肝細胞中第九型基質金屬蛋白酶凝血酶素結構抑制LPS誘導之IL-6分泌

Abstract

肝炎是對病源入侵重要的防禦機制。一旦動物體的免疫系統持衡被打破，發炎反應將會轉變為危險的慢性肝炎，進而可能發展為肝硬化。脂多醣 (Lipopolysaccharide, LPS) 是革蘭氏陰性菌外膜的核心成分，被認為是肝炎發展的重要因素。腸道細菌過度生長和腸道通透性增加會導致過量的LPS進入肝臟，刺激促發炎細胞因子和基質金屬蛋白酶-9 (MMP9) 的表現。許多研究結果顯示，MMP9在肝臟發炎中起關鍵的保護作用。 MMP9是一種分泌性明膠酶，屬於基質金屬蛋白酶家族（MMPs）的一員。本研究旨在闡明MMP9在LPS誘導的發炎中的潛在保護作用。 我們的數據顯示，與野生型小鼠相比，MMP9基因剔除（KO）小鼠在LPS誘導後，肝臟中白介素-6（IL-6）的濃度顯著提高。推測是肝臟中的主要細胞－肝細胞和屬於巨噬細胞的Kupffer cells 中的MMP9調控了LPS誘導的發炎反應。在體外研究中，我們使用MMP9 KO RAW264.7細胞研究MMP9減緩LPS誘導發炎的機制。 在LPS誘導後，MMP9 KO細胞中的IL-6分泌量顯著高於野生型組。然而在建立了Tet-On誘導MMP9-overexpression的 RAW264.7細胞株，多西環素（doxycycline）誘導的MMP9未能緩解LPS誘導的IL-6。因此，巨噬細胞並不會透過MMP9調控IL-6的表現。接下來，透過小鼠的肝細胞株Hepa1-6細胞測試了MMP9調節肝細胞的發炎反應。我們建立了Tet-On誘導的MMP9 Hepa1-6細胞，發現多西環素誘導的MMP9降低了LPS刺激的IL-6蛋白質表現。這項發現顯示MMP9下調LPS誘導肝細胞的發炎反應。我們進一步建立了催化結構（Catalytic domain）突變的MMP9和凝血酶（hemopexin domain or PEX domain）結構缺失的MMP9，結合Tet-On系統，以闡明哪個結構在調節LPS誘導的IL-6表現中扮演關鍵角色。結果顯示，MMP9的凝血酶結構若產生缺失便無法降低IL-6的分泌。這因此，MMP9調控發炎反應，特別是IL-6，凝血酶結構域是不可或缺的。 綜上所述，我們證明了肝細胞可透過MMP9的活化來建立抗發炎反應。我們也確定了凝血酶結構對於MMP9下調發炎反應是必要的。

Liver inflammation is one of the essential defenses against intruders. Once the homeostasis is impaired, it turns into hazardous chronic hepatitis, which may evolve into cirrhosis. Lipopolysaccharide (LPS), a central component of the outer membrane of Gram-negative bacteria, is considered a vital factor in developing hepatitis. The overgrowth of intestinal bacteria and increasing intestinal permeability cause excess LPS to enter the liver and stimulate the expressions of pro-inflammatory cytokines and matrix metalloproteinase-9 (MMP9). Numerous studies showed that MMP9 plays a critical protective role in hepatic inflammation. MMP9, a secretory gelatinase, is a matrix metalloproteinase family (MMPs) member with a zinc exonuclease. The primary function of MMP9 is the degradation and remodeling of the extracellular matrix proteins (ECMs). A recent study has suggested that MMP9 may have significant anti-inflammatory potential. The study aimed to investigate the potential protective roles of MMP9 against LPS-induced inflammation. Our data showed that MMP9 knockout (KO) mice had significantly higher hepatic interleukin-6 (IL-6) levels than wild-type mice did after LPS induction. We hypothesized that hepatocytes and hepatic local macrophages-Kupffer cells may be the primary cells responsible for regulating LPS-induced inflammation via MMP9. In the in vitro study, we used MMP9 KO RAW264.7 cells to examine how MMP9 attenuates LPS-induced inflammatory responses. The IL-6 level in MMP9 KO cells was significantly higher than in wild-type groups after LPS induction. In addition, we established Tet-On inducible MMP9 RAW264.7 cells and found that overexpressed MMP9 through doxycycline induction did not affect mRNA and protein levels of IL-6 induced by LPS. We concluded that gene and protein expression of IL-6 in macrophages were not dependent on MMP9. Next, we tested the role of MMP9 in hepatocytes by using the mouse hepatic cell line Hepa1-6 cells. We produced Tet-On inducible MMP9 Hepa1-6 cells and found that overexpression of MMP9 attenuated the increase in IL-6 protein levels caused by LPS. This finding suggests that the MMP9 downregulates LPS-induced inflammatory responses in hepatocytes. We further established two Tet-on inducible cell lines with catalytic domain mutation and hemopexin (PEX) domain deletion to clarify which domain of MMP9 is responsible for the regulation of MMP9 in LPS-induced IL-6 expression. The results showed that the deletion of the PEX domain in MMP9 failed to attenuate IL-6 secretion. This result indicates that MMP9-regulated inflammatory response, specifically IL-6, is through the PEX domain. In summary, we demonstrated that MMP9 established anti-inflammatory function mainly in the hepatocytes. We also reported that the PEX domain of MMP9 is necessary for MMP9 to down-regulate the inflammatory response.