Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration.

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS: Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Host miR-129-5p reverses effects of ginsenoside Rg1 on morphine reward possibly mediated by changes in B. vulgatus and serotonin metabolism in hippocampus.

Morphine addiction is closely associated with dysbiosis of the gut microbiota. miRNAs play a crucial role in regulating intestinal bacterial growth and are involved in the development of disease. Ginsenoside Rg1 exhibits an anti-addiction effect and significantly improves intestinal microbiota disorders. In pseudo-germfree mice, supplementation with Bacteroides vulgatus (B. vulgatus) synergistically enhanced Rg1 to alleviate morphine addiction. However, it is currently unknown the relationship between fecal miRNAs in morphine-exposed mice and their potential modulation of gut microbiome, as well as their role in mediating the resistance of ginsenoside Rg1 to drug addiction. Here, we studied the fecal miRNA abundance in mice treated with morphine to explore the different miRNAs expressed, their association with B. vulgatus and their role in the amelioration of morphine reward by ginsenoside Rg1. Our results indicated ginsenoside Rg1 attenuated the significant increase in miR-129-5p expression observed in the feces of morphine-treated mice. The miR-129-5p, specifically, inhibited the growth of B. vulgatus by modulating the transcript of the site-tag BVU_RS11835 and increased the levels of 5-hydroxytryptophan and indole-3-carboxaldehyde in vitro. Subsequently, we noticed that oral administration of synthetic miR-129-5p increased 5-HT levels in the hippocampus and inhibited the reversal effect of ginsenoside Rg1 both on the relative abundance of B. vulgatus in the feces and CPP effect induced by morphine exposure. In short, Ginsenoside Rg1 might play an indirect role in remodeling the B. vulgatus against morphine reward by suppressing miR-129-5p expression. These results highlight the role of miR-129-5p and B. vulgatus in morphine reward and the anti-morphine addiction of ginsenoside Rg1.

Rhynchophylline inhibits methamphetamine dependence via modulating the miR-181a-5p/GABRA1 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Uncaria rhynchophylla (Miq.) Miq. ex Havil. is a plant species that is routinely devoted in traditional Chinese medicine to treat central nervous system disorders. Rhynchophylline (Rhy), a predominant alkaloid isolated from Uncaria rhynchophylla (Miq.) Miq. ex Havil., has been demonstrated to reverse methamphetamine-induced (METH-induced) conditioned place preference (CPP) effects in mice, rats and zebrafish. The precise mechanism is still poorly understood, thus further research is necessary. AIM OF STUDY: This study aimed to investigate the role of miRNAs in the inhibitory effect of Rhy on METH dependence. MATERIALS AND METHODS: A rat CPP paradigm and a PC12 cell addiction model were established. Microarray assays were used to screen and identify the candidate miRNA. Behavioral assessment, real-time PCR, dual-luciferase reporter assay, western blotting, stereotaxic injection of antagomir/agomir and cell transfection experiments were performed to elucidate the effect of the candidate miRNA and intervention mechanism of Rhy on METH dependence. RESULTS: Rhy successfully reversed METH-induced CPP effect and the upregulated miR-181a-5p expression in METH-dependent rat hippocampus and PC12 cells. Moreover, suppression of miR-181a-5p by antagomir 181a reversed METH-induced CPP effect. Meanwhile, overexpression of miR-181a-5p by agomir 181a in combination with low-dose METH (0.5 mg/kg) elicited a significant CPP effect, which was blocked by Rhy through inhibiting miR-181a-5p. Finally, the result demonstrated that miR-181a-5p exerted its regulatory role by targeting γ-aminobutyric acid A receptor α1 (GABRA1) both in vivo and in vitro. CONCLUSION: This finding reveals that Rhy inhibits METH dependence via modulating the miR-181a-5p/GABRA1 axis, which may be a promising target for treatment of METH dependence.

Isoalantolactone Induces Cell Cycle Arrest, Apoptosis and Autophagy in Colorectal Cancer Cells.

