Single-dose methamphetamine administration impairs ORM retrieval in mice via excessive DA-mediated inhibition of PrLGlu activity.

Methamphetamine (METH), an abused psychostimulant, impairs cognition through prolonged or even single-dose exposure, but animal experiments have shown contradictory effects on memory deficits. In this study we investigated the effects and underlying mechanisms of single-dose METH administration on the retrieval of object recognition memory (ORM) in mice. We showed that single-dose METH administration (2 mg/kg, i.p.) significantly impaired ORM retrieval in mice. Fiber photometry recording in METH-treated mice revealed that the activity of prelimbic cortex glutamatergic neurons (PrLGlu) was significantly reduced during ORM retrieval. Chemogenetic activation of PrLGlu or glutamatergic projections from ventral CA1 to PrL (vCA1Glu-PrL) rescued ORM retrieval impairment. Fiber photometry recording revealed that dopamine (DA) levels in PrL of METH-treated mice were significantly increased, and micro-infusion of the D2 receptor (D2R) antagonist sulpiride (0.25 µg/side) into PrL rescued ORM retrieval impairment. Whole-cell recordings in brain slices containing the PrL revealed that PrLGlu intrinsic excitability and basal glutamatergic synaptic transmission were significantly reduced in METH-treated mice, and the decrease in intrinsic excitability was reversed by micro-infusion of Sulpiride into PrL in METH-treated mice. Thus, the impaired ORM retrieval caused by single-dose METH administration may be attributed to reduced PrLGlu activity, possibly due to excessive DA activity on D2R. Selective activation of PrLGlu or vCA1Glu-PrL may serve as a potential therapeutic strategy for METH-induced cognitive dysfunction.

Phosphorylation of STAT3 at Tyr705 contributes to TFEB-mediated autophagy-lysosomal pathway dysfunction and leads to ischemic injury in rats.

We previously reported that permanent ischemia induces marked dysfunction of the autophagy-lysosomal pathway (ALP) in rats, which is possibly mediated by the transcription factor EB (TFEB). However, it is still unclear whether signal transducer and activator of transcription 3 (STAT3) is responsible for the TFEB-mediated dysfunction of ALP in ischemic stroke. In the present study, we used AAV-mediated genetic knockdown and pharmacological blockade of p-STAT3 to investigate the role of p-STAT3 in regulating TFEB-mediated ALP dysfunction in rats subjected to permanent middle cerebral occlusion (pMCAO). The results showed that the level of p-STAT3 (Tyr705) in the rat cortex increased at 24 h after pMCAO and subsequently led to lysosomal membrane permeabilization (LMP) and ALP dysfunction. These effects can be alleviated by inhibitors of p-STAT3 (Tyr705) or by STAT3 knockdown. Additionally, STAT3 knockdown significantly increased the nuclear translocation of TFEB and the transcription of TFEB-targeted genes. Notably, TFEB knockdown markedly reversed STAT3 knockdown-mediated improvement in ALP function after pMCAO. This is the first study to show that the contribution of p-STAT3 (Tyr705) to ALP dysfunction may be partly associated with its inhibitory effect on TFEB transcriptional activity, which further leads to ischemic injury in rats.

Repeated oxytocin treatment during abstinence inhibited context- or restraint stress-induced reinstatement of methamphetamine-conditioned place preference and promoted adult hippocampal neurogenesis in mice.

