Liraglutide With Metformin Therapy Ameliorates Hepatic Steatosis and Liver Injury in a Mouse Model of Non-alcoholic Steatohepatitis.

BACKGROUND/AIM: Non-alcoholic fatty liver disease is a major cause of liver-related morbidity and mortality. Metformin is a widely used medication and may have additional benefits beyond glycemic control. Liraglutide, a novel treatment for diabetes and obesity, also has beneficial effects on non-alcoholic steatohepatitis (NASH). Metformin and liraglutide have both benefited NASH treatment. However, no study has reported the effects of combination therapy with liraglutide and metformin on NASH. MATERIALS AND METHODS: We investigated the in vivo effects of metformin and liraglutide on NASH in a methionine/choline-deficient (MCD) diet-fed C57BL/6JNarl mouse model. Serum triglyceride, alanine aminotransferase and alanine aminotransferase levels were documented. Histological analysis was performed according to the NASH activity grade. RESULTS: After treatment with liraglutide and metformin, body weight loss improved, and the liver/body weight ratio decreased. The metabolic effects and liver injury improved. Liraglutide and metformin alleviated MCD-induced hepatic steatosis and injury. Histological analysis revealed that NASH activity was reduced. CONCLUSION: Our results provide evidence for the anti-NASH activity of liraglutide in combination with metformin. Liraglutide with metformin may offer the potential for a disease-modifying intervention for NASH.

Curcumin suppresses cell proliferation and triggers apoptosis in vemurafenib-resistant melanoma cells by downregulating the EGFR signaling pathway.

Melanoma is a malignant tumor with aggressive behavior. Vemurafenib, a BRAF inhibitor, is clinically used in melanoma, but resistance to melanoma cytotoxic therapies is associated with BRAF mutations. Curcumin can effectively inhibit numerous types of cancers. However, there are no reports regarding the correlation between curcumin and vemurafenib-resistant melanoma cells. In this study, vemurafenib-resistant A375.S2 (A375.S2/VR) cells were established, and the functional mechanism of the epidermal growth factor receptor (EGFR), serine-threonine kinase (AKT), and the extracellular signal-regulated kinase (ERK) signaling induced by curcumin was investigated in A375.S2/VR cells in vitro. Our results indicated that A375.S2/VR cells had a higher IC50 concentration of vemurafenib than the parental A375.S2 cells. Moreover, curcumin reduced the viability and confluence of A375.S2/VR cells. Curcumin triggered apoptosis via reactive oxygen species (ROS) production, disruption of mitochondrial membrane potential (ΔΨm), and intrinsic signaling (caspase-9/-3-dependent) pathways in A375.S2/VR cells. Curcumin-induced apoptosis was also mediated by the EGFR signaling pathway. Combination treatment with curcumin and gefitinib (an EGFR inhibitor) synergistically potentiated the inhibitory effect of cell viability in A375.S2/VR cells. The present study provides new insights into the therapy of vemurafenib-resistant melanoma and suggests that curcumin might be an encouraging therapeutic candidate for its drug-resistant treatment.

Resveratrol inhibited the metastatic behaviors of cisplatin-resistant human oral cancer cells via phosphorylation of ERK/p-38 and suppression of MMP-2/9.

Cisplatin resistance is a major clinical problem in the clinical management of oral squamous cell carcinoma (OSCC) patients. Resveratrol is a natural phytoestrogen with antitumor activities. Whether resveratrol can overcome cisplatin resistance and prevent metastasis in OSCC cells is not known. In this study, we first examined the anti-metastatic capacity of resveratrol and then explored the underlying mechanisms using a cisplatin-resistant human OSCC cell line (CAR). The results demonstrated that at a non-toxic dose range (25 to 75 µM), 24-hr treatment of resveratrol was able to suppress the migration and invasion capacities of CAR cells dose dependently. Interestingly, 50 µM resveratrol treatment could significantly down-regulate the expression of the phosphorylated forms of ERK and p-38, in addition to those of MMP-2 and MMP-9. At the same time, the expression levels of phosphorylated ERK together with those unphosphorylated forms of ERK, p38, and JNK were all insignificantly altered. In conclusion, the signaling cascade for resveratrol's suppression of cisplatin-resistant human oral cancer CAR cells was revealed and summarized. Also the rapid effectiveness in suppressing metastatic behaviors of drug-resistant oral cancer cells of non-toxic resveratrol might extend its application to the drug-resistant oral cancer treatment in the near future. PRACTICAL APPLICATIONS: Based on the evidence we provided in the study, we have proposed a model recording the possible pathway for resveratrol inhibiting the metastasis of cisplatin-resistant oral cancer cells. We suppose this signaling pathway may work in other cancer cell lines, and can be helpful in full understanding of the drug-resistance.