Colorectal cancer (CRC) is an aggressive cancer. Isoalantolactone (IATL) has been reported to exert cytotoxicity against various cancer cells, but not CRC. In this study, we explored the anti-CRC effects and mechanism of action of IATL in vitro and in vivo. Our results demonstrated that IATL inhibited proliferation by inducing G0/G1 phase cell cycle arrest, apoptosis and autophagy in CRC cells. Repression of autophagy with autophagy inhibitors chloroquine (CQ) and Bafilomycin A1 (Baf-A1) enhanced the anti-CRC effects of IATL, suggesting that IATL induces cytoprotective autophagy in CRC cells. Mechanistic studies revealed that IATL lowered protein levels of phospho-AKT (Ser473), phospho-mTOR (Ser2448), phospho-70S6K (Thr421/Ser424) in CRC cells. Inhibition of AKT and mTOR activities using LY294002 and rapamycin, respectively, potentiated the inductive effects of IATL on autophagy and cell death. In vivo studies showed that IATL suppressed HCT116 tumor growth without affecting the body weight of mice. In consistent with the in vitro results, IATL lowered protein levels of Bcl-2, Bcl-XL, phospho-AKT (Ser473), phospho-mTOR (Ser2448), and phsopho-70S6K (Thr421/Ser424), whereas upregulated protein levels of cleaved-PARP and LC3B-II in HCT116 tumors. Collectively, our results demonstrated that in addition to inhibiting proliferation, inducing G0/G1-phase cell cycle arrest and apoptosis, IATL initiates cytoprotective autophagy in CRC cells by inhibiting the AKT/mTOR signaling pathway. These findings provide an experimental basis for the evaluation of IATL as a novel medication for CRC treatment.

Effects of Pereskia aculeate Miller Petroleum Ether Extract on Complete Freund's Adjuvant-Induced Rheumatoid Arthritis in Rats and its Potential Molecular Mechanisms.

Objective: To investigate the therapeutic effect of petroleum ether extract of P. aculeate Miller (PEEP) on rheumatoid arthritis (RA). Methods: In vitro: The Cell Counting Kit-8 (CCK-8) was used to detect cell activity and select the optimal concentration of the extract; the effective site was screened by nitric oxide (NO) colorimetric method and Q-PCR method; the expression of p38, p-p38, p-MK2, and Tristetraprolin (TTP) in RAW 264.7 cells were detected by Western blot. In vivo: The rat model was established by complete Freund's adjuvant (CFA). The different doses of PEEP on CFA rats were observed with life status, paw swelling, spleen index, X-ray, Hematoxylin eosin (HE) staining; the secretion of Tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and Prostaglandin E2 (PGE2) were detected by Enzyme linked immunosorbent assay (ELISA); the expressions of p38, p-p38, p-MK2, and TTP in the ankle joints of CFA rats were detected by Western blot. Result: In vitro: PEEP, Ethyl Acetate Extract of P. aculeate Miller (EEEP), N-butanol Extract of P. aculeate Miller (BEEP) have no toxic effects on RAW264.7 macrophages. PEEP, EEEP, and BEEP reduce the secretion of NO in RAW264.7 cells induced by lipopolysaccharide (LPS), only PEEP significantly inhibited the mRNA expression levels of inflammatory factors TNF-α and IL-6; PEEP-dependently reduce the secretion of TNF-α and IL-6, decrease the expression of p-p38 and p-MK2, and the level of TTP phosphorylation in LPS-induced RAW264.7 cells. In vivo: PEEP improve the living conditions of CFA rats, reduce foot swelling, spleen index, bone surface erosion and joint space narrowing; reduce the formation of synovial cells, inflammatory cells and pannus in the foot and ankle joints. PEEP reduce the secretion of TNF-α, IL-6, PGE2 in rat serum, downregulate the expression of p-p38 and p-MK2 in the ankle joint, and reduce the phosphorylation of TTP. Conclusion: PEEP improve the living conditions of CFA rats, reduce the degree of foot swelling, protect immune organs, reduce inflammatory cell infiltration, cartilage damage, pannus formation, reduce inflammation and RA damage. The mechanism through regulating the signal pathway of p38 mitogen-activated protein kinase (p38/MAPK), which reduces the release of TNF-α, IL-6, and PGE2 in the serum.

