目的：通过观察右美托咪定（Dexmedetomidine, DEX）对两种因素诱导的肝损伤模型小鼠的影响，探讨右美托咪定对小鼠肝损伤的保护作用，并试图阐明其可能作用机制。

方法：第一部分采用胆管结扎法（Bile Duct Ligation, BDL）建立胆汁淤积性肝纤维化小鼠模型，将40只C57/BL6小鼠随机分成四组，每组10只，分为正常对照组（CON组，假手术+腹腔注射等量生理盐水），模型组（MOD组，BDL+腹腔注射等量生理盐水），阳性对照组（PC组，BDL+腹腔注射秋水仙碱0.2mg/kg），右美托咪定组（DEX组，BDL+腹腔注射右美托咪定10μg/kg）。CON组只打开腹腔不结扎，禁食不禁水12h，麻醉后处死小鼠。采集小鼠血清，微板法测定血清谷丙转氨酶（Alanine Transaminase，ALT）、谷草转氨酶（Aspartate Transaminase，AST）含量。采用ELISA法检测小鼠血清中Ⅲ型前胶原（Procollagen Type Ⅲ ，PC-Ⅲ）、Ⅳ型胶原（Type Ⅳ collagen，Ⅳ-C）和肝组织中肿瘤坏死因子-ɑ(Tumor Necrosis Factor-ɑ, TNF-ɑ)、白介素-6（Interleukin-6, IL-6）、白介素-1β（Interleukin-1β，IL-1β）的水平。测定小鼠肝脏组织中超氧化物歧化酶（Superoxide Dismutase, SOD）、丙二醛（Malondialdehyde , MDA）、谷胱甘肽（Glutathione，GSH）、谷胱甘肽过氧化物酶（Glutathione peroxidase，GSH-PX）的活性。分别进行HE、Masson、天狼猩红染色，观察肝组织病理形态变化。利用Western blot法检测小鼠肝脏组织中磷脂酰肌醇3-激酶（Phosphatidylinositol3-kinases，PI3K）、蛋白激酶B （Protein kinase B，AKT）、核因子E2相关因子（Erythroid transcription factor NF-E2，Nrf2）、血红素加氧酶-1（Heme oxygenase-1,HO-1）、核转录因子-κB（Nuclear factor kappa-B，NF-κB）、TNF-ɑ、IL-6蛋白因子表达水平。

第二部分采用对乙酰氨基酚（Acetaminophen, APAP）诱导形成药物性肝损伤小鼠模型。将40只C57/BL6小鼠随机分成四组，每组10只，分为正常对照组（CON组，腹腔注射等量生理盐水），模型组（MOD组，腹腔注射APAP 200mg/kg），阳性对照组（PC组，腹腔注射APAP200mg/kg+秋水仙碱0.2mg/kg），右美托咪定组（DEX组，腹腔注射APAP200mg/kg+右美托咪定10μg/kg）。禁食不禁水12h，麻醉后处死小鼠。采集小鼠血清，微板法测定ALT、AST含量。测定小鼠肝组织中SOD、MDA、GSH、GSH-PX活性。采用ELISA法检测血清中TNF-α、IL-6、IL-1β的水平。利用HE染色观察肝组织病理形态改变。利用Western blot法检测模型小鼠肝脏组织中PI3K、AKT、Nrf2、HO-1和NF-κB、TNF-ɑ、IL-6蛋白表达水平。

结果：

1、BDL模型，与CON组比较，MOD组小鼠脏器指数明显增高，血清中ALT和AST水平升高（P<0.05)，PC-III和IV-C含量升高（P<0.05)，GSH、MDA水平升高（P<0.05)，SOD、GSH-PX活性降低（P<0.05)，TNF-α、IL-6、IL-1β水平明显升高（P<0.05）。与MOD组比较，DEX组小鼠脏器指数明显降低，血清中ALT和AST水平降低（P<0.05)，PC-III和IV-C含量降低（P<0.05），SOD、GSH-PX活性增高（P<0.05），GSH、MDA含量减少（P<0.05），TNF-α、IL-6、IL-1β明显降低（P<0.05）。HE染色显示，CON组肝组织形态结构正常，MOD组肝细胞水肿，炎症浸润和局灶坏死，DEX组细胞水肿减轻，仅见少量炎症细胞浸润。Masson染色显示，MOD组肝组织纤维增多，DEX组肝组织纤维减少。天狼猩红染色显示，MOD组肝组织纤维增多，DEX组肝组织纤维减少。Western blot检测结果显示，与CON组比较，MOD组PI3K/AKT蛋白表达水平升高（P<0.05），Nrf2/HO-1蛋白表达水平降低（P<0.05），P65、IKBα、TNF-α、IL-6蛋白表达水平增高（P<0.05）。与MOD组比较，DEX组降低模型小鼠肝组织中PI3K/AKT蛋白表达水平（P<0.05），提高Nrf2/HO-1蛋白表达水平（P<0.05），降低模型小鼠肝组织中P65、IKBα、TNF-α、IL-6蛋白表达水平（P<0.05）。