Propensity to relapse, even after long-term abstinence, is a crucial feature of methamphetamine (METH) abuse. We and other laboratories have reported that acute treatment of oxytocin (OXT), a hormone and neuropeptide, could inhibit reinstatement of METH seeking in animal studies. However, the effects of repeated OXT treatment on METH reinstatement as well as underlying mechanisms are still unclear. In the present study, the effects of repeated OXT treatment during abstinence on context- or restraint stress-induced reinstatement were investigated using the mice conditioned place preference (CPP) paradigm. After three intermittent injections of METH (2 mg/kg, i.p.) to induce CPP, mice received a daily bilateral intra-hippocampus injection of OXT (0.625, 1.25 or 2.5 µg) for 8 consecutive days before the context- or restraint stress-induced reinstatement test. Meanwhile, adult hippocampal neurogenesis (AHN) level was detected using immunostaining. To further clarify the role of AHN underlying OXT's effects on METH-CPP reinstatement, temozolomide (TMZ, 25 mg/kg, i.p.) was employed to deplete AHN prior to OXT treatment. The data showed that repeated OXT treatment (1.25 and 2.5 µg, intra-hippocampus) significantly inhibited both context- and restraint stress-induced METH-CPP reinstatement and concomitantly promoted AHN in a dose-dependent manner. Notably, TMZ pre-treatment markedly abolished all the above-mentioned effects of OXT, suggesting that AHN was closely involved in OXT's inhibition on reinstatement induced by both triggers. Taken together, the present study indicated that repeated OXT treatment during abstinence could inhibit both context- and restraint stress-induced METH-CPP reinstatement possibly by promoting AHN in mice, which provided a better understanding for OXT's beneficial effects on METH addiction.

Agonistic analog of growth hormone-releasing hormone promotes neurofunctional recovery and neural regeneration in ischemic stroke.

Ischemic stroke can induce neurogenesis. However, most stroke-generated newborn neurons cannot survive. It has been shown that MR-409, a potent synthetic agonistic analog of growth hormone-releasing hormone (GHRH), can protect against some life-threatening pathological conditions by promoting cell proliferation and survival. The present study shows that long-term treatment with MR-409 (5 or 10 µg/mouse/d) by subcutaneous (s.c.) injection significantly reduces the mortality, ischemic insult, and hippocampal atrophy, and improves neurological functional recovery in mice operated on for transient middle cerebral artery occlusion (tMCAO). Besides, MR-409 can stimulate endogenous neurogenesis and improve the tMCAO-induced loss of neuroplasticity. MR-409 also enhances the proliferation and inhibits apoptosis of neural stem cells treated with oxygen and glucose deprivation-reperfusion. The neuroprotective effects of MR-409 are closely related to the activation of AKT/CREB and BDNF/TrkB pathways. In conclusion, the present study demonstrates that GHRH agonist MR-409 has remarkable neuroprotective effects through enhancing endogenous neurogenesis in cerebral ischemic mice.

Hippocampal neurogenesis interferes with extinction and reinstatement of methamphetamine-associated reward memory in mice.

Drugs of abuse, including morphine and cocaine, can reduce hippocampal neurogenesis (HN). Whereas promotion of HN is being increasingly recognized as a promising strategy for treating morphine and cocaine addiction. The present study is focused on exploring the changes of HN during methamphetamine (METH) administration and further clarify if HN is involved in METH-associated reward memory. After successfully establishing the conditioned place preference (CPP) paradigm to simulate the METH-associated reward memory in C57BL/6 mice, we observed that HN was significantly inhibited during METH (2 mg/kg, i. p.) administration and returned to normal after the extinction of METH CPP, as indicated by the immunostaining of bromodeoxyuridine (BrdU) and doublecortin (DCX) in the hippocampus. To promote/inhibit HN levels, 7,8-dihydroxyflavone (DHF), a small tyrosine kinase receptor B (TrkB) agonist and temozolomide (TMZ), an alkylating agent, were administered intraperitoneally (i.p.), respectively. The data showed that either DHF (5 mg/kg, i. p.) or TMZ (25 mg/kg, i. p.) pre-treatment before METH administration could significantly prolong extinction and enhance reinstatement of the reward memory. Notably, DHF treatment after METH administration significantly facilitated extinction and inhibited METH reinstatement, while TMZ treatment resulted in opposite effects. The present study indicated that METH administration could induce a temporal inhibitory effect on HN. More importantly, promotion of HN after the acquisition of METH-associated reward memory, but not inhibition of HN or promotion of HN before the acquisition of reward memory, could facilitate METH extinction and inhibit METH reinstatement, indicating the beneficial effect of HN on METH addiction by erasing the according reward memory.

