第九型基質金屬蛋白酶 (MMP-9) 在LPS誘發肝發炎反應中的角色

Abstract

敗血症是一種危害人類健康的疾病，有許多因素會導致敗血症，但最主要的原因是細菌入侵血液循環中。脂多醣 (lipopolysaccharide, LPS)是格蘭氏陰性菌細胞壁上的成分之一，被視為內毒素，會引發強烈的免疫反應，導致感染性肝損傷或衰竭。LPS 除了會誘導發炎反應外，也會增加第九型基質金屬蛋白酶 (Matrix metalloproteinase 9，MMP-9) 的表現量。先前研究指出，MMP-9 缺失會增加小鼠腹部敗血症的嚴重性，特別是發炎及肝損傷的程度；臨床研究也指出MMP-9及TIMP-1在血液中的比例與敗血症造成的死亡率相關；在MC3T3-E1成骨細胞株中，MMP-9能夠調控LPS的訊息傳遞路徑。綜上所述，鑒於肝臟是生物體內最重要的解毒器官，我們假設MMP-9在LPS誘導的肝臟發炎及損傷可能扮演著保護的角色。 本研究旨在探討MMP-9是否參與調控LPS誘導的肝損傷及發炎反應中。試驗分成兩個部分，活體試驗 (In vivo) 分成四個組別，使用C57BL/6 (WT)及MMP-9tm1Tvu/J (KO) 15週齡之公鼠，腹腔注射4.5 mg/kg的LPS或食鹽水，24小時後進行犧牲採樣。血液生化值的結果顯示，LPS會誘導高血脂，且KO組在LPS注射後血脂量較WT組高 (P<0.05)，顯示其血脂代謝異常；然而肝損傷指標AST及ALT，WT及KO組在LPS注射後並沒有顯著的差異；此外，在H&E染色下，LPS會誘導油滴堆積以及免疫細胞浸潤的情況，肝臟三酸甘油脂的含量也顯示相同趨勢，然而在WT及KO組間並沒有觀察到顯著的差異。總上所述，LPS誘導的肝臟損傷及脂質堆積並不會因為MMP-9的剔除而變得更為嚴重。在肝臟發炎反應部分，免疫組織染色的結果顯示，LPS誘導的巨噬細胞浸潤的現象在兩組別並沒有顯著差異，然而在KO組別，LPS誘導的促發炎因子IL-6及LPS受體CD14的基因表現量表現顯著高於WT組 (P<0.05)。為了探究其原因，針對LPS訊息傳遞路徑的蛋白質進行測定，結果顯示KO組在LPS處理後，NF-κB，為LPS訊息傳遞路徑中重要的轉錄因子，其蛋白質水平顯著的高於其他組別 (P<0.01)，而LPS的受體TLR4及CD14在處理LPS後表現量皆顯著降低，並在WT及KO組中並沒有顯著差異。 細胞實驗 (In vitro) 部分，使用LPS處理RAW264.7小鼠巨噬細胞株，來模擬肝臟中庫氏細胞 (Kupffer cell) 接收LPS後的反應。經24小時的LPS處理能夠顯著增加MMP-9的蛋白質表現量以及酵素活性，並使用MMP-9 siRNA來抑制MMP-9的表現量。然而結果顯示，無論是siRNA組或是對照組，由LPS所誘導的發炎反應皆沒有顯著的差異；此外，利用MMP-9抑制劑來抑制細胞MMP-9的酵素功能後，亦無對LPS造成的發炎反應造成影響。而初代培養肝細胞的結果顯示，MMP-9 KO會增加LPS誘導的促發炎基因表現，然而目前仍需要多次重覆來確定結果的穩定性。 綜合以上結果，MMP-9 KO小鼠在LPS處理下，雖然不會有較嚴重的肝損傷情況，卻有較高的發炎反應，並且可能是透過影響LPS訊息傳遞路徑而導致；而從細胞實驗的結果推測，(1) MMP-9可能不是透過截切單一細胞的受器來影響LPS誘導的發炎反應進程；(2) 細胞實驗僅抑制部分MMP-9表現，而沒有將MMP-9基因剔除，因此結果與活體試驗不符；或是(3) MMP-9是影響肝細胞的LPS訊息傳遞。然而，詳細機制仍需更多實驗才能進一步確認。

