microphysiometer. Treatment of nociceptin/orphanin FQ increased extracellular acidification rate significantly.

Low-dose morphine elicits ventilatory excitant and depressant responses in conscious rats: Role of peripheral <i>µ</i>-opioid receptors

The systemic administration of morphine affects ventilation via a mixture of central and peripheral actions. The aims of this study were to characterize the ventilatory responses elicited by a low dose of morphine in conscious rats;to determine whether tolerance develops to these responses;and to determine the potential roles of peripheral μ-opioid receptors (μ-ORs) in these responses. Ventilatory parameters were monitored via unrestrained whole-body plethysmography. Conscious male Sprague-Dawley rats received an intravenous injection of vehicle or the peripherally-restricted μ-OR antagonist, naloxone methiodide (NLXmi), and then three successive injections of morphine (1 mg/kg) given 30 min apart. The first injection of morphine in vehicle-treated rats elicited an array of ventilatory excitant (i.e., increases in frequency of breathing, minute volume, respiratory drive, peak inspiratory and expiratory flows, accompanied by decreases in inspiratory time and end inspiratory pause) and inhibitory (i.e., a decrease in tidal volume and an increase in expiratory time) responses. Subsequent injections of morphine elicited progressively and substantially smaller responses. The pattern of ventilatory responses elicited by the first injection of morphine was substantially affected by pretreatment with NLXmi whereas NLXmi minimally affected the development of tolerance to these responses. Low-dose morphine elicits an array of ventilatory excitant and depressant effects in conscious rats that are subject to the development of tolerance. Many of these initial actions of morphine appear to involve activation of peripheral μ-ORs whereas the development of tolerance to these responses does not.

Determination of structure-activity relationships between fentanyl analogs and human μ-opioid receptors based on active binding site models

Fentanyl is a potent and widely used clinical narcotic analgesic, as well as a highly selective IJ-opioid agonist. The present study established a homologous model of the human μ-opioid receptor; an intercomparison of three types of μ-opioid receptor protein sequence homologous rates was made. The secondary receptor structure was predicted, the model reliability was assessed and verified using the Ramachandran plot and ProTab analysis. The predictive ability of the CoMFA model was further validated using an external test set. Using the Surflex-Dock program, a series of fentanyl analog molecules were docked to the receptor, the calculation results from Biopolymer/SitelD showed that the receptor had a deep binding area situated in the extracellular side of the transmembrane domains （TM） among TM3, TM5, TM6, and TMT. Results suggested that there might be 5 active areas in the receptor. The important residues were Asp147, Tyr148, and Tyr149 in TM3, Trp293, and His297 in TM6, and Trp318, His319, Ile322, and Tyr326 in TM7, which were located at the 5 active areas. The best fentanyl docking orientation position was the piperidine ring, which was nearly perpendicular to the membrane surface in the 7 TM domains. Molecular dynamic simulations were applied to evaluate potential relationships between ligand conformation and fentanyl substitution.

Immunohistochemical identification of dynorphin A and Kappa opioid receptor-1 in the digestive system of scallop Chlamys farreri

Little is known about the roles of dynorphin and Kappa opioid receptor(KOR) in mollusks. In this study, we aim to determine the distribution of dynorphin A and KOR-1 in the digestive system of the scallop Chlamys farreri. Using immunohistochemical staining, we confirmed the expression of dynorphin A and KOR-1 in the digestive system of C. farreri. Dynorphin A immunopositive cells were identified in intestine and hepatopancreas. In intestine, small and spherical dynorphin A immunopositive cells(4–9 μm in diameter) were scattered among the long columnar epithelial cells(ECs). In hepatopancreas, cells containing masses(5–14 μm in diameter) of dynorphin A immunopositive products were observed in epithelium of acinis. These immunopositive cells may be synthetic and/or secretory cells of dynorphin A. Dynorphin A immunoreactive products were commonly observed in epithelium and connective tissue(CT) of labial palps, mouth labia and stomach, which presented in forms of grains, fibers or flakes. KOR-1 immunoreactive material was observed in ECs and CTs of labial palps, mouth labia and stomach, intestine and hepatopancreas. The distribution of both dynorphin A and KOR-1 in the digestive organs suggests an involvement of dynorphin via KOR-1 in the functional regulation of the digestive system of C. farreri.

