Two major kinase-mediated signaling pathways are activated following TLR4 stimulation by LPS. These include the MAPKs and IkB kinase complexes, which lead to activation of AP-1 and NF-kB transcription factors, respectively. Here we show that LPSup regulated expression of Dusp1 was significantly up regulated by CBD but not by THC. An increase in the expression of Dusp1 was also observed following incubation of resting BV-2 cells with CBD, but not with THC. A similar CBD mediated regulation of mRNA level was observed for Dusp8, another negative regulator of MAPKs. The IPA interactome analysis has linked this CBD-stimulated expression of Dusp8, via negative modulation of p38 MAPK, to the attenuation of LPS mediated induction of NFAT5, a member of the inflammatory transcription complex. On the other hand, the LPS-stimulated expression of Dusp2 , a key positive regulator of the inflammatory MAPKs signaling, was significantly repressed by CBD as well as by THC. Moreover, CBD but not THC decreased basal levels of Dusp2/Pac-1 mRNA in resting BV-2 cells. IPA and interactome analysis indicate that pretreatment with CBD, but less so with THC, result in attenuation of LPS stimulated activation of NF-kB and its dependent gene transcription pathways. Indeed, our previous data show that CBDdecreases the activity of the NFkB signaling pathway in BV-2 microglial cells via the partial reversal of the LPS-induced degradation of IRAK-1 intermediate kinase, thus reversing IkB degradation and reducing NF-kB p65 subunit phosphorylation. This effect of CBD in decreasing NF-kB signaling activity is in line with the CBD-induced diminished transcription of NF-kBdependent genes, e.g., IL-1b and IL-6. We and others previously reported that BV-2 cells express CB1, CB2, GPR55, GPR18 and TRPV2, G protein-coupled receptors and a channel that are known to interact with cannabinoids.

Using gene array analysis,procona florida container we have observed that the relative levels of CB1, CB2, GPR18 and TRPV2 as well as of the fatty acid amide hydrolase gene transcripts were not significantly affected by the cannabinoid treatments and their levels did not exceed the 2-fold induction or 50% reduction by either CBD or THC treatment. On the other hand, we show here that LPS markedly down regulates CB2 and GPR55 and that this down regulation is not affected by either CBD or THC pretreatment. This result is in agreement with our previous report showing that LPS markedly down regulates CB2 and GPR55 mRNAs in BV- 2 microglial cells and in microglial primary cultures.A relationship between CBD-mediated oxidative stress response and glutathione depletion was previously reported . More recently, we showed that CBD-specific gene expression profile in BV-2 cells displays changes normally occurring under either nutrient limiting conditions or proteasome inhibition, and that are attributed to activation of GCN2/eIF2a/ p8/ATF4/CHOP-Trib3 pathway leading to autophagy as well as to apoptotic cell death. The Trib3 gene product seem to be of high importance to the CBD effect due to its ability to serve as a master regulator of an array of pathways including AP-1, ER stress, Akt/PKB and NF-kB. Trib3 expression is significantly up regulated by CBD as well as by THC and as observed here, remains up regulated after LPS treatment . According to these gene array studies and the qPCR results, LPS by itself does not significantly affect the expression of Trib3 mRNA. IPA interactome analysis of the microarrays data reveals an interaction between the CBD-up regulated Trib3 and the NF-kB transcription factor pathway . This interaction seems to be responsible for the attenuation by CBD of the transcription of many proinflammatory genes. There are several indications suggesting interaction between these two pathways. First, a direct interaction between p65/RelA and Trib3 protein which induces inhibition of PKA dependent p65 phosphorylation, was described. Second, Trib3 protein can negatively regulate the serinethreonine kinase Akt/PKB, a downstream effector of PI3K that has been implicated in the potentiation of NF-kB-induced transcription of proinflammatory mediators.

