高迁移率族蛋白B1在肝脏慢性炎症与纤维化中的作用
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摘要
研究背景：
肝纤维化（Hepaticfibrosis）这一病理过程是各种慢性肝病进展为肝硬化的共同途径。肝纤维化最终发展为肝硬化和肝癌的比率甚高，高达25％～40％，而肝硬化的并发症和肝癌的死亡率是严重危害人们健康的世界性问题。肝纤维化是一个具有可逆性的病理过程，因此，阻止肝纤维化的进展是改善肝病预后的重要策略。目前研究认为，慢性炎症反应是肝纤维化发生发展的重要因素。近期的研究表明，高迁移率族蛋白B1（Highmobilitygroupproteinbox1，HMGB1）作为典型的内源性危险相关分子（Danger-associatedmolecularpatterns，DAMPs），在肝损伤引发的炎症反应中起着重要的作用。针对HMGB1在肝纤维化中作用的研究，引发了学者们极大的兴趣。因此，我们旨在观察HMGB1对肝脏慢性炎症反应和纤维化反应的作用。
目的：
通过观察高迁移率组蛋白1（Highmobilitygroupboxprotein1，HMGB1）对肝纤维化小鼠的相关炎症因子和纤维化因子的影响，探讨HMGB1在肝脏慢性炎症和纤维化中的作用。
方法：
第一部分实验：昆明雄性小鼠40只，采用CCL-4灌胃法制备肝纤维化模型，随机分为正常对照组（1ul/g生理盐水灌胃，每5天一次）、2周模型组、4周模型组、6周模型组（1ul/gCCL4油溶液灌胃，每5天一次）。各组动物处理前12小时禁食，摘除眼球法取血，ELISA法检测血清中透明质酸（HA）、Ⅰ型胶原蛋白（Col-Ⅰ）、Ⅲ型前胶原蛋白（PⅢP）和肿瘤坏死因子α（TNF-α）、白介素6（IL-6）的含量；分别对肝组织病理切片进行HE染色和天狼猩红染色；Westernblot法检测肝组织HMGB1的表达水平。
第二部分实验：昆明雄性小鼠20只，采用CCL4灌胃法制备肝纤维化模型，随机分为模型组（1ul/gCCL4油溶液灌胃，每5天一次）、干预组（1ul/gCCL4油溶液灌胃，50ug/mouseHMGB1-Ab和5ug/g丙酮酸乙酯腹腔注射,每5天一次）。各组动物处理前12小时禁食，摘除眼球法取血，ELISA法检测血清中HA、Col-Ⅰ、PⅢP和IL-6、TNF-α的含量；分别对肝组织病理切片进行HE染色和天狼猩红染色；半定量RT-PCR法检测小鼠肝组织中TGF-β、α-SMA和MMP-9mRNA的表达。
结果：
1、建立小鼠肝纤维化模型。6周模型组病理切片显示肝细胞变性坏死比较严重，伴有炎症细胞浸润，并出现肝纤维组织增生；ELISA法检测血清胶原蛋白含量结果显示，正常对照组血清中HA、Col-Ⅰ、PⅢP含量较少；2周、4周、6周模型组血清中HA、Col-Ⅰ、PⅢP含量均较正常对照组明显增加(P＜0.05)；6周模型组血清中HA、Col-Ⅰ、PⅢP含量均较4周模型组明显增加(P＜0.05)。
2、CCL4致肝纤维化模型IL-6、TNF-α和HMGB1的变化。ELISA法检测正常对照组血清中IL-6、TNF-α含量较少；2周、4周、6周模型组血清中IL-6、TNF-α含量均较对照组明显增加(P＜0.05)；6周组血清中IL-6、TNF-α含量均较4周组明显增加(P＜0.05)。Western-blot结果表明正常对照组肝组织内无HMGB1表达，肝纤维化过程中肝组织HMGB1的表达随着CCL4损伤周期的延长而逐渐升高，与正常对照组比较，2周、4周、6周模型组的HMGB1表达量明显增加，有统计学意义（P<0.05）；与4周模型组比较，6周HMGB1的表达量显著增加（P<0.05）。
3、拮抗HMGB1可降低血清中IL-6、TNF-α的含量。ELISA法检测各组动物血清中炎症因子含量结果显示，与模型组比较，干预组动物血清中IL-6、TNF-α的含量明显减少（P<0.05）。
4、拮抗HMGB1可降低肝纤维化程度。HE染色显示，模型组肝细胞弥漫性脂肪变性，中央静脉周围肝细胞灶状坏死，炎症细胞浸润，汇管区周围纤维化，纤维间隔形成。干预组肝细胞少量的脂肪变性，炎症细胞浸润减少，汇管区有纤维产生，但无间隔形成。天狼猩红染色结果显示模型组肝组织内胶原纤维向肝小叶延伸，并形成纤维间隔，与模型组相比较，干预组肝组织内胶原纤维显著下降（0.57％±0.21％vs1.02％±0.33％；P<0.05）。ELISA法检测各组动物血清中胶原蛋白含量结果显示，与模型组比较，干预组动物血清中HA、Col-Ⅰ、PⅢP的含量明显减少（P<0.05）。RT-PCR结果显示与模型组小鼠肝脏组织TGF-β、α-SMA的表达量相比较，干预组小鼠肝脏组织这些因子的表达量均明显降低（P<0.05），同时MMP-9mRNA的表达量增高。
结论：1.CCl4致肝纤维化动物模型中，HMGB1和相关炎症因子IL-6、TNF-α的表达增高.2.HMGB1的促纤维化作用与促炎作用有关。拮抗HMGB1后，相关炎症因子的浓度降低，致纤维化因子减少，肝纤维化程度减轻。
Background:
Hepatic fibrosis is the common pathological process of a variety of chronic liver disease to cirrhosis, and is an important part of influencing the prognosis of chronic liver disease. The rate of liver fibrosis to cirrhosis and liver cancer is very high(25%～40%), and these diseases are becoming serious threats to people's health worldwide. Hepatic fibrosis is a reversible pathological process, so holding back the progress of hepatic fibrosis is an important strategy to improve the liver