Thienorphine induces analgesia by binding κ -and δ-, or by partially binding μ-opioid receptor,thus further regulating cAMP-PKA activity

OBJECTIVE Thienorphine,a new oripavine derivative,has shown to possess stronger antinociceptive effects and better oral bioavailability compared to buprenorphine.The present study examines the effect of thienorphine on c AMP-dependent protein kinase A(PKA) activity in CHO cells expressing μ-,κ-,δ-and ORL1 receptors.In addition,we further examined its analgesic effect in vivo.METHODS The effect of thienorphine on cA MP-dependent PKA redistribution and cA MP inhibition were analyzed in CHO-PKAcatEGFP cells.PKA redistribution assays in CHO-PKAcatEGFP cells stably expressing μ-,κ-,δ-and ORL1 receptors were analyzed by high-throughput screening system to elucidate the efficacy of agonists or antagonists on opioid receptors.Moroever,the antinociceptive effects of thienorphine in vivo were examined using hot plate test.RESULTS Briefly,the maximum inhibition of thienorphine on PKA activity was about 36%,100%,100%and 12% in CHO-μ/κ/δ/ORL1-PKAcatE GFP cel s,respectively.In addition,thienorphine concentrationdependently inhibited the PKA activity with EC50 value of(22.7±18.1) nmol·L^(-1) in CHO-κ-PKAcatE GFP cels and(12.4±7.7) nmol·L^(-1) in CHO-δ-PKAcatE GFP cells.Thienorphine induced approximately 50%antinociceptive effect in mice lacking μ receptors compared to their wild-type controls(P<0.05).Also,the κ and δ selective antagonist nor-binaltorphimine,naltrindole decreased approximately 50%-60% in % MPE of theinorphine in μ-KO mice,respectively.The ORL1 receptor selective antagonist J113397 had no effect in %MPE of theinorphine in μ-KO mice.CONCLUSION Thienorphine induces analgesia through bindingκ-and δ-,or by partially binding μ-opioid receptor,thus further regulating the cAMP-PKA activity.Therefore,thienorphine may be used in acute or chronic pain with minimal addictive potential.

MODULATION OF A δ-AND C-FIBER EVOKED RESPONSES OF NOCICEPTIVE NEURONS IN THE SUPERFICIAL AND THE DEEPER DORSAL HORN OF THE MEDULLA:ROLE OF OPIOID RECEPTORS(μ, δ_1, δ_2)