Pseudoginsenoside-F11 ameliorates okadiac acid-induced learning and memory impairment in rats via modulating protein phosphatase 2A.

We have reported that pseudoginsenoside-F11 (PF11) can significantly improve the cognitive impairments in several Alzheimer's disease (AD) models, but the mechanism has not been fully elucidated. In the present study, the effects of PF11 on AD, in particular the underlying mechanisms related with protein phosphatase 2A (PP2A), were investigated in a rat model induced by okadaic acid (OA), a selective inhibitor of PP2A. The results showed that PF11 treatment dose-dependently improved the learning and memory impairments in OA-induced AD rats. PF11 could significantly inhibit OA-induced tau hyperphosphorylation, suppress the activation of glial cells, alleviate neuroinflammation, thus rescue the neuronal and synaptic damage. Further investigation revealed that PF11 could regulate the protein expression of methyl modifying enzymes (leucine carboxyl methyltransferase-1 and protein phosphatase methylesterase-1) in the brain, thus increase methyl-PP2A protein expression and indirectly increase the activity of PP2A. Molecular docking analysis, structural alignment and in vitro results showed that PF11 was similar in the shape and electrostatic field feature to a known activator of PP2A, and could directly bind and activate PP2A. In conclusion, the present data indicate that PF11 can ameliorate OA-induced learning and memory impairment in rats via modulating PP2A.

CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage.

Background and Purpose: CAPN1 (calpain1)­an intracellular Ca2+-regulated cysteine protease­can be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined. Methods: In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucose­deprived primary neuron in vitro model. Results: CAPN1 was activated in the cortex of permanent middle cerebral occlusion­operated rats and oxygen-glucose deprivation­exposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage. Conclusions: The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemia­mediated autophagy-lysosomal pathway defects and neuronal damage.

Oxytocin signaling in the treatment of drug addiction: Therapeutic opportunities and challenges.

Drug addiction is one of the leading causes of mortality worldwide. Despite great advances were achieved in understanding the neurobiology of drug addiction, the therapeutic options are severely limited, with poor effectiveness and serious side effects. The neuropeptide oxytocin (OXT) is well known for its effects on uterine contraction, sexual/maternal behaviors, social affiliation, stress and learning/memory by interacting with the OXT receptor and other neuromodulators. Emerging evidence suggests that the acute or chronic exposure to drugs can affect the OXT system. Additionally, OXT administration can ameliorate a wide range of abused drug-induced neurobehavioral changes. Overall, OXT not only suppresses drug reward in the binge stage of drug addiction, but also reduces stress responses and social impairments during the withdrawal stage and, finally, prevents drug/cue/stress-induced reinstatement. More importantly, clinical studies have also shown that OXT can exert beneficial effects on reducing substance use disorders of a series of drugs, such as heroin, cocaine, alcohol, cannabis and nicotine. Thus, the present review focuses on the role of OXT in treating drug addiction, including the preclinical and clinical therapeutic potential of OXT and its analogs on the neurobiological perspectives of drugs, to provide a better insight of the efficacy of OXT as a clinical addiction therapeutic agent.

Pseudoginsenoside-F11 attenuates cognitive dysfunction and tau phosphorylation in sporadic Alzheimer's disease rat model.