This negative regulation of Akt activity by the highly induced Trib3 gene product could point to the mechanism for the CBD-mediated regulation of LPS-stimulated gene expression. Indeed, the effect of CBD treatment on a number of LPS-stimulated genes as reported here is reminiscent of the effects described for the PI3K inhibitor and for the NFkB inhibitor in the murine macrophage cell line RAW264.7 activated with LPS. Both PI3K and NFkB signaling pathways exert important roles in gene expression in response to LPS, but they are not overlapping. Specifically, treatment with CBD repressed a number of typical proinflammatory genes stimulated by LPS, which are known to be NFkB dependent and of other genes including Csf3, Il-1b, Il-1a and Cox2/Ptgs2, which are under the control of both PI3K and NFkB pathways. Finally, Trib3 was documented to interfere with the inflammatory MAPK signaling via direct interaction with MEK-1 and MKK7 leading to attenuation of AP-1 mediated transcriptional activity in cancer HeLa cells. AP-1 is a transcription factor involved in the regulation of inflammation-mediated cellular functions and has been shown to be inhibited by Nrf2-activating agents. Indeed, our IPA network analysis indicates that the observed decrease in mRNA levels for a number of genes is probably related to a reduction in AP-1 dependent transcription. Additionally, according to these IPA results, this repression is reinforced by combined treatments of CBD and LPS as observed by the induction of FosL1 gene product, another negative regulator of AP-1 . Trib3 has been shown to down regulate PPARc transcription and serve as a potent negative regulator of adipocyte differentiation and PPARc is a molecular target for CBD that could be involved in mediating transcriptional effects in BV-2 microglial cells. Indeed, CBD has been shown to bind to PPARc in vitro as well as to activate its transcriptional activity in 3T3L1 fibroblast and in HEK293 transfected cells. In addition, Necela et al., described a regulatory feedback loop in which PPARc represses NF-kB-mediated inflammatory signaling in unstimulated macrophages.

Moreover, they show that upon activation of TLR4 in LPS-stimulated macrophages, NF-kB drives down PPARc expression. These results are in agreement with our results showing that LPS highly down regulates the expression of Pparg1 and Pparg2 in BV-2 cells. The profiles of CBD-induced gene expression with either resting or LPS-activated BV-2 cells, show that CBD stimulates the transcription of several anabolic genes encoding amino acid bio-synthetic enzymes, amino acid transporters and aminoacyltRNA synthetases known to be activated by ATF4, a basic leucine zipper transcription factor, that is increased when cultured cells are deprived of amino acids or subjected to endoplasmic reticulum stress . The divergent types of stress converge on a single event—phosphorylation of the translation initiation factor eIF2a, resulting in a general translational pause followed by selective increase in ATF4 mRNA translation and subsequent stimulation of expression of ATF4 target genes. Many of the CBD-affected transcripts are indeed classified as Nrf2-mediated oxidative stress response genes, including enzymes involved in the biosynthesis of glutathione. Thus, the observed CBD-mediated induction of ATF4-dependent anabolic genes may serve to replenish the amino acids reduced during the elevated turnover of GSH . The mechanism underlying CBD action presumably engages generation of ROS which in turn depletes intracellular GSH. Perturbations in redox tone and GSH levels activate the ‘‘phase 2 response’’, a mechanism used by cells to mitigate oxidative stress. As we have previously shown, many of the ‘‘phase 2’’ gene products are significantly up regulated by CBD. Our present results show that CBD, and less so THC, have immunosuppressive and protective activities that are reminiscent of other clinically applied drugs such as glucocorticoids ,procona London container rexinoids and synthetic triterpenoids. GCs are immunomodulatory agents known to act as suppressive and protective mediators against inflammation. GCs are known to clear antigens by stimulating cell trafficking as well as scavenger systems and matrix metalloproteinases while they stop cellularimmune responses by inhibiting antigen presentation and T cell activation. Synthetic oleanane triterpenoids were shown to be highly effective in many in vivo models in the prevention and treatment of cancer and other diseases with an inflammatory component. Molecular targets of SO include KEAP1 , PPARc, IkB kinase, TGF-b signaling and STAT signaling. SO are among the most potent known inducers of the phase 2 response both in vivo and in vitro and affect the expression of several key cell cycle proteins . In some cancer cells, SO signal through PPARc to inhibit proliferation. The rexinoids bind almost exclusively to the RXRs and are involved in regulation of development, cell proliferation, differentiation and apoptosis. Because RXRs heterodimerize with other receptors , rexinoids modulate the actions of many steroid-like molecules that control metabolism and cellular energetics. In view of these results, triterpenoids and rexinoids are defined as multifunctional drugs. Their targets are either regulatory proteins that control the activity of transcription factors or transcription factors themselves . These complex modulatory activities exerted by GCs, rexinoids and SO display a panorama of effects that closely resembles the complex actions of CBD.Preventing and reducing risky opioid misuse among older adolescents and young adults is critical given that peak misuse rates and associated morbidity and mortality coincide with this developmental period.