The present study was designed to investigate the effects of intravenously administered agonists and antagonists at μ(DAMGO, naloxone,)δ1 (DPDPE,BNTX)andδ2(DELT, NTB)opioid receptors on the Aδ-and C-fiber evoked responses of nociceptive neurons in the superficial and the deeper dorsal horn of the rat medulla.Extracellular single unit recording were made from 70 nociceptive neurons(28 NS,42 WDR) in the superficial dorsal horn and 37 nociceptive neurons(4 NS,33 WDR)in the deeper dorsal horn.All these neurons had an ipsilateral orofacial mechanoreceptive field and majority of these neurons had no spontaneous activity. The latencies for the C fiber evoked responses ranged from 34～105 msec whereas for Aδfiber-evoked responses it ranged from 3～22msec. A clear separation was observed between early and late responses of evoked by Cand Aδ-fiber.Application of DPDPE,DELT and DAMGO produced inhibitory effects on the Aδ-and C-fiber evoked responses of nociceptive neurons in the superficial and thedeeper dorsal horn.By comparison,the inhibition was more pronounced on the C-fiber evoked response than on the Aδ-fiber evoked response,and DAMGO produced a stronger inhibitory action than both DELT and DPDPE. Additionally,DPDPE produced facilitation, or inhibition followed by facilitation on the Aδ-and C-response and the effect had longer latency and longer time course.DPDPE also induced completely oppsite effects on the Aδ-and C-fiber evoked responses.Although the facilitation was observed,the effect was not dose-dependent. Application of BNTX (0.4～1mg/kg),a δ1 receptor antagonist,produced antagonism of DPDPE in 88%(7/8) neurons. Application of the doses (0.7～1mg/kg) of BTB,δ2-receptor antagonist,resulted in antagonism of both DELT and DPDPE. The inhibition of DELT on Aδ-response was antagonized by doses (0.3～1mg/kg)of NTB in 100% (14/14)neurons while the antagonism on C-response was in 79%(11/14) neurons.The effect produced by DPDPE was antagonized by the doses (0.7～1mg/kg) of NTB in 100%(4/4) neurons. However,a smaller dose of NTB(0.3mg/kg)which and antagonize the effect of DELT,did not antagonize the effect of DPEPE in 100%(4/4) neurons. The inhibitory action of DAMGO on Aδ-and C-fiber evoked responses was completely antagonized by naloxone(0. 2mg/kg) in 100% (6/6) neurons. These results suggest that:①μ-and δ-opioid receptors play an important role in modulating Aδ-and Cfiber evoked responses of nociceptive neurons in the superficial and the deeper dorsal horn of the rat medulla; ② The inhibitory action produced by DPDPE, DELT and DAMGO was more pronounced on the C-fiber evoked excitation and indicates that the agonists produce more predominant inhibition on the responses of dorsal horn neurons to noxious stimuli; ③ activation of either δ1-orδ2-opioid receptors produces inhibitory actions on Aδ- and C-response of nociceptive neurons in the superficial and the deeper dorsal horn of the medullal;DPDPE and DELT act at different δ-opioid receptor subtypes in the rat rnedulla; ⑤i.v.-administered NTB can distinguish δ-opioid receptor subtypes in a limited dose range.When administered i. v., 0. 3mg/kg of NTB is selective for δ2-opioid receptor.

Effects of Peripherally Acting Opioid Ligands on Central Opioid Receptors and <i>β</i>-Endorphin Release in Stressed Rats

Using the radioreceptor binding assay, μ-opioid receptor (MOR) affinity in the midbrain of stressed rats was higher than in naive controls. MOR density in the rat frontal cortex was reduced after stress. Intragastric administration of the MOR antagonist naloxone methiodide was followed by an increase in the number of MORs in the frontal cortex. However, the MOR agonist loperamide significantly decreased the density of MORs in the frontal cortex and midbrain of naive animals. Loperamide and naloxone methiodide were shown to prevent an increase in MOR affinity and a decrease in MOR density in the midbrain of rats after restraint stress. The restraint stress was accompanied by an increase in the release of β-endorphin (BE) in the ventral tegmental area (VTA) of control rats. After administration, loperamide slightly decreased the release of BE, naloxone methiodide significantly increased the release of BE in the cingulate cortex (CC) of untreated animals, while drugs had no effect on the release of BE in the VTA. The drugs significantly increased the extracellular level of BE in the CC of stressed animals. Loperamide abolished the increase in the stress-induced release of BE in the VTA. By contrast, naloxone methiodide significantly increased the release of BE in the VTA of stressed rats. Our data indicated that activation of peripheral MORs induces depression of the central part of the μ-opioid system, but suppression of peripheral MOR activity induces activation of the central μ-opioid system, the interaction of which can be modulated by stress.

Functional expression of opioid receptor-like receptorand its endogenous specific agonist nociceptin/orphanin FQ during mouse embryogenesis

Expression of opioid receptor-like receptor (ORL1)and its endogenous peptide agonist nociceptin/orphaninFo (N/OFQ) during mouse embryogenesis have been investigated. Transcripts of ORL1 and N/OFQ were detected by RT-PCR in mouse brain of day 8 embryo (E8)and the expression continued afterwards. Northern blotanalysis revealed abundant expression of ORL1 at postnatal day 1 (P1) and N/OFQ at E17 and P1 in the brain butnone was detected in other embryonic tissues. The presence of functional ORL1 in mouse embryonic brain wasalso confirmed by specific binding of [3H] N/OFQ (kd=1.3±0.5 nM and Bmax = 72±9 fmol/mg protein) as wellas by N/OFQ-stimulated G protein activation.