We previously reported that pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, significantly ameliorated Alzheimer's disease (AD)-associated cognitive defects in APP/PS1 and SAMP8 mice by inhibiting Aß aggregation and tau hyperphosphorylation, suggesting a potential therapeutic effect of PF11 in the treatment of AD. In the present study we further evaluated the therapeutic effects of PF11 on relieving cognitive impairment in a rat model of sporadic AD (SAD). SAD was induced in rats by bilateral icv infusion of streptozotocin (STZ, 3 mg/kg). The rats were treated with PF11 (2, 4, 8 mg·kg-1·d-1, ig) or a positive control drug donepezil (5 mg·kg-1·d-1, ig) for 4 weeks. Their cognitive function was assessed in the nest building, Y-maze, and Morris water maze tests. We showed that STZ icv infusion significantly affected the cognitive function, tau phosphorylation, and insulin signaling pathway in the hippocampus. Furthermore, STZ icv infusion resulted in significant upregulation of the calpain I/cyclin-dependent protein kinase 5 (CDK5) signaling pathway in the hippocampus. Oral administration of PF11 dose-dependently ameliorated STZ-induced learning and memory defects. In addition, PF11 treatment markedly reduced the neuronal loss, protected the synapse structure, and modulated STZ-induced expression of tau phosphorylation by regulating the insulin signaling pathway and calpain I/CDK5 signaling pathway in the hippocampus. Donepezil treatment exerted similar beneficial effects in STZ-infused rats as the high dose of PF11 did. This study highlights the excellent therapeutic potential of PF11 in managing AD.

Receptor-targeting nanomaterials alleviate binge drinking-induced neurodegeneration as artificial neurotrophins.

The distinguished properties of nanomaterials promote us to explore whether their intrinsic activities would be beneficial to disease treatment. Furthermore, understanding the molecular mechanism is thereby crucial for biomedical applications. Here, we investigate the therapeutic effects of single-walled carbon nanotubes (SWNTs) in a rat model of binge alcohol-induced neurodegeneration. With selection from four types of SWNT structures, bundled SWNTs (bSWNTs) facilitated the recovery of learning and memory via enhancing neuroprotection and neuroregeneration. We screened the potential target for bSWNTs, and found that bSWNTs have the abilities to directly interact with neurotrophic receptors, especially tropomyosin-related kinase B (TrkB). Moreover, similar to the actions of endogenous neurotrophins, bSWNTs could trigger the dimerization and phosphorylation of TrkB, while these conformational changes resulted in activating their downstream signals involved in neuroprotection and neuroregeneration. With relatively clear mechanisms, these "artificial neurotrophins" provide a proof-of-concept example as an efficiently therapeutic strategy for the treatment of neurodegenerative diseases.

Lipopolysaccharide induces neuroinflammation in microglia by activating the MTOR pathway and downregulating Vps34 to inhibit autophagosome formation.

BACKGROUND: Microglial activation is a prominent feature of neuroinflammation, which is present in almost all neurodegenerative diseases. While an initial inflammatory response mediated by microglia is considered to be protective, excessive pro-inflammatory response of microglia contributes to the pathogenesis of neurodegeneration. Although autophagy is involved in the suppression of inflammation, its role and mechanism in microglia are unclear. METHODS: In the present study, we studied the mechanism by which lipopolysaccharide (LPS) affects microglial autophagy and the effects of autophagy on the production of pro-inflammatory factors in microglial cells by western blotting, immunocytochemistry, transfection, transmission electron microscopy (TEM), and real-time PCR. In a mouse model of neuroinflammation, generated by intraventricular injection of LPS (5 µg/animal), we induced autophagy by rapamycin injection and investigated the effects of enhanced autophagy on microglial activation by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. RESULTS: We found that autophagic flux was suppressed in LPS-stimulated N9 microglial cells, as evidenced by decreased expression of the autophagy marker LC3-II (lipidated form of MAP1LC3), as well as increased levels of the autophagy adaptor protein SQSTM1. LPS significantly decreased Vps34 expression in N9 microglial cells by activating the PI3KI/AKT/MTOR pathway without affecting the levels of lysosome-associated proteins and enzymes. More importantly, overexpression of Vps34 significantly enhanced the autophagic flux and decreased the accumulation of SQSTM1 in LPS-stimulated N9 microglial cells. Moreover, our results revealed that an LPS-induced reduction in the level of Vps34 prevented the maturation of omegasomes to phagophores. Furthermore, LPS-induced neuroinflammation was significantly ameliorated by treatment with the autophagy inducer rapamycin both in vitro and in vivo. CONCLUSIONS: These data reveal that LPS-induced neuroinflammation in N9 microglial cells is associated with the inhibition of autophagic flux through the activation of the PI3KI/AKT/MTOR pathway, while enhanced microglial autophagy downregulates LPS-induced neuroinflammation. Thus, this study suggests that promoting the early stages of autophagy might be a potential therapeutic approach for neuroinflammation-associated diseases.

