探討β-Lapachone對於發炎反應誘發之纖維化的抑制效果

Abstract

緒論 發炎反應於許多疾病中都扮演重要角色，亦可能導致組織或器官發生纖維化，包括肺、心、肝、腎等都有所影響。文獻已知刺激巨噬細胞發炎，會導致組織的纖維化，然而對於纖維母細胞的發炎相關機制目前尚不清楚。組織裡的膠原蛋白大部分經由纖維母細胞製造，因此我們推測，抑制纖維母細胞發炎，應可降低其膠原蛋白過量生產，減少纖維化發生。過去的研究發現β-Lapachone(β-Lap)是一種具有多種藥理功能的植物萃取物，透過NQO1調控對細胞之影響。我們認為倘若β-Lap能夠抑制發炎反應，或許可透過減緩細胞產生過量的膠原蛋白，降低組織纖維化之程度。 材料方法 本研究以脂多醣(LPS，Lipopolysaccharide)誘發小鼠胚胎纖維母細胞(NIH3T3)發炎，並以結晶紫染色法、西方墨點法與細胞螢光染色法，檢測發炎的指標蛋白COX-2 (Cyclooxygenase-2)與NF-κB(Nuclear factor kappa-light-chain-enhancer of activated B cells)確認細胞的發炎情況，並且觀察第一型與第三型膠原蛋白以及α-SMA (Alpha smooth actin)的含量變化，以了解發炎是否影響纖維母細胞產生細胞外基質的能力。另外本研究也檢驗β-Lap是否能透過抑制發炎反應，降低纖維母細胞產生膠原蛋白，進而抑制組織之纖維化。 實驗結果 本研究之纖維母細胞在藥物處理前經過5小時starvation，在0.1 μg/mL LPS與0.1或0.5 μM β-Lap分別處理或合併處理之下，不會造成細胞死亡。我們發現給予LPS處理可造成纖維母細胞的COX-2及p-p65 (NF-κB subunit)蛋白表現量上升，並利用TLR4的抑制劑CLI-095確認LPS是透過TLR4引起細胞發炎。此外，我們亦利用NF-kB與COX-2抑制劑加以確認分子訊號途徑。我們另發現給予細胞β-Lap處理能夠抑制由LPS所引致的發炎反應。若將細胞長時間處理LPS，則細胞之第一型及第三型膠原蛋白過量表現，若同時給予細胞β-Lap則可加以抑制。亦觀察到LPS會造成細胞內NAD+/NADH比例下降，給予β-Lap處理則能夠使比例回升。 結論 纖維母細胞透過LPS直接刺激，會產生發炎反應，進一步引發膠原蛋白增生；而給予細胞β-Lap處理，能夠有效抑制LPS所引致之發炎反應與膠原蛋白增生。根據本研究結果，β-Lap很有潛力作為臨床上發炎造成之纖維化的治療藥物。

Introduction Inflammatory response plays an important role in many diseases, and it could also cause fibrosis in many tissues or organs, which include heart, lung, liver and kidney. Fibrosis is led by excess collagen and extracellular matrix (ECM) which produced by fibroblasts. Most researches about inflammation are focused on macrophages, but the inflammation on fibroblasts is under the mist. Therefore the study aimed to study the inflammation induced by LPS in fibroblasts. It is important to inhibit the inflammation-induced fibrosis by blocking the collagen production in fibroblasts. β-Lapachone (β-Lap) is a kind of pharmacodynamics complex extracted from Tabebuia avellanedae, and the studies show the effective function of β-Lap might be regulated by NQO1 enzyme(NAD(P)H:Quinone Oxidoreductase 1 ). Accordingly, we assume that if β-Lap could reduce the inflammation on fibroblasts, it might decrease the situation fibrosis through lowering the overwhelming collagen product in fibroblasts. Material and Methods NIH3T3 cell line was used in this study with different concentrations of β-Lap under lipopolysaccharide (LPS) treatment, the inflammation inducer, to observe the mechanisms of LPS-induced inflammation in fibroblasts and whether β-Lap could protect cells from inflammation-induced collagen production and fibrosis. We examined the cell viability by crystal violet and observe the protein expression of COX-2 (Cyclooxygenase-2), NF-κB(Nuclear factor kappa-light-chain-enhancer of activated B cells), α-SMA (Alpha smooth actin), COL1A1 and COL3A1 (type I and type III collagen) by western blotting and immunocytochemistry(ICC). Results In our study all the drugs we used in the experiments would not affect the viability of fibroblasts. We found that COX-2 and p-p65 (NF-κB subunit) expression increased and collagen products were enhanced through LPS stimulation of LPS. And CLI-095, TLR4 inhibitor, was used to confirm the specific receptor of LPS on fibroblasts. The study also checked the pathway of inflammation through the inhibitor of NF-kB and COX-2. In addition, we found that β-Lap could reduce the level of inflammation and fibrogenesis induced by LPS. Besides, the study also demonstrated that LPS lowered the ratio of NAD+/NADH, and the treatment of β-Lap could retain it. Conclusions Direct stimulation of LPS lead inflammation in fibroblasts, and induced overwhelming collagen production. The treatment of β-Lap could reduce the inflammation and fibrogenesis significantly. In consequence, β-Lap has the huge potential to become the clinical treatment on the inflammation-induced fibrosis.