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  • 學位論文

RXR-alpha 在敗血症鼠合併肝內膽汁淤積中所扮演的角色

Role of RXR-alpha in Intrahepatic Cholestasis during Polymicrobial Sepsis in Rats

指導教授 : 許勝光
共同指導教授 : 許勤(Chin Hsu)

摘要


敗血症致死主要是因多重器官衰竭(multiple organ failure),而肝臟被認為是體內維持能量產生與代謝的重要器官,故當敗血症時肝功能會嚴重受損,以致引發起其他器官合併衰竭。敗血症時肝臟也會呈現肝內膽汁淤積、膽汁流量及膽鹽的運送功能都會下降,因而導致膽鹽的堆積情形發生。在先前研究結果顯示,在敗血症晚期時,有9個差異性基因在肝臟衰竭中扮演重要的角色,其中3αHSD和kan-1/rBAT是有參與膽酸合成之主要酵素且rBAT為CLP敗血症之肝內膽酸淤積時特異性表現下降。FXR/RXRα結合至IR-1序列上,雖理論上能調控rBAT和膽酸運送因子BSEP的表現,但敗血症6小時的時候,只有rBAT具有特異性的下降,而在Hepatocyte primary culture實驗中發現給Dexamethasone可以促使RXRα表現上升,且rBAT的變化是隨著RXRα表現呈現正相關性之變化。除了FXR/RXRα外,許多文獻指出RXRα和其他parter會共同調節膽酸代謝合成?〝M膽酸的運送因子,如:RXRα/FXR會調控rBAT、BSEP;RXRα/RAR會調控MRP2、NTCP;RXRα/LXR會調控CYP7A1及RXRα/PXR會調控 OATP1。所以,我們有興趣想瞭解在早期敗血症6小時,RXRα表現下降時,在調控參與膽酸合成代謝和運送之各因子中所扮演的角色為何呢?是否只對某些因子的調控呈現特異性的表現? 本研究實驗動物模式是以大白鼠進行盲腸結紮穿刺手術(Cecal ligation and puncture,CLP)來為引發敗血症,然後測試出治療敗血症老鼠的藥物Dexamethasone之最適當劑量和時間點,依據我們初步的結果顯示,在CLP後4.5小時給於Dexamethasone(0.01mg /kg ) 的敗血症鼠存活率是最好的,所以,我們即將以此條件作為CLP6h+Dexa組的治療。實驗分組為:偽手術組(Sham)、偽手術+Dexamethasone組(Sham+Dexa)、盲腸結紮穿刺手術組6小時(CLP6h)和盲腸結紮穿刺手術6小時+Dexamethasone組(CLP6h+Dexa)四組進行實驗,而CLP6h+Dexa組在CLP後4.5小時給dexamethasone治療,在CLP後6小時分別偵測各組實驗老鼠的膽汁引流量、膽鹽的濃度變化情形,再運用即時定量反轉錄聚合?﹞狨?(Real-time RT-PCR)測定CLP6組、CLP6h+Dexa組其RXRα和所調控的CYP7A1、rBAT、BSEP、MRP2、NTCP、OATP之mRNA表現量;用西方點墨法(Western blot)觀察CLP6h組、CLP6h+Dexa組,RXRα和所調控的CYP7A1、rBAT、BSEP、MRP2、NTCP、OATP之蛋白質表現量,並運用EMSA去觀察RXRα/FXR結合到IR-1序列上的結合能力,以瞭解RXRα/FXR結合到IR-1序列上之活性。運用分離的肝細胞(hepatocyte cell)進行體外初代培養(Primary culture)實驗,用siRNA技術阻斷RXRα基因後,再運用即時定量反轉錄聚合?﹞狨釧M西方點墨法探討RXRα和所調控的CYP7A1、rBAT、BSEP、MRP2、NTCP、OATP之mRNA和蛋白質的表現量。 活體內(In vivo)實驗結果顯示○1Dexa.可以提升敗血症老鼠的存活率和膽汁流量及膽鹽濃度。○2 Dexa.可促使敗血症老鼠肝臟內的RXRα和rBAT 的mRNA及蛋白質表現量增加;其CYP7A1、MRP2、NTCP、BSEP、OATP1則不變○3 Dexa.可促使敗血症老鼠肝臟內RXRα/FXR在rBAT promote IR-1上的結合能力增加。 在活體分離出初代培養(Primary culture)結果顯示:1. 敗血症老鼠肝臟內的RXRα、rBAT、CYP7A1、MRP2和NTCP的mRNA及蛋白質表現量會下降2. Dexa.對敗血症老鼠肝臟內的RXRα和rBAT 之mRNA及蛋白質表現量有特異性增加3.當RXRα抑制後,研究發現受RXRα所調控的rBAT、CYP7A1、MRP2、NTCP、BSEP和OATP1 mRNA表現都有下降;其中rBAT、CYP7A1、MRP2和NTCP蛋白質表現也有下降,但以RXRα和rBAT的下降為最明顯。 綜合以上結果,知道在敗血症後4.5小時,給於0.01mg/kg BW的Dexamethasone治療可以提升老鼠的存活率,促使膽汁淤積的情形改善使得膽汁流量和運送膽鹽濃度上升,同時也增加RXRα蛋白質的表現量。同時,發現RXRα調節膽鹽的代謝合成和運送因子中,主要對rBAT的調控具有特異敏感性的調節。所以,我們希望這樣的新發現能作為敗血症早期的發現及治療功效之指標,進而防止敗血症持續惡化。

