| Opioids are the most commonly used analgesics in clinic and have a strong analgesic effect.However,when opioids exert their powerful analgesic effect,they are also accompanied by drug addiction,tolerance,respiratory depression,constipation and other side effects,which greatly limit their clinical use.Therefore,how to make opioid drugs produce strong analgesic with less side effects has become the focus of researchers.The classical opioid receptors are divided into three types: μ,δ and κ[3].When opioids activate μ opioid receptors(MOR),Gαi/o and Gβγ subunits become dissociated,thus leading to inhibition of adenylyl cyclase(AC)and opening of Na+,K+ ion channels.After being activated,the opioid receptor will be phosphorylated by GPCR kinases(GRKs)and sequentially result in β-arrestin2 recruitment,receptor endocytosis,and additional kinase-driven signaling events.Until now,many proteins have been found to participate in the regulation of MOR.They regulate the function of MOR by altering the modification of MOR,affecting the downstream signaling of MOR or affecting one of the steps in the process of MOR activation.Previous study in our laboratory showed that A20 binding inhibitor of NF-κB activation 1(ABIN-1)could regulate the function of MOR.We further found that ABIN-1 always cooperated with TNF alpha induced protein 3(A20)in many signaling pathways by searching literatures.Current studies have shown that A20 played important roles in inflammation by inhibiting inflammation-related signaling pathways,and A20 also related to the occurrence of a variety of immune diseases.However,there is few researches about the function of A20 in the central nervous system.Recently,it has been reported that mi R-873a-5p could affect morphine tolerance by targeting A20,but the mechanism is still unclear.Therefore,we hypothesized that A20 could affect the function of MOR that the same as ABIN-1.Accordingly,in this project,we intend to unravel whether A20 could affect the function of MOR and further explore its possible mechanism,which will contribute to design and synthesis of opioid analgesics with less side effects.Objective Comfirming whether A20 could affect the function of MOR and unravel its possible mechanism,which will help designing new analgesics with less side effects.Methods1.Explore the effect of A20 on morphine analgesia and tolerance by using hot plate and hot tail flick methods 1.1 Morphine analgesia experiment by using hot plate test After overexpression or knockdown of A20 in the spinal cord of mice,the changes of pain response time were compared by using hot plate test to reflect the change of morphine analgesia,and the effect of A20 on morphine analgesia was evaluated.1.2 Morphine chronic tolerance experiment by using hot plate test After overexpression or knockdown of A20 in the spinal cord of mice,the changes of morphine analgesia were compared in the chronic tolerance model by using hot plate test,and the effect of A20 on morphine chronic tolerance was evaluated.1.3 Morphine analgesia experiment by using hot tail flick test After overexpression or knockdown of A20 in the spinal cord of mice,the changes of the reaction time of tail flick by using hot tail flick test were used to reflect the change of morphine analgesia,and the effect of A20 on morphine analgesia was evaluated.1.4 Morphine chronic tolerance experiment by using hot tail flick test After overexpression or knockdown of A20 in the spinal cord of mice,the changes of morphine analgesia were compared in the chronic tolerance model by using hot tail flick test,and the effect of A20 on morphine chronic tolerance was evaluated.1.5 Morphine acute tolrance experiment by using hot plate test A20 was overexpressed in the spinal cord of mice,the changes of morphine analgesia were compared in the morphine acute tolerance model by using hot plate test,and the effect of A20 on morphine acute tolerance was evaluated.2.Effect of A20 on the AC-c AMP-PKA signaling pathway and phosphorylation of MOR S375 2.1 Effect of A20 on cyclic adenosine monophosphate(c AMP)of the MOR downstream After overexpression or knockdown or rescuing A20 in the MOR-293 T cells individually,the changes of c AMP were detected by using the LANCE c AMP Assay and Glo Sensor c AMP Assay,and the effect of A20 on c AMP of the MOR downstream was evaluated.2.2 Effect of A20 on Protein kinase A(PKA)redistribution of the MOR downstream After overexpression A20 or A20 C103 A mutants in the MOR-PKA-Green fluorescent protein(GFP)-CHO cells,the redistribution of fluorescent PKA in cells was analyzed by high content analysis instruments to evaluate the effect of A20 on PKA of the MOR downstream.2.3 Effect of A20 on phosphorylation of MOR S375 After overexpressed or knockdown of A20 in the MOR-CHO cells,the effect of A20 on the phosphorylation of MOR S375 was analyzed by Western blot.3.A20 enhanced the function of MOR through the mechanism of inhibiting the recruitment of β-arrestin2 which affected endocytosis of MOR 3.1 A20 interacting with β-arrestin2 3.1.1 Co-immunoprecipitation A20 interacting with β-arrestin2 After overexpression of β-arrestin2 and A20 in the 293 T cells,the interaction of A20 and β-arrestin2 was detected by using co-immunoprecipitation experiment.3.1.2 His-Pull down After of overexpression of A20 in the 293 T cells,the interaction between A20 and His purified β-arrestin2 was detected by using co-immunoprecipitation experiment.3.1.3 Nano Bit Assay After overexpression of A20 and β-arrestin2 with luciferase fragments in the 293 T cells,the interaction between A20 and β-arrestin2 was studied by detecting the change of luminescence value in the system.3.1.4 Immunofluorescence After tranfection of A20 and β-arrestin2 which