Approaches towards fighting the COVID­19 pandemic (Review).

The coronavirus disease 2019 (COVID­19) outbreak, which has caused >46 millions confirmed infections and >1.2 million coronavirus related deaths, is one of the most devastating worldwide crises in recent years. Infection with COVID­19 results in a fever, dry cough, general fatigue, respiratory symptoms, diarrhoea and a sore throat, similar to those of acute respiratory distress syndrome. The causative agent of COVID­19, SARS­CoV­2, is a novel coronavirus strain. To date, remdesivir has been granted emergency use authorization for use in the management of infection. Additionally, several efficient diagnostic tools are being actively developed, and novel drugs and vaccines are being evaluated for their efficacy as therapeutic agents against COVID­19, or in the prevention of infection. The present review highlights the prevalent clinical manifestations of COVID­19, characterizes the SARS­CoV­2 viral genome sequence and life cycle, highlights the optimal methods for preventing viral transmission, and discusses possible molecular pharmacological mechanisms and approaches in the development of anti­SARS­CoV­2 therapeutic agents. In addition, the use of traditional Chinese medicines for management of COVID­19 is discussed. It is expected that novel anti­viral agents, vaccines or an effective combination therapy for treatment/management of SARS­CoV­2 infection and spread therapy will be developed and implemented in 2021, and we would like to extend our best regards to the frontline health workers across the world in their fight against COVID­19.

Sensitivity of allyl isothiocyanate to induce apoptosis via ER stress and the mitochondrial pathway upon ROS production in colorectal adenocarcinoma cells.

Allyl isothiocyanate (AITC), a bioactive phytochemical compound that is a constituent of dietary cruciferous vegetables, possesses promising chemopreventive and anticancer effects. However, reports of AITC exerting antitumor effects on apoptosis induction of colorectal cancer (CRC) cells in vitro are not well elucidated. The present study focused on the functional mechanism of the endoplasmic reticulum (ER) stress­based apoptotic machinery induced by AITC in human colorectal cancer HT­29 cells. Our results indicated that AITC decreased cell growth and number, reduced viability, and facilitated morphological changes of apoptotic cell death. DNA analysis by flow cytometry showed G2/M phase arrest, and alterations in the modulated protein levels caused by AITC were detected via western blot analysis. AITC also triggered vital intrinsic apoptotic factors (caspase­9/caspase­3 activity), disrupted mitochondrial membrane potential, and stimulated mitochondrial­related apoptotic molecules (e.g., cytochrome c, apoptotic protease activating factor 1, apoptosis­inducing factor, and endonuclease G). Additionally, AITC prompted induced cytosolic Ca2+ release and Ca2+­dependent ER stress­related signals, such as calpain 1, activating transcription factor 6α, glucose­regulated proteins 78 and 94, growth arrest­ and DNA damage­inducible protein 153 (GADD153), and caspase­4. The level of reactive oxygen species (ROS) production was found to induce the hallmark of ER stress GADD153, proapoptotic marker caspase­3, and calpain activity after AITC treatment. Our findings showed for the first time that AITC induced G2/M phase arrest and apoptotic death via ROS­based ER stress and the intrinsic pathway (mitochondrial­dependent) in HT­29 cells. Overall, AITC may exert an epigenetic effect and is a potential bioactive compound for CRC treatment.

Metformin triggers the intrinsic apoptotic response in human AGS gastric adenocarcinoma cells by activating AMPK and suppressing mTOR/AKT signaling.

