Inhibitory Effects Of Lentivirus-mediated RNA Interference On Spinal IL-33 Gene In Rat Adjuvantinduced Arthritis

Part I Inhibitory effects of lentivirus-mediated RNA interference on IL-33 gene in cultured rat glioma cells.ObjectiveTo construct lentiviral vectors encoding short-hairpin RNAs(sh RNAs) targeting IL-33 and transfect it into glioma cell to silence IL-33 gene and to observe its inhibitory effect.Methods1.Three si-RNA sequences and a negative control were designed according to IL-33 conquence of rats. The complementary DNA containing both sense and antisense DNA oligos of the targeting sequence was synthesized and cloned into the GV118 vector, which were digested by HpaⅠand XhoⅠ. The recombined vector was confirmed by PCR and DNA sequencing and cotransfected with p Helper1.0 and p Helper 2.0 packaging plasmids into 293 T cells, then the virus titer was measured.2.The recombinant lentivirus was transfected into glioma cell C6. The transfection efficiencies were observed in each group.3.Morphologic change of C6 cells were observed under invert microscop. The expression level of IL-33 m RNA in C6 cells was detected by Real-time PCR.RESULTS1. DNA sequencing results demonstrated that the inserted sequences were correct. The titer of viruses are shown below. LV-IL33-RNAi(1): 1×109TU/ml; LV-IL33-RNAi(2):8×108TU/ml; LV-IL33-RNAi(3):1×109TU/ml.2. Recombinant lentivirus could be successfully transfected into C6 cells. Compared to the negative control group, LV-IL-33-RNAi(1),LV-IL-33-RNAi(2) or LV-IL-33-RNAi(3)groups showed a lower expression level of IL-33 m RNA.The efficiency of LV-IL33-RNAi(2) group was up to 66.5%( P<0.05).ConclusionThe lentivirus RNAi vector targeting rat IL-33 gene has been constructed successfully. It may down-regulate IL-33 expression in glioma cell C6. Taken together, our reults suggest that the lentiviral vector derived sh RNA approach shows a great promise for the study of functional IL-33 gene expression in nervous system.Part II Activation of spinal IL-33 contributes to peripheral inflammation and hyperalgesia in rat adjuvant-induced arthritisObjectiveIt is known that noxious stimuli from inflamed tissues may increase the excitability of spinal dorsal horn neurons(central sensitization),which can signal back and contribute to peripheral inflammation. However, the underlying mechanisms have yet to be fully defined. A number of recent studies indicate that spinal IL-33 is involved in central sensitization as well as pain-related behavior. Thus, our aim was to determine whether IL-33 also can facilitate a peripheral in ammatory response in rat adjuvantinduced arthritis(AIA).Methods1. Arthritis was induced by CFA inoculation of the rats on day 0. The animals received intrathecal the the lentiviral construct encoding either the IL-33 sh RNA sequence or the control ones on day2. Pain-related behavior and paw swelling were assessed at baseline(1 day before LV-sh RNA-IL-33 injection). Measurements were also obtained at 6h, 12 h, 24 h, 2d, 3d, 4d, 7d,10 d,14d and 21 days following the injection.2.The expression of spinal IL-33 on day1,day2,day3,day4,day7,day,10,day14,day21 was assayed by Western blot analysis,And the spinal IL-33 of CFA+LV-IL-33-RNAi and CFA+LVIL-33-NC on day14 and day21 were measured.On the day 21, joint histopathological changes were evaluated.Results1.CFA group showed an obvious increase in mechanical hyperalgesia, thermal hyperalgesia and paw edema compare to sham group( P<0.05).2.Peripheral Inflammation induced an increase in IL-33 expression. The expression of spinal IL-33 in LV-IL-33-RNAi decreased on day14 and day21,but not in LV-IL-33-NC( P<0.05).ConclusionPeripheral Inflammation induced an increase in IL-33 expression. Spinally delivered LV-sh RNA-IL-33 knocked down IL-33 expression. Thus, inhibition of spinal IL-33 expression may provide a potential treatment to manage painful inflammatory disorders.