Sepsis is a disease that endangers human health. There are many factors that can lead to sepsis, but the main reason is the invasion of bacteria into the blood circulation. Lipopolysaccharide (LPS), one of the components on the cell wall of Gram-negative bacteria, is regarded as an endotoxin that triggers a strong immune response, leading to infectious liver damage or failure. Besides the inflammatory response, LPS also increased the expression of matrix metalloproteinase 9 (MMP-9). Previous studies have shown that MMP-9 deletion impairs defenses against abdominal sepsis in mice and increases liver damage and inflammation. Clinical studies have also pointed out that the MMP-9/TIMP-1 ratio of the blood is related to mortality caused by sepsis. In MC3T3-E1 cell, MMP-9 can regulate LPS signaling pathway. Taken together, given that the liver is the primary organ that processes LPS in organisms, we hypothesized that MMP-9 might play a protective role in LPS-induced liver inflammation and injury. The aim of this study is to investigate whether MMP-9 is involved in the regulation of LPS-induced liver injury and inflammation. The experiment was divided into two parts. The in vivo experiment was divided into four groups, C57BL/6 (WT) and MMP-9tm1Tvu/J (KO) 15-week-old male mice were intraperitoneally injected with 4.5 mg/kg LPS or saline, sacrificing and sampling were performed 24 hours later. The results of blood biochemistry showed that LPS induced hyperlipidemia, and the blood TG level in the KO group was higher than the WT group (P<0.05); however, there was no significant difference in liver injury marker AST and ALT between both genotypes after LPS injection; in addition, H&E staining showed that LPS induced oil droplet accumulation and immune cell infiltration, and increased liver triglyceride content, but no significant difference was observed between both genotypes. Taken together, LPS-induced liver injury and lipid accumulation are not aggravated by the depletion of MMP-9. As for inflammatory response, the result of immunohistochemical staining showed that there is no significant difference in LPS-induced macrophage infiltration between both genotypes. However, the gene expression of LPS-induced pro-inflammatory cytokine IL-6 and LPS receptor CD14 were significantly higher in the KO group than WT group (P<0.05). In order to explore the reason, the proteins in the LPS signaling pathway were measured. The results showed that after LPS treatment, NF-κB, an important transcription factor of the LPS signaling pathway, had a significantly higher expression level in the liver of KO group than others (P<0.01). The expression levels of LPS receptors TLR4 and CD14 were significantly decreased after LPS treatment, and there was no significant difference between both genotypes. In in vitro part, RAW264.7 mouse macrophage cell line was treated with LPS to simulate the response of Kupffer cells in the liver after receiving LPS. After 24 hours of LPS treatment, the protein expression and enzyme activity of MMP-9 significantly increased, and MMP-9 siRNA was used to inhibit the expression of MMP-9. However, qPCR results showed no significant difference in the inflammatory response induced by LPS between the siRNA group and the control group. In addition, MMP-9 inhibitors, which can inhibit the enzymatic function of MMP-9, also did not affect the inflammatory response caused by LPS. The results of primary hepatocytes showed that MMP-9 KO increased the expression of pro-inflammatory genes induced by LPS, however, multiple repetitions are still needed to determine the stability of the results. To sum up, MMP-9 KO mice did not develop severer liver damage under LPS treatment, but had a higher liver inflammatory response, which may be due to regulation of the LPS signaling pathway. The in vitro results indicate that (1) MMP-9 may not affect the progression of LPS-induced inflammatory response by merely receptors cleavage; (2) the MMP-9 gene was not deleted completely in in vitro experiments, so the results were inconsistent with in vivo experiments; or (3) MMP-9 affects the LPS signal transduction of hepatocytes. However, the detailed mechanism still needs more experiments to be further confirmed.