disease prognosis. Recognized chronic inflammatory response is an important factor in hepatic fibrosis. Recent studies show that the High mobility group protein box1(HMGB1), as a typical endogenous Danger-associated molecular patterns(DAMPs), plays an important role in the inflammatory response of the liver damage. The study of the role of HMGB1in liver fibrosis, led to great interest of scholars. Therefore, we aim to observe the role of HMGB1in chronic hepatic inflammatory response and fibrosis reaction.
Objective:
To investigate the role of High Mobility Group Box Protein1in the chronic hepatic inflammation and fibrosis reaction through observe the expression of inflammatory cytokines and fibrosis factor.
Methods:
The first part of the experiment:Forty male Kunming mice, the ccl4gavage preparation of liver fibrosis were randomly divided into three groups:the normal control group,1ul/g saline was administered every five days;2weeks model group,4weeks model group,6weeks model group, ccl4(1ul/g,diluted with corn oil) was administered every five days. Each mice was sacrificed by removal of the eye method. The parameters of plasma'HA, Col-Ⅰ, PⅢP and IL-6, TNF-α were measured by ELISA. The morphological changes of the liver tissue were observed with HE staining and Sirius red staining. The level of HMGB1in liver tissue were measured by western blot.
The second part of the experiment:Twenty male Kunming mice,the ccl4gavage preparation of liver fibrosis were randomly divided into two groups:the model group, ccl4(1ul/g,diluted with corn oil) was administered every five days; the intervention group, ccl4(1ul/g, diluted with corn oil),50ug/mouse HMGB1-Ab,5ug/g ethyl pyruvate were administered by gavage or intraperitoneal injection every five days. Each mice was sacrificed by removal of the eye method. The parameters of plasma'HA, Col-Ⅰ, PⅢP and IL-6, TNF-α were measured by ELISA. The morphological changes of the liver tissue were observed with HE staining and Sirius red staining. The level of HMGB1in liver tissue were measured by western blot. The level of TGF-β,α-SMA和MMP-9mRNA were detected by RT-PCR.
Results:
1. Successfully established liver fibrosis model. In6weeks model group, liver cells degenerate and necrosis, inflammatory cells infiltrate and liver fibrous tissue proliferate. The levels of HA, Col-Ⅰ, PⅢP in the normal control group was very low in serum measured by ELISA. Compared with the normal control group, the levels of them in2weeks model group,4weeks model group and6weeks model group were remarkably increased (P<0.05), meanwhile, they in6weeks group were significantly higher than4weeks group(P<0.05).
