HMGB1參與Oxaliplatin誘發周邊神經病變之研究

Abstract

研究背景 化學治療所引起的副作用，諸如化學治療引起的周邊神經病變 (chemotherapy-induced peripheral neuropathy, CIPN) 為癌症治療常見的不良反應。目前已有許多文獻探討CIPN的成因，破壞神經系統結構並引起CIPN的致病機理是多因素的，包括與免疫過程和神經發炎有關。在大部分細胞中，高遷移率族蛋白1(high mobility group box 1 protein, HMGB1)存在於細胞核中並參與許多細胞核內反應。HMGB1可經由主動或被動釋放至細胞外，可作為損傷相關分子模式(damage associated molecular patterns, DAMPs)，針對危險訊息警告周邊細胞與免疫系統引發發炎反應，於CIPN中可能扮演重要的角色。 研究目的 本論文欲探討巨噬細胞內HMGB1是否經由乙醯化修飾後主動釋放?是否參與CIPN？HATs抑制劑是否可影響HMGB1釋放？另外，巨噬細胞與神經細胞在交互影響下，探討其中可能影響神經發炎的因子。 研究方法 在鉑類製劑中oxaliplatin的CIPN發生率高達90%，因此本研究以oxaliplatin作為引發神經痛反應之藥物。利用西方墨點法偵測HMGB1、MCP-1蛋白質表現量，並利用傷口癒合實驗測試巨噬細胞RAW264.7遷移能力。 研究結果 結果顯示加入15 μM的oxaliplatin培養24小時會使巨噬細胞RAW264.7與BV-2的釋放HMGB1蛋白質表現量增加2.7與3.9倍 (p=0.039；p=0.01)，達顯著差異。在巨噬細胞RAW264.7中，預先加入HMGB1抑制劑甘草素(glycyrrhizin) 500 μM培養24小時，再更換成15 μM的oxaliplatin培養24小時，發現隨甘草素濃度上升至500 μM可以顯著降低HMGB1蛋白質的釋放至0.3倍 (p<0.001)。另外，加入15 μM的oxaliplatin於RAW264.7細胞培養12或24小時後釋放的HMGB1蛋白質中乙醯化增加14.7及16.1倍 (p<0.001；p<0.001)，大幅顯著上升，使HMGB1蛋白可經由巨噬細胞主動釋放至細胞外。預先加入HATs抑制劑薑黃素(curcumin) 50 μM培養24小時再更換成15 μM的oxaliplatin培養24小時，發現薑黃素濃度增加至50 μM可以降低40% HMGB1蛋白質釋放(p<0.001)，且釋放的乙醯化蛋白質量明顯降低至0.5倍 (p<0.001)。接著再探討免疫細胞所釋放的HMGB1如何影響神經細胞。研究發現加入oxaliplatin的神經細胞N2a會釋出單核細胞趨化蛋白-1(monocyte chemoattractant protein-1；MCP-1)。將此神經細胞N2a的上清液加入巨噬細胞RAW264.7培養，會使RAW264.7細胞遷移能力顯著上升，暗示可能藉由MCP-1之分泌而使得免疫細胞聚集至神經細胞周遭釋放HMGB1，導致神經發炎反應的產生。而這經由MCP-1影響HMGB1釋放增加的現象，加入50 μM 的curcumin可逆轉HMGB1過度釋放，使HMGB1表現量降低70% (p<0.001)。 結論 本研究證明於給予適當濃度之oxaliplatin會促使巨噬細胞主動釋放乙醯化HMGB1，這項機轉可能使得神經產生發炎反應。薑黃素在本研究亦發現可隨濃度上升，抑制HMGB1乙醯化能力越強，使得釋放至細胞外HMGB1表現量大幅下降。經由本研究結果，未來能以此機制為基礎找出可以用作預防或治療CIPN的標靶藥物。

Background Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating adverse effect. However, the molecular mechanisms underlying CIPN are still unclear. Accumulating evidence supports that neuropathic pain is associated with neuroinflammation. High mobility group Box 1 protein (HMGB1) is a nuclear protein that acts as a DNA chaperone and participates in a number of activities in the nucleus. HMGB1 can exit the cell through two different pathways: passive or active release. HMGB1 can also translocate to the extracellular medium, where it acts as a damage associated molecular pattern (DAMP) molecule that alerts nearby cells and the immune system to immediate danger, triggering inflammation. Several lines of evidence suggest that HMGB1 may play pronociceptive role in CIPN. Objective We investigated the effect of oxaliplatin on the active release pathway of immune cells which participate in CIPN through acetylated HMGB1, and the influence of HATs inhibitors. Methods The incidence rate of CIPN by oxaliplatin is up to 90%, much higher than by other chemotherapeutic agents. Oxaliplatin was introduced to study the CIPN on immune cell and nerve cell interactions. Protein levels of HMGB1 and MCP-1 were detected using Western blot, and the migration ability of macrophage RAW264.7 was detected by the wound healing assay. Results The extracellular release of HMGB1 protein increased by 2.7 and 3.8 folds, respectively, when macrophage RAW264.7 cells and glial BV-2 cells were administrated with 15 μM oxaliplatin for 24 hours. When RAW264.7 cells were pretreated with 500 μM glycyrrhizin, known as a HMGB1 inhibitor, for 24h, the oxaliplatin-induced extracellular HMGB1 protein was significantly reduced to 0.3 fold. The extracellular release of HMGB1 protein was also inhibited by 50 μM curcumin, an HAT inhibitor, in RAW264.7 cells. Acetylation of HMGB1 protein was significantly detected in extracellular medium of RAW264.7 cells (p<0.001). Accordingly, the acetylated HMGB1 protein was reduced by 50 μM curcumin in RAW264.7 cells. On the other hand, it is interesting to know the way how the macrophage RAW264.7 cells approach nerve cells for further action on the neuron through HMGB1. Co-culture of macrophage RAW264.7 cells and nerve N2a cells resulted in production of profound amount of extracellular HMGB1 and monocyte chemoattractant protein-1 (MCP-1), compared to the uni-cell cultures. The MCP-1 protein was detected from oxaliplatin-treated N2a cell culture medium. The wound healing assay showed the MCP-1-containing supernatant of oxaliplatin-treated N2a cell culture facilitated migration of macrophages RAW264.7. It implies that the secreted MCP-1 may serve as a chemoattractant for macrophages aggregating toward the nerve cells and triggering HMGB1 release from macrophage, which evokes ultimate neuroinflammation. Conclusion Given clinically therapuetical concentration of oxaliplatin to macrophage enables actively extracellular release of acetylated HMGB1 which may be involved in neuroinflammation. Curcumin inhibits HMGB1 acetylation and reduces the amount of extracellular HMGB1 so as to become a reversal of CIPN.