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NLRP3炎性体在胆总管结扎诱导的肝纤维化大鼠模型中的表达特点
DOI: 10.3969/j.issn.1001-5256.2021.09.020
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：周蒙恩、陈懿榕等负责课题设计，资料分析，撰写论文; 张娜、晏旎、阙任烨等参与收集数据，修改论文; 李勇等负责拟定写作思路，指导撰写文章并最后定稿。
Expression of NLRP3 inflammatory body in a rat model of liver fibrosis induced by common bile duct ligation
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摘要:
目的 探讨NLRP3炎性体在胆总管结扎(BDL)大鼠肝纤维化过程中的表达特点，探索NLRP3炎性体与肝纤维化的关系。 方法 将65只SD大鼠随机分为假手术组(n=15)、BDL模型组(n=50)。分别于第3、7、14、21、28天，处死10只模型组大鼠，同时处死3只假手术组大鼠。检测血清ALT、AST、DBil、TBil、TBA、ALP水平，对肝组织行HE、Masson、天狼星红-苦味酸染色，评估各组肝纤维化程度，免疫组化检测肝组织α平滑肌肌动蛋白(αSMA)、TGFβ1的表达水平，Western Blot、qRT-PCR检测肝组织NLRP3炎性体的表达水平，ELISA法检测肝组织炎症IL-1β水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。 结果 与假手术组比较，各BDL模型组大鼠血清ALT、AST、DBil、TBil、TBA、ALP水平均升高(P值均＜0.05)，肝组织IL-1β水平均升高(P值均＜0.05)，并于第3天达到最高，其后有所降低。与假手术组比较，随时间延长，BDL大鼠模型肝纤维化评分明显增加(P值均＜0.05)，免疫组化显示αSMA、TGFβ1表达逐渐增加(P值均＜0.05)，Western Blot、qRT-PCR显示肝组织NLRP3炎性体蛋白表达逐渐增加(P值均＜0.05)，并于第14天后保持稳定。 结论 BDL大鼠模型肝功能损伤持续存在，肝组织病理学存在肝炎-肝纤维化-肝硬化的动态演变，NLRP3炎性体处于持续活化状态，可能在肝纤维化过程中发挥重要作用。 -
关键词:
- 肝硬化 /
- 胆汁淤积 /
- NLR家族, 热蛋白结构域包含蛋白3 /
- 大鼠, Sprague-Dawley
Abstract:Objective To investigate the expression of NLRP3 inflammatory body in the process of liver fibrosis in a rat model of common bile duct ligation (BDL) and the association of NLRP3 inflammatory body with liver fibrosis. Methods A total of 65 Sprague-Dawley rats were randomly divided into sham-operation group with 15 rats and BDL model group with 50 rats. On days 3, 7, 14, 21, and 28, 10 rats in the model group and 3 rats in the sham-operation group were sacrificed. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBil), total bilirubin (TBil), total bile acid (TBA), and alkaline phosphatase (ALP) were measured, and HE staining, Masson staining, and sirius red-picric acid staining were performed for liver tissue to evaluate liver fibrosis degree. Immunohistochemistry was used to measure the expression levels of alpha-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) in liver tissue, Western blot and qRT-PCR were used to measure the expression level of NLRP3 inflammatory body, and ELISA was used to measure the level of the inflammatory factor interleukin-1β (IL-1β) in liver tissue. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for further comparison between two groups. Results Compared with the sham-operation group, the BDL model group had significant increases in the serum levels of ALT, AST, DBil, TBil, TBA, and ALP (all P < 0.05) and the level of IL-1β in liver tissue (P < 0.05), which reached the highest level on day 3 and then decreased. Compared with the sham-operation group over time, the BDL group had a significant increase in liver fibrosis score (P < 0.05); immunohistochemistry showed gradual increases in the expression of SMA-α and TGF-β1 (P < 0.05), and Western blot and qRT-PCR showed a gradual increase in the protein expression of NLRP3 inflammatory body in liver tissue (P < 0.05), which remained stable after day 14. Conclusion Liver injury exists persistently in a rat model of BDL, and liver histopathology shows the dynamic evolution of hepatitis, liver fibrosis, and liver cirrhosis. NLRP3 inflammatory body is in a state of continuous activation and may play an important role in the process of liver fibrosis. -
图 1 大鼠肝脏组织HE染色(×200)
注：a，假手术组; b，BDL 3 d; c，BDL 7 d; d，BDL 14 d; e，BDL 21 d; f，BDL 28 d。