Ginsenoside Rg1 mitigates morphine dependence via regulation of gut microbiota, tryptophan metabolism, and serotonergic system function.

BACKGROUND: Morphine dependence, a devastating neuropsychiatric condition, may be closely associated with gut microbiota dysbiosis. Ginsenoside Rg1 (Rg1), an active ingredient extracted from the roots of Panax ginseng C.A. Meyer, has potential health-promoting effects on the nervous system. However, its role in substance use disorders remains unclear. Here, we explored the potential modulatory roles of Rg1 in protection against morphine dependence. METHODS: Conditioned place preference (CPP) was used for establishing a murine model of morphine dependence. 16S rRNA gene sequencing and metabolomics were performed for microbial and metabolite analysis. Molecular analysis was tested for evaluating the host serum and brain responses. RESULTS: Rg1 prevented morphine-induced CPP in mice. The 16S rRNA gene-based microbiota analysis demonstrated that Rg1 ameliorated morphine-induced gut microbiota dysbiosis, specifically for Bacteroidetes. Moreover, Rg1 also inhibited gut microbiota-derived tryptophan metabolism and reduced the serotonin, 5-hydroxytryptamine receptor 1B (5-HTR1B), and 5-hydroxytryptamine receptor 2 A (5-HTR2A) levels. However, the Rg1-induced amelioration of CPP was not observed in mice when their gut microbiome was depleted by non-absorbable antibiotics. Subsequently, gavage with Bacteroides vulgatus increased the abundance of Bacteroidetes. B. vulgatus supplementation synergistically enhanced Rg1-alleviated morphine-induced CPP in mice with microbiome knockdown. Co-treatment with B. vulgatus and Rg1 produced suppressive effects against morphine dependency by inhibiting tryptophan metabolism and reducing the serotonin and 5-HTR1B/5-HTR2A levels. CONCLUSIONS: The gut microbiota-tryptophan metabolism-serotonin plays an important role in gut-brain signaling in morphine disorders, which may represent a novel approach for drug dependence treatment via manipulation of the gut microbial composition and tryptophan metabolite.

Decrease of morphine-CPP by sinomenine via mediation of tyrosine hydroxylase, NMDA receptor subunit 2B and opioid receptor in the zebrafish brain.

To study the effects of sinomenine on conditioned place preference (CPP) zebrafish induced by morphine and expression levels of intracephalic tyrosine hydroxylase (TH), NMDA receptor subunit 2B (NR2B), µ-opioid receptor (zfmor) and δ-opioid receptors (zfdor1 and zfdor2), morphine (40mg/kg) was administrated to zebrafish and the effect of CPP was detected in these zebrafish treated with sinomenine. The expression of TH and NR2B was detected by immunohistochemistry; and the mRNA expression of opioid receptors zfmor, zfdor1 and zfdor2 in the zebrafish brain was assayed by RT-qPCR. In the CPP test, morphine induced significant behavioral alteration, while pretreatment with sinomenine or methadone, resulted in decreased activity time in the morphine-paired compartment significantly. Morphine also increased the integral optical density value of TH- and NR2B-positive cells in the zebrafish brain, and reduced the amount of opioid receptors. However, the compound sinomenine could attenuate these effects. These findings demonstrate that sinomenine (80mg/kg) decreased the CPP effects of zebrafish induced by morphine significantly, downregulated expression of TH and NR2B, and upregulated µ-opioid (zfmor) and δ-opioid (zfdor1 and zfdor2) receptor expression in the CPP zebrafish brains.

Extracellular Vesicle-Encapsulated miR-183-5p from Rhynchophylline-Treated H9c2 Cells Protect against Methamphetamine-Induced Dependence in Mouse Brain by Targeting NRG1.