N2 neutrophils may participate in spontaneous recovery after transient cerebral ischemia by inhibiting ischemic neuron injury in rats.

Neutrophils have been traditionally considered as the major mediators of harmful inflammatory responses in ischemic stroke, whereas accumulating evidence indicates that neutrophils can be polarized into an N2 phenotype. Similar to M2 microglia, N2 neutrophils contribute to resolution of inflammation and may participate in neuroprotection. However, it remains unclear whether N2 neutrophils protect ischemic neurons and whether they are associated with long-term outcomes after transient cerebral ischemia in rats. The present study proved that N2 neutrophils protected against oxygen glucosedeprivation/re-oxygenation (OGD/R)-induced primary cortical neuron injury via brain-derived neurotrophic factor/tropomyosin-related kinase B (BDNF/TrkB) signaling. In addition, in vivo studies revealed that transient middle cerebral artery occlusion (tMCAO)-induced injury exhibited spontaneous recovery over time in rats. Moreover, neutrophils could infiltrate the ipsilateral brain parenchyma from the periphery after transient cerebral ischemia. Pearson's correlation analysis indicated that the proportion of N2 neutrophils in ipsilateral brain parenchyma was negatively correlated with the number of degenerating neurons, modified Neurological Severity Score (mNSS), brain water content and infarct volume, and positively correlated with the number of surviving neurons and grip strength. In summary, the present study shows that N2 neutrophils likely participate in spontaneous recovery after transient cerebral ischemia by inhibiting ischemic neuron damage in rats, which indicates that N2 neutrophils may represent promising therapeutic target for promoting recovery after ischemic stroke.

Pseudoginsenoside-F11 Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Suppressing Neutrophil Infiltration and Accelerating Neutrophil Clearance.

Pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, has been reported to have anti-inflammatory properties, but the effects of PF11 on acute lung inflammation were unknown. The present study aimed to investigate the protective effects and potential mechanisms of PF11 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in male BALB/c mice. After being treated with PF11 (3, 10, and 30 mg/kg, intravenous) once a day for 3 consecutive days, the mice were challenged by intratracheal instillation of LPS, and then their lung tissues and bronchoalveolar lavage fluid (BALF) were collected for further analysis. The results showed that PF11 attenuated LPS-induced ALI, with alleviated histopathological damage, decreased lung wet/dry weight ratio, and reduced protein concentration and inflammatory cells number in BALF. Moreover, PF11 reversed the LPS-induced increases of mRNA expression and protein levels of interleukin-6, tumor necrosis factor-α, and interleukin-1ß. Meanwhile, PF11 decreased LPS-induced myeloperoxidase activity and neutrophil infiltration in lung tissue by reducing the expression of macrophage inflammatory protein-2 and intercellular adhesion molecule-1, as well as enhanced neutrophil clearance by accelerating neutrophils apoptosis and their phagocytosis by alveolar macrophages. In conclusion, these results indicated that PF11 significantly attenuated LPS-induced ALI through suppressing neutrophil infiltration and accelerating neutrophil clearance, suggesting its potential in the treatment of ALI.

Neuronal-targeted TFEB rescues dysfunction of the autophagy-lysosomal pathway and alleviates ischemic injury in permanent cerebral ischemia.