2、APAP模型，与CON组比较，MOD组血清中ALT和AST水平升高（P<0.05)，GSH、MDA水平升高（P<0.05)，SOD、GSH-PX活性降低（P<0.05)，TNF-α、IL-6、IL-1β水平明显增高（P<0.05）。与MOD相比，DEX组血清中ALT和AST水平降低（P<0.05），SOD、GSH-PX含量增高（P<0.05），GSH、MDA含量减少（P<0.05），TNF-α、IL-6、IL-1β明显降低（P<0.05）。HE染色显示，CON组肝组织形态结构正常，MOD组肝细胞水肿，炎症浸润和局灶坏死，DEX组细胞水肿减轻，仅见少量炎症细胞浸润。Western blot检测结果显示，与CON组比较，MOD组PI3K/AKT蛋白表达水平增高（P<0.05），Nrf2/HO-1蛋白表达水平降低（P<0.05），P65、IKBα、TNF-α、IL-6蛋白表达水平增高（P<0.05）。与MOD组比较，DEX组降低模型小鼠肝组织中PI3K/AKT蛋白表达水平（P<0.05），提高NrF2/HO-1蛋白表达水平（P<0.05）。DEX组降低模型小鼠肝组织中P65、IKBα、TNF-α、IL-6蛋白表达水平（P<0.05）。

结论：

1、右美托咪定对胆汁淤积小鼠肝纤维化和APAP致药物性肝损伤小鼠的肝脏损伤有改善作用。

2、右美托咪定对两种因素致肝损伤模型小鼠肝脏的保护作用机制，可能与其能够降低体内氧化应激和炎症水平有关,并通过调节PI3K/AKT/Nrf2和NF-κB/IKBα/TNF信号通路发挥一定作用。

关键词：右美托咪定；肝纤维化；胆汁淤积；药物性肝损伤；氧化应激；炎症反应

Objective: To observe the effect of dexmedetomidine on liver injury model mice induced by two factors, this paper discusses the protective effect of dexmedetomidine on liver injury in mice, and tries to clarify its possible mechanism.

Methods: In the first part, the mouse model of cholestatic hepatic fibrosis was established by bile duct ligation (BDL). Forty C57/BL6 mice were randomly divided into four groups. They were divided into Control group (CON group，sham operation+ intraperitoneal injection of the same amount of normal saline), Model group (MOD group, bdl+ intraperitoneal injection of the same amount of normal saline), Positive control group (PC group, bdl + intraperitoneal injection of colchicine 0.2mg/kg), Dexmedetomidine control group (DEX group, bdl + intraperitoneal injection of dexmedetomidine 10μg/kg). The CON group only open the abdominal cavity without ligation. Fasting can not help water for 12 hours. The mice were killed after anesthesia. The serum of mice was collected and the contents of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by microplate method. ELISA was used to detect type Ⅲ procollagen (PC-Ⅲ), type Ⅳ collagen (Ⅳ-C) in mouse serum and tumor necrosis factor in liver tissue- ɑ (Tumor Necrosis Factor-ɑ, TNF-ɑ)、 Interleukin-6 (IL-6), interleukin-1β（IL-1β）. The activities of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase (GSH PX) in mouse liver were measured. He, Masson and Sirius red staining were performed to observe the pathological changes of liver tissue. Western blot was used to detect the expression level of phosphatidylinositol 3-kinases (PI3K), protein kinase B (AKT), nuclear factor E2 (Nrf2), heme oxygenase-1 (HO-1) and nuclear transcription factor in mouse liver-κB（Nuclear factor kappa-B, NF-κB）、TNF- ɑ、IL-6 protein factor.

