霍氏格里蒙菌熱穩定性溶血素造成活體內外肝臟毒性的探討

Abstract

霍氏格里蒙菌(G. hollisae)已經被證實會產生熱穩定性溶血素(Gh-TDH)。以往的研究指出此毒素在人體會經由腸胃吸收，造成消化道的傷害，尤其在小腸是格外劇烈。然而，熱穩定性溶血素除了會在小腸吸收外，更有可能經由門脈循環(portal system)的靜脈回流侵犯肝臟造成二次傷害，但此關係卻從未被研究過。在本研究中，我們將進行熱穩定性溶血素造成活體內外肝臟毒性(in vitro and in vivo hepatotoxicity)的探討。我們使用純化的G. hollisae recombinant thermostable direct hemolysin (Gh-rTDH)來進行肝毒性的分析。在活體外培養老鼠肝臟細胞株(FL83B cell line)和人類正常肝臟細胞(primary human liver cell)來測試此毒素後發現，此毒素對老鼠和人類的肝臟細胞均造成劇烈的傷害。在接觸此毒素後，肝臟細胞出現細胞質流失和皺縮等(loss of cell cytoplasm with cell shrinkage)型態上的改變，並且最終造成細胞死亡。細胞毒性測試(MTT assay)的結果顯示當毒素濃度超過10-6 μg/ml則開始顯著造成肝細胞死亡，作用時間越長或毒素的濃度越高，則毒殺肝細胞的作用就更為明顯(dose and time dependent)。Gh-rTDH在結合異硫氰酸螢光素(fluorescein isothiocyanate-conjugated)之後被用來定位細胞受侵犯的部位，我們發現此毒素起初會圍繞肝細胞，最後將進入細胞內並且侵犯細胞核造成細胞死亡。另外，在活體內的實驗中，我們讓若干數量的小鼠(BALB/c)口服單次且不同劑量的熱穩定性溶血素或霍氏格里蒙菌，並且進行抽血分析以評估其肝臟功能。結果發現這些老鼠的肝臟酵素(glutamic-oxaloacetic transaminase / glutamic-pyruvic transaminase)、膽紅素(bilirubin)、白蛋白(albumin)、球蛋白(globulin)在餵食毒素或細菌後均發生顯著異常，肝臟酵素在餵食後第八小時上升至最高峰。病理切片則證實無論是餵食毒素或直接餵食細菌，在肝臟的門脈周圍區域(periportal area)皆會遭受明顯的破壞，毒素或細菌的濃度越高，破壞越大。除此之外，此毒素對於心臟、腎臟並沒有造成顯著的傷害。最後，我們使用正子照影18F-FDG PET (positron emission tomography)/CT (computer tomography) scan來活體評估老鼠在餵食毒素或細菌前後之肝臟代謝功能及復原情形。結果發現老鼠在餵食毒素或細菌後，其代謝功能有顯著下降，並且在七天後可逐漸回復其功能。總結來說，本研究首次證實霍氏格里蒙菌之熱穩定性溶血素的確具有活體內外之肝臟毒性，肝臟的門脈周圍區域是此毒素主要的侵犯部位，肝臟代謝功能可在七天後逐漸回復。

G. hollisae thermostable direct hemolysin (Gh-TDH) is produced by most strains of G. hollisae. This toxin has been reported to be absorbed in the intestines in humans. In addition to being absorbed by the intestine, TDH may also cause secondary injury to the liver via effects on the venous return of the portal system. However, a relationship between the TDH and the liver has not been reported or analyzed. In this study, we analyzed the hepatotoxicity of TDH in vivo and in vitro to provide insights into the acute injury and recovery stages of THD-induced hepatotoxicity in living animals. Liver cells (primary human non-cancer cell and FL83B mouse cells) were treated and mice (BALB/c) were fed with this toxin to investigate its hepatotoxicity. In this study, G. hollisae recombinant thermostable direct hemolysin (Gh-rTDH) was purified for further analysis. Morphological examination and cytotoxicity assays using liver cells were also performed. The morphological changes included cell detachment and a loss of cell cytoplasm with cell shrinkage. The MTT assay revealed that the cytoviability of liver cells decreased in proportion to the concentration of Gh-rTDH over different treatment durations (dose and time dependent.). Moreover, we noted that Gh-rTDH damaged liver cells in vitro when the concentration of Gh-rTDH exceeded 10-6 μg/ml. Fluorescein isothiocyanate-conjugated toxin was used to analyze the localization of this protein in liver cells. Cellular localization showed that the toxin was initially located around the cellular margins and subsequently entered the nucleus. Mice were subjected to liver function measurements and liver biopsies following toxin treatment and wild-type bacterial infection. Liver function measurements and liver biopsies of the mice following treatment with toxin or infection with wild-type Grimontia hollisae showed elevated levels of transaminases and damage to the periportal area, respectively. PET (positron emission tomography)/CT (computed tomography) images were taken to assess liver metabolism during acute injury and recovery. The PET/CT images revealed that the reconstruction of the liver continued for at least one week after exposure to a single dose of the toxin or bacterial infection. In conclusion, the hepatotoxicity of Gh-TDH was firstly demonstrated. The damage was located in the periportal area of the liver, and the liver became functionally insufficient.