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Objective In this study,we attempted to investigate whether Electroacupuncture(EA)could promote the autophagy function in macrophages of inflammatory skin tissues by activating CB2 receptor,thus relieving inflammatory pain induced by CFA in mice,and whether activation of CB2 receptor in NR8383 macrophages cell line can simulate the effect of EA on the autophagy function and mitochondrial damage.Methods Inflammatory pain model was induced by CFA injection into the planta the hind paw of wildtype and CB2 knockout mice.EA or sham EA was applied on the left Huantiao(GB30)and Yanglingquan(GB34)sites.Thermal hyperalgesia was determined with the Hargreaves test.Mechanical sensitivity was assessed with von Frey filaments.NR8383 microphage cell line was used to study the effect of CB2 activation on macrophage function induced by CFA.The expression level of autophagy protein LC3 and p62 in wildtype and CB2 knockout mice skin tissue and NR8383 cell line were determined by Western blot.And flow cytometry analysis was applied to detect damaged mitochondria and mitochondrial superoxide.Results CFA significantly reduced the thermal and mechanical pain threshold in both wildtype and CB2 knockout mice,comparing with the vehicle control groups(P<0.01).EA significantly inhibited thermal and mechanical hyperpathia induced by CFA in wildtype mice(P<0.05),but had no effect on CB2 knockout mice with CFA(P>0.05).CFA significantly increased the expression of p62 protein and decreased LC3-II/I ratio,which was inversed by EA in wildtype mice but wasn't affected by EA in CB2 knockout mice.CFA increased the expression of p62 protein and decreased LC3-II/I ratio in NR8383 cell line,which were inversed by CB2 agonist AM1241.CFA increased mitochondria damage,which were then attenuated by CB2 agonist AM1241.Conclusion The analgesic effect of EA on inflammatory pain induced by CFA was mediated by activation of CB2 receptor,which promoted the autophagy function and the clearance of damaged mitochondria in macrophage.
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Objective To establish a method for detection of serum urocanic acid (UCA) by high performance liquid chromatography (HPLC),and explore the clinical significance of serum UCA concentration for children acute leukemia.Methods The chromatographic conditions of HPLC were set up and optimized,and the linearity of standard curve,precision,accuracy and stability were validated.Then the serum from ninety acute leukemia children and ninety non-tumor blood disease children was collected,the concentration of serum UCA was detected with HPLC,and the differences of two groups were compared to study the clinical significance of UCA in children acute leukemia.Results The HPLC method for detecting serum UCA was successfully established and optimized.The standard curves of trans-UCA and cis-UCA both showed good linearities(R2=0.999 6 and 0.999 9) at the condition of the mobile phase of acetonitrile-20 mmol/L KH2PO4,pH 3.7(5:95,V/V),flow rate of 1.2 mL/min,detection wavelength of 264 nm in HPLC.The relative standard deviation RSD％ of intra-assay and inter-assay were lower than 5％.Compared with non-tumor blood disease,the serum concentration of cis urocanic acid (cis-UCA) and trans urocanic acid (trans-UCA) of children with acute leukemia were significantly increased (P＜0.001).Compared with cis-UCA,trans-UCA was more valuable for risk classification of acute lymphoblastic leukemia (ALL).Conclusions HPLC is a good technology to titrate of UCA in serum.The concentration of serum UCA in children with acute leukemia may provide the clues for diagnosis and prognosis,with important clinical significance.