Nationally, past-year prescription opioid use ranges from 19.7% for ages 16-17 to 28.2% for ages 26-29, whereas past-year opioid misuse ranges from 3.4% to 5.8%, for these age groups respectively.Nearly one in three adolescents who report prescription opioid misuse by age 18 transition to heroin use in young adulthood.AYAs who misuse opioids are at increased risk for adverse health outcomes6 such as fatal/non-fatal injury, overdose, and opioid use disorder, warranting approaches designed to mitigate these consequences. U.S. emergency departments have over 130 million visits annually and the ED is a key location to bridge the divide to increase access to services and connection to the larger health system among at-risk AYAs who often are not continually connected to healthcare providers.Despite the current US opioid crisis, early interventions for AYAs focused on preventing opioid misuse/opioid use disorder are generally lacking in healthcare settings and research has called for more robust strategies, including those that use health coaching, focused on opioids.Although ED-based brief motivational interventions delivered by counselors of varying training backgrounds reduce other substance use/consequences among AYAs, their impact when tailored for AYA opioid misuse remains to be seen, lending to the focus of this trial. Our emphasis on tailored motivational interventions is underscored by prior work finding primary efficacy of a single-session brief motivational intervention in reducing opioid misuse and overdose risk behaviors in adult ED patients.We also demonstrated the secondary efficacy of ED and primary care-based brief interventions on reducing AYAs’ prescription drug misuse . We blended and packaged the content from these promising interventions as an initial intervention strategy in this trial. Our delivery approach for this early intervention is designed to increase the likelihood of implementation in busy medical settings, by using remote health coaches , allowing for real-time personalization and maximizing shelf-life to adapt to this rapidly changing crisis, with limited impact on ED staff. Due to the short-term and modest effects of the prior interventions mentioned above, we are also testing a strategy wherein health coaches are providing MI-based interventions for 30 days post-ED visit using a chat-based web portal. This portal mirrors the messaging style, and increasingly the function, of health systems’ patient portals, which promotes future implementation and scalability. Indeed, our prior work demonstrated the potential of this approach by delivering motivational interviewing-based 23 content in a portal-like platform to enhance motivation to seek mental health services for suicide prevention and others have used a portal to deliver alcohol-related feedback.After evaluating feasibility and acceptability of our ED-initiated interventions among AYAs screening positive for recent prescription opioid use with at least one risk factor or prescription or illicit opioid misuse we initiated a 2 x 2 factorial randomized controlled trial . The primary aim of the RCT is to evaluate the efficacy of 1) an intake condition of a remote health coach-delivered single session brief motivational intervention vs. a control condition of an enhanced usual care community resource brochure; and, 2) post-intake health coach-delivered portal-like messaging via a web portal over 30 days or EUC delivered at 30 days post-intake. Testing the relative efficacy of the health coach session, health coach session combined with the portal, or the portal intervention alone has high potential for public health impact by identifying the most effective combination of strategies to reduce primary outcomes of opioid misuse severity.