Metformin is commonly used to treat patients with type 2 diabetes and is associated with a decreased risk of cancer. Previous studies have demonstrated that metformin can act alone or in synergy with certain anticancer agents to achieve anti­neoplastic effects on various types of tumors via adenosine monophosphate­activated protein kinase (AMPK) signaling. However, the role of metformin in AMPK­mediated apoptosis of human gastric cancer cells is poorly understood. In the current study, metformin exhibited a potent anti­proliferative effect and induced apoptotic characteristics in human AGS gastric adenocarcinoma cells, as demonstrated by MTT assay, morphological observation method, terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase­3/7 assay kits. Western blot analysis demonstrated that treatment with metformin increased the phosphorylation of AMPK, and decreased the phosphorylation of AKT, mTOR and p70S6k. Compound C (an AMPK inhibitor) suppressed AMPK phosphorylation and significantly abrogated the effects of metformin on AGS cell viability. Metformin also reduced the phosphorylation of mitogen­activated protein kinases (ERK, JNK and p38). Additionally, metformin significantly increased the cellular ROS level and included loss of mitochondrial membrane potential (ΔΨm). Metformin altered apoptosis­associated signaling to downregulate the BAD phosphorylation and Bcl­2, pro­caspase­9, pro­caspase­3 and pro­caspase­7 expression, and to upregulate BAD, cytochrome c, and Apaf­1 proteins levels in AGS cells. Furthermore, z­VAD­fmk (a pan­caspase inhibitor) was used to assess mitochondria­mediated caspase­dependent apoptosis in metformin­treated AGS cells. The findings demonstrated that metformin induced AMPK­mediated apoptosis, making it appealing for development as a novel anticancer drug for the treating gastric cancer.

Synergistic inhibitory effects of cetuximab and curcumin on human cisplatin-resistant oral cancer CAR cells through intrinsic apoptotic process.

Cetuximab, an epidermal growth factor receptor (EGFR)-targeting monoclonal antibody (mAb), is a novel targeted therapy for the treatment of patients with oral cancer. Cetuximab can be used in combination with chemotherapeutic agents to prolong the overall survival rates of patients with oral cancer. Curcumin is a traditional Chinese medicine, and it has been demonstrated to have growth-inhibiting effects on oral cancer cells. However, information regarding the combination of cetuximab and curcumin in drug-resistant oral cancer cells is lacking, and its underlying mechanism remains unclear. The purpose of the present study was to explore the oral anticancer effects of cetuximab combined with curcumin on cisplatin-resistant oral cancer CAR cell apoptosis in vitro. The results demonstrated that combination treatment synergistically potentiated the effect of cetuximab and curcumin on the suppression of cell viability and induction of apoptosis in CAR cells. Cetuximab and curcumin combination induced apoptosis and dramatically increased caspase-3 and caspase-9 activities compared with singular treatment. Combination treatment also markedly suppressed the protein expression levels of EGFR and mitogen-activated protein kinases (MAPKs) signaling (phosphorylation of ERK, JNK and p38). The results demonstrated that co-treatment with cetuximab and curcumin exerts synergistic oral anticancer effects on CAR cells through the suppression of the EGFR signaling by regulation of the MAPK pathway.

Ursolic acid elicits intrinsic apoptotic machinery by downregulating the phosphorylation of AKT/BAD signaling in human cisplatin­resistant oral cancer CAR cells.

Oral squamous cell carcinoma (OSCC) is a type of cancer with high morbidity and mortality rates worldwide; it also demonstrates chemotherapeutic resistance. Triterpenoid ursolic acid has been shown to exhibit various biological activities and anticancer effects in several preclinical studies. In our previous study, human cisplatin­resistant oral cancer CAR cells were established, and the present study aimed to further examine the effects of ursolic acid on CAR cells. The results revealed that ursolic acid inhibited CAR cell viability, as determined using a 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide assay. Ursolic acid­induced cell death was mediated through a caspase­dependent pathway, determined with the pan­caspase inhibitor, z­VAD­fmk. Ursolic acid also increased the activities of caspase­3 and caspase­9 in CAR cells, determined by a colorimetric assay. Specifically, the production of reactive oxygen species and loss of mitochondrial membrane potential, detected by flow cytometry, were observed in the ursolic acid­treated CAR cells. The apoptosis­associated signaling showed that ursolic acid decreased the phosphorylation of AKT (Ser473) and B­cell lymphoma 2 (Bcl­2)­associated agonist of cell death (BAD; Ser136), and the protein levels of Bcl­2 and Bcl­extra large (Bcl­xL), and increased the expression of BAD and Bcl­2­associated X (Bax) protein in CAR cells. In summary, the results supported the potential application of ursolic acid against drug­resistant oral carcinoma and to improve oral anticancer efficacy in the near future.