Methamphetamine (Meth) is a highly addictive substance and the largest drug threat across the globe. There is evidence to indicate that Meth use has serious damage on central nervous system (CNS) and heart in several animal and human studies. However, the connection in the process of Meth addiction between these two systems has not been determined. Emerging data suggest that extracellular vesicles (EVs) carrying behavior-altering microRNA (miRNAs) play a crucial role in cell communication between CNS and peripheral system. Rhynchophylline (Rhy), an antiaddictive alkaloid, was used to protect the brain and heart from Meth-induced damage, which has caught our attention. Here, we used Meth-dependent conditioned place preference (CPP) animal model and cell model to verify the protective effect of Rhy-treated EVs. Further, small RNA sequencing analysis, qPCR, dual-luciferase reporter assay, and transfection test were used to identify the key EVs-encapsulated miRNAs, isolated from cultured H9c2 cells with different treatments, involved in the therapeutic effect and the underlying mechanisms of Rhy. The results demonstrate that Rhy-treated EVs exert protective effects against Meth dependence through the pathway of miR-183-5p-neuregulin-1 (NRG1). Our collective findings provide novel insights into the roles of EVs miRNAs in Meth addiction and support their potential application in the development of novel therapeutic approaches.

Curcumin Enhances Radiosensitization of Nasopharyngeal Carcinoma via Mediating Regulation of Tumor Stem-like Cells by a CircRNA Network.

Circular RNAs (circRNAs) are involved in cancer development via inhibition of miRNAs, which are associated with differentiation, proliferation, migration, and carcinogenicity. Curcumin has antioxidant and anti-cancer properties, and it has also been used as a radiosensitizer. In this study, we explored the potential relationships among curcumin, circRNAs, and nasopharyngeal carcinoma (NPC). We compared the differences in circRNA levels in NPC cell lines after radiotherapy and after treatment with curcumin, using a high-throughput microarray. Further, a circRNA-miRNA-mRNA interaction network between radiation resistance NPC cell lines and tumor stem cells was constructed by applying bioinformatics. Finally, it was demonstrated by reverse transcription-quantitative polymerase chain reaction assay and wound healing assay that curcumin could enhance radiosensitization of NPC cell lines via mediating regulation of tumor stem-like cells by the "hsa_circRNA_102115"-"hsa-miR-335-3p"-"MAPK1" interaction network.

Expression of miR-133a-5p and ROCK2 in Heart in Methamphetamine-Induced Rats and Intervention of Rhynchophylline.

INTRODUCTION: Rhynchophylline, as a traditional Chinese medicine, was used for the treatment of drug addiction. OBJECTIVE: To investigate miRNAs expression profile in the rat hearts of methamphetamine dependence and the intervention mechanisms of rhynchophylline. MATERIALS AND METHODS: This study detected the expression profile of miRNAs in the methamphetamine-induced rat hearts by microarray and verified the expression of miR-133a-5P and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) protein. RESULTS: After conditioned place preference training, methamphetamine significantly increased the time spent in the drug-paired compartment, while rhynchophylline and MK-801 could reduce the time. Cluster analysis results of miRNAs showed that compared with the control group, the expression of miR-133a-5p in methamphetamine-induced rat hearts was decreased significantly; rhynchophylline could significantly increase the expression of miR-133a-5p. The result was verified by real-time polymerase chain reaction. The results of target gene predictive software and related research showed that ROCK2 protein may be the target gene of miR-133a-5p. The immunohistochemistry results of heart tissues showed that the expression of ROCK2 protein was significantly upregulated in the methamphetamine group and downregulate in the rhynchophylline group; the difference between the MK-801 group and the methamphetamine group was not significant. The result of western blot was consistent with the immunohistochemistry. CONCLUSION: The active ingredient of Chinese herbal medicine rhynchophylline can effectively inhibit the formation of methamphetamine-dependent conditional place preference (CPP) effect in rats to some extent. MiR-133a-5p may participate in the cardioprotective effects of CPP rats by targeting ROCK2.

