RoMS Continuously Activates Dopamine Receptors To Alleviate LPS-induced Liver Injury In Mice Via β-arrestin2 Dependent Akt/NF-κB Pathway

Objective: Dopamine（DA）,an important neurotransmitter in the brain,plays an important role in the interaction of the central nervous system and the immune system.Many studies showed that dopamine receptors（DRs）agonists have anti-inflammatory effects.Lipopolysaccharide（LPS）,an immune activator,can activate immune signaling pathways in liver macrophages and induce inflammatory damage in the liver.Rotigotine,a non-ergot dopamine receptor agonist,mainly actives DRD2,dopamine 1 receptor and dopamine 3 receptor.Rotigotine extended-release microspheres（RoMS）are a sustainedrelease formulation that can release sustainably rotigotine for more than 7 days after a single dose of RoMS administration.Based on the regulatory relationship between DRs signal and the immune system,several previous studies have demonstrated the antiinflammatory effects of DA or DRs agonists in some central or peripheral immunerelated diseases.However,the effect of continuous activation of DRs on inflammatory liver injury is unclear,therefore this study aimed to investigate whether RoMS can attenuate the LPS induced inflammatory liver injury in mice.Methods: According to the drug release characteristics of RoMS,this study was performed at 3 or 7 days after RoMS administration.C57BL/6 mice were randomly divided into the following 8 groups: control group,model group,dexamethasone（DXM）group,RoMS 5 mg/kg group,RoMS 10 mg/kg group,RoMS 20 mg/kg group,DRD2-specific blocker raclopride（R121）group,RoMS 5 mg/kg + R121 group.Mice in the control,model,DXM,and R121 groups were injected with CMC-Na via intramuscular injection.The mice in the RoMS group were injected with RoMS by intramuscular injection,and mice in the RoMS 5 mg/kg+R121 group were injected intraperitoneally with R121 at 30 minutes before RoMS administration.At 2.5 days or 6.5 days after RoMS injection,mice were injected intraperitoneally with LPS（10 mg/kg）except the mice in control group.At 12 h after LPS injection,all mice were anesthetized and whole blood was collected.Then,serum was isolated and the levels of AST,ALT and proinflammatory cytokines（TNF-α and IL-6）were measured.The livers of mice were isolated,of which the right lobe part of the liver was used for histopathological examination,and the rest of the liver was frozen at-80℃ for assaying the expression of protein of NF-κB signaling pathway.Results:（1）Compared with the control group,the serum AST and ALT levels of mice in the model group were significantly increased（P < 0.01）.Compared with the model group,the serum AST and ALT levels of mice in the DXM group were significantly reduced（P < 0.01,P < 0.05）;the serum AST and ALT levels of mice in the RoMS 5mg/kg,10 mg/kg and 20 mg/kg groups were significantly decreased at 3 or 7 days after the pre-administration of RoMS（P < 0.05）;the serum AST and ALT levels of mice in the DRD2-specific blocker R121 group were not changed.Compared with the RoMS 5mg/kg group,the serum AST and ALT levels of mice in the RoMS 5 mg/kg+R121 group were significantly increased（P < 0.01）.（2）H＆E staining showed there were no abnormalities in the liver tissues of the control group mice.The livers of mice in the model group showed abnormal structures,such as extensive hemorrhage,massive hepatocellular edema and inflammatory cell infiltration.Compared to the model group,these abnormalities in the liver were significantly improved by pre-administration of RoMS for 3 and 7 days,such as slight hemorrhage,reduced inflammatory cell infiltration,and partial hepatocellular edema around the portal area and central vein.Liver damage was not improved in mice of the R121 group.Liver tissue damage was aggravated in mice in the RoMS + R121 group when compared with the RoMS 5 mg/kg group.（3）The levels of TNF-α and IL-6 in the model group increased after LPS exposure compared with the control group（P < 0.01）.The levels of TNF-α and IL-6 were significantly decreased in the DXM group compared with the model group（P < 0.01）.The levels of TNF-α and IL-6 were also significantly decreased at 3 and 7 days after RoMS preadministration（P < 0.01）.Serum TNF-α and IL-6 were not significantly altered in the R121 group of mice.Compared with the RoMS 5 mg/kg group,the serum TNF-α and IL-6 levels were significantly increased in the RoMS 5 mg/kg+R121 group mice（P < 0.01）.（4）The expression of TLR4 and IL-6 in liver tissues of mice in the model group was increased after LPS injection（P < 0.01,P < 0.05）.Pre-administration of RoMS reduced the expression of TLR4 and IL-6 in liver tissues.In contrast,R121 administration abolished the effect of RoMS on TLR4 and IL-6 expression.The phosphorylation levels of Akt,IκBα,and NF-κBp65 were increased in mouse liver tissues after LPS injection compared to the control group（P < 0.01,P < 0.05）.However,the phosphorylation levels of Akt,IκBα,and NF-κBp65 decreased after 3 and 7 days of RoMS pre-administration.R121 partially abolished the effect of RoMS on the protein expression of Akt/NF-κB signaling pathway.Liver injury induced by LPS decreased the expression of DRD2 andβ-arrestin2.However,RoMS increased the expression of DRD2 and β-arrestin2.R121 abolished the effect of RoMS on DRD2 and β-arrestin2 expression in mice.These results suggest that RoMS can negatively regulate the Akt/NF-κB signaling pathway by binding to β-arrestin2.These results mentioned above suggest that RoMS can continuously activate DRs and inhibit the secretion of pro-inflammatory cytokines,thereby attenuating the inflammatory response induced by LPS,and the protective effect of RoMS can last for 7 days,at least in part.Conclusion: The present study showed that RoMS can exert a protective effect in the LPS-induced liver injury via the activation of DRs.Activation of DRs alleviates the inflammatory liver injury by regulating the Akt/NF-κB pathway through binding to β-arrestin2.To elucidate the part mechanism of RoMS in treating LPS-induced liver injury in mice.This finding suggests that DRs may be drug targets exerting anti-inflammatory effects.Innovation: In this study,continuous activation of DRs was achieved with RoMS,and used LPS-induced liver injury as a model.It was demonstrated that continuous activation of DRs had a protective effect on LPS-induced inflammatory liver injury,which was achieved by activating DRs that negatively regulated the Akt/NF-κB signaling pathway through β-arrestin2.