Pterostilbene modulates the suppression of multidrug resistance protein 1 and triggers autophagic and apoptotic mechanisms in cisplatin-resistant human oral cancer CAR cells via AKT signaling.

Pterostilbene is a natural polyphenolic compound that is primarily found in fruits, such as blueberries and has a similar structure to resveratrol. Pterostilbene exhibits antioxidant, anti-inflammatory and antitumor activity but the effects of pterostilbene on drug-resistant oral cancer cells and its underlying mechanisms of action have not yet been explored. Therefore, the present study was performed to clarify the anticancer effects of pterostilbene on cisplatin-resistant human oral cancer CAR cells. The results demonstrated that CAR cells exhibited marked shrinkage, cell membrane breakage and autophagic vacuole formation following treatment with pterostilbene. Pterostilbene also effectively inhibited cell viability and suppressed cell confluence in a time- and concentration-dependent manner. Probing with acridine orange, monodansylcadaverine and LysoTracker Red demonstrated that the number of acidic vesicular organelles was increased, indicating increased autophagy. Furthermore, Heochst 33342 staining determined that DNA condensation, a characteristic of apoptosis, was enhanced following treatment with pterostilbene. Furthermore, pterostilbene upregulated mRNA levels of LC3-II and Atg12, as well as the expression of Atgs/Beclin-1/LC3-associated signaling, suggesting that it enhances autophagy. The autophagy inhibitors 3-methyladenine and chloroquine were used to confirm that pterostilbene induces autophagy. It was also determined that pterostilbene triggered caspase-dependent apoptosis by directly testing DNA breakage and using the pan-caspase inhibitor carbobenzoxyvalyl-alanyl-aspartyl fluoromethyl ketone. The results demonstrated that pterostilbene mediates the apoptosis of CAR cells via the intrinsic apoptotic cascade. In addition, pterostilbene inhibited MDR1 expression and the phosphorylation of AKT on the Ser473 site in CAR cells. Therefore, pterostilbene may elicit an oral anticancer response in drug-resistant cells and may be used as a chemotherapeutic adjuvant to treat patients with oral cancer.

Kaempferol inhibits angiogenic ability by targeting VEGF receptor-2 and downregulating the PI3K/AKT, MEK and ERK pathways in VEGF-stimulated human umbilical vein endothelial cells.

Anti-angiogenesis is one of the most general clinical obstacles in cancer chemotherapy. Kaempferol is a flavonoid phytochemical found in many fruits and vegetables. Our previous study revealed that kaempferol triggered apoptosis in human umbilical vein endothelial cells (HUVECs) by ROS­mediated p53/ATM/death receptor signaling. However, the anti­angiogenic potential of kaempferol remains unclear and its underlying mechanism warranted further exploration in VEGF­stimulated HUVECs. In the present study, kaempferol significantly reduced VEGF­stimulated HUVEC viability. Kaempferol treatment also inhibited cell migration, invasion, and tube formation in VEGF­stimulated HUVECs. VEGF receptor­2 (VEGFR­2), and its downstream signaling cascades (such as AKT, mTOR and MEK1/2­ERK1/2) were reduced as determined by western blotting and kinase activity assay in VEGF­stimulated HUVECs after treatment with kaempferol. The present study revealed that kaempferol may possess angiogenic inhibition through regulation of VEGF/VEGFR­2 and its downstream signaling cascades (PI3K/AKT, MEK and ERK) in VEGF-stimulated endothelial cells.

The molecular mechanism of contrast-induced nephropathy (CIN) and its link to in vitro studies on iodinated contrast media (CM).