2. The changes of IL-6, TNF-α and HMGB1in ccl4induced liver fibrosis model. The levels of IL-6, TNF-α in the normal control group was very low in serum measured by ELISA. Compared with the normal control group, the levels of them in4weeks model group and6weeks model group were remarkably increased (P<0.05), meanwhile, they in6weeks group were significantly higher than4weeks group(P<0.05). Wester-blot results show that no HMGB1expression in the liver tissue of normal control group, and in the process of liver fibrosis, the higher HMGB1is expressed in liver tissue,the longer ccl4is administered. Compared with the normal control group, the levels of HMGB1in4weeks model group and6weeks model group were significantly increased (P<0.05), meanwhile, they in6weeks group were much higher than4weeks group(P<0.05).
3. Antagonistic HMGB1can reduce the degree of liver fibrosis. In model group, liver cells steatosis and necrosis, inflammation cells infiltrate, liver fibrous tissue proliferate. Liver cells of intervention group have a small amount of steatosis,inflammatory cells infiltration,but no liver fibrosis formation in tissue. Antagonistic HMGB1can cause the marked decrease of liver fibrosis tissue by Sirius red staining (0.57%±0.21%vs1.02%±0.33%; P<0.05) and significantly reversed liver fibrosis. Compared with model group, the levels of HA, Col-Ⅰ, PⅢP in intervention group were remarkably decreased (P<0.05). Compared with model group, The mRNA expression of TGF-β, α-SMA in intervention group were significantly downregulated (P<0.05), however, the mRNA expression of MMP-9was upregulated(P<0.05).
4. Antagonistic HMGB1can reduce the content of IL-6, TNF-α. Compared with model group, the levels of IL-6, TNF-α in intervention group were remarkably decreased (P<0.05).
Conclusion:
1. In the CC14induced liver fibrosis model, the levels of HMGB1and inflammatory factors IL-6,TNF-a are significantly increased.
2. There are relationship between fibrogenic role and proinflammatory role in HMGB1. HMGB1antibody can reduce the lever of the inflammatory factors and fibro-genic cytokines in mice with liver fibrosis.
肝纤维化（Hepaticfibrosis）这一病理过程是各种慢性肝病进展为肝硬化的共同途径。肝纤维化最终发展为肝硬化和肝癌的比率甚高，高达25％～40％，而肝硬化的并发症和肝癌的死亡率是严重危害人们健康的世界性问题。肝纤维化是一个具有可逆性的病理过程，因此，阻止肝纤维化的进展是改善肝病预后的重要策略。目前研究认为，慢性炎症反应是肝纤维化发生发展的重要因素。近期的研究表明，高迁移率族蛋白B1（Highmobilitygroupproteinbox1，HMGB1）作为典型的内源性危险相关分子（Danger-associatedmolecularpatterns，DAMPs），在肝损伤引发的炎症反应中起着重要的作用。针对HMGB1在肝纤维化中作用的研究，引发了学者们极大的兴趣。因此，我们旨在观察HMGB1对肝脏慢性炎症反应和纤维化反应的作用。
目的：
通过观察高迁移率组蛋白1（Highmobilitygroupboxprotein1，HMGB1）对肝纤维化小鼠的相关炎症因子和纤维化因子的影响，探讨HMGB1在肝脏慢性炎症和纤维化中的作用。
