敲减Blimp1基因表达对CCl4诱导的小鼠肝纤维化模型早期肝损伤的保护作用

doi:10.19723/j.issn.1671-167X.2025.04.016

摘要/Abstract

摘要：

目的: 探讨敲减转录因子B淋巴细胞诱导成熟蛋白1(B lymphocyte induced maturation protein 1, Blimp1)基因对四氯化碳(carbon tetrachloride, CCl₄)诱导的小鼠肝纤维化模型早期肝损伤的保护作用。方法: 采用C57BL/6小鼠腹腔注射5%(体积分数)CCl₄橄榄油溶液制备肝纤维化小鼠模型，采用小鼠腹腔注射短发夹RNA (short hairpin RNA, shRNA)腺相关病毒(adeno-associated virus, AAV)敲减Blimp1基因表达。将小鼠随机分为3组，空白实验组(n=10)，无意义RNA对照组(n=10)和Blimp1敲减组(n=10)。CCl₄诱导的小鼠肝纤维化模型制备27 d后取材，通过Western blot和real-time PCR检测小鼠肝组织Blimp1蛋白、α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、Ⅰ型胶原蛋白(collagen type Ⅰ alpha 1, COL1A1)、Ⅲ型胶原蛋白(collagen type Ⅲ alpha 1, COL3A1)及其mRNA表达水平；测定各组小鼠血清中天门冬氨酸氨基转移酶(aspartate transaminase, AST)、丙氨酸氨基转移酶(alanine transaminase, ALT)水平；采用苏木素-伊红染色、Masson染色和天狼星红染色法鉴定小鼠肝组织的病理变化及肝纤维化程度。结果: 与空白实验组相比，无意义RNA对照组小鼠肝脏Blimp1蛋白表达水平显著升高(2.036±0.244, t=3.690, P=0.002)，Blimp1敲减组小鼠Blimp1蛋白表达降低至基础水平(0.783±0.249, t=6.223, P=0.003)。与无意义RNA对照组小鼠血清ALT [(1 957.8±633.6) U/L]和AST [(1 808.8±260.1) U/L]相比，Blimp1敲减组小鼠血清ALT [(894.0±360.1) U/L, t=3.998, P=0.003]和AST [(820.0±100.6) U/L, t=6.141, P=0.004]水平均显著降低，肝组织炎性细胞浸润减少、纤维化程度减轻，肝脏α-SMA(0.676±0.064, t=7.930, P=0.001)、COL1A1(1.426±0.143, t=6.364, P=0.003)、COL3A1(1.124±0.198, t=3.440, P=0.026)蛋白表达水平降低，且mRNA表达与蛋白水平变化一致。结论: Blimp1在CCl₄诱导的小鼠肝纤维化中发挥重要作用，敲减Blimp1表达有利于保护小鼠的早期肝损伤。

关键词: 肝纤维化, 肝损伤, 动物模型, B淋巴细胞诱导成熟蛋白1

Abstract:

Objective: To explore the protective effect of knock-down the expression of B lymphocyte induced maturation protein 1 (Blimp1) gene on early liver injury in carbon tetrachloride (CCl₄)-induced mouse model of liver fibrosis. Methods: C57BL/6 mice were intraderitoneal injected with 5% CCl₄ olive oil solution to create mouse model of hepatic fibrosis. The expression of Blimp1 gene in the mice was reduced by intraderitoneal injection of short hairpin RNA (shRNA) adeno-associated virus (AAV). The mice were randomly divided into 3 groups: blank test group (n=10), CCl₄+AAV-shRNA-NC group (n=10) and CCl₄+AAV-shRNA-Blimp1 group (n=10). After 27 days of preparation of the CCl₄ mouse model, animal materials were carried out. Western blot and real-time PCR were used to detect the levels of Blimp1, α-smooth muscle actin (α-SMA), collagen type Ⅰ alpha 1 (COL1A1), collagen type Ⅲ alpha 1 (COL3A1), and their mRNA expression levels of liver tissue in each group. The serum of each group was separated to measure aspartate transaminase (AST) and alanine transaminase (ALT) by automatic biochemical analyzer. The pathological changes of liver tissue and the degree of liver fibrosis in the mice were detected by pathological staining including hematoxylin-eosin staining, Masson, and Sirius red. Results: The expression levels of Blimp1 protein in the liver of CCl₄+AAV-shRNA-NC group (2.036±0.244, t=3.690, P=0.002) were significantly increased than that of the blank test group. In the CCl₄+AAV-shRNA-Blimp1 group, the expression of Blimp1 protein decreased to the basal level (0.783±0.249, t=6.223, P=0.003). Compared with the serum levels of ALT [(1 957.8±633.6) U/L] and AST [(1 808.8±260.1) U/L] in the CCl₄+AAV-shRNA-NC group, the serum levels of ALT [(894.0±360.1) U/L, t=3.998, P=0.003] and AST [(820.0±100.6) U/L, t=6.141, P=0.004] in the CCl₄+AAV-shRNA-Blimp1 group were significantly decreased. The pathological results of the CCl₄+AAV-shRNA-Blimp1 group showed that compared with the CCl₄+AAV-shRNA-NC group, the infiltration of inflammatory cells in the liver tissue was reduced and the degree of fibrosis was alleviated. The level of α-SMA (0.676±0.064, t=7.930, P=0.001), COL1A1 (1.426±0.143, t=6.364, P=0.003) and COL3A1 (1.124±0.198, t=3.440, P=0.026) of liver in the CCl₄+AAV-shRNA-Blimp1 group were significantly decreased than that of CCl₄+AAV-shRNA-NC group, and the mRNA expression levels were altered as well as their protein levels. Conclusion: Blimp1 plays an important role in CCl₄-induced liver fibrosis in mice, and knock-down the expression of Blimp1 gene is beneficial to protect early liver injury in mice.

Key words: Liver fibrosis, Liver injury, Animal models, B lymphocyte induced maturation protein 1

中图分类号:

R575.2

秦秋实, 李蕊, 周妍希, 张玥, 韩铭, 朱鏐娈. 敲减Blimp1基因表达对CCl₄诱导的小鼠肝纤维化模型早期肝损伤的保护作用[J]. 北京大学学报（医学版）, 2025, 57(4): 727-734.

Qiushi QIN, Rui LI, Yanxi ZHOU, Yue ZHANG, Ming HAN, Liuluan ZHU. Protective effect of knock-down the expression of Blimp1 gene on early liver injury in CCl₄-induced mouse model of liver fibrosis[J]. Journal of Peking University (Health Sciences), 2025, 57(4): 727-734.