Iodinated contrast media (iodinated CM) have increased ability to absorb x-rays and to visualize structures that normally are impossible to observe in a radiological examination. The use of iodinated CM may destory renal function, commonly known as contrast-induced nephropathy (CIN), which can result in acute renal failure (ARF). This review article mainly focuses on the following areas: (1) classifications of iodinated CM: ionic or non-ionic, high-osmolarity contrast media (HOCM), low-osmolarity contrast media (LOCM) and iso-osmolarity contrast media (IOCM); (2) an introduction to the physical and chemical properties of the non-ionic iodinated CM; (3) the management of anaphylactic reaction by iodinated CM; (4) a suggested single injection of adult doses and maximum dose for non-ionic iodinated CM; (5) the molecular mechanism of contrast-induced nephropathy (CIN); (6) In vitro studies on iodinated CM. Based on above information, this review article provide an insight for understanding the drug safety of iodinated CM.

YC-1 induces G0/G1 phase arrest and mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells.

Oral cancer is a serious and fatal disease. Cisplatin is the first line of chemotherapeutic agent for oral cancer therapy. However, the development of drug resistance and severe side effects cause tremendous problems clinically. In this study, we investigated the pharmacologic mechanisms of YC-1 on cisplatin-resistant human oral cancer cell line, CAR. Our results indicated that YC-1 induced a concentration-dependent and time-dependent decrease in viability of CAR cells analyzed by MTT assay. Real-time image analysis of CAR cells by IncuCyte™ Kinetic Live Cell Imaging System demonstrated that YC-1 inhibited cell proliferation and reduced cell confluence in a time-dependent manner. Results from flow cytometric analysis revealed that YC-1 promoted G0/G1 phase arrest and provoked apoptosis in CAR cells. The effects of cell cycle arrest by YC-1 were further supported by up-regulation of p21 and down-regulation of cyclin A, D, E and CDK2 protein levels. TUNEL staining showed that YC-1 caused DNA fragmentation, a late stage feature of apoptosis. In addition, YC-1 increased the activities of caspase-9 and caspase-3, disrupted the mitochondrial membrane potential (AYm) and stimulated ROS production in CAR cells. The protein levels of cytochrome c, Bax and Bak were elevated while Bcl-2 protein expression was attenuated in YC-1-treated CAR cells. In summary, YC-1 suppressed the viability of cisplatin-resistant CAR cells through inhibiting cell proliferation, arresting cell cycle at G0/G1 phase and triggering mitochondria-mediated apoptosis. Our results provide evidences to support the potentially therapeutic application of YC-1 on fighting against drug resistant oral cancer in the future.

Resveratrol-induced autophagy and apoptosis in cisplatin-resistant human oral cancer CAR cells: A key role of AMPK and Akt/mTOR signaling.

Resveratrol is known to be an effective chemo-preventive phytochemical against multiple tumor cells. However, the increasing drug resistance avoids the cancer treatment in oral cavity cancer. In this study, we investigated the oral antitumor activity of resveratrol and its mechanism in cisplatin-resistant human oral cancer CAR cells. Our results demonstrated that resveratrol had an extremely low toxicity in normal oral cells and provoked autophagic cell death to form acidic vesicular organelles (AVOs) and autophagic vacuoles in CAR cells by acridine orange (AO) and monodansylcadaverine (MDC) staining. Either DNA fragmentation or DNA condensation occurred in resveratrol-triggered CAR cell apoptosis. These inhibitors of PI3K class III (3-MA) and AMP-activated protein kinase (AMPK) (compound c) suppressed the autophagic vesicle formation, LC3-II protein levels and autophagy induced by resveratrol. The pan-caspase inhibitor Z-VAD-FMK attenuated resveratrol-triggered cleaved caspase-9, cleaved caspase-3 and cell apoptosis. Resveratrol also enhanced phosphorylation of AMPK and regulated autophagy- and pro-apoptosis-related signals in resveratrol-treated CAR cells. Importantly, resveratrol also stimulated the autophagic mRNA gene expression, including Atg5, Atg12, Beclin-1 and LC3-II in CAR cells. Overall, our findings indicate that resveratrol is likely to induce autophagic and apoptotic death in drug-resistant oral cancer cells and might become a new approach for oral cancer treatment in the near future.

Long-lasting alterations in 5-HT2A receptor after a binge regimen of methamphetamine in mice.

