Dataset for: Macrophage migration inhibitory factor mediates protease activated receptor 4 induced bladder pain through urothelial high mobility group box 1

Objectives: Macrophage migration inhibitory factor (MIF) mediates pain although the mechanisms are not well understood. Urothelial activation of protease activated receptor 4 (PAR4) results in urothelial MIF release, urothelial high mobility group box 1 (HMGB1) release and bladder pain in mice without bladder inflammation. All three effects are prevented by MIF inhibition while intravesical disulfide HMGB1 alone can induce bladder pain. The current study utilizes genetic MIF deletion to determine whether MIF mediates PAR4-induced bladder pain and is upstream of HMGB1-induced bladder pain. Methods: Wild type (C57/BL6) and MIF knockout (KO) mice were treated with intravesical PAR4 activating peptide or disulfide HMGB1 and tested for abdominal mechanical hypersensitivity at baseline (before treatment) and 24 hours after injection. Micturition parameters and bladder histology were examined after behavioral test. Real-time PCR and western blotting measured HMGB1 mRNA and protein levels in the bladders of naïve wild type and MIF KO mice, while immunofluorescence measured HMGB1 protein levels in the urothelium of both strains. Results: Intravesical PAR4 activation resulted in abdominal mechanical hypersensitivity in wild type mice but not MIF KO mice. Intravesical disulfide HMGB1 induced abdominal mechanical hypersensitivity in both strains. Neither treatment resulted in significant changes in micturition or bladder histology in either strain. HMGB1 mRNA and protein levels were higher in MIF KO mouse bladders and the urothelium of MIF KO bladder had greater immunostaining than the wild type strain. Conclusion: MIF is a pivotal molecule mediating PAR4-induced bladder pain and regulating urothelial HMGB1 production and release to elicit bladder pain.

研究目的：巨噬细胞迁移抑制因子（Macrophage migration inhibitory factor，MIF）可介导疼痛，但相关作用机制尚未完全阐明。蛋白酶激活受体4（Protease activated receptor 4，PAR4）的尿路上皮激活可诱导无膀胱炎症的小鼠出现尿路上皮MIF释放、尿路上皮高迁移率族蛋白B1（High mobility group box 1，HMGB1）释放以及膀胱疼痛。上述三类效应均可通过MIF抑制加以阻断，而仅经膀胱内给予二硫键HMGB1即可独立诱导膀胱疼痛。本研究采用基因敲除MIF（MIF knockout，MIF KO）小鼠模型，旨在明确MIF是否介导PAR4诱导的膀胱疼痛，且是否位于HMGB1诱导膀胱疼痛的上游调控通路。方法：选取野生型（C57/BL6）与MIF基因敲除（KO）小鼠，经膀胱内给予PAR4激活肽或二硫键HMGB1，分别在给药前（基线状态）与给药后24小时检测小鼠的腹部机械痛敏情况。行为学检测结束后，对小鼠的排尿参数与膀胱组织学标本进行检测。采用实时荧光定量PCR（Real-time PCR）与蛋白质印迹法（western blotting）检测未造模野生型及MIF KO小鼠膀胱组织中HMGB1的mRNA与蛋白水平；采用免疫荧光染色法（immunofluorescence）检测两种小鼠品系尿路上皮内的HMGB1蛋白表达水平。结果：膀胱内激活PAR4可在野生型小鼠中诱导腹部机械痛敏，但在MIF KO小鼠中未出现该效应。经膀胱内给予二硫键HMGB1则可在两种小鼠品系中均诱发腹部机械痛敏。两种给药处理均未对两种小鼠的排尿参数或膀胱组织学造成显著改变。MIF KO小鼠膀胱组织中HMGB1的mRNA与蛋白水平均高于野生型小鼠，且其尿路上皮的HMGB1免疫荧光染色强度也强于野生型小鼠。结论：MIF是介导PAR4诱导膀胱疼痛的关键分子，同时可通过调控尿路上皮HMGB1的产生与释放来诱发膀胱疼痛。