Dihydroartemisinin Inhibits Morphine-mediated Microglia Activation

Objectives:Morphine has been widely used as a classical opioid analgesic,however,long-term use of morphine can result in analgesic tolerance,thus heavily restricting its application in clinical practice.It was found that the activation of microglia acts as a critical player in morphine tolerance.Recently,there has been an increasing interest in the exploration of drugs that exsert suppressive effects on microglia activation.Artemisinin,which is commonly known as a firstline antimalarial agent,also possesses anti-inflammatory and anti-tumor properties.Previous studies have demonstrated that dihydroartemisinin(DHA)can inhibit the activation of microglia induced by lipopolysaccharide(LPS).Nevertheless,it still remains obscure whether DHA can extenuate morphine tolerance through the inhibition of microglia activation.In view of this,this study was designed,aiming to investigate the efficacy of DHA in the activation of microglia-mediated by morphine as well as discuss its hidden mechanism,thus providing a preliminary exploration of whether DHA can alleviate morphine tolerance.Methods:In order to figure out the relationship among DHA,microglia activation and morphine tolerance,we comprehensively and systematically investigated the efficacy of DHA in microglia activation and morphine tolerance through implementing a series of in vitro experiments.An in vitro morphine tolerance model was established by administrating BV-2microglial cells with morphine and minocycline was utilized as the positive control group.Initially,BV-2 microglial cells were administrated with varying concentrations of DHA(5,10,20 and 40μM)and cell viability was assessed by CCK-8 to determine the optimal concentration of DHA given to BV-2 microglial cells.In morphine-mediated BV-2 microglial cells with different concentrations of DHA pre-treatment,western blot was applied for the measurement of IBa-1 and ELISA was adopted to evaluate the levels of inflammatory factors,including IL-1β,IL-6 and TNF-α,intending to identify the role that DHA acts in the inhibition of morphine-mediated microglial activation in vitro.With the application of qRT-PCR,the role that miR-16 plays in morphine-mediated microglia activation was further investigated.In addition,the effects of DHA on microglial activation were observed using miR-16 inhibitor to treat BV-2 microglial cells.According to the Starbase database,miR-16 can bind to TLR4,which was subsequently confirmed by the luciferase report assay.Moreover,the activation of the TLR4/NF-κB signaling pathway in BV-2 microglial cells by DHA was observed in vitro using miR-16 inhibitor.Results:Compared with the Control group,the viability of BV-2 microglial cells was reduced with the increase of DHA concentration.DHA with a concentration of 40μM contributed to the lowest cell viability(P<0.01).Notably,the inhibitory effects of DHA with a concentration of20μM on the viability of morphine-pretreated cells were similar to that of minocycline,in this way,20 μM DHA was chosen to treat BV-2 microglial cells in the following experiments.In morphine-mediated BV-2 microglial cells with different doses of DHA pretreatment,the protein level of IBa-1 was declined in a concentration-dependent manner(P<0.01-0.001).In comparison with the Control group,the levels of IL-1β,IL-6 and TNF-α were markedly elevated by morphine administration(P<0.001).Compared with the Morphine group,DHA treatment dose-dependently diminished the levels of IL-1β,IL-6 and TNF-α(P<0.05-0.001).Besides,morphine administration greatly ascended the mRNA levels of IL-1β,IL-6 and TNF-α in contrast with the Control group(P<0.001),which were then rapidly declined by DHA administration when compared to the Morphine group(P<0.05-0.001).It was also discovered that minocycline could cut down the mRNA levels of IL-1β,IL-6 and TNF-α in cells(P<0.001).Results obtained from qRT-PCR revealed that morphine treatment conspicuously descended miR-16 level in BV-2 microglial cells when compared to the Control group(P<0.001),which was then reversed by DHA with varying concentrations(P<0.05-0.001).According to ELISA,miR-16 inhibitor could greatly reduce the suppressive effects of DHA on the activation of BV-2 microglial cells(P<0.001).Luciferase report assay showed the binding sites of TLR4 and the miR-16.In contrast with Control group,morphine remarkably enhanced the protein level of TLR4(P<0.001);in comparison with Morphine group,DHA rapidly reduced TLR4 expression in BV-2 microglial cells(P<0.001),which was partially abolished by miR-16 inhibitor(P<0.001).What’s more,morphine conspicuously ascended NF-κB expression in contrast with the Control group(P<0.05);in comparison with the Morphine group,DHA hugely cut down NF-κB expression(P<0.05)while miR-16 inhibitor exhibited opposite effects on NF-κB in morphine-mediated BV-2 microglial cells with DHA administration(P<0.05).Conclusion:This study was the first to reveal that DHA suppressed microglia activation via regulating miR-16 expression and blocking TLR4/NF-κB signaling pathway.It was also uncovered that DHA effectively attenuates the development of chronic morphine tolerance by inhibiting microglia activation and neuroinflammation,which provides a theoretical basis for the alleviation of morphine tolerance.Elsewhere,this study also offers novel targets and insights into reducing the side effects of morphine as well as expanding its application in clinic.