Dopamine D₁- and D₂-Receptors in Immunostimulation under Activation of Mu-Opioid Receptors in Mice with Different Psychoemotional States

The purpose of the present study was to analyze the effect of activation of mu-opioid receptors (mu-OR) on the immune response under blockade of postsynaptic D1-and D2-receptors in mice of the C57BL/6J strain displaying either aggressive or depressive-like behaviors in the social conflict model. It is shown that activation of activation of mu-OR with a highly selective agonist DAGO (100 μg/kg) increased significantly IgM-immune response not only in C57BL/6J mice with an unchanged psychoemotional state but also in mice displaying aggressive or depressive-like behaviors in the social stress model (10 days of agonistic confrontations). Selective blockade of DA receptors of the D1-type with SCH-23390 (1.0 mg/kg with DAGO administration) caused a more pronounced elevation of IgM-immune response than DAGO alone while DAGO effect was completely blocked by prior administration of D2-receptor antagonist haloperidol (1.0 mg/kg). At the same time, both SCH-23390 and haloperidol prevented the immune response increase induced by DAGO injection in mice engaged in aggressive or depressive-like behaviors. Thus, in animals not subjected to social stress DAGO-induced immunostimulation is provided only by D2-receptors, whereas in animals with altered psychoemotional state mu-opioid immunostimulation is mediated by both types of DA receptors—D1 and D2. These data provide evidence for different impacts of the main subtypes of DA receptors in the mediation of immunomodulating effects of mu-opioid system under normal and stressful conditions.

κ-阿片受体激动剂U50488H通过调节巨噬细胞极化减轻体外循环致大鼠急性肺损伤的研究

目的探讨κ-阿片受体(KOR)激动剂U50488H是否通过调节巨噬细胞极化减轻体外循环(CPB)引发的大鼠急性肺损伤(ALI)。方法将24只成年雄性清洁级SD大鼠(50~450 g)随机分为Sham组(假手术)、CPB组(CPB)和U50488H组(KOR激动剂+CPB),每组8只。U50488H组于CPB前30 min静脉注射1.5 mg/kg U50488H。分别于CPB后0、1、2 h行动脉血气分析,计算肺泡-动脉氧分压差(A-aDO_(2))和呼吸指数(RI)。3组大鼠均在停CPB后2 h处死,取完整右肺下叶。采用重力法测定血管外肺水(EVLW),采用苏木精-伊红(HE)染色观察肺组织形态学变化。采用酶联免疫吸附试验(ELISA)检测血浆酯多糖(LPS)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-4(IL-4)含量。采用免疫荧光法测定大鼠肺组织iNOS和CD206水平。结果与Sham组比较,CPB组大鼠EVLW和TNF-a、血浆IL-6水平及肺组织iNOS表达增高,血浆IL-4水平和肺组织CD206表达降低,差异有统计学意义(P<0.05)。CPB后0、1、2 h,CPB组的A-aDO_(2)和RI、LPS高于Sham组,U50488H组的A-aDO_(2)和RI、LPS低于CPB组,差异有统计学意义(P<0.05)。CPB组大鼠出现严重肺损伤和肺泡内充血/出血,并伴有广泛的炎症细胞浸润,U50488H组肺损伤显著减轻。结论KOR激动剂U50488H可促进CPB后大鼠肺巨噬细胞向M2表型极化,减轻炎症反应,增加抗炎因子释放,进而减少CPB后ALI的发生。

