BNTX用於治療全身性紅斑性狼瘡之研究

Abstract

7-Benzylidenenaltrexone (BNTX)是一種δ1型鴉片類受體(δ1 opioid receptor，簡稱DOR1)的拮抗劑。先前的研究指出BNTX能抑制免疫細胞增殖和存活，但是它的作用機制不明，也未曾用於治療免疫疾病。本研究目的以MRL/lpr紅斑性狼瘡鼠驗證BNTX是否有療效，並探討其作用機制。我們在小鼠17週齡已呈現狼瘡腎炎時分別自腹腔給予2, 5或10 mg/kg/day BNTX 14天做療效評估。結果顯示，BNTX能夠改善老鼠的臉部蝴蝶斑、掉髮與關節腫等外觀狀況，甚至使鬍鬚再長出。與對照組小鼠比較，BNTX各劑量均能有效降低脾臟與淋巴結腫大，且不影響體重。進一步分析發現BNTX顯著減少脾臟和骨髓中分泌IgG、IgM和dsDNA-IgG及-IgM自體免疫抗體的漿細胞數目和血清抗體效價。最重要的是BNTX能夠顯著改善狼瘡腎炎，包括:降低蛋白尿、減少IgM免疫複合體沉積腎絲球和腎小管間質、維護腎絲球型態與大小和緩解腎纖維化。在作用機制方面，初步發現BNTX能使FcγRIIB表達上升，這與FcγRIIB能透過與免疫複合體傳遞訊息抑制B細胞增殖和促進B細胞凋亡，減少免疫複合體在腎臟沉積吻合。有趣的是，BNTX這些作用並不須透過δ1型鴉片類受體，因為DOR1基因敲落並不影響FcγRIIB上升。而且，廣效型δ型鴉片類受體拮抗劑naltrindole和全鴉片類受體拮抗劑naloxone均不影響FcγRIIB表達。這樣的結果顯示BNTX對於B細胞的抑制機制具特異性，值得進一步研究找出作用蛋白。總結，本研究提供多項證據顯示BNTX對紅斑性狼瘡鼠之腎炎具有療效，而減少自體免疫漿細胞與促進FcγRIIB表達誘導細胞凋亡有關。由於目前紅斑性狼瘡治療仍以類固醇為主，副作用大。近年來，採用專一性抗體選擇性去除B細胞以降低免疫複合體生成，已證明能緩解狼瘡腎炎並延長病人壽命。因此，以低劑量BNTX減少B細胞，若機制明確，應有臨床應用價值。

7-Benzylidenenaltrexone (BNTX) is a δ1 opioid receptor antagonist. BNTX is known to be immuosuppressive but its mechanism remains elusive, thereby restricting its development for clinical use. In this study, we sought to investigate the potential therapeutic effects in MRL/lpr lupus mice. We treated mice of 17 weeks old when nephritis was evident by intraperitoneal injection of BNTX at 2, 5 and 10 mg/kg/day respectively for 14 days. A five-parameter scoring system was used to evaluate and monitor disease activities of lupus before and after treatment with vehicle or BNTX. We found that the signs and symptoms of lupus mice improved most significantly in including butterfly-like rashes, alopecia, sparse whiskers and arthritis after BNTX treatment. We further found that BNTX at various doses all significantly reduced the degree of splenomegaly and lymphadenopathy. The body weight of BNTX-treated mice remained unaltered, suggesting a safe dose range of BNTX in our experiments. Moreover, BNTX treatment dramatically decreased the numbers of IgG- and IgM-antibody-secreting cells (ASCs) and more importantly the anti-dsDNA-IgG and -IgM ASCs in the spleen and bone marrow, respectively. Concomitantly, serum titers of autoantibodies also decreased in BNTX-treated mice but not in control mice. Importantly, BNTX ameliorated lupus nephritis, including proteinuria, immune complex deposition in glomeruli and interstitial fibrosis. To investigate the mechanisms of BNTX in B cells, we found that BNTX could up-regulate the expression levels of FcγRIIB, which is a crucial negative regulator of B cells. Because FcγRIIB can trigger apoptosis of B cells via immune complexes, this explains the BNTX-mediated reduction of ASCs in MRL/lpr mice. Interestingly, knockdown of DOR1 gene did not affect FcγRIIB expression induced by BNTX. In contrast, natrindole, a non-selective δ opioid receptor antagonist, and naloxone, a pan-opioid receptor antagonist, did not affect the expression level of FcγRIIB. These results suggest a unique role of BNTX in the upregulation of FcγRIIB but its underlying mechanisms require further investigation. Because recent studies suggest a selective depletion of B cells via antibodies and histone deacetylation inhibitors can alleviate lupus nephritis, BNTX may be an additional promising candidate using this strategy. Further studies to elucidate the molecular mechanisms of BNTX in immune cells shall facilitate its clinical application for the treatment of systemic lupus erythematosus.