Aloperine induces apoptosis and G2/M cell cycle arrest in hepatocellular carcinoma cells through the PI3K/Akt signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) ranks third among the most common causes of cancer-related deaths worldwide. The chemotherapy for HCC is still insufficient, so far. In searching for effective anti-HCC agents from traditional Chinese medicine, we discovered that aloperine (ALO), a quinolizidine alkaloid from Sophora alopecuroides L., exerts anti-HCC activities. However, the effects of ALO on HCC have been rarely studied, and its underlying mechanisms remain unknown. PURPOSE: This study aims to evaluate the anti-HCC activities of ALO and explore its underlying mechanisms. METHODS: MTT assay and colony formation assay were used to investigate the anti-proliferative effects of ALO on human HCC Hep3B and Huh7 cells. Hoechst 33258 staining was used to observe the morphological changes of cells after ALO treatment. Flow cytometry was used to analyze apoptosis induction, the collapse of the mitochondrial membrane potential and cell cycle distribution. Western blotting was used to examine the expression levels of proteins associated with apoptosis and cell cycle arrest, and key proteins in the PI3K/Akt signaling pathway. Small interfering RNA (siRNA) transfection was used to investigate the role of Akt in ALO-induced apoptosis and cell cycle arrest. Zebrafish tumor model was used to evaluate the anti-HCC effects of ALO in vivo. RESULTS: ALO inhibited the proliferation of Hep3B and Huh7 cells. ALO induced apoptosis in HCC cells, which was accompanied by the loss of mitochondrial potential, the release of cytochrome c into cytosol, as well as the increased cleavages of caspase-9, caspase-3 and PARP. Moreover, ALO induced G2/M cell cycle arrest by downregulating the expression levels of cdc25C, cdc2 and cyclin B1. In addition, ALO inhibited activation of the PI3K/Akt signaling pathway by decreasing the expression levels of p110α, p85, Akt and p-Akt (Ser473). Further study showed that inhibition of Akt by siRNA augmented ALO-mediated apoptosis and G2/M cell cycle arrest in HCC cells. Critically, ALO inhibited the growth of Huh7 cells in vivo. CONCLUSION: We first demonstrated that ALO induced apoptosis and G2/M cell cycle arrest in HCC cells through inhibition of the PI3K/Akt signaling pathway. This study provides a rationale for ALO as a potential chemotherapeutic agent for HCC.

Sinomenine Protects Against Morphine Dependence through the NMDAR1/CAMKII/CREB Pathway: A Possible Role of Astrocyte-Derived Exosomes.

Sinomenine is a nonaddictive alkaloid used to prevent morphine dependence, even thoughits mechanism isnot fully understood. Astrocytes aggravate the pathological process in their neighboring cellsthrough exosomes in central nervous system diseases. However, the effect of sinomenine on astrocyte-derived exosomes for the amelioration of morphine dependence has not been reported yet. In this study, we found that sinomenine prevented the morphine-induced conditionedplace preference in mice. Sinomenine reduced the levels of cAMP and intracellular Ca2+ in morphine-treated SH-SY5Y cells. Moreover, sinomenine inhibited the expressions of p-NMDAR1/NMDAR1, p-CAMKII/CAMKII, and p-CREB/CREB in the hippocampusof morphine-dependent mice and SH-SY5Y cells. Furthermore, we found that sinomenine inhibitedthe morphine-induced activation of astrocytesin vivo and in vitro. Afterwards, exosomes were isolated from cultured primary astrocytes treated with phosphate buffer saline (PBS, ctl-exo), morphine (mor-exo), or morphine and sinomenine (Sino-exo). Subsequently, morphine-treated SH-SY5Y cells were treated with ctl-exo, mor-exo, and Sino-exo. Results showed that Sino-exo reduced the level of cAMP, intracellular Ca2+, and the expression of p-CAMKII/CAMKII and p-CREB/CREB in morphine-treated SH-SY5Y cells. In conclusion, we demonstrated that sinomenine exhibited protective effects against morphine dependencein vivo and in vitro through theNMDAR1/CAMKII/CREB pathway. Sinomenine-induced alterationof the function of astrocyte-derived exosomes may contribute to the antidependence effects of sinomenine in morphine dependence.

Expression of miRNAs in Serum Exosomes versus Hippocampus in Methamphetamine-Induced Rats and Intervention of Rhynchophylline.