Mounting attention has been focused on defects in macroautophagy/autophagy and the autophagy-lysosomal pathway (ALP) in cerebral ischemia. TFEB (transcription factor EB)-mediated induction of ALP has been recently considered as the common mechanism in ameliorating the pathological lesion of myocardial ischemia and neurodegenerative diseases. Here we explored the vital role of TFEB in permanent middle cerebral artery occlusion (pMCAO)-mediated dysfunction of ALP and ischemic insult in rats. The results showed that ALP function was first enhanced in the early stage of the ischemic process, especially in neurons of the cortex, and this was accompanied by increased TFEB expression and translocation to the nucleus, which was mediated at least in part through activation by PPP3/calcineurin. At the later stages of ischemia, a gradual decrease in the level of nuclear TFEB was coupled with a progressive decline in lysosomal activity, accumulation of autophagosomes and autophagy substrates, and exacerbation of the ischemic injury. Notably, neuron-specific overexpression of TFEB significantly enhanced ALP function and rescued the ischemic damage, starting as early as 6 h and even lasting to 48 h after ischemia. Furthermore, neuron-specific knockdown of TFEB markedly reversed the activation of ALP and further aggravated the neurological deficits and ischemic outcome at the early stage of pMCAO. These results highlight neuronal-targeted TFEB as one of the key players in the pMCAO-mediated dysfunction of ALP and ischemic injury, and identify TFEB as a promising target for therapies aimed at neuroprotection in cerebral ischemia. Abbreviations: AAV, adeno-associated virus; AIF1/IBA1, allograft inflammatory factor 1; ALP, autophagy-lysosomal pathway; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; CsA, cyclosporin A; GFAP, glial fibrillary acidic protein; LAMP, lysosomal-associated membrane protein; LC3, microtubule-associated protein 1 light chain 3; MAP2, microtubule-associated protein 2; mNSS, modified Neurological Severity Score; MTOR, mechanistic target of rapamycin kinase; OGD, oxygen and glucose deprivation; pMCAO, permanent middle cerebral artery occlusion; RBFOX3/NeuN, RNA binding fox-1 homolog 3; SQSTM1, sequestosome1; TFEB, transcription factor EB; TTC, 2,3,5-triphenyltetrazolium chloride.

Potential role of the ecto-5'-nucleotidase in morphine-induced uridine release and neurobehavioral changes.

There is growing evidence that uridine may act as an endogenous neuromodulator with a potential signaling role in the central nervous system in addition to its function in pyrimidine metabolism. We previously found that acute morphine treatment significantly increased uridine release in the dorsal striatum of mice, indicating that uridine may contribute to morphine-induced neurobehavioral changes. In the present study, we analyzed the mechanism involved in morphine-induced uridine release and the role of uridine in morphine-induced neurobehavioral changes. Uridine release in the dorsal striatum of mice was assessed by in vivo microdialysis coupled with high performance liquid chromatography (HPLC) after morphine treatment. Western blotting and immunofluorescence were used to evaluate the expression of uridine-related proteins. Morphine-induced neurobehavioral changes were assessed by locomotor activity, behavioral sensitization and conditioned place preference (CPP) test. The expression of NT5E, an extracellular enzyme involved in formation of nucleosides, including uridine, was specifically knocked down in the dorsal striatum of mice using adeno-associated virus (AAV)-mediated short hairpin RNA (shRNA). The results indicated that both acute and chronic morphine administration significantly increased uridine release in the dorsal striatum, and this was associated with upregulation of NT5E but not other uridine-related proteins. Inhibition of NT5E with APCP or shRNA markedly inhibited morphine-induced uridine release in the dorsal striatum and related neurobehavioral changes, including hyperlocomotor activity, behavioral sensitization and CPP. Our data give a better understanding of the contribution of NT5E to morphine-induced uridine release and neurobehavioral changes, and identify NT5E as a potential target for treating morphine abuse.

Suppressing autophagy enhances disulfiram/copper-induced apoptosis in non-small cell lung cancer.

