The Experimental Study On The Analgesic Effect And Mechanisms Of Agmatine On The Inflammatory Pain

Part one The analgesic effect of agmatine on inflammatorypain and its influence on the analgesic effect of morphine Objective To evaluate the analgesic effect of agmatine on the model of formalin-evoked nociception and its influence on the analgesic effect of morphine. To investigate whether the mechanisms of analgesic effect of agmatine is related to activation of imidazoline recepter. Methods Male SD rats weighing 180-220g were randomly classified into groups. Rats were injected subcutaneously with 50 μL of 5% formalin solution into the plantar surface of the right hind paw. During the next 60min, the time spent in licking and biting the injected paw was counted continuously every 5 min and the weighted pain score was recorded in 5-min intervals. Agmatine was administered subcutaneously (s.c.) 20 min or intraperitoneally (i.p.) 15min before intraplantar injection of formalin. Morphine (1mg/kg, s.c.) was injected 15min before the formalin injection and idazoxan was given 5 min prior to agmatine. Results Intraplantar subcutaneous injection of formalin produced a typical biphasic nocifensive response. Intraperitoneally pretreatment with agmatine dose dependently inhibited the second, but not the first phase of the nocifensive and hyperalgesic response (P<0.01). Agmatine administered subcutaneously has much weaker anti-nocifensive effect. In phase II, but not phase I, agmatine (i.p. or s.c.) pretreatment together with morphine had significantly higher analgesic effect than the individual compounds by themselves (P<0.05). Idazoxan (5mg/kg, i.p.) did not affect the nocifensive responses when administered alone, nor did it attenuate the antinociceptive effect of agmatine (P>0.05). However, idazoxan inhibited the analgesic effect of morphine, so attenuated the synergic effect of agmatine and morphine (P<0.01). Conclusion Agmatine induces a dose-dependent analgesia and enhances morphine analgesia in the second but not the first phase of formalin-induced nocifension. Imidazoline recepter may not to play a major role in the mechanism of the analgesic effect ofagmatine on inflammatory pain.Part two Mechanisms of the analgesic effect of agmatine on inflammatory painExperiment 1 Effects of agmatine on basal and K* evoked release of endogenous neurotransmitters from rat spinal cord slices Objective To investigate the effects of agmatine on basal and K+ evoked release of endogenous glutamate and gamma-aminobutyric acid (GAB A) from rat spinal cord slices so as to explore the possible mechanism of the analgesic effect of agmatine in fore-receptor level. Methods Spinal cord slices were prepared from SD rats weighing 180-220g and were randomly divided into 5 groups. Immediately after each slice was cut, it was transferred into artifical cerebrospinal fluid (ACSF) which was continually bubbled with 95% O₂ and 5% CO₂ and maintained at 37℃ for equilibration for 40 min. The incubation medium (0.5mL) was renewed every 10min. The supernatant within 10 min of incubation was collected and measured as basal release levels. To investigate the effects of agmatine on basal and K⁺ evoked release of glutamate and GAB A, the incubation medium was replaced with ACSF (0.5mL) containing agmatine at concentrations of 0, 1, 10, 100, 1000 umol/L and incubated for 10 min and the supernatant was collected. Then the slices were incubated with 50mmol/L KC1 (Na⁺ reduced to maintain osmolarity) containing the same concentrations of agmatine for another 10 min. The concentrations of glutamate and GAB A in collected supernatants were determined by high performance liquid chromatography (HPLC). Results Agmatine at concentrations of 1, 10, 100, 1000 umol/L had no effect on the basal release of glutamate and GABA from spinal cord slices. Potassium (50 mmol/L) evoked a 1.77±0.75 and 3.99± 1.15-fold increase in glutamate and GABA levels, respectively. Agmatine 1-1000 umol/L had no effect on the K⁺ evoked release of glutamate and GABA contents. Conclusion The mechanism of the analgesic effect of agmatine may not be associated with inhibitting glutamate release nor increasing the GABA content. Experiment 2 Effects of agmatine on hippocampal neuronal calcium overload evoked by glutamate or KC1Objective To investigate the effects of agmatine on basal [Ca²⁺]; level and glutamate or K⁺ evoked increase of [Ca²⁺]i from primarily cultured rat hippocampal neurons so as to explore the effects of agmatine on N-methyl-D-aspartate (NMDA) receptor and voltage-dependent calcium ion channel (VDCC). Methods Hippocampal neurons of neonatal rat were cultured in vitro for 10 days, then the culture medium were loaded with agmatine at concentrations of 0, 1, 10, 100, 1000 umol/L, and then the neurons were exposed to 300 umol/L glutamate or 50 mmol/L KC1. Fluo-3, a fluorescent probe, was used imaging of intracellular calcium in laser scanning confocal microscope (LSCM) to measure the real-time changes of [Ca²⁺]i evoked by glutamate or KC1 and the influence of agmatine on the changes. Result [Ca²⁺]i was increased in the presence of 300 umol/L glutamate or 50 mmol/L KC1. Though agmatine at