OBJECTIVE: To compare the expressions of miRNAs (microRNAs) in serum exosomes and in hippocampus and to provide insights into the miRNA-mediated relationship between peripheral and central nervous systems in the presence of methamphetamine. METHODS: Published results on conditioned place preference (CPP) in rats conditioned by methamphetamine were replicated. The expressions of miRNAs in serum exosomes and hippocampus were determined by gene-chip sequencing. We then predicted the potential target genes of selected, differentially expressed (DE) miRNAs and then carried out functional analysis of these target genes. We also verified our results by RT-qPCR. RESULTS: Methamphetamine reward could greatly increase the activity time and distance in the intrinsically nonpreferred side of the behavioral apparatus compared with control rats (P < 0.01). Rhynchophylline treatment significantly counteracted these changes (P < 0.01). Methamphetamine-induced CPP upregulated 23 miRNAs (log2 fold change [FC] > 1, P < 0.01) in serum exosomes, whereas rhynchophylline treatment could downregulate these miRNAs (log2 FC < -1, P < 0.01). Analysis of hippocampal miRNAs profiles found 22 DE miRNAs (log2 FC > 1 or <-1, P < 0.01). When methamphetamine induced CPP, 11 of those miRNAs were upregulated, whereas rhynchophylline treatment could downregulate these miRNAs. The other 11 miRNAs behaved in the opposite way. We selected six DE miRNAs from each of serum exosomes and hippocampus for target gene prediction and functional analysis. We found that, in both, the DE miRNAs and their target genes may be related to neuronal information transmission and synaptic transmission. CONCLUSIONS: Rhynchophylline blocked the alteration of behavior and the expression of some DE miRNAs induced by methamphetamine. The biological functions of these DE miRNAs target genes are correlated between serum exosomes and hippocampus. As to these biological processes and pathways which are involved in the development of addiction at multiple stages, we speculate that these DE miRNAs in serum exosomes and hippocampus are closely related to methamphetamine addiction.

Expression of microRNAs in the serum exosomes of methamphetamine-dependent rats vs. ketamine-dependent rats.

Drug abuse is a public health and social problem. A number of studies have reported that drug addiction is associated with microRNAs (miRNAs). By comparing the expression of miRNAs in the serum exosomes of methamphetamine-dependent and ketamine-dependent rats, the aim of the present study was to provide insights into the miRNA-mediated associations between the two groups. Published results on conditioned place preference (CPP) in rats conditioned by methamphetamine and ketamine were replicated. The expression of miRNAs in serum exosomes were determined by gene-chip sequencing. The potential target genes of differentially expressed (DE) co-miRNAs were predicted in the methamphetamine and ketamine rats, then functional analysis of their target genes was performed. Methamphetamine and ketamine reward greatly increased the activity time and distance in the intrinsically non-preferred side of the behavioral apparatus when compared with controlled rats (P<0.01). In addition, methamphetamine upregulated the expression of 276 miRNAs and downregulated 25 miRNAs, while ketamine only downregulated the expression of 267 miRNAs. Ten DE co-miRNAs in the two model groups were identified. Functional analysis revealed that DE co-miRNAs are involved in the development of addiction at different stages, and their target genes were enriched in 'vesicular transport', 'amphetamine addiction', 'dopaminergic synapse' and 'GABAergic synapse'. Therefore, it was suggested that these co-miRNAs may have a strong association with drug addiction, and they may be involved in the different addiction processes, which partly explains methamphetamine and ketamine addiction.

Rhynchophylline Downregulates Phosphorylated cAMP Response Element Binding Protein, Nuclear Receptor-related-1, and Brain-derived Neurotrophic Factor Expression in the Hippocampus of Ketamine-induced Conditioned Place Preference Rats.

