肥胖病人非酒精性脂肪肝疾病與發炎因子 : 探討 Tumor Necrosis Factor-alpha, Leptin 和 Adiponectin 的角色

肥胖病人非酒精性脂肪肝疾病與發炎因子 : 探討 Tumor Necrosis Factor-alpha, Leptin 和

Adiponectin 的角色

 肥胖是心血管疾病，腦中風，糖尿病及癌症的重要危險因子；高血壓、高血脂症、高膽固醇症、糖尿病及脂肪肝被認為是新陳代謝症候群（ metabolic synd rome ）。很多研究顯示，肥胖是一種慢性、低度發炎疾病（ low grade inflammation ），發炎因子在各種新陳代謝症候群中扮演重要的角色。非酒精性脂肪 肝疾病（ nonalcoholic fatty liver disease, NAFLD ）是一群因脂肪囤積而引起廣泛性肝臟細胞破壞引起疾病的統稱，它的病程包括單純性脂肪肝（ liver steat osis ）、脂肪性肝臟發炎（ steatohepatitis ）、肝臟纖維化（ liver fibrosis ）及肝硬化（ liver cirrhosis ）。確切的診斷及嚴重度之評斷需藉助病理切片檢查。

非酒精性脂肪肝疾病的致病機轉仍未完全明瞭，目前最被人接受的是“兩次撞擊”的假說，“第一次撞擊（ first hit ）”是脂肪囤積在肝臟細胞，與胰島素 抗性（ insulin resistance ）有密切關係。大部份的病人可能只有單純脂肪肝卻沒有伴隨發炎反應，這種疾病的病程是比較良性的。但是若合併發炎反應，即 進入“第二次撞擊（ second hit ）”，它所引發的發炎反應就變得很複雜，牽涉到很多不正常細胞激素產生（ abnormal cytokine production ）及氧化壓力反 應（ oxidative stress ）的作用，其中最著名的細胞激素是 adiponectin ， tumor necrosis factor -?? ( TNF-??) 和 leptin 。 TNF-? 悇 O 一種前發炎因子（ proinfla mmatory cytokine ）。 Leptin 則與肝臟纖維化有關。 Adiponectin 是目前被研究較多的 cytokine ，它具有明顯抗發炎性（ anti-inflammatory effect ）及胰島 素敏感化（ insulin sensitivity ）的作用，與 NAFLD 的發生有密切的關連。

非酒精性脂肪肝疾病的致病機轉與發炎因子應有明顯的關係，本研究以 BMI?d35 以上的台灣肥胖病人肝臟組織，分析 Leptin ， TNF-? 恁 AAdiponectin 及 Adiponectin Receptor II （ adipoRII ）的表現與相互間的關係，藉此探討非酒精性脂肪肝疾病的致病機轉。同時以病理組織學的變化，佐以臨床資料與血液 生化檢查結果（年齡、性別，腰臀比、血糖、血脂、肝功能指數），研究 Leptin ， TNF-? 恁 AAdiponectin 及 Adiponectin Receptor II 與肝臟受損程度，肝 臟纖維化與臨床資料的相關性。

本研究包含四十位肥胖病人，共 12 位男性， 28 位女性，平均年齡為 29.43 ± 9.19 歲，平均 BMI 為 44.79 ± 5.37 kg/m2 。四十位肥胖病人中有 30 位（ 75

% ）診斷為非酒精性脂肪性肝發炎（ nonalcoholic steatohepatitis ， NASH ）。我們將病人分成三組作研究：第一組為非酒精性脂肪性肝發炎（ nonalcoholic steatohepatitis ， NASH ）合併 0-1 期肝臟纖維化，共 11 位病人。第二組為非酒精性脂肪性肝發炎合併 2-4 期肝臟纖維化，共 19 位病人。第三組包括 10 位病人，因只有單純脂肪肝卻沒有伴隨發炎反應，被歸類為對照組。臨床抽血資料顯示， aspartate transaminase (AST) （ p =0.000 ）、 alanine transamina se (ALT) （ p =0.000 ）、 γ-glutamyl transferase (GGT) （ p =0.045 ） 和 hemoglobin A1c (HbA1c) （ p =0.001 ） 在三組病人中具有統計學上的差異。

在病理切片組織學方面，總共 36 （ 90% ）位病人發現單純脂肪囤積在肝臟細胞。肝臟細胞汽球化退化（ ballooning degeneration ）存在於所有的肝臟發炎