Autophagy, a cellular survival mechanism, is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy, thus decreasing drug-induced apoptosis. Disulfiram (DSF), a drug widely used to control alcoholism, possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models, and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells. The present study is designed to investigate whether DSF/Cu can induce autophagy in NSCLC cells and to determine the relationship between autophagy and apoptosis. First, we observed that DSF/Cu induced significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells, accompanied by the formation of vacuoles in the cytoplasm. Next, we investigated the levels of autophagic markers, including LC3II (microtubule-associated protein 1 light chain 3), ATG5 and p62 with or without chloroquine by Western blot. In addition, we observed co-localization of LC3 and the lysosomal protein LAMP2 (lysosome-associated membrane protein 2) after treatment with DSF/Cu. The results showed that DSF/Cu induced autophagy. Finally, we demonstrated that DSF/Cu-induced apoptosis was greatly enhanced when autophagy was suppressed with Atg5 siRNA or 3-MA in NSCLC cells. This synergistic effect of DSF/Cu and 3-MA was further confirmed in the NSCLC xenograft model. Taken together, our results show that DSF/Cu stimulates autophagy in NSCLC cells, which is an impediment to DSF/Cu-induced apoptosis.

Uridine attenuates morphine-induced conditioned place preference and regulates glutamate/GABA levels in mPFC of mice.

Several lines of evidence suggest that uridine, as a neuromodulator, plays an important role in drug addiction. We previously found that uridine circumvents morphine-induced behavioral sensitization by decreasing the extracellular dopamine levels in the dorsal striatum. In the present study, the effects of uridine on morphine-induced conditioned place preference (CPP) and the possible roles of glutamate and GABA in the stress-induced reinstatement of CPP were investigated. First, the effects of uridine (1, 10 and 100mg/kg, i.p.) on the four defined phases - acquisition, expression, extinction and reinstatement (drug priming and restraint stress) - of morphine-induced CPP were studied. The results showed that pretreatment with uridine significantly blocked the acquisition and expression phases of CPP. Additionally, uridine also facilitated CPP extinction and inhibited stress-induced CPP reinstatement, although it failed to affect drug-induced CPP reinstatement. Since glutamatergic and GABAergic systems are both involved in CPP reinstatement, the extracellular levels of glutamate and GABA in the mPFC during the stress-induced CPP reinstatement were determined using in vivo microdialysis. The results showed that uridine attenuated the stress-induced glutamate increase in the mPFC without influencing the basal glutamate levels, and increased the levels of extracellular GABA in the mPFC both under normal physiological conditions and after the stress stimulus. Thus, our results indicate that uridine depresses the stress-induced reinstatement of CPP, simultaneously regulating glutamatergic and GABAergic neurotransmission in the mPFC. The present work provides further understanding of the role of uridine in morphine-induced neurobehavioral changes.

Primary renal lymphoma: A case report and literature review.

Primary renal lymphoma (PRL) is a rare disease, with no more than 70 cases reported in the literature. The present study reports the case of a 70-year-old woman with PRL. The patient was asymptomatic, however, a mass on the right kidney was identified incidentally during routine physical examination. Computed tomography revealed a mass in the right kidney that was 3.6 cm in diameter. Subsequently, right nephrectomy was performed. The histological evaluation of the nephrectomy specimen showed diffuse large B-cell non-Hodgkin's lymphoma. The patient was treated with 6-8 cycles of a cyclophosphamide, epirubicin, vindesine and dexamethasone regimen. Follow-up examination performed after 2 months of treatment revealed no evidence of local recurrence. The present study also reviewed 49 cases of PRL that have been reported since 1989. It was found that a shorter survival time was experienced by patients with bilateral PRL (mean, 21 months) compared with unilateral PRL (mean, 68 months). A shorter survival time was also experienced by patients who were treated with chemotherapy only (mean, 15.8 months) compared with those who were treated with combination chemotherapy and surgery (mean, 49.4 months).

Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics.