BACKGROUND: Addiction to ketamine is becoming a serious public health issues, for which there exists no effective treatment. Rhynchophylline (Rhy) is an alkaloid extracted from certain Uncaria species that is well known for both its potent anti-addictive and neuroprotective properties. Increasing evidence supports the contributions of cAMP response element binding protein (CREB), nuclear receptor-related-1 (Nurr1), and brain-derived neurotrophic factor (BDNF) in modulating neural and behavioral plasticity which was induced by addictive drugs. OBJECTIVE: To investigate the effects of Rhy on the behavior and the levels of phosphorylated CREB (p-CREB), Nurr1, and BDNF in the hippocampus of ketamine-induced conditioned place preference (CPP) rats. MATERIALS AND METHODS: CPP paradigm was used to establish the model of ketamine-dependent rats and to evaluate the effect of Rhy on ketamine dependence. The expressions of p-CREB, Nurr1, and BDNF were tested by Western blotting and immunohistochemistry. RESULTS: We observed that Rhy can reverse the behavior preference induced by ketamine CPP training. At the same time, expression of p-CREB, Nurr1, and BDNF, which was significantly increased by ketamine, was restored in the Rhy -treated group. CONCLUSION: This study indicates that Rhy can reverse the reward effect induced by ketamine in rats and the mechanism can probably be related to regulate the hippocampal protein expression of p-CREB, Nurr1, and BDNF. SUMMARY: P-CREB, Nurr1 and BDNF play an important role in the formation of ketamine-induced place preference in ratsRhynchophylline reversed the expression of p-CREB, Nurr1 and BDNF which was activated by ketamine in the hippocampusRhynchophylline demonstrates the potential effect of mediates ketamine induced rewarding effect. Abbreviations used: Rhy: Rhynchophylline; CREB: cAMP response element binding protein; Nurr1: Nuclear receptor-related-1; BDNF: Brain-derived neurotrophic factor; CPP: Conditioned place preference; NMDA: N-methyl-D-aspartic acid; METH: Methamphetamine; CNS: Central nervous system; PFA: Paraformaldehyde; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; LTP: long-term potentiation.

Effects of rhynchophylline on the hippocampal miRNA expression profile in ketamine-addicted rats.

In the past few years, ketamine, a noncompetitive NMDA antagonist, has been widely abused worldwide as a new type of synthetic drug, severely affecting the physical and mental health of ketamine abusers. Previous studies have suggested that rhynchophylline can alleviate drug abuse and reverse the conditioned place preference caused by the abuse. MicroRNAs (miRNAs) are important factors regulating gene expression and are involved in the drug addiction process. The hippocampus is a critical area in the brain involved in causing drug addiction. However, the hippocampal miRNA expression profile and the effects of rhynchophylline on miRNA expression during ketamine abuse have not been reported. Thus, this study analyzed the hippocampal miRNA expression profile during ketamine-dependence formation and the effects of rhynchophylline on the differential expression of miRNAs induced by ketamine. The results of microarray analysis suggested that the expression levels of miR-331-5p were significantly different among three groups (the control, ketamine, and ketamine + rhynchophylline groups). miR-331-5p levels were significantly decreased in the ketamine model group and were upregulated in the ketamine + rhynchophylline group. Bioinformatics analysis of miR-331-5p and the 3' UTR of nuclear receptor related 1 protein (Nurr1) identified binding sites and showed downregulation, and the overexpression of miR-331-5p in hippocampal tissues showed that miR-331-5p is a negative transcription regulatory factor of Nurr1. Interestingly, we found that the downstream protein of Nurr1, brain-derived neurotrophic factor (BDNF), showed identical expression trends in the hippocampus as Nurr1. However, the transcription of the protein upstream of Nurr1, cyclic adenosine monophosphate response element-binding protein (CREB), did not show any significant differences between the ketamine group and the ketamine + rhynchophylline group. However, after rhynchophylline intervention, p-CREB showed significant differences between the ketamine and the ketamine + rhynchophylline groups. In summary, miR-331-5p is a key regulatory factor of Nurr1, and rhynchophylline can participate in the process of resistance to ketamine addiction through the miR-331-5p/Nurr1/BDNF pathway or inhibition of CREB phosphorylation.

Use of an UHPLC-MS/MS Method for Determination of Kuraridin and Characterization of Its Metabolites in Rat Plasma after Oral Administration.