（ steatohepatitis ）病人。三十九 (97.5%) 位病人表現肝小葉發炎（ lobular inflammation ），有 11 位 （ 27.5% ）表現輕度、 13 位（ 32.5% ）表現中度 和 15 位（ 37.5% ）表現嚴重程度肝小葉發炎。四十位肥胖病人中，沒有任何一位進展到肝硬化。本研究無法偵測到肝臟 leptin 的 mRNA 表現。另一方面

，不管是 TNF-α/GAPDH cDNA ， adiponectin/GAPDH cDNA 或是 adipoRII /GAPDH cDNA 的肝臟 mRNA 表現，也沒有達到統計學上的差異。

臨床病理相關性方面，我們發現單純脂肪囤積在肝臟細胞與血糖、 AST 、 ALT 、 GGT 、三酸甘油脂和 HbA1c 有關 ; 卻和高密度脂蛋白（ high density li poprotein ）成負相關。肝臟細胞汽球化退化（ ballooning degeneration ） 與血糖、 AST 、 ALT 和 HbA1c 也有相關性。另一方面，肝小葉發炎（ lobular i nflammation ）和 AST 、 ALT 和 HbA1c 有關。至於肝臟纖維化則和 AST 、 ALT 和 C-peptide 有關。 Adiponectin/GAPDH cDNA 和尿酸成負相關性 ; T NF-α/GAPDH cDNA ratio 則與單純脂肪囤積在肝臟細胞有關。免疫組織染色方面， leptin 的免疫染色表現與 C-peptide 、 門脈區發炎（ portal inflammatio n ）有相關性。 AdipoRII 與收縮壓成負相關性 ; 與 Mallory body 、 glycogenated nuclei 成正相關性。 TNF-α 的免疫染色表現和 AST 、血紅素、肝小葉發 炎、門脈區發炎和 NAS activity 有關。而 adiponectin 的免疫染色表現則沒有達到統計學上的相關性。

胰島素抗性（ insulin resistance ）和人體對胰島素敏感度降低是非酒精性脂肪肝疾病重要特徵。在本研究中，我們無法釐清 TNF-α 、 leptin 、 adiponectin

、 adipoRII 和單純脂肪囤積在肝臟細胞、 肝臟發炎和肝臟纖維化的關係，未來需要更大、更深入的研究來探討非酒精性脂肪肝疾病與肝臟纖維化的致病 機轉。我們的研究也透露出胰島素抗性（ insulin resistance ），不管是肝臟或是週邊的胰島素抗性，似乎比不正常細胞激素產生（ abnormal cytokine produc tion ）與發炎因子扮演更重要的角色。

Inflammatory Cytokines in Nonalcoholic Fatty Liver Disease in Obese Patients: Role of Tumor Necrosi

s Factor-alpha, Leptin and Adiponectin



Obesity is a chronic inflammatory condition and obesity has been claimed one of risk factors for cardiovascular disease, stroke, diabetes mellitus and cancers. The metabolic syndrome in cludes hypertension, hyperlipidemia, diabetes mellitus and fatty liver disease. Nonalcoholic fatty liver disease (NAFLD) is considered a hepatic manifestation of systemic metabolic syndr ome. NAFLD is a wide spectrum of liver damage, ranging from simple steatosis, to nonalcoholic steatohepatitis (NASH) and cirrhosis on the basis of longstanding fatty change of the live r. While NASH implies a risk of progressive liver disease, simple steatosis is regarded as a benign condition.

The “two hit hypothesis” has become an important theoretical framework for understanding the pathogenesis of NAFLD. The “first hit” is liver fat accumulation in hepatocytes, linked wi th insulin resistance. In the majority of patients with excess liver fat or scarce hepatic inflammation, this condition will be termed simple steatosis. However, a “second hit” may trigger th e necroinflammatory response characterizing NASH through abnormal cytokine production and oxidative stress. The mediated abnormal cytokine production including adiponectin, lepti n and tumor necrosis factor -? 恁 ]TNF-? 恁 ^. TNF-?? is a proinflammatory cytokine, whereas leptin has fibrogenetic effect. In contrast, adiponectin play antidiabetic and anti-inflammat ory role that acts as a fascinating mediator linking adipose tissue, insulin resistance, and inflammation. Adiponectin may have protective effect on the development of NAFLD, potentiall y antagonizing the effect of TNF-??.