In order to improve the delivery efficiency of microRNA (miRNA or miR)-145, the present study examined several factors which may affect cationic liposome (CL)-based transfection, including the hydration medium used for the preparation of liposomes, the quantity of the plasmid, the molar ratio of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP)/cholesterol (chol), or DOTAP/chol, and the weight ratio of DOTAP/DNA. In order to enhance the transfection efficiency, protamine was selected as a DNA-condensing agent to form liposome­protamine­DNA (LPD) ternary complexes. An agarose gel retardation assay was used to examine the DNA binding affinity of the CLs. Following transfection, GFP fluorescence images were captured and flow cytometry was performed to determine the transfection efficiency. Furthermore, an MTT assay was performed to determine the cytotoxicity of the liposome complexes. The final optimal conditions were as follows: 5% glucose as the hydration medium, a molar ratio of DOTAP/chol at 3:1 for the preparation of CLs, a weight ratio of DOTAP/protamine/DNA of 3:0.5:1, with 8 µg plasmid added for the preparation of the LPD complexes. In vitro, the LPD complexes exhibited an enhanced transfection efficiency and low cytotoxicity, which indicated that the presented LPD vector enhanced the transfection efficiency of the CLs. The HepG2 cells were found to have the lowest expression levels of miR­145 out of the cell lines tested (A549, BGC-823, HepG2, HeLa, LoVo and MCF-7). Following the transient transfection of the HepG2 cells with miR­145, the results revealed that the overexpression of miR­145 inhibited the proliferation of the HepG2 cells and downregulated the expression of cyclin-dependent kinase 6 (CDK6), cyclinD1, c-myc, and Sp1 transcription factor (Sp1). In conclusion, in this study, we optimized a liposome­based delivery system for the efficient delivery of miR­145 into cancer cells. This may provide a foundation for further research into the use of miR­145 in anticancer therapeutics.

Dual inhibition of COX-2/5-LOX blocks colon cancer proliferation, migration and invasion in vitro.

Inflammation is emerging as a new hallmark of cancer. Arachidonic acid (AA) metabolism, the family of cyclooxygenases (COXs) and lipoxygenase (LOX) play important roles in AA-related inflammatory cascades. In 94 colorectal cancer samples collected from the Han population, the immunohistochemical results indicated that 68% of the patients with colorectal cancer had a co-expression of both COX-2 and 5-LOX, while both displayed low expression in the matched normal tissues. In cell lines, three colorectal cancer cell lines exhibited high expression of COX-2 and 5-LOX. During stable silencing of the expression of COX-2 or 5-LOX in LoVo cancer cells, we found that downregulation of either COX-2 or 5-LOX significantly diminished the growth, migration and invasion of the colon cancer cells and specifically, downregulation of COX-2 could elicit upregulation of 5-LOX protein and vice versa. The above results suggested that the simultaneous blocking of COX-2 and 5-LOX activity may bring more potential benefits in managing the progression of colon cancer. Therefore, we sought to explore the effectiveness of a dual COX-2/5-LOX inhibitor darbufelone on the proliferation, migration, invasion and apoptosis of colon cancer cells, as well as the underlying mechanism of action. The results indicated that darbufelone significantly decreased the proliferative and invasive abilities of the colon cancer cells, in a dose-dependent manner. During the study of the related mechanisms, we found an upregulation of p27 and downregulation of cyclin D1 as well as CDK4 after darbufelone treatment, which indicated that darbufelone could arrest the cell cycle of LoVo cells at the G0/G1 phase. Furthermore, the activation of caspase-3 and -9, upregulation of Bax and downregulation of Bcl-2 demonstrated the occurrence of apoptosis by darbufelone. Finally, darbufelone also prevented the migration and invasion of LoVo cells, which may be ascribed to the upregulation of E-cadherin and ZO-1. In summary, our data suggest that the inhibition of both COX-2/5-LOX may be an effective therapeutic approach for colon cancer management, particularly for those patients with high expression of COX-2/5-LOX.