並列摘要


sepsis is an illness after infecting and the death rate is to achieve 40% ~ 60%. Multiple organs failure continues to be the primary cause of death after sepsis. The liver is thought to be one of the major organs responsible for the initiation of multiple organ failure because of its central role in metabolism. Hepatic dysfunction in sepsis is characterized by apoptosis、mitochondria Damage 、hyperbilirumia (Kim et al., 2000) 、impaired uptake and secretion of hepatocyte transport of bile acids and organic anions 、increased GOT, GPT and intra-hepatic cholestasis. The nuclear receptor RXRα(retinoid X receptor) has three family members (RXRα, RXRβ,?z RXRγ ) and RXRα is highly expressed in liver. There is substantial evidence that RXRα virtually participates in all aspects of hepatic function including metabolism of cholesterol, fatty acid, carbohydrate, bile acid and transporter activities. Heterodimer of RXRα with special nuclear receptor (CAR, FXR, LXR, PPAR, or PXR) controls the expression of?n proteins which involve in bile acid metabolism , such as bile acid synthesis enzyme(CYP7A1、rBAT) and bile salt transporters (BSEP、NTCP、MRP2、OATP1).[10].Through suppression subtractive hybridization (SSH) technique, we have been identified that two genes, 3α-HSD and BAT, were down regulated in late septic liver. Both 3α-HSD and rBAT are the key enzymes in bile acid synthesis and metabolism.We find that decreased rBAT expression was a specific character in sepsis-associated intra-cholestasis and RXRα was the key transcription factor. In primary hepatocyte culture, RXRα agonist dexamethasone (Dexa) can increase the protein content of RXRα and binding activity of IR-1(Shock aceepted). It is interesting for us to know the roles of RXRα in the regulation of bile acid metabolism during sepsis, in vivo. In this study, we identified the best treated time and dose of Dexamethason which can improve the survival rate of CLP-induced septic rats. The volume of bile flow and concentration of bile salt were analyzed. Tissue extract (nuclear, cytosolic, and membrane)、Western blot (protein expression)、Real-time PCR (mRNA expression)、EMSA(protein and DNA binding activity) 、siRNA transfection techniques were used .The results showed that: Dexamethason (0.01mg/Kg) improves the survival rates of CLP-induced septic rat. Dexamethason can increase the expression of RXRα protein in CLP-induced septic rat. Dexamethason treated can increase the volume of bile flow and the concentration of bile salt in bile duct of septic rats. The protein and mRNA expressions of bile acid synthesis enzymes (CYP7A1、rBAT) were decreased during sepsis and Dexa can improve the expression of rBAT but not CYP7A1. Both BSEP and OATP1 were unaffected by sepsis and Dexa treated. The mRNA, but not protein, expression of NTCP and MRP2 were decreased during sepsis. The protein and mRNA expression of rBat was significantly enhanced while RXRα protein increased, but not BSEP、NTCP、MRP2、OATP1 and CYP7A1 in septic rats. DNA binding activity of RXRα/FXR to IR-1 was also increased in septic rats after Dexa treatment. RXRα and rBAT protein expression showed downregulation after RXRα siRNA transfection. Take all the results together, both rBAT and RXRα decreased during sepsis and the expression of rBAT was significantly enhanced while RXRα protein increased after Dexamethason treated. It is suggested that rBAT and RXRα might be developed as a diagnostic and prognostic markers of sepsis

並列關鍵字

RXR-alpha intrahepatic cholestasis sepsis

參考文獻


1.Bolder, U., et al., Hepatocyte transport of bile acids and organic anions in endotoxemic rats: impaired uptake and secretion. Gastroenterology, 1997. 112(1): p. 214-25.
2.Tavakoli, H. and L. Mela, Alterations of mitochondrial metabolism and protein concentrations in subacute septicemia. Infect Immun, 1982. 38(2): p. 536-41.
3.Kim, C.Y., et al., Protective effect of melatonin on methylmercury-Induced mortality in mice. Tohoku J Exp Med, 2000. 191(4): p. 241-6.
4.Wang, K., Y. Wang, and Z. Yang, [Evaluation of the treatment effect for bruxism by using myomonitor and occlusal splint]. Zhonghua Kou Qiang Yi Xue Za Zhi, 1998. 33(5): p. 300-2.
5.Jao, H.C., et al., The decrease of PKCalpha is associated with hepatic apoptosis at early and late phases of polymicrobial sepsis. Shock, 2001. 15(2): p. 130-4.

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