The repeated administration of methamphetamine (MA) to animals in a single-day 'binge' dosing regimen produces damage to dopamine and serotonin terminals and psychosis-like behaviours similar to those observed in MA abusers. The present study aimed to examine the effects of MA binge exposure on 5-HT2A receptors, the subtype of serotonin receptors putatively involved in psychosis. ICR male mice were treated with MA (4 × 5 mg/kg) or saline at 2 h intervals. Recognition memory and social behaviours were sequentially evaluated by a novel location recognition test, a novel object recognition test, a social interaction and a nest-building test to confirm the persistent cognitive and behavioural impairments after this dosing regimen. Subsequently, a hallucinogenic 5-HT2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI)-induced head-twitch, molecular and electrophysiological responses were monitored. Finally, the levels of 5-HT2C, 5-HT1A, 5-HT2A and mGlu2 receptors in the medial prefrontal cortex were determined. MA binge exposure produced recognition memory impairment, reduced social behaviours, and increased DOI-induced head-twitch response, c-Fos and Egr-2 expression and field potentials in the medial prefrontal cortex. Furthermore, MA binge exposure increased 5-HT2A and decreased mGlu2 receptor expression in the medial frontal cortex, whereas 5-HT2C and 5-HT1A receptors were unaffected. These data reveal that the increased behavioural, molecular and electrophysiological responses to DOI might be associated with an up-regulation of 5-HT2A receptors in the medial prefrontal cortex after MA binge exposure. Identifying the biochemical alterations that parallel the behavioural changes in a mouse model of MA binge exposure may facilitate targeting therapies for treatment of MA-related psychiatric disorders.

N-acetylcysteine modulates hallucinogenic 5-HT(2A) receptor agonist-mediated responses: behavioral, molecular, and electrophysiological studies.

N-acetylcysteine (NAC) has been reported to reverse the psychotomimetic effects in the rodent phencyclidine model of psychosis and shown beneficial effects in treating patients with schizophrenia. The effect of NAC has been associated with facilitating the activity of cystine-glutamate antiporters on glial cells concomitant with the release of non-vesicular glutamate, which mainly stimulates the presynaptic metabotropic glutamate receptor subtype 2 receptors (mGluR2). Recent evidence demonstrated that functional interactions between serotonin 5-HT2A receptor (5-HT(2A)R) and mGluR2 are responsible to unique cellular responses when targeted by hallucinogenic drugs. The present study determined the effects of NAC on hallucinogenic 5-HT(2A)R agonist (±)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-elicited behavioral and molecular responses in mice and DOI-evoked field potentials in the mouse cortical slices. NAC significantly attenuated DOI-induced head twitch response and expression of c-Fos and Egr-2 in the infralimbic and motor cortex and suppressed the increase in the frequency of excitatory field potentials elicited by DOI in the medial prefrontal cortex. In addition, the cystine-glutamate antiporter inhibitor (S)-4-carboxyphenylglycine (CPG) and the mGluR2 antagonist LY341495 reversed the suppressing effects of NAC on DOI-induced head twitch and molecular responses and increased frequency of excitatory field potentials, supporting that NAC attenuates the 5-HT(2A)R-mediated hallucinogenic effects via increased activity of cystine-glutamate antiporter followed by activation of mGluR2 receptors. These findings implicate NAC as a potential therapeutic agent for hallucinations and psychosis associated with hallucinogen use and schizophrenia.

Cocaine- and amphetamine-regulated transcript (CART) peptide activates ERK pathways via NMDA receptors in rat spinal cord dorsal horn in an age-dependent manner.