In the second part, the mouse model of drug-induced liver injury was induced by acetaminophen (APAP). Forty C57/BL6 mice were randomly divided into four groups with 10 mice in each group. They were divided into normal control group (CON group, intraperitoneal injection of the same amount of normal saline), model group (MOD group, intraperitoneal injection of APAP 200mg/kg), positive control group ( PC group，intraperitoneal injection of APAP 200mg/kg and colchicine 0.2mg/kg) and dexmedetomidine group (DEX group, intraperitoneal injection of APAP 200mg/kg and dexmedetomidine 10μg/kg). Fasting can not help water for 12 hours. The mice were killed after anesthesia. The serum of mice was collected and the contents of ALT and AST were measured. The activities of SOD, MDA, GSH and GSH-PX in mouse liver were measured. The level of  TNF- α、IL-6、IL-1β were detected by ELISA. HE staining was used to observe the pathological changes of liver tissue. Western blot was used to detect the expression level of PI3K、Akt、Nrf2、HO-1、NF-κB、TNF- ɑ、IL-6.

Results:

1、BDL model, compared with MOD group, the organ index，the levels of ALT and AST in serum, the contents of pc-Ⅲ and IV-C, GSH and MDA in mod group increased significantly (P<0.05)，the activities of SOD and GSH-PX decreased(P<0.05)，the level of TNF- α、IL-6、IL-1β increased significantly (P<0.05). Compared with MOD group, the organ index, the level of ALT and AST in serum, the contents of pc-Ⅲ and IV-C, GSH and MDA in mod group decreased significantly (P<0.05), the activities of SOD and GSH-PX increased(P<0.05), the level of TNF- α、IL-6、IL-1β decreased significantly (P<0.05). HE staining showed that the morphological structure of liver tissue was normal in CON group, hepatocyte edema, inflammatory infiltration and focal necrosis in MOD group, and cell edema was reduced in DEX group, with only a small amount of inflammatory cell infiltration. Masson staining showed that the liver tissue fibers increased in mod group and decreased in DEX group. Sirius red staining showed that liver tissue fibers increased in mod group and decreased in DEX group. Western blot showed that compared with con group, the expression level of PI3K/Akt protein in mod group increased (P<0.05), the expression level of Nrf2/HO-1 protein decreased (P <0.05), the expression level of p65、IKB α、TNF- α、IL-6 protein increased (P<0.05). Compared with MOD group, DEX group decreased the expression level of PI3K/Akt protein in liver tissue of model mice (P<0.05), increased the expression level of Nrf2/HO-1 (P<0.05), the expression level of p65、IKB α、TNF- α、IL-6 decreased (P<0.05).

2、APAP model, compared with con group, the serum levels of ALT and AST in mod group increased (P<0.05), the levels of GSH and MDA increased (P<0.05), the activities of SOD and GSH-PX decreased (P<0.05),the level of TNF-α、IL-6、IL-1β increased significantly (P<0.05). Compared with MOD group, the serum ALT and AST level in DEX group decreased (P<0.05), the contents of SOD and GSH-PX increased (P<0.05), the contents of GSH and MDA decreased (P<0.05), the level of TNF- α、IL-6、IL-1β significantly decreased (P<0.05). HE staining showed that the morphological structure of liver tissue was normal in CON group, hepatocyte edema, inflammatory infiltration and focal necrosis in MOD group, and cell edema was reduced in DEX group, with only a small amount of inflammatory cell infiltration. Western blot showed that compared with CON group, the expression level of PI3K/Akt protein in MOD group increased (P<0.05), the expression level of Nrf2/HO-1 protein decreased (P<0.05), the expression level of p65、IKBα、TNF- α、IL-6 protein increased (P<0.05). Compared with MOD group, DEX group decreased the expression level of PI3K/Akt protein in liver tissue of model mice (P<0.05) and increased the expression level of Nrf2/HO-1 protein (P<0.05). DEX group decreased the level of p65、IKBα、TNF- α、IL-6 protein (P<0.05).

Conclusion: 

1、Dexmedetomidine can improve the liver function of hepatic fibrosis in cholestatic mice and liver injury in APAP induced drug-induced mice.

2、The protective mechanism of dexmedetomidine on liver injury model mice caused by two factors be related, which may be reducing the level of oxidative stress and the production of inflammatory factors. Dexmedetomidine demonstrated a protective effect on liver damage potentially via activation of the PI3K/Akt/Nrf2 and NF-κB/IKBα/TNF-α signaling pathway.

Keywords: dexmedetomidine; hepatic fibrosis; cholestasis; drug induced liver injury; oxidative stress; inflammatory reaction

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