探討脂多醣影響血腦屏障上轉運蛋白表現之機制及轉運蛋白與神經毒性物質之作用關係

Abstract

血腦屏障(Blood-brain barrier)是體循環血液與中樞神經系統的分界，阻止許多物質進入中樞神經系統，包括藥物與毒物。腦微血管內皮細胞組成有如表皮細胞般的細胞間緊密連結(Tight junction; TJ) 因此造就了屏障的特性，使大部分物質進出腦部需要轉運蛋白的協助而無法經由細胞間隙進入。許多溶質轉運蛋白(Solute carrier; SLC)表達於腦微血管內皮細胞上，包括有機陽離子轉運蛋白(organic cation transporters; OCTs; SLC22A1-2)及漿膜單胺轉運蛋白(plasma membrane monoamine transporter; PMAT; SLC29A4)。 本實驗將腦部的微血管分別從Swiss小鼠、C57BL/6 (B6)小鼠和Wistar大鼠分離，偵測其上Octs及Pmat的表現量。並偵測在以脂多醣 (lipopolysacchride; LPS)處理前及處理後轉運蛋白表達量的變化。我們以免疫螢光染色(immunofluorescence) 和反轉錄即時定量聚合酶連鎖反應(reverse transcription-quantitative polymerase chain reaction; RT-qPCR) 來偵測其上的蛋白及mRNA的表達。而我們也進一步以體外模型探討LPS對Octs及Pmat的調控是否受星狀細胞(astrocyte)及周邊細胞(pericyte)所影響。另外，我們也利用過度表達Octs及Pmat的MDCK細胞株來探討內生性神經毒物1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),N-methyl-(R)-salsolinol(N-methyl-(R)SAL)及 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-benzyl-TIQ)的動力學性質。 研究結果顯示，Oct1及Oct2的mRNA相較於腦皮質，集中表現於B6小鼠的腦微血管。Oct1、Oct2及Pmat的蛋白則與內皮細胞的標記蛋白(markers)，如CD31(cluster of differentiation 31)、第四型膠原蛋白(Collagen IV)共位表現(co-localized)。體內試驗結果顯示，Oct1、Oct2及Pmat的蛋白表現量在給予LPS後，短期與長期分別是上升與下降。體外試驗結果顯示，在給予急性LPS刺激下，星狀細胞對於轉運蛋白表達量的調控扮演重要的角色。最後，動力學實驗方面發現人類OCT1可以運輸1-benzyl-TIQ 及N-methyl-(R)SAL；人類PMAT 可以運送 1-benzyl-TIQ及MPTP。 總而言之，Oct1-2及Pmat表達於B6小鼠及Wistar 大鼠的腦微血管，但並未表達於Swiss小鼠的腦微血管。急性與長期LPS的刺激對Oct1、Oct2及Pmat的表達量，有相反的調控。根據結果可以推測，這些轉運蛋白表達量的改變會改變血腦屏障對神經毒性物質N-methyl-(R)SAL、 1-benzyl-TIQ 及MPTP的運送，進而影響所造成之毒性。

The blood–brain barrier (BBB) is the interface separating the circulation blood from the brain extracelluar fluid in central nervous system (CNS). The BBB prevents brain uptake of many substrates, including pharmaceuticals and toxins. This property arises from the epithelial-like tight junctions formed by the brain microvascular endothelial cells (BMECs). Several solute carrier (SLC) families are expressed on BMECs, including SLC22 and SLC29, mediating the transportation of a variety of substances. In this study, the expression of organic cation transporters (Octs; slc22A1-2) and plasma membrane monoamine transporter (Pmat; slc29a4) were measured in brain microvessels (BMV) isolated from Swiss mice, C57BL/6 (B6) mice and Wistar rats before and after lipopolysaccharide(LPS) treatment. Immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to measure the expression of mRNA and protein, respectively. The effects of LPS on the regulation of Oct1, Oct2 and Pmat were further investigated in BMECs with or without co-culture with astrocyte and pericyte. Also, the kinetics properties of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),N-methyl-(R)-salsolinol(N-methyl-(R)SAL) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline(1-benzyl-TIQ), were measured in MDCK cells expressing human OCTs or PMAT. The results showed that the mRNA of Oct1 and Oct2, but not Oct3, were enriched in BMV of B6 mice (compare to cerebral cortex). Oct1, Oct2 and Pmat proteins were co-localized with endothelial markers (CD31, collagen IV, and p-glycoprotein). Protein levels of Oct1, Oct2 and Pmat protein levels were increased and reduced, respectively, upon acute and long-term LPS treatment. Result of in vitro BMECs co-culturing system showed that astrocyte play a role on the up-regulation of Octs and Pmat after acute LPS treatment. Finally, the kinetic study showed that human OCT1 can transport both 1-benzyl-TIQ and N-methyl-(R)SAL. Human PMAT can transport 1-benzyl-TIQ and MPTP. In conclusion, Oct1-2 and Pmat were expressed in BMV of C57BL/6 (B6) mice and Wistar rats, but not of Swiss mice. Acute and long-term LPS treatment regulated Oct1-2 and Pmat expression, but in a different manner. These data suggests that the change in the expression of Octs and Pmat at BMV may change BBB transport of N-methyl-(R)SAL, 1-benzyl-TIQ and MPTP.