κ-阿片受体激动剂对体外循环大鼠肺细胞焦亡和NLRP3炎症小体的影响

目的通过观察κ-阿片受体(KOR)激动剂U50488H对心肺转流(CPB)大鼠肺组织NLRP3炎症小体和细胞焦亡的影响,探讨U50488H减轻CPB致急性肺损伤(ALI)的可能机制。方法24只成年雄性清洁级SD大鼠,350~450 g,随机分为假手术组(Sham组)、CPB组和CPB+KOR激动剂组(U50448H组),每组8只。U50448H组于CPB前30 min静脉注射1.5 mg/kg U50488H。分别于CPB后0 h、1 h和2 h各时点行动脉血气分析,计算肺泡-动脉氧分压差(AaDO2)和呼吸指数(RI)。三组大鼠均在停CPB后2 h时处死,取完整右肺下叶,采用重力法测定血管外肺水(EVLW),HE染色观察肺组织形态学变化。采用ELISA方法检测血浆脂多糖(LPS)、肿瘤坏死因子-α(TNF-α)、白介素(IL)-6和IL-4的含量,Western blot测定肺组织GSDMD-C、GSDMD-N和NLRP3、半胱氨酸天冬氨酸酶前体(pro-Caspase-1)蛋白的表达。结果CPB组大鼠在CPB后0 h、1 h和2 h各时点的AaDO2和RI、LPS明显高于Sham组(P<0.05);U50488组三个时点的AaDO2和RI、LPS明显低于CPB组(P<0.05)。与Sham组比较,CPB组大鼠EVLW、血浆TNF-a和IL-6、肺组织GSDMD-C、GSDMD-N和NLRP3、pro-Caspase-1表达明显增高,血浆IL-4水平明显降低,其差异有统计学意义(P<0.05);而U50488组上述指标与Sham组无明显差异(P>0.05)。CPB组大鼠出现了严重的肺损伤和肺泡内充血/出血,并伴有广泛的炎症细胞浸润,U50448H组肺损伤明显减轻。结论KOR激动剂U50488H对CPB所致ALI期间NLRP3炎性小体诱导的细胞焦亡具有抑制作用,从而减轻了CPB后的肺损伤。

A non-opioid pathway for dynorphin-caused spinal cord injury in rats

Intrathecal injection of dynorphin into rats via subarachnoid catheter induces damage to spinal cord tissue and motor function. Injection of the kappa opioid receptor antagonist nor-binaltorphine, or the excitatory amino acid N-methyl-D-aspartate receptor antagonist MK-801 into rats alleviated the pathological changes of dynorphin-caused spinal cord tissue injury and reduced the acid phosphatase activity in the spinal cord. The experimental findings indicate that there are opioid and non-opioid pathways for dynorphin-induced spinal cord injury, and that the non-opioid receptor pathway may be mediated by the excitatory amino acid N-methyl-D-aspartate receptor.

Cyclooxygenase-2 and epithelial growth factor receptor up-regulation during progression of Barrett's esophagus to adenocarcinoma

AIM： To investigate the expression of cyclooxygenase-2 （COX-2） and epithelial growth factor receptor （EGFR） throughout the progression of Barrett＇s esophagus （BE）. METHODS： COX-2 and EGFR protein expressions were detected by using immunohistochemical method. A detailed cytomorphological changes were determined. Areas of COX-2 and EGFR expression were quantified by using computer Imaging System. RESULTS： The expressions of both COX-2 and EGFR increased along with the progression from BE to esophagus adenocarcinoma （EAC）. A positive correlation was found between COX-2 expression and EGFR expression. CONCLUSION： COX-2 and EGFR may be cooperative in the stepwise progression from BE to EAC, thereby leading to carcinogenesis.