图/表 6

表1

图1

图2

图3

图4

图5

参考文献 27

1	Marcellin P , Kutala BK . Liver diseases: A major, neglected global public health problem requiring urgent actions and large-scale screening[J]. Liver Int, 2018, 38 (Suppl 1): 2- 6.
2	Ramachandran P , Dobie R , Wilson-Kanamori JR , et al. Resolving the fibrotic niche of human liver cirrhosis at single-cell level[J]. Nature, 2019, 575 (7783): 512- 518.
3	Friedman SL , Pinzani M . Hepatic fibrosis 2022:Unmet needs and a blueprint for the future[J]. Hepatology, 2022, 75 (2): 473- 488.
4	Rockey DC , Bell PD , Hill JA . Fibrosis: A common pathway to organ injury and failure[J]. N Engl J Med, 2015, 372 (12): 1138- 1149.
5	Shapiro-Shelef M , Lin KI , McHeyzer-Williams LJ , et al. Blimp-1 is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B cells[J]. Immunity, 2003, 19 (4): 607- 620.
6	Minnich M , Tagoh H , Bönelt P , et al. Multifunctional role of the transcription factor Blimp-1 in coordinating plasma cell differentiation[J]. Nat Immunol, 2016, 17 (3): 331- 343.
7	Bankoti R , Ogawa C , Nguyen T , et al. Differential regulation of effector and regulatory T cell function by Blimp1[J]. Sci Rep, 2017, 7 (1): 12078.
8	Shin H , Blackburn SD , Intlekofer AM , et al. A role for the transcriptional repressor Blimp-1 in CD8(+) T cell exhaustion during chronic viral infection[J]. Immunity, 2009, 31 (2): 309- 320.
9	Savitsky D , Cimmino L , Kuo T , et al. Multiple roles for Blimp-1 in B and T lymphocytes[J]. Adv Exp Med Biol, 2007, 596, 9- 30.
10	Kim SJ , Zou YR , Goldstein J , et al. Tolerogenic function of Blimp-1 in dendritic cells[J]. J Exp Med, 2011, 208 (11): 2193- 2199.
11	Gateva V , Sandling JK , Hom G , et al. A large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus[J]. Nat Genet, 2009, 41 (11): 1228- 1233.
12	Ellinghaus D , Zhang H , Zeissig S , et al. Association between variants of PRDM1 and NDP52 and Crohn ' s disease, based on exome sequencing and functional studies[J]. Gastroenterology, 2013, 145 (2): 339- 347.
13	Boi M , Zucca E , Inghirami G , et al. PRDM1/BLIMP1:A tumor suppressor gene in B and T cell lymphomas[J]. Leuk Lymphoma, 2015, 56 (5): 1223- 1228.
14	Li N , Fan X , Wang X , et al. PRDM1 levels are associated with clinical diseases in chronic HBV infection and survival of patients with HBV-related hepatocellular carcinoma[J]. Int Immunopharmacol, 2019, 73, 156- 162.
15	Scholten D , Trebicka J , Liedtke C , et al. The carbon tetrachloride model in mice[J]. Lab Anim, 2015, 49 (Suppl 1): 4- 11.
16	Bárcena C , Aran G , Perea L , et al. CD5L is a pleiotropic player in liver fibrosis controlling damage, fibrosis and immune cell content[J]. EBioMedicine, 2019, 43, 513- 524.
17	Pellicano AJ , Spahn K , Zhou P , et al. Collagen characterization in a model of nonalcoholic steatohepatitis with fibrosis: A call for development of targeted therapeutics[J]. Molecules, 2021, 26 (11): 3316.
18	Fan W , Liu T , Chen W , et al. ECM1 prevents activation of transforming growth factor β, hepatic stellate cells, and fibrogenesis in mice[J]. Gastroenterology, 2019, 157 (5): 1352- 1367.
19	Friedman SL . Hepatic stellate cells: Protean, multifunctional, and enigmatic cells of the liver[J]. Physiol Rev, 2008, 88 (1): 125- 172.
20	Xu XY , Ding HG , Li WG , et al. Chinese guidelines on management of hepatic encephalopathy in cirrhosis[J]. World J Gastroenterol, 2019, 25 (36): 5403- 5422.
21	Cao Y , Mai W , Li R , et al. Macrophages evoke autophagy of hepatic stellate cells to promote liver fibrosis in NAFLD mice via the PGE2/EP4 pathway[J]. Cell Mol Life Sci, 2022, 79 (6): 303.
22	Kiziltas S . Toll-like receptors in pathophysiology of liver diseases[J]. World J Hepatol, 2016, 8 (32): 1354- 1369.
23	Seki E , De Minicis S , Osterreicher CH , et al. TLR4 enhances TGF-beta signaling and hepatic fibrosis[J]. Nat Med, 2007, 13 (11): 1324- 1332.
24	Genestier L , Taillardet M , Mondiere P , et al. TLR agonists selectively promote terminal plasma cell differentiation of B cell subsets specialized in thymus-independent responses[J]. J Immunol, 2007, 178 (12): 7779- 7786.
25	Ko YA , Chan YH , Liu CH , et al. Blimp-1-mediated pathway promotes type Ⅰ IFN production in plasmacytoid dendritic cells by targeting to interleukin-1 receptor-associated kinase M[J]. Front Immunol, 2018, 9, 1828.
26	Dewidar B , Meyer C , Dooley S , et al. TGF-β in hepatic stellate cell activation and liver fibrogenesis-updated 2019[J]. Cells, 2019, 8 (11): 1419.
27	Flores-Contreras L , Sandoval-Rodríguez AS , Mena-Enriquez MG , et al. Treatment with pirfenidone for two years decreases fibrosis, cytokine levels and enhances CB2 gene expression in patients with chronic hepatitis C[J]. BMC Gastroenterol, 2014, 14, 131.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