concentrations of 1¹000 umol/L displayed no significantly effect on basal [Ca²⁺]i, it inhibited [Ca²⁺]i elevation caused by 300 umol/L glutamate, an agonist of the NMDA receptor. The maximal changing rate of fluorescence intensity were reduced from 372% to 349%, 327%, 297%, 233%, respectively. However, only 1000 umol/L agmatine but not lower concentrations could inhibit calcium overload evoked by 50 mmol/L KC1 which induced the opening of the VDCC. Conclusion The mechanism of the analgesic effect of agmatine on inflammatory pain might be partially related to the inhibition of [Ca²⁺]i overload via the NMDA receptor calcium ion channels. Experiment 3 Effect of agmatine on the change of protein kinase C gamma expression in the rat spinal cord in the formalin testObjective To validate the changes in the expression of protein kinase C gamma (PKCγ) in the dorsal horn of the spinal cord in the inflammatory pain and study the effect of agmatine on the PKCγ activation and translocation. Methods Male SD rats weighing 180-220g were randomly divided into 3 groups: (1) control.intraplantar injection of 50 μL saline; (2) formalin group: intraplantar injection of 50 μL of 5% formalin; (D agmatine-formalin group: 160mg/kg agmatine was administered intraperitoneally 15 min before formalin injection. The animals were decapitated at 10 min after intraplantar injection and L—(4,5) spinal cords were dissected. Immunohistochemistry, immunofluorescence and western blot analyses were used to observe the localization of PKCγ in the dorsal horn of spinal cord and the changes of its expression and the effect of agmatine on the changes of their expression. Results PKCγimmunoreactivity was concentrated in the ventral part of lamina II and the border between laminae II and III. Formalin-induced nociception up-regulated the expression of membrane-associated PKCγ (P<0.01). The increases were seen bilaterally, but were more prominent ipsilaterally (1.56 fold vs. 1.19 fold). Pretreatment with 160mg/kg agmatine significantly inhibited the up-regulation of PKCγ in the membrane fraction (P<0.01). On the other hand, the content of PKCγ in the cytosolic fraction in formalin-induced animals was almost the same as that in controls nor did agmatine has any effect on it (P>0.05). Conclusion Up-regulation and translocation of PKCy in the plasma membrane following formalin stimulation of the hind paw suggested that PKCy may play a role in the central mechanism of hyperalgesia that follows peripheral inflammation. The inhibition of PKCy expression and translocation may contribute to the antinociceptive effects of agmatine.Experiment 4 Effect of agmatine on the change of pERK expression in the rat spinal cord in the formalin testObjective To investigate the changes in the expression of phosphorylated extracellular signal-regulated protein kinase (pERK) in the dorsal horn of the spinal cord in the model of formalin-induced inflammatory pain and study the effect of agmatine on the pERK expression. Methods Twenty-four male SD rats weighing 180-220g were randomly divided into 3 groups. The method was the same as experiment 3. The animals were decapitated at 8 min after intraplantarinjection and L_4,5 spinal cords were dissected and coronal sections (4 μm) were cut. Immunohistochemistry was used to observe the changes of pERK expression in the dorsal horn of spinal cord. Results Injection of 5% formalin subcutaneously in the hindpaw of rats induced an increase in pERK expression in neurons in the superficial dorsal horn on the ipsilateral side of the L_4,5 spinal cord (P<0.01). The pERK-labeled neurons were predominantly localized in laminae I and II, and the pERK was present in the nucleus and cytoplasm. pERK had little basal expression and no induction was found in the contratateral side. Pretreatment with 160mg/kg agmatine significantly inhibited formalin-induced spinal pERK increase (P<0.01). Conclusion pERK activation may underlie spinal nociceptive processing and secondary hyperalgesia after formalin inflammation and may contribute to the antinociceptive effects of agmatine.Experiment 5 Effect of agmatine on the change of pCREB expression in the rat spinal cord in the formalin testObjective To investigate the changes in the expression of phosphorylated cAMP response element binding protein (pCREB) in the spinal cord in the model of formalin-induced inflammatory pain and study the effect of agmatine on the pCREB expression. Methods The method was the same as experiment 3. The animals were decapitated at 20 min after intraplantar injection and L_4,5 spinal cords were dissected and homogenized. Western blot analyses were used to observe the changes of pCREB expression in the spinal cord. Results Phosphorylated CREB increased in rat spinal cord as a response to formalin-induced nociception (P<0.01). The increase was seen bilaterally but was greater on the ipsilateral side (1.55 fold) compared to the contralateral side (1.15 fold). Pretreatment with agmatine could markedly suppress the phosphorylation (P<0.01). Conclusion pCREB up-regulation may be responsible for the development of hyperalgesia induced by formalin and may play a role in the analgesic effect of agmatine.