were connected to fluorescent proteins with different colors in the 293 T cells,the interaction between A20 and β-arrestin2 was evaluated by observing the distribution of fluorescence in the cells.3.2 The effect of A20 on the recruitment of β-arrestin2 3.2.1 High content analysis After overexpression of A20 in the MOR-β-arrestin2-GFP-CHO cells,the effect of A20 on the recruitment of β-arrestin2 was evaluated by using high content analysis of fluorescent.3.2.2 Nano Bit Assay After overexpression of A20,MOR and β-arrestin2 with luciferase fragments in the 293 T cells,the effect of A20 on the recruitment of β-arrestin2 was evaluated by detecting the changes of the luminescence value of the system.3.3 Effect of A20 on MOR receptor endocytosis 3.3.1 MOR on membrane was labeled with fluorescent antibody After overexpression of A20 in the SNAP-MOR-CHO cells,MORs on cell membrane were labeled with fluorescent antibodies,and the effect of A20 on MOR endocytosis was evaluated by detecting changes of fluorescence value.3.3.2 Isolation of cell membrane proteins After overexpression of A20 in the spinal cord of mice or in the SNAP-MOR-CHO cells individually,the membrane proteins were separated,and the effect of A20 on MOR endocytosis was evaluated by detecting the MOR content in the membrane.3.4 Effect of knockdownt β-arrestin2 on the function of A20 3.4.1 β-arrestin2 knockout mice The A20 was overexpressed in the spinal cord of β-arrestin2 knockout mice,and the analgesic effect of morphine was measured by hot plate test to evaluate the effect of knockout β-arrestin2 on the function of A20.3.4.2 Glo Sensor c AMP Assay A20 was overexpressed in β-arrestin2 knockdown MOR-293 T cells or β-arrestin2 was knocked down in A20 overexpressed MOR-293 T cells,the effects of knockdownt β-arrestin2 on the function of A20 was evaluated by using hot plate test and Glo Sensor c AMP Assay.Results 1.A20 enhanced morphine analgesia,inhibited acute tolerance but not chronic tolerance 1.1 A20 enhanced the analgesic effect of morphine Hot plate test results showed that the analgesic percentage of mice overexpressed A20 group is significantly higher than the control group,indicating that A20 could enhance the analgesic effect of morphine.The analgesic percentage of mice in the group of knockdown A20 was decreased compared with the control group,indicating that knockdown A20 weakened the analgesic effect of morphine.1.2 A20 did not affect the chronic tolerance of morphine Results of the hot plate test and the hot radiation tail flick test showed that the mice developed morphine chronic tolerance after overexpression or knockdown A20,indicating that A20 had no effect on the formation of chronic tolerance.1.3 A20 inhibited acute morphine tolerance Hot plate test results showed that after high doses of morphine treatment,the mice developed acute morphine tolerance,but the degree of acute tolerance of mice overexpressed A20 was less than the control group,indicating that A20 could inhibit the formation of morphine acute tolerance.2.A20 enhanced the AC-c AMP-PKA signaling pathway of the of MOR downstream and inhibited S375 phosphorylation 2.1 A20 enhanced the inhibitory effect of MOR on the c AMP-PKA pathway The results of LANCE c AMP Assay and Glo Sensor c AMP Assay showed that overexpression of A20 enhanced the inhibition of MOR on its downstream c AMP,and knockdown of A20 weakened this inhibition,indicating that A20 could enhance MOR’s inhibition of c AMP.The results of PKA redistribution experiments showed that overexpression of A20 could enhance the inhibition of MOR on PKA,and overexpression of A20 C103 A mutant had no such effect.2.2 A20 inhibited phosphorylation of MOR S375 Results showed that overexpressed A20 could inhibit the phosphorylation of MOR S375.After knocking down A20,the phosphorylation of MOR was increased.These results indicating that A20 could inhibit phosphorylation of MOR S375.3.A20 enhanced the function of MOR through the mechanism of inhibiting the recruitment of β-arrestin2 which affected endocytosis of MOR 3.1 A20 interacted with β-arrestin2 The results of co-immunoprecipitation and Nano Bit Assay showed that there was an interaction between A20 and β-arrestin2,and the interaction was further enhanced when treated with agonist.Immunofluorescence results showed that A20 and β-arrestin2 co-localized in the cells.3.2 A20 inhibited β-arrestin2 recruitment High content analysis and Nano Bit Assay results showed that the recruitment of β-arrestin2 in the A20 overexpressed group was significantly less than the control group,indicating that A20 could inhibit the recruitment of β-arrestin2.3.3 A20 inhibited MOR endocytosis The results of fluorescent antibody-labeled membrane protein assay showed that the number of MOR on the A20 overexpressed cell membrane increased significantly compared with the control group,indicating that A20 inhibited the endocytosis of MOR.3.4 A20 could not affect the fuction of MOR whenknocking down β-arrestin2 Results showed that after knocking down β-arrestin2 both in mice or in the cells,A20 could no longer enhance the function of MOR.It is indicated that A20 enhanced the function of MOR through β-arresin2.Conclusions 1.A20 could enhance the analgesic effect of morphine and inhibit the formation of morphine acute tolerance,but not the chronic tolerance of morphine.2.A20 could enhance the agonist-induced inhibitory effect of MOR on the ACc AMP-PKA signaling pathway.3.A20 could inhibit β-arrestin2 recruitment by interacting with β-arrestin2,thereby inhibit the endocytosis of MOR and increase the amount of MOR on the cell membrane.4.A20 affected the function of MOR through β-arrestin2. |
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