Mechanism Of The Chinese Medicine Compound Huanglian Decoction Against Morphine Tolerance Based On Network Pharmacology And Microglia Activition

ObjectiveMorphine is a classic opioid and the most widely used.The major limitation of long-term morphine use is its gradual loss of efficacy,known as morphine tolerance.Huanglian Decoction is characterized by multiple components,targets and pathways,but its pharmacological mechanism in preventing and treating morphine tolerance remains unclear.In this study,effective compositions and possible targets of Huanglian Decoction were filtrated according to network pharmacological strategies.We established morphine tolerance model and morphine-mediated microglia cell activation model,so as to reveal the pharmacological mechanism of effective components of Huanglian Decoction in preventing and treating morphine tolerance.MethodsPart Ⅰ: Potential targets related to Huanglian decoction and morphine tolerance were identified from series databases.GO and KEGG enrichment analysis were performed for related genes and targets.The protein interaction network(PPI)diagram,Venny diagram,composition target signal pathway network diagram of Huanglian Decoction and morphine tolerance were constructed by String platform,Cytoscape and other programs.Auto Dock Vina software was used to conduct molecular docking of activated compositions and nuclear targets of Huanglian Decoction.The mechanism of effective components of Huanglian decoction against morphine tolerance was studied under the guidance of network pharmacology.Part Ⅱ:(1)Morphine-induced acute tolerance model: mice received intraperitoneal berberine at doses of 2.5,5.0,and 10 mg/kg;30 minutes later,subcutaneous morphine 10mg/kg was injected each hour for nine continuous hours.Morphine 10 mg/kg alone was administered at 24 and 48 hours.(2)Morphine-induced chronic tolerance model: mice received intraperitoneal berberine 2.5,5.0,and 10 mg/kg;30 min later,morphine was administered subcutaneously at 10 mg/kg for 8 days.On the ninth day,morphine 10 mg/kg was given alone.(3)Morphine-induced established tolerance model: mice were injected subcutaneously with morphine 10 mg/kg once a day for eight consecutive days.Berberine2.5 mg/kg was administered on day one,four,and seven and morphine 10 mg/kg alone on day nine.The baseline latency and post-treatment latency were determined by the hot plate test,and the maximum possible analgesic effect(MPAE)was calculated.At the spinal cord level,NOS activity,NO,TNF-α and IL-1β content were measured by the method of ELISA.The expression of protein CD86 presenting the activation of microglia was detected by Western blot.Part Ⅲ: BV2 cells were cultured in vitro and morphine-mediated microglia cell activation model were established.Cells were randomly into five groups: normal group,morphine group,and berberine low,medium,high dose groups(5,10,20 μmol/L).After incubating berberine for 2 h,all groups were given morphine 200 μmol/L for 24 h except for control group.The viability of BV2 cells was examined by the method of CCK-8.The morphology of BV2 cells was examined by microscope,the apoptosis of BV2 cells was viewed by Hoechst dye solution,oxidative stress of BV2 cells was observed by ROS probe;pro-inflammatory mediators(IL-1β,TNF-α)anti-inflammatory mediator(IL-10)levels were detected by ELISA.Western Blot assay was used to verify expression levels of protein CD86,NF-κB,Bax,Bcl-2,and cleaved casepase3.ResultsPart Ⅰ: Through network pharmacological screening and analysis,twenty-two active ingredients and ninety-six targets of Huanglian decoction were screened,among which the key components were berberine,quercitrin,kaempferol,stigmasterol,oxyberberine,canadine and the key targets were IL-1β、TNF-α、ROS.The main pathways involved include NF-κB,cell apoptosis signaling transduction pathway.The results of computer simulation of molecular docking proved that active ingredients of Huanglian Decoction had stable binding ability with the key target of morphine tolerance.Berberine,as a potential pharmacologically active ingredient,was used for the next verification.Part Ⅱ: Berberine 2.5 and 5.0 mg/kg could prolong the analgesic time of morphine(P<0.05).In acute and chronic morphine tolerance models,berberine could inhibit the decrease of % MPAE and baseline latency(P<0.05).In the established tolerance model,berberine could rapidly reverse the decreased % MPAE(P<0.05).The combination of berberine and morphine on day one could effectively inhibit morphine tolerance.Compared with the morphine group,the protein expression of CD86 and the levels of i NOS,NO,IL-1β,TNF-α of the berberine groups in the spinal cord were significantly decreased(P<0.05).Part Ⅲ: Compared with the morphine group,the cell viability of the berberine groups were significantly declined(P<0.05).The release of ROS was decreased obviously(P<0.05).The number of early apoptosis of BV2 cells was reduced(P<0.05).The content of pro-inflammatory glial mediators(IL-1β and TNF-α)were reduced,while the content of anti-inflammatory glial mediators(IL-10)were increased obviously(P<0.05).The expression of protein CD86,NF-κB and pro-apoptotic protein(Bax and cleaved-casepase3)were obviously reduced,whereas those of Bcl-2 increased(P <0.05).ConclusionBerberine was screened out to be the active compounds in Huanglian decoction,and could prolong the analgesic time of morphine and inhibit the development of morphine tolerance and hyperalgesia.Berberine could inhibit the activation of microglia,NF-κB signaling pathway,and reduce the release of pro-inflammatory cytokines.In addition,berberine could inhibit oxidative stress and apoptosis of microglia cells,which could be the one of the mechanisms of Huanglian decoction for treating morphine tolerance.