Activation of extracellular signal-regulated kinase (ERK) cascade in the spinal cord dorsal horn may contribute to pain hypersensitivity. Our recent study showed that cocaine- and amphetamine-regulated transcript peptide fragment 55-102 (CARTp) increased the levels of phosphoserine 896 and phosphoserine 897 on the N-methyl-d-aspartate (NMDA) receptor NR1 subunit (pNR1-ser896 and pNR1-ser897) via protein kinase A (PKA) and protein kinase C (PKC) signaling pathways leading to increases in NMDA receptor function in spinal cord dorsal horn neurons. Because NMDA receptor, PKC, and PKA signaling pathways may participate in ERK activation, we examined the effects of CARTp on ERK activation in spinal cord dorsal horn neurons in vitro. Western blot analysis showed a significant increase in the level of phosphorylated (activated) ERK (pERK) in the dorsal part of the spinal cord slices after incubation of the slices with CARTp (300nM). Co-administration of CARTp with an NMDA receptor antagonist, MK801 or AP5, or an ERK inhibitor PD98059 blocked the increase in the level of pERK. Interestingly, the increase in the level of pERK by CARTp was observed in postnatal week 3 (W3) and postnatal week 4 (W4), but not in postnatal week 2 (W2) rats. The age-related responses were also noted by CARTp-induced increases in the levels of pNR1-ser896 and pNR1-ser897. In the in vitro electrophysiological study, CARTp increased the amplitude of NMDA-mediated depolarizations in spinal substantia gelatinosa neurons of W3 and W4 rats, but not W2 rats. The results suggest that CARTp activation of ERK signals via the NMDA receptor in the spinal cord dorsal horn was age-dependent.

Potentiation of spinal NMDA-mediated nociception by cocaine- and amphetamine-regulated transcript peptide via PKA and PKC signaling pathways in rats.

Our previous study showed that cocaine- and amphetamine-regulated transcript peptide fragment 55-102 (CARTp) specifically potentiated spinal N-methyl-D-aspartate (NMDA)-mediated nociceptive transmission in vivo and in vitro. The cellular mechanisms underlying CARTp potentiation of NMDA receptor function remains unclear. The present study was carried out to test the hypothesis that CARTp changes the phosphorylated state of NMDA receptors by activating intracellular signals and subsequently increasing the function of NMDA receptors. We found that the potentiating effect of CARTp on spinal NMDA-induced hyperalgesia in rats was reduced by intrathecal pretreatment with KT5720 (a selective PKA inhibitor) or GF109206X (a selective PKC inhibitor), but was increased by pretreatment with calyculin A (a protein phosphatase inhibitor). In the in vitro electrophysiological study, CARTp potentiation of NMDA-induced depolarizations was blocked by superfusion of PKA or PKC inhibitor applied 10 min before the application of CARTp. The levels of phosphoserine 897 on the NR1 subunit (pNR1-ser897) and phosphoserine 896 on the NR1 subunit (pNR1-ser896) in the dorsal horn of spinal lumbar segments significantly increased following CARTp superfusion in vitro or intrathecal injection in vivo. The increases in pNR1-ser897 and pNR1-ser896 in the in vivo and in vitro studies were inhibited by pretreatment with KT5720 and GF109206X, respectively. The results provide the first evidence that CARTp increases the phosphorylation of NMDA receptor NR1 subunit via activation of PKA and PKC signals, which may play a crucial role in CARTp regulation of spinal NMDA receptor-mediated nociceptive responses.

Potentiation of spinal N-methyl-D-aspartate-mediated nociceptive transmission by cocaine-regulated and amphetamine-regulated transcript peptide in rats.

The present study examined the effects of cocaine-regulated and amphetamine-regulated transcript peptide (CARTp) fragment 55-102, on N-methyl-D-aspartate (NMDA)-mediated nociceptive transmission in vivo and in vitro. In-vivo experiments were conducted in Sprague-Dawley rats to evaluate the effects of CARTp on thermal hyperalgesia induced by NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Intrathecal NMDA (1, 2, 4 nmol) or AMPA (0.5, 1, 2 nmol) dose-dependently decreased the tail-flick latency. Intrathecal CARTp was without effect on the tail-flick latency. Interestingly, it significantly enhanced NMDA-induced, but not AMPA-induced, nociceptive effects. The in-vitro effects of CARTp on NMDA-induced or AMPA-induced depolarizations in substantia gelatinosa neurons were studied in rat spinal cord slices. CARTp (100, 300 nM), which caused no significant change of membrane potentials, increased the amplitude of NMDA-induced depolarizations in substantia gelatinosa neurons with little effect on AMPA-induced depolarizations. The present study demonstrates that exogenously applied CARTp selectively facilitates NMDA receptor-mediated nociceptive transmission.