The Effects And Mechanisms Of Peaonol And ZBD-2 In Chronic Inflammatory Pain Induced By Complete Freund’s Adjuvant In Mice

ObjectiveChronic pain is a common symptom in Department of orthopedics.Since the central regulation mechanism is not well-known,the treatment is very difficult.The root bark of Paeonia suffruticosa(Paeoniaceae),a traditional Chinese herb Cortex Moutan,has been used as an analgesic and anti-inflammatory drug,and also has a beneficial effect in prevention and treatment of thromboembolic diseases.Paeonols are the major anti-inflammatory compound of the radix of P.suffruticosa.But the mechanical of analgesic in chronic pain in central is unknown.ZBD-2 is a novel ligand for TSPO synthesized in our laboratory(Chinese patent number 201210047188.6).The activation of TSPO by ZBD-2 attenuates chronic pain-induced anxiety-like behaviors by regulating the balance between GABAergic and glutamatergic transmission.However,less is known about the effects of paeonol and ZBD-2 on the development of anxiety in animals with chronic pain.In present study,we designed to investigate the anxiolytic effects of paeonol and ZBD-2 in mice with chronic pain induced by CFA,and clarify the underlying mechanisms.Method Method of Paeonol attenuate chronic inflammatory pain induced by complete Freund’s adjuvant(CFA)in mice:Mouse model of chronic inflammatory pain was induced by an injection of 10μl of 50% CFA into the plantar surface of the left hind paws of mice.The mechanical hypersensitivity threshold was evaluated by using a set of von Frey filaments with the up-down method.Thermal threshold of mice was measured by hot plate experiment.The distance travelled and time in the center areas of mice were recorded by a camera in open field.Effects of paeonol on levels of synaptic proteins expression in the ACC were analyzed by Western Blot.The expression of S6 and p-s6 in ACC were detected by immunofluorescence.Anxiolytic-like effects of translocator protein(TSPO)ligand ZBD-2 in an animal model of chronic pain:Mouse model of CFA-induced chronic inflammatory pain was induced by an injection of 10μl of 50% CFA into the plantar surface of the left hind paws of mice.The mechanical hypersensitivity threshold was evaluated by using a set of von Frey filamentswith the up-down method.Thermal threshold of mice was measured by hot plate experiment.A dial thickness gauge was used to measure the ipsilateral paw thickness of CFA injection of the mice.The number of entries in opened and closed arms as well as time spent in each arm were recorded by a camera in elevated plus maze(EPM).The distance travelled and time in the center areas of mice were recorded by a camera in open field.Effects of ZBD-2 on levels of synaptic proteins expression in the BLA were Analyzed by Western Blot.We performed whole-cell patch-clamp recordings and recorded miniature excitatory postsynaptic currents(m EPSC)and the miniature inhibitory postsynaptic currents(m IPSC)in the pyramidal neurons of BLA after three weeks since the CFA injection.ResultTreatment of paeonol notably increased paw withdrawal threshold of mechanical allodynia and the paw withdrawal latency of the thermal hyperalgesia.CFA injection or the subsequent treatment of paeonol did not change the total travel distance as compared with the control mice in the open field test.It showed that paeonol reversed the up-regulation of NR2B-containing NMDARs in CFA injected mice in the ACC,while no effect on the NR2 A expression.Furthermore the expression of Glu R1,p-Glu R1-Ser831 and Ca MKIIαwere significantly reduced,but no significant changes in p-Glu R1-Ser845 expression after paeonol administration in CFA mice.no significant differences were observed in the levels of GABA A-α2.paeonol reversed the up-regulation of p-m TOR,p-S6 K,p-S6 and p-4EBP in CFA-injected mice in the ACC,However,the CFA injection and paeonol did not produce significant changes in the levels of total m TOR,S6 K,S6 and 4EBP.The immunohistochemistry results confirm the changes of S6 and p S6 in ACC.The western blotting result also showed that paeonol reversed the up-regulation of p-ERK1/2 and p-CREB in CFA-injected mice in the ACC.However,the CFA injection and paeonol did not produce significant changes in the levels of total ERK1/2 and CREB.We found that anxiolytic doses of ZBD-2(0.15 and 1.5 mg/kg)did not reduce mechanical allodynia and thermal hyperalgesia in CFA mouse;Paw edema was increased after CFA-injected,ZBD-2 did not change edema in CFA-injected hindpaws.In the EPM test,mice spent less time in the open arms,and the number of open arm entries decreased after injected with CFA 21 days,the time in the central area was also decreased in the open field(OF)tests,ZBD-2 can reversed the above result dose dependently.However,the totalnumber of arm entries in the EPM tests and the total distance traveled in the OF tests did not change significantly,as compared with the controls.Levels of TSPO in the BLA were increased on day 21 after hindpaw CFA injection,treatments of ZBD-2 significantly decreased the upregulation of TSPO in the BLA.Treatment with ZBD-2 significantly reversed the up-regulation of NR2A-and NR2B-containing NMDARs,Glu R1 receptors,and the excitatory synaptic protein Ca MKII in the BLA from CFA-injected mice.CFA and ZBD-2 did not affect the levels of GABAA-α2 and GAD67 in BLA.Treated with ZBD-2down-regulated the frequency and amplitude in m EPSC.Frequency of m IPSC could not detect significant differences among the mice from different treatment groups.However,treated with ZBD-2(1.5 mg/Kg)could reversed the up-regulated of amplitude in m IPSC in CFA mice.ConclusionIn the present study,Paeonol(80 mg/Kg)was produced significant analgesic effects in the animal model of inflammatory pain.We found that paeonol reversed the up-regulated the levels of NR2 B,Ca MKIIα,Glu R1,p-Glu R1-Ser831,ERK/CREB and m TOR pathway proteins in CFA mice in ACC,but have no effects on NR2 A,p-Glu R1-Ser845 and GABAA-α2.These dates indicating that paeonol reduces chronic inflammation pain induced by CFA-treated at least partially through regulating the AMPA,NMDARs,m TOR and ERK/CREB signaling pathways.Anxiety-like behaviors was found in mice after injected CFA 21 days.as the glutamine pathway proteins were changed,the balance between excitatory synaptic transmission and inhibitory synaptic transmission was also changed.Our findings suggested that activation of TPSO with ZBD-2 induced notable anxiolytic effects in mice with chronic pain,but did not alter the nociceptive threshold and the inflammation.The anxiolytic effects of ZBD-2 were related to activation of TSPO receptor and rebalance the glutamine and GABA transmission in the BLA.