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Aim To study the function of VR1 in chronic pain, to construct VR1 siRNA expression vectors and to study their silencing effect in the DRG neurons of rats were detected.Methods The hairpin sequences of siRNAs targeting VR1 gene of rat were designed, and two pairs of oligonucleotide sequence were synthesized. The annealed oligonucleotide fragments were cloned into linearized pRNAT-U6.2/Lenti expression vector and identified by PCR and DNA sequencing.Then, they were co-transfected by lipofectamine into 293T cells.The silencing effects of the lentivector-mediated VR1 siRNAs on the expression of VR1 mRNA were determined by RT-PCR after intrathecal injection in rats.Results DNA sequencing showed that the oligonucleotide fragments were correctly cloned into linearized pRNAT-U6.2/Lenti expression vector and the expression of VR1 mRNA in L4-L6 DRG neurons was inhibited significantly by pRNAT-U6.2/Lenti-siVR1 after intrathecal injection in rats.Conclusion The lentivector-mediated siRNAs are successfully constructed and they inhibit the expression of VR1 mRNA in the DRG neurons of rats, which may provide a potential tool for the further study and treatment of chronic pain.
RESUMO
Objective To investigate whether emodin(1,3,8-trihydroxy-6-methylanthraquinone) can attenuate inflammatory response in rats' lungs with acute necrotic pancreatitis (ANP). Method Acute necrotic pancreatitis model was induced by injection of 3% sodium taurocholic acid into the subcapsular of pancreas and emodin was administered by intestine perfusion. Plasma amylase of 3, 6 and 12 h with acute necrotic pancreatitis was measured together with the detection of IL-1β, IL-6 and IL-10 mRNA expressions in rats' lungs by semi-quantitative RT-PCR. Immunohistochemistry was detected the expression of IL-1β converting enzyme (ICE) in the rats' lungs. Result Plasma amylase of 3, 6 and 12 h with acute necrotic pancreatitis groups are obviously high as compared with normal group(P＜0.05). Plasma amylase was (1 611.20±218.72)IU/L in normal group. Plasma amylase of 3, 6 and 12 h with acute necrotic pancreatitis groups were (1 981.40±56.81)IU/L, (3 287.40±612.37)IU/L and (4 914.60±746.82)IU/L. Plasma amylase of 3, 6 and 12 h with acute necrotic pancreatitis after treatment with emodin groups were obviously low as compared with acute necrotic pancreatitis groups. The plasma amylase was(1 617.20±136.80)IU/L,(2 323.40±318.19) IU/L and (2 670.20±390.03)IU/L respectively. The study showed that the mRNA expression of pro-inflammatory cytokin IL-1β and the expression of IL-6, as well as the expression of IL-1β converting enzyme(ICE) were decreased and IL-10 was increased. Conclusion The study demonstrates that emodin plays an important role in reducing plasma amylase level. Emodin exerts anti-inflammatory effects in acute necrotic pancreatitis rats' lungs by downregulating the mRNA expression of IL-1β and IL-6 and upregulating the mRNA expression of IL-10.