shRNA	Oligonucleotides (5′-3′)
shRNA-NC	CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTT
shRNA-Blimp1-1	AAAAGGTGCAGCCTTTATGAGTCCTCGAGGACTCATAAAGGCTGCACC
shRNA-Blimp1-2	AAAACTCTCGACAGCAAATGGTTCTCGAGAACCATTTGCTGTCGAGAG
shRNA-Blimp1-3	AAAAGCAGGATTACCCAAGAATACTCGAGTATTCTTGGGTAATCCTGC

[1]	柯涵炜, 王起, 许克新. 优化环磷酰胺剂量在间质性膀胱炎/膀胱疼痛综合征啮齿动物模型中的应用[J]. 北京大学学报（医学版）, 2024, 56(5): 908-912.
[2]	王磊,金香淑,董慧君,欧国敏,赖鑫源,庄辉,李彤,向宽辉. 基于COL1A1启动子和增强型绿色荧光蛋白基因建立人肝星状细胞活化的细胞模型[J]. 北京大学学报（医学版）, 2023, 55(5): 876-885.
[3]	孟令玮,李雪,高胜寒,李悦,曹瑞涛,张毅,潘韶霞. 三种方法建立大鼠种植体周炎模型的比较[J]. 北京大学学报（医学版）, 2023, 55(1): 22-29.
[4]	梁秀睿,闪雪纯,关晶,张锐,杨静,张怡,金佳琦,张誉馨,徐凡,傅继华. 高血糖诱导肝星状细胞5-羟色胺降解在2型糖尿病致肝脏炎症和纤维化时的作用[J]. 北京大学学报（医学版）, 2022, 54(6): 1141-1150.
[5]	朱琳,张维宇,许克新. 环磷酰胺诱导SD大鼠膀胱疼痛综合征模型的有效性[J]. 北京大学学报（医学版）, 2022, 54(4): 735-740.
[6]	王贵红,左婷,李然,左正才. 瑞巴派特在大鼠痛风性关节炎急性发作中的作用[J]. 北京大学学报（医学版）, 2021, 53(4): 716-720.
[7]	胡卫国, 王晓峰, 徐涛, 李建兴, 陈亮, 于澄钒, 黄晓波. 纳米细菌大鼠肾结石模型初步建立及成石因素分析[J]. 北京大学学报（医学版）, 2010, 42(4): 433-435.
[8]	邱永祥, 阮明, 贾凤兰, 邱飞婵, 李雪婷, 尚兰琴, 张宝旭. 金属硫蛋白对秋水仙碱致肝毒性的保护作用[J]. 北京大学学报（医学版）, 2005, 37(5): 558-559.
[9]	于洁, 张芳婷, 万汇涓, 叶静, 龙霞, 房家智. 体外模拟肝损伤环境下人脐血细胞向类肝细胞的转化[J]. 北京大学学报（医学版）, 2005, 37(4): 402-405.

敲减Blimp1基因表达对CCl₄诱导的小鼠肝纤维化模型早期肝损伤的保护作用

Protective effect of knock-down the expression of Blimp1 gene on early liver injury in CCl₄-induced mouse model of liver fibrosis

RICH HTML

PDF (PC)

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 27

相关文章 9

Metrics

本文评价

推荐阅读 0