Although the participation of inflammatory mediators and cytokines in the pathogenesis of NAFLD is widely recognized, their relative important in Taiwanese obese patients is not fulfill ed. In this study, we analyzed the clinicopathology of morbidly obese patients (BMI?d35) from liver biopsied specimens. To further elucidate the role of inflammation in NAFLD, we eva luated mRNA and protein expression of leptin, TNF-??, and anti-inflammatory factor adiponectin and adiponectin receptor II (adopoRII) in liver tissues of NAFLD patients.

Our study enrolled 40 obese patients, including 12 men and 28 women with mean age 29.43 ± 9.19 years, and the mean BMI was 44.79 ± 5.37 kg/m2. Histologic evidence of NASH (NA S 5) was present in 30 patients (75%). Of these 30 patients, 11 patients were grouped as NASH with stage 0-1 mild liver fibrosis and 19 patients were grouped as NASH with stage 2-4 ≧ advanced liver fibrosis. The other 10 patients had normal liver histology or simple steatosis only without necroinflammation (NAS<3) and they were considered as control group. The ser um levels of alanine transaminase (ALT) (p =0.000), aspartate transaminase (AST) (p =0.000), γ-glutamyl transferase (GGT) (p =0.045) and hemoglobin A1c (HbA1c) (p =0.001) have st atistically difference in these three groups. Only AST was independently different after multivariate logistic regression analysis.

Hepatic steatosis was present in 36 patients (90%). Ballooning degeneration of hepatocytes was occurred in all NASH group. Lobular inflammation found in 39 patients (97.5%). Of these 39 patients, 11 (27.5%), 13 (32.5%) and 15 (37.5%) presented with mild, moderate and severe lobular inflammation. Regarding the stage of fibrosis, only one patient presented without fi brosis. No obvious stage 4 fibrosis (or liver cirrhosis) was noted in any of the cases. The mRNA of leptin was undetectable in the liver tissue of any cases in our study. TNF-α/GAPDH cD NA ratio was highest in patients without NASH, while the adiponectin/GAPDH cDNA ratio tended to be higher in NASH with advanced liver fibrosis. In contrast, the adipoRII/GAPDH c DNA ratio was lower in NASH with advanced liver fibrosis. All the mRNA levels of the studied genes have not reached significantly different. Immunohistochemistry, adiponectin and a dipoRII staining were less pronounced in liver sinusoids in NASH group compared with non-NASH group. In contrast, leptin staining was more pronounced in liver tissues of NASH gro up with advanced liver fibrosis. However, no significant difference between three groups was noted.

The clinicopathological correlation demonstrated that the degree of hepatic steatosis was best correlated with fasting sugar, AST, ALT, GGT, triglyceride and HbA1c. Inverse relationship was noted between hepatic steatosis and high density lipoprotein. Independent predictors of ballooning degeneration were serum fasting sugar, AST, ALT and HbA1c. The best predictors of lobular inflammation were AST, ALT and HbA1c. The predictors for liver fibrosis were AST, ALT and C-peptide. In addition, the predictor for adiponectin/GAPDH cDNA ratio was i nversely correlated to uric acid level; and the predictor for TNF-α/GAPDH cDNA ratio was related to liver steatosis. Immunohistochemistry study showed that the best predictors of lepti n expression were correlated with C-peptide and portal inflammation. AdipoRII immunohistochemical stain was inversely related with systolic blood pressure; and positively associated Mallory body and glycogenated nuclei. Finally, the predictors for TNF-α immunohistochemical expression were AST, hemoglobin, lobular inflammation, portal inflammation and NAS a ctivity. No significantly predictor of adiponectin immunohistochemical expression was noted.

Insulin resistance and reduced whole-body insulin sensitivity are major characteristics of NAFLD. In our study, we could not completely address the complex association between TNF-α,

leptin and adiponectin/adipoRII, insulin, liver steatosis, necroinflammation, and liver fibrosis. Therefore, the possibility that all these adipocytokines could influence necroinflammation a

nd fibrosis, finally to the functional severity in NASH, is not completely understood. Larger studies are needed to fully establish the pathogenesis and mechanisms of fat accumulation in t

he liver and the progression to fibrosis. Finally, the relationship between hepatic and peripheral insulin resistance and the development of hepatic steatosis should be clarified in future res

earch.