Epigenetic suppression of potassium-chloride co-transporter 2 expression in inflammatory pain induced by complete Freund's adjuvant (CFA).

BACKGROUND: Multiple mechanisms contribute to the stimulus-evoked pain hypersensitivity that may be experienced after peripheral inflammation. Persistent pathological stimuli in many pain conditions affect the expression of certain genes through epigenetic alternations. The main purpose of our study was to investigate the role of epigenetic modification on potassium-chloride co-transporter 2 (KCC2) gene expression in the persistence of inflammatory pain. METHODS: Persistent inflammatory pain was induced through the injection of complete Freund's adjuvant (CFA) in the left hind paw of rats. Acetyl-histone H3 and H4 level was determined by chromatin immunoprecipitation in the spinal dorsal horn. Pain behaviour and inhibitory synaptic function of spinal cord were determined before and after CFA injection. KCC2 expression was determined by real time RT-PCR and Western blot. Intrathecal KCC2 siRNA (2 µg per 10 µL per rat) or HDAC inhibitor (10 µg per 10 µL per rat) was injected once daily for 3 days before CFA injection. RESULTS: Persistent inflammatory pain epigenetically suppressed KCC2 expression through histone deacetylase (HDAC)-mediated histone hypoacetylation, resulting in decreased inhibitory signalling efficacy. KCC2 knock-down caused by intrathecal administration of KCC2 siRNA in naïve rats reduced KCC2 expression in the spinal cord, leading to sensitized pain behaviours and impaired inhibitory synaptic transmission in their spinal cords. Moreover, intrathecal HDAC inhibitor injection in CFA rats increased KCC2 expression, partially restoring the spinal inhibitory synaptic transmission and relieving the sensitized pain behaviour. CONCLUSION: These findings suggest that the transcription of spinal KCC2 is regulated by histone acetylation epigenetically following CFA. SIGNIFICANCE: Persistent pain suppresses KCC2 expression through HDAC-mediated histone hypoacetylation and consequently impairs the inhibitory function of inhibitory interneurons. Drugs such as HDAC inhibitors that suppress the influences of persistent pain on the expression of KCC2 may serve as a novel analgesic.

Intrathecal clonidine decreases spinal nitric oxide release in a rat model of complete Freund's adjuvant induced inflammatory pain.

A long-lasting antihyperalgesic effect has been demonstrated for intrathecal (IT) clonidine, an alpha2-adrenergic agonist. In the present study, the mechanism and antihyperalgesic effects of IT clonidine were examined post-treatment in a rat model of Complete Freund's Adjuvant (CFA)-induced inflammatory hyperalgesia. Using a chronic model of spinal cord dialysis, we examined the effect of the adjuvant-induced inflammation on spinal release of nitric oxide (NO) and the development of chronic pain and assessed the antinociceptive effects and mechanisms of the alpha2-adrenergic agonist, clonidine (IT). Chronic, persistent inflammatory pain was induced by left hind paw injection of 0.3 ml CFA prepared in a mixture with Mycobacterium butyricum. Rats were randomly assigned to groups receiving IT clonidine in discrete doses of 1, 10 or 50 microg, 3 or 24 hr post-inflammation. Measurement of total NOx (NO + NO2- + NO3-) was used to determine NO release into the cerebrospinal fluid. Rat thermal antinociception was assessed using a radiant heat thermal hyperalgesia model. CFA injection resulted in significant thermal hyperalgesia throughout the four days of observation. A dose-dependent suppression of thermal hyperalgesia and spinal NO release was observed after IT clonidine treatment. Evidence from this CFA-induced inflammatory pain model suggests that clonidine's spinal antihyperalgesic mechanisms act through inhibition of spinal NO release.

[Study of platelet aggregation affected by 8 classical recipes in rabbit].

Arachidonic acid can induce platelet aggregation in rabbits in vitro. The experimental model was designed to observe inhibitory effect of 8 classical recipes of activating blood circulation to remove the stasis on platelet aggregation in vitro. The results showed that each one of 8 classical recipes has inhibitory effect on platelet aggregation in vitro in various degree. Among them, Gexia Zhuyu Tang (GZT), Shentong Zhuyu Tang (SZT) and Shaofu Zhuyu Tang (SFZT) were more effective. The results suggested that inhibitory effect of platelet aggregation produced by 8 classical recipes might be one of mechanisms of blood stasis treatment.

[Effects of rhubarb on arachidonic acid metabolism of the renal medulla in the rabbit].

The effects of rhubarb on arachidonic acid metabolism in renal medulla of rabbits were studied in vitro with radiochromatography and radioautography. The results showed that radioactive agent was markedly reduced in the rhubarb group and significant difference vs the control was observed (P less than 0.01). The results indicated that rhubarb blocked the biosynthesis of renal medulla PGE2 and PGF2 alpha in a dose-dependent manner. Also the rhubarb inhibited the biosynthesis of PGA2 and TXB2. It was suggested that the site of action of the rhubarb is at cyclooxygenase level.

Effect of cholesterol feeding on tissue lipid perioxidation, glutathione peroxidase activity and liver microsomal functions in rats and guinea pigs.

The effect of cholesterol feeding on liver and aortic nonenzymatic lipid peroxidation and glutathione peroxidase activities, and on liver microsomal NADPH-dependent lipid peroxidation, codeine hydroxylation and cytochrome P-450 levels was examined in rats and guinea pigs. One percent cholesterol was added to a casein-sucrose-soybean oil basal diet for rats or a stock diet with 2% soybean oil for guinea pigs. The effect of vitamin E and cholestyramine was also examined in some experiments. Cholesterol feeding increased the rate of lipid peroxidation in liver and aortic homogenate both in rats and guinea pigs when fed non-vitamin E supplemented basal diets. Vitamin E supplementation prevented the increase in the aorta, but not as completely in the liver in rats, while the reverse was true in guinea pigs. The effect of cholestyramine was dependent on the level of vitamin E in the diet. Cholesterol feeding decreased glutathione peroxidase activities in rats and guinea pigs. In guinea pigs, cholesterol feeding also markedly decreased liver microsomal NADPH-dependent lipid peroxidation, codein hydroxylation and cytochrome P-450 levels especially when fed non-vitamin E supplemented basal diets. In rats, cholesterol feeding reduced liver microsomal NADPH-dependent lipid peroxidation and in some cases, increased microsomal codeine hydroxylation activities, but had no effect on microsomal cytochrome P-450 levels. Vitamin E supplementation increased liver and serum cholesterol levels in guinea pigs, but had no such effect in rats. Results of this study indicate that cholesterol feeding can result in various metabolic alterations in rats and guinea pigs. The implication of these alterations in atherogenesis requires further investigations.