方法：
第一部分实验：昆明雄性小鼠40只，采用CCL-4灌胃法制备肝纤维化模型，随机分为正常对照组（1ul/g生理盐水灌胃，每5天一次）、2周模型组、4周模型组、6周模型组（1ul/gCCL4油溶液灌胃，每5天一次）。各组动物处理前12小时禁食，摘除眼球法取血，ELISA法检测血清中透明质酸（HA）、Ⅰ型胶原蛋白（Col-Ⅰ）、Ⅲ型前胶原蛋白（PⅢP）和肿瘤坏死因子α（TNF-α）、白介素6（IL-6）的含量；分别对肝组织病理切片进行HE染色和天狼猩红染色；Westernblot法检测肝组织HMGB1的表达水平。
第二部分实验：昆明雄性小鼠20只，采用CCL4灌胃法制备肝纤维化模型，随机分为模型组（1ul/gCCL4油溶液灌胃，每5天一次）、干预组（1ul/gCCL4油溶液灌胃，50ug/mouseHMGB1-Ab和5ug/g丙酮酸乙酯腹腔注射,每5天一次）。各组动物处理前12小时禁食，摘除眼球法取血，ELISA法检测血清中HA、Col-Ⅰ、PⅢP和IL-6、TNF-α的含量；分别对肝组织病理切片进行HE染色和天狼猩红染色；半定量RT-PCR法检测小鼠肝组织中TGF-β、α-SMA和MMP-9mRNA的表达。
结果：
1、建立小鼠肝纤维化模型。6周模型组病理切片显示肝细胞变性坏死比较严重，伴有炎症细胞浸润，并出现肝纤维组织增生；ELISA法检测血清胶原蛋白含量结果显示，正常对照组血清中HA、Col-Ⅰ、PⅢP含量较少；2周、4周、6周模型组血清中HA、Col-Ⅰ、PⅢP含量均较正常对照组明显增加(P＜0.05)；6周模型组血清中HA、Col-Ⅰ、PⅢP含量均较4周模型组明显增加(P＜0.05)。
2、CCL4致肝纤维化模型IL-6、TNF-α和HMGB1的变化。ELISA法检测正常对照组血清中IL-6、TNF-α含量较少；2周、4周、6周模型组血清中IL-6、TNF-α含量均较对照组明显增加(P＜0.05)；6周组血清中IL-6、TNF-α含量均较4周组明显增加(P＜0.05)。Western-blot结果表明正常对照组肝组织内无HMGB1表达，肝纤维化过程中肝组织HMGB1的表达随着CCL4损伤周期的延长而逐渐升高，与正常对照组比较，2周、4周、6周模型组的HMGB1表达量明显增加，有统计学意义（P<0.05）；与4周模型组比较，6周HMGB1的表达量显著增加（P<0.05）。
3、拮抗HMGB1可降低血清中IL-6、TNF-α的含量。ELISA法检测各组动物血清中炎症因子含量结果显示，与模型组比较，干预组动物血清中IL-6、TNF-α的含量明显减少（P<0.05）。
4、拮抗HMGB1可降低肝纤维化程度。HE染色显示，模型组肝细胞弥漫性脂肪变性，中央静脉周围肝细胞灶状坏死，炎症细胞浸润，汇管区周围纤维化，纤维间隔形成。干预组肝细胞少量的脂肪变性，炎症细胞浸润减少，汇管区有纤维产生，但无间隔形成。天狼猩红染色结果显示模型组肝组织内胶原纤维向肝小叶延伸，并形成纤维间隔，与模型组相比较，干预组肝组织内胶原纤维显著下降（0.57％±0.21％vs1.02％±0.33％；P<0.05）。ELISA法检测各组动物血清中胶原蛋白含量结果显示，与模型组比较，干预组动物血清中HA、Col-Ⅰ、PⅢP的含量明显减少（P<0.05）。RT-PCR结果显示与模型组小鼠肝脏组织TGF-β、α-SMA的表达量相比较，干预组小鼠肝脏组织这些因子的表达量均明显降低（P<0.05），同时MMP-9mRNA的表达量增高。
结论：1.CCl4致肝纤维化动物模型中，HMGB1和相关炎症因子IL-6、TNF-α的表达增高.2.HMGB1的促纤维化作用与促炎作用有关。拮抗HMGB1后，相关炎症因子的浓度降低，致纤维化因子减少，肝纤维化程度减轻。
Background:
Hepatic fibrosis is the common pathological process of a variety of chronic liver disease to cirrhosis, and is an important part of influencing the prognosis of chronic liver disease. The rate of liver fibrosis to cirrhosis and liver cancer is very high(25%～40%), and these diseases are becoming serious threats to people's health worldwide. Hepatic fibrosis is a reversible pathological process, so holding back the progress of hepatic fibrosis is an important strategy to improve the liver disease prognosis. Recognized chronic inflammatory response is an important factor in hepatic fibrosis. Recent studies show that the High mobility group protein box1(HMGB1), as a typical endogenous Danger-associated molecular patterns(DAMPs), plays an important role in the inflammatory response of the liver damage. The study of the role of HMGB1in liver fibrosis, led to great interest of scholars. Therefore, we aim to observe the role of HMGB1in chronic hepatic inflammatory response and fibrosis reaction.