Effects of ω-3 fatty acids on toll-like receptor 4 and nuclear factor-κB p56 in lungs of rats with severe acute pancreatitis

AIM To determine the effects of ω-3 fatty acids(ω-3FA) on the toll-like receptor 4(TLR4)/nuclear factor κB p56(NF-κBp56) signal pathway in the lungs of rats with severe acute pancreatitis(SAP).METHODS A total of 56 Sprague-Dawley rats were randomly divided into 4 groups: control group, SAP-saline group, SAP-soybean oil group and SAP-ω-3FA group. SAP was induced by the retrograde infusion of sodium taurocholate into the pancreatic duct. The expression of TLR4 and NF-κBp56 in the lungs was evaluated by immunohistochemistry and Western blot analysis. The levels of inflammatory cytokines interleukin-6 and tumor necrosis factor-alpha in the lungs were measured by enzyme-linked immunosorbent assay. RESULTS The expression of TLR4 and NF-κBp56 in lungs and of inflammatory cytokines in serum significantly increased in the SAP group compared with the control group(P < 0.05), but was significantly decreased in the ω-3FA group compared with the soybean oil group at 12 and 24 h(P < 0.05).CONCLUSION During the initial stage of SAP, ω-3FA can efficiently lower the inflammatory response and reduce lung injury by triggering the TLR4/NF-κBp56 signal pathway.

Effect of ω-3 Polyunsaturated Fatty Acid on Toll-like Receptors in Patients with Severe Multiple Trauma

This study examined the effects of ω-3 polyunsaturated fatty acid（ω-3PUFA） on the expression of toll-like receptor 2（TLR2）,toll-like receptor 4（TLR4） and some related inflammatory factors in peripheral blood mononuclear cells（PBMCs） of patients with early-stage severe multiple trauma.Thirty-two patients who were admitted to the Department of Traumatic Surgery,Tongji Hospital（Wuhan,China） between May 2010 and November 2010,and diagnosed as having severe multiple trauma with a injury severity score（ISS） no less than 16,were enrolled in the study and divided into two groups at random（n=16 in each）：ω-3PUFA group and control group in which routine parenteral nutrition supplemented with ω-3PUFA or not was administered to the patients in two groups for consecutive 7 days.Peripheral blood from these patients was collected within 2 h of admission（day 0）,and 1,3,5 and 7 days after the nutritional support.PBMCs were isolated and used for detection of the mRNA and protein expression of TLR2 and TLR4 by using real-time PCR and flow cytometry respectively,the levels of NF-κB by quantum dots-based immunofluorescence assay,the levels of TNF-α,IL-2,IL-6 and COX-2 by ELISA,respectively.The results showed that the mRNA and protein expression of TLR2 and TLR4 in PBMCs was significantly lower in ω-3PUFA group than in control group 5 and 7 days after nutrition support（both P0.05）.The levels of TNF-α,IL-2,IL-6 and COX-2 were found to be substantially decreased in PBMCs in ω-3PUFA group as compared with control group at 5th and 7th day（P0.05 for all）.It was concluded that ω-3PUFA can remarkably decrease the expression of TLR2,TLR4 and some related inflammatory factors in NF-κB signaling pathway in PBMCs of patients with severe multiple trauma,which suggests that ω-3PUFA may suppress the excessive inflammatory response meditated by the TLRs/NF-κB signaling pathway.

Optogenetics-induced activation of glutamate receptors improves memory function in mice with Alzheimer’s disease

Optogenetics is a combination of optics and genetics technology that can be used to activate or inhibit specific cells in tissues. It has been used to treat Parkinson’s disease, epilepsy and neurological diseases, but rarely Alzheimer’s disease. Adeno-associated virus carrying the CaMK promoter driving the optogenetic channelrhodopsin-2 (CHR2) gene (or without the CHR2 gene, as control) was injected into the bilateral dentate gyri, followed by repeated intrahippocampal injections of soluble low-molecular-weight amyloid-β1–42 peptide (Aβ1–42). Subsequently, the region was stimulated with a 473 nm laser (1–3 ms, 10 Hz, 5 minutes). The novel object recognition test was conducted to test memory function in mice. Immunohistochemical staining was performed to analyze the numbers of NeuN and synapsin Ia/b-positive cells in the hippocampus. Western blot assay was carried out to analyze the expression levels of glial fibrillary acidic protein, NeuN, synapsin Ia/b, metabotropic glutamate receptor-1a (mGluR-1a), mGluR-5, N-methyl-D-aspartate receptor subunit NR1, glutamate receptor 2, interleukin-1β, interleukin-6 and interleukin-10. Optogenetic stimulation improved working and short-term memory in mice with Alzheimer’s disease. This neuroprotective effect was associated with increased expression of NR1, glutamate receptor 2 and mGluR-5 in the hippocampus, and decreased expression of glial fibrillary acidic protein and interleukin-6. Our results show that optogenetics can be used to regulate the neuronal-glial network to ameliorate memory functions in mice with Alzheimer’s disease. The study was approved by the Animal Resources Committee of Jinan University, China (approval No. LL-KT-2011134) on February 28, 2011.