Kuraridin is an active natural prenylated flavonoid ingredient originating from the well-known traditional Chinese medicine Sophora flavescens Ait., that possesses various bioactivities, such as antitumor activity, PLCγ1 inhibitory activity, glycosidase inhibitory activity, etc. However, there is no report on the plasma metabolic profile and pharmacokinetic study of kuraridin. The current study was designed to use an ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method for the quantification and characterization metabolites in rat plasma after oral administration of kuraridin. A liquid-liquid extraction method with ethyl acetate-acetonitrile (1:3) was used to extract the kuraridin from rat plasma samples. The chromatographic separation was carried out on a Hypersil GOLD UHPLC C18 column equipped with a C18 guard cartridge using a gradient elution with organic solvent-water as mobile phase. Based on comparing the retention times with reference standards or on the basis of MS2 fragmentation behaviors, a total of 19 metabolites were identified or tentatively characterized from rat plasma. Under the optimized conditions, the method showed good linearity (r² > 0.99) over the ranges of 1-500 ng/mL for kuraridin. The inter- and intra-day precisions were less than 8.95%, and the accuracy was in the range of -6.27-6.48%. The recovery of kuraridin ranged from 90.1% to 100.4%. The developed UHPLC-MS/MS method was thus successfully applied in the qualitative of metabolites and quantitative analysis of kuraridin in rat plasma.

In vitro and in vivo anti-malignant melanoma activity of Alocasia cucullata via modulation of the phosphatase and tensin homolog/phosphoinositide 3-kinase/AKT pathway.

Alocasia cucullata, a Chinese herb, has been used as an anticancer treatment in southern China. Phosphatase and tensin (PTEN), is a tumor suppressor gene and the loss of PTEN expression may activate the phosphoinositide-3-kinase (PI3K)/AKT signaling pathway which play a key role in tumors formation and progression. In this study, we evaluated the anti-melanoma effect and the underlying mechanism of 50% ethanolic extract of A. cucullata (EAC) in vitro and in vivo. Using MTT, wound healing, and transwell assays, we found that EAC suppressed the proliferation, migration, and invasion of melanoma cells (B16-F10, A375 and A2058) in a dose-dependent manner. We also found that EAC suppresses B16-F10 tumor growth in a xenografted mouse model. Western blot analysis revealed that the expression level of PTEN was up-regulated, and phosphorylation of PI3K and AKT reduced in B16-F10 cells and tumor tissues after EAC treatment. No significant differences were observed in PI3K and AKT expression. Moreover, immunohistochemistry showed that the number of PTEN-positive cells in tumor tissues increased and that of p-AKT-positive cells decreased with EAC treatment, corroborating the western blot results. Our data reveal that EAC can inhibit malignant melanoma in vitro and in vivo and suggest that its anti-tumor effect is associated with modulation of the PTEN/ PI3K/AKT signaling pathway. In summary, our findings highlight a promising herbal remedy for the treatment of malignant melanoma, which warrants further study.

Effect of Sinomenine on the Morphine-Dependence and Related Neural Mechanisms in Mice.

Evidence suggests that the dopamine receptor rate-limiting enzyme, tyrosine hydroxylase (TH), and the glutamate receptor, N-methyl-D-aspartate receptor 2B (NR2B), contribute to morphine dependence. Previous studies show that chronic exposure to morphine changes the expression of opioid receptors. In this study, we focus on the effects of sinomenine on morphine-dependent mice and its related neural mechanisms. Conditioned place preference (CPP) mouse model was established using morphine (9 mg/kg, s.c.), and their expression levels of TH and NR2B were observed by immunohistochemistry. Moreover, their mu opioid receptor (MOR) and delta opioid receptor (DOR) contents were assessed using quantitative reverse transcription polymerase chain reaction. Results showed that high sinomenine dose (80 mg/kg) effectively attenuated the behavior of CPP mice and reversed increased expression levels of TH and NR2B induced by morphine. Moreover, compared with the morphine group, sinomenine up-regulated the content of MOR to a normal level but did not significantly affect the DOR expression. In summary, these data indicate that sinomenine can inhibit morphine dependence by increasing the expression levels of TH, NR2B, and MOR in the mouse brain; however, DOR may not contribute to this effect.