Objective:
To investigate the role of High Mobility Group Box Protein1in the chronic hepatic inflammation and fibrosis reaction through observe the expression of inflammatory cytokines and fibrosis factor.
Methods:
The first part of the experiment:Forty male Kunming mice, the ccl4gavage preparation of liver fibrosis were randomly divided into three groups:the normal control group,1ul/g saline was administered every five days;2weeks model group,4weeks model group,6weeks model group, ccl4(1ul/g,diluted with corn oil) was administered every five days. Each mice was sacrificed by removal of the eye method. The parameters of plasma'HA, Col-Ⅰ, PⅢP and IL-6, TNF-α were measured by ELISA. The morphological changes of the liver tissue were observed with HE staining and Sirius red staining. The level of HMGB1in liver tissue were measured by western blot.
The second part of the experiment:Twenty male Kunming mice,the ccl4gavage preparation of liver fibrosis were randomly divided into two groups:the model group, ccl4(1ul/g,diluted with corn oil) was administered every five days; the intervention group, ccl4(1ul/g, diluted with corn oil),50ug/mouse HMGB1-Ab,5ug/g ethyl pyruvate were administered by gavage or intraperitoneal injection every five days. Each mice was sacrificed by removal of the eye method. The parameters of plasma'HA, Col-Ⅰ, PⅢP and IL-6, TNF-α were measured by ELISA. The morphological changes of the liver tissue were observed with HE staining and Sirius red staining. The level of HMGB1in liver tissue were measured by western blot. The level of TGF-β,α-SMA和MMP-9mRNA were detected by RT-PCR.
Results:
1. Successfully established liver fibrosis model. In6weeks model group, liver cells degenerate and necrosis, inflammatory cells infiltrate and liver fibrous tissue proliferate. The levels of HA, Col-Ⅰ, PⅢP in the normal control group was very low in serum measured by ELISA. Compared with the normal control group, the levels of them in2weeks model group,4weeks model group and6weeks model group were remarkably increased (P<0.05), meanwhile, they in6weeks group were significantly higher than4weeks group(P<0.05).
2. The changes of IL-6, TNF-α and HMGB1in ccl4induced liver fibrosis model. The levels of IL-6, TNF-α in the normal control group was very low in serum measured by ELISA. Compared with the normal control group, the levels of them in4weeks model group and6weeks model group were remarkably increased (P<0.05), meanwhile, they in6weeks group were significantly higher than4weeks group(P<0.05). Wester-blot results show that no HMGB1expression in the liver tissue of normal control group, and in the process of liver fibrosis, the higher HMGB1is expressed in liver tissue,the longer ccl4is administered. Compared with the normal control group, the levels of HMGB1in4weeks model group and6weeks model group were significantly increased (P<0.05), meanwhile, they in6weeks group were much higher than4weeks group(P<0.05).
3. Antagonistic HMGB1can reduce the degree of liver fibrosis. In model group, liver cells steatosis and necrosis, inflammation cells infiltrate, liver fibrous tissue proliferate. Liver cells of intervention group have a small amount of steatosis,inflammatory cells infiltration,but no liver fibrosis formation in tissue. Antagonistic HMGB1can cause the marked decrease of liver fibrosis tissue by Sirius red staining (0.57%±0.21%vs1.02%±0.33%; P<0.05) and significantly reversed liver fibrosis. Compared with model group, the levels of HA, Col-Ⅰ, PⅢP in intervention group were remarkably decreased (P<0.05). Compared with model group, The mRNA expression of TGF-β, α-SMA in intervention group were significantly downregulated (P<0.05), however, the mRNA expression of MMP-9was upregulated(P<0.05).
4. Antagonistic HMGB1can reduce the content of IL-6, TNF-α. Compared with model group, the levels of IL-6, TNF-α in intervention group were remarkably decreased (P<0.05).
Conclusion:
1. In the CC14induced liver fibrosis model, the levels of HMGB1and inflammatory factors IL-6,TNF-a are significantly increased.
2. There are relationship between fibrogenic role and proinflammatory role in HMGB1. HMGB1antibody can reduce the lever of the inflammatory factors and fibro-genic cytokines in mice with liver fibrosis.
引文
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