Estrogen affects neuropathic pain through upregulating N-methyl-D-aspartate acid receptor 1 expression in the dorsal root ganglion of rats

Estrogen affects the generation and transmission of neuropathic pain,but the specific regulatory mechanism is still unclear.Activation of the N-methyl-D-aspartate acid receptor 1（NMDAR1） plays an important role in the production and maintenance of hyperalgesia and allodynia.The present study was conducted to determine whether a relationship exists between estrogen and NMDAR1 in peripheral nerve pain.A chronic sciatic nerve constriction injury model of chronic neuropathic pain was established in rats.These rats were then subcutaneously injected with 17β-estradiol,the NMDAR1 antagonist D（-）-2-amino-5-phosphonopentanoic acid（AP-5）,or both once daily for 15 days.Compared with injured drug na？ve rats,rats with chronic sciatic nerve injury that were administered estradiol showed a lower paw withdrawal mechanical threshold and a shorter paw withdrawal thermal latency,indicating increased sensitivity to mechanical and thermal pain.Estrogen administration was also associated with increased expression of NMDAR1 immunoreactivity（as assessed by immunohistochemistry） and protein（as determined by western blot assay） in spinal dorsal root ganglia.This 17β-estradiol-induced increase in NMDAR1 expression was blocked by co-administration with AP-5,whereas AP-5 alone did not affect NMDAR1 expression.These results suggest that 17β-estradiol administration significantly reduced mechanical and thermal pain thresholds in rats with chronic constriction of the sciatic nerve,and that the mechanism for this increased sensitivity may be related to the upregulation of NMDAR1 expression in dorsal root ganglia.

Increased Cellular Invasion and Proliferation via Estrogen Receptor after 17-<i>β</i>-Estradiol Treatment in Breast Cancer Cells Using Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC)

17-β-estradiol (estrogen) is a steroid hormone important to human development;however, high levels of this molecule are associated with increased risk of breast cancer primarily due to estrogen’s ability to bind and activate the estrogen receptor (ER) and initiate gene transcription. Currently, estrogen mechanisms of action are classified as genomic and non-genomic and occur in an ER-dependent and ER-independent manner. In this study, we examine estrogen signaling pathways, by measuring changes in protein expression as a function of time of exposure to estrogen in both ER-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. Using a robust experimental design utilizing isotopic labeling, two-dimensional LC-MS, and bioinformatics analysis, we report genomic and non-genomic ER regulated estrogen responsive proteins. We find a little over 200 proteins differentially expressed after estrogen treatment. Cell proliferation, transcription, actin filament capping and cell to cell signaling are significantly enriched in the MCF-7 cell line alone. Translational elongation and proteolysis are enriched in both cell lines. Subsets of the proteins presented in this study are for the first time directly associated with estrogen signaling in mammary carcinoma cells. We find that estrogen affected the expression of proteins involved in numerous processes that are related to tumorigenesis such as increased cellular division and invasion in an ER-dependent manner. Moreover, we identified negative regulation of apoptosis as a non-genomic process of estrogen. This study complements gene expression studies and highlights the need for both genomic and proteomic analyses in unraveling the complex mechanisms by which estrogen affects progression of breast cancer.