图 2 大鼠肝脏组织Masson染色(×200)
注：a，假手术组; b，BDL 3 d; c，BDL 7 d; d，BDL 14 d; e，BDL 21 d; f，BDL 28 d。
图 3 大鼠肝脏组织苦味酸-天狼星染色(×200)
注：a，假手术组; b，BDL 3 d; c，BDL 7 d; d，BDL 14 d; e，BDL 21 d; f，BDL 28 d。
图 4 免疫组化检测αSMA在大鼠肝组织中的表达(×200)
注：a，假手术组; b，BDL 3 d; c，BDL 7 d; d，BDL 14 d; e，BDL 21 d; f，BDL 28 d。
图 5 免疫组化检测TGFβ1在大鼠肝组织中的表达(×200)
注：a，假手术组; b，BDL 3 d; c，BDL 7 d; d，BDL 14 d; e，BDL 21 d; f，BDL 28 d。
表 1 各组大鼠血清AST、ALT、ALP、DBil、TBA、TBil指标比较
组别 动物数(只) AST(U/L) ALT(U/L) ALP(U/L) DBil(μmol/L) TBA(μmol/L) TBil(μmol/L) 假手术组 15 91.05±6.68 30.60±13.03 169.70±37.65 0.07±0.08 16.68±5.25 0.42±0.13 BDL3 d 10 808.80±159.91) 154.00±63.771) 666.40±139.701) 141.60±24.221) 383.60±185.101) 151.60±32.611) BDL7 d 10 233.10±11.861)2) 31.40±5.681)2) 388.20±74.171)2) 61.66±15.401)2) 122.30±19.511)2) 71.51±16.261)2) BDL14 d 10 234.20±12.081)2) 25.10±9.191)2) 347.70±63.381)2) 70.42±25.001)2) 136.20±26.021)2) 72.78±24.281)2) BDL21 d 10 237.90±9.621)2) 41.10±12.111)2) 408.40±61.851)2) 74.14±11.051)2) 140.00±21.651)2) 77.21±22.151)2) BDL28 d 10 237.30±9.711)2) 63.10±23.871)2)4) 466.80±109.001)2)4) 84.19±13.661)2) 141.50±73.971)2) 88.96±9.931)2)3) F值 170.9 31.49 43.92 94.50 26.54 71.10 P值 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 ＜0.01 注：与假手术组相比，1)P＜0.05;与BDL3 d相比，2)P＜0.05;与BDL7 d相比，3)P＜0.05; 与BDL14 d相比，4)P＜0.05。 
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表 2 免疫组化半定量及肝纤维化评分
组别 动物数(只) αSMA表达强度(IOD/area)×100 TGFβ1表达强度(IOD/area)×100 肝纤维化评分 假手术组 15 1.16±0.42 1.05±0.38 0.00±0.00 BDL3 d 10 1.75±0.73 1.59±0.57 1.30±0.671) BDL7 d 10 4.04±0.771)2) 4.35±1.071)2) 2.30±0.951)2) BDL14 d 10 5.99±0.781)2)3) 6.70±0.991)2)3) 3.60±1.171)2)3) BDL21 d 10 7.12±0.611)2)3)4) 7.92±1.381)2)3)4) 4.80±1.401)2)3)4) BDL28 d 10 8.31±1.761)2)3)4)5) 9.11±1.551)2)3)4)5) 5.10±1.201)2)3)4) F值 114.60 118.90 51.41 P值 ＜0.01 ＜0.01 ＜0.01 注：与假手术组相比，1)P＜0.05;与BDL3 d相比，2)P＜0.05;与BDL7 d相比，3)P＜0.05; 与BDL14 d相比，4)P＜0.05;与BDL21 d相比，5)P＜0.05。
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表 3 NLRP3炎性体蛋白表达
组别 动物数(只) NLRP3(NLRP3/β-actin) ASC(ASC/β-actin) caspase1(caspase1/β-actin) 假手术组 15 1 1 1 BDL3 d 10 1.18±0.07 1.78±0.121) 1.74±0.141) BDL7 d 10 1.60±0.171)2) 2.18±0.151)2) 2.35±0.131)2) BDL14 d 10 2.07±0.141)2)3) 2.71±0.171)2)3) 2.80±0.161)2)3) BDL21 d 10 2.21±0.161)2)3) 2.76±0.181)2)3) 2.82±0.161)2)3) BDL28 d 10 2.24±0.131)2)3) 2.78±0.161)2)3) 2.78±0.171)2)3) F值 55.90 73.89 89.55 P值 ＜0.01 ＜0.01 ＜0.01 注：与假手术组相比，1)P＜0.05;与BDL3 d相比，2)P＜0.05;与BDL7 d相比，3)P＜0.05。
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表 4 NLRP3炎性体mRNA表达
组别 动物数(只) NLRP3(NLRP3/β-actin) ASC(ASC/β-actin) caspase1(caspase1/β-actin) 假手术组 15 1 1 1 BDL3 d 10 1.73±0.181) 1.59±0.221) 1.52±0.191) BDL7 d 10 2.16±0.321)2) 2.12±0.291)2) 1.80±0.241)2) BDL14 d 10 3.03±0.321)2)3) 2.74±0.291)2)3) 2.61±0.241)2)3) BDL21 d 10 2.97±0.231)2)3) 2.78±0.231)2)3) 2.71±0.241)2)3) BDL28 d 10 3.03±0.181)2)3) 2.79±0.201)2)3) 2.77±0.191)2)3) F值 177.50 141.20 173.00 P值 ＜0.01 ＜0.01 ＜0.01 注：与假手术组相比，1)P＜0.05;与BDL3 d相比，2)P＜0.05;与BDL7 d相比，3)P＜0.05。
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表 5 各组肝组织IL-1β水平
组别 动物数(只) IL-1β(pg/mg) 假手术组 15 45.81±4.77 BDL3 d 10 139.90±18.381) BDL7 d 10 55.83±10.251)2) BDL14 d 10 79.76±12.921)2)3) BDL21 d 10 70.52±8.621)2)3) BDL28 d 10 100.60±8.691)2)3)4)5) F值 106.10 P值 ＜0.01 注：与假手术组相比，1)P＜0.05;与BDL3 d相比，2)P＜0.05;与BDL7 d相比，3)P＜0.05;与BDL14 d相比，4)P＜0.05;与BDL21 d相比，5)P＜0.05。
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