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1，25（OH）2D3改善胆碱缺乏氨基酸饮食诱导的非酒精性脂肪性肝炎大鼠模型肝脏炎症的机制分析
DOI: 10.12449/JCH250210
伦理学声明：动物的实验流程严格遵循中国实验动物管理协会的伦理原则，并于2023年9月11日经由延边大学实验动物福利伦理委员会审批，批号：YD202309110024。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：朱海洋负责分析数据、撰写论文和参与课题设计；崔京淑负责病理实验数据统计；杨柳、周梦婷、童戬参与实验操作、数据统计，修改论文；韩红梅负责课题设计、拟定写作思路，指导撰写文章并最后定稿。
Mechanism of 1，25（OH）2D3 improving liver inflammation in a rat model of nonalcoholic steatohepatitis induced by choline-deficient L-amino acid-defined diet
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摘要:
目的 分析1，25（OH）2D3对非酒精性脂肪性肝炎（NASH）大鼠模型肝脏过氧化物酶体增殖物激活受体γ（PPAR-γ）水平以及肝巨噬细胞表型、肝脏炎症的影响，初步探讨1，25（OH）2D3改善NASH肝脏炎症的相关机制。 方法 将24只SPF级Wistar大鼠适应性喂养1周后，随机分为4组。正常组（n=6）：给予胆碱充足的氨基酸（CSAA）饮食；正常+1，25（OH）2D3组（n=6）：给予CSAA饮食+1，25（OH）2D3干预；模型组（n=6）：给予胆碱缺乏的氨基酸（CDAA）饮食；模型+1，25（OH）2D3组（n=6）：给予CDAA饮食+1，25（OH）2D3干预。1，25（OH）2D3剂量为5 μg/kg，经腹腔注射，每周2次，共12周。检测每组大鼠血清AST、ALT水平；观察肝组织病理学严重程度，评估SAF评分。检测肝组织M1型、M2型肝巨噬细胞，分析肝巨噬细胞表型变化；并检测肝组织TNF-α、IL-1β、IL-4、IL-10水平以及PPAR-γ的mRNA水平。组间比较采用双因素方差分析，进一步比较采用LSD-t多重检验，相关性分析采用Pearson相关法。 结果 与正常组比较，CDAA饮食诱导的NASH模型AST和ALT水平均升高（P值分别为0.019、<0.001），肝组织病理学SAF评分（P<0.001）、M1型肝巨噬细胞表达水平（P<0.001）、M1/M2表型比值（P=0.001）、TNF-α表达水平显著增加（P<0.001），IL-4表达水平降低（P=0.025）；1，25（OH）2D3显著降低了CDAA饮食诱导NASH大鼠血清ALT水平（P<0.001）、肝组织病理学SAF评分（P<0.001）、M1型肝巨噬细胞水平（P<0.001）、M1/M2型比值（P=0.001）、IL-1β水平（P<0.001），增加了M2型肝巨噬细胞水平（P=0.017）、IL-10水平（P=0.039）、IL-4水平（P<0.001）、PPAR-γ水平（P=0.016）。CDAA饮食诱导的NASH模型和1，25（OH）2D3在大鼠血清AST和ALT水平（P值分别为0.007、0.008）、肝组织病理学SAF评分（P<0.001）、M1型肝巨噬细胞水平（P<0.001）、M2型肝巨噬细胞水平（P=0.008）、M1/M2表型比值（P=0.005）、TNF-α水平（P<0.001）、IL-10水平（P=0.038）、IL-4水平（P<0.001）、PPAR-γ水平（P=0.009）中有显著交互作用。相关性分析结果示：PPAR-γ与肝巨噬细胞M1/M2表型比值呈负相关（r=-0.415，P=0.044）；与M2型肝巨噬细胞、IL-10、IL-4均呈正相关（r值分别为0.435、0.433、0.532，P值分别为0.033、0.035、0.007）。 结论 1，25（OH）2D3激活PPAR-γ调控肝巨噬细胞表型转变，改善NASH肝脏炎症。 -
关键词:
- 非酒精性脂肪性肝炎 /
- 骨化三醇 /
- 炎症 /
- PPARγ /
- 大鼠， Wistar
Abstract:Objective To investigate the effect of 1，25（OH）2D3 on the level of peroxisome proliferator-activated receptor-γ （PPAR-γ） in the liver， the phenotype of hepatic macrophages， and liver inflammation in a rat model of nonalcoholic steatohepatitis （NASH）， as well as the mechanism of 1，25（OH）2D3 improving liver inflammation. Methods After 1 week of adaptive feeding， 24 specific pathogen-free Wistar rats were randomly divided into normal group ［choline-supplemented L-amino acid-defined （CSAA） diet］， normal+1，25（OH）2D3 group ［CSAA diet+1，25（OH）2D3］， model group ［choline-deficient L-amino acid-defined diet （CDAA） diet］， and model+1，25（OH）2D3 group ［CDAA diet+1，25（OH）2D3］， with 6 rats in each group. The dose of 1，25（OH）2D3 was 5 μg/kg for intraperitoneal injection twice a week for 12 weeks. The serum levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） were measured， liver histopathology was observed， and SAF score was assessed. M1 hepatic macrophages and M2 hepatic macrophages were measured to analyze in the change in the phenotype of hepatic macrophages， and ELISA was used to measure the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） in liver tissue， and qPCR was used to measure the mRNA level of PPAR-γ. The two-factor analysis of variance was use for comparison between groups， and the least significant difference t-test was used for further comparison； the Pearson method was used for correlation analysis. Results Compared with the normal group， the model rats with CDAA diet-induced NASH had significant increases in the serum levels of AST and ALT （P=0.019 and P<0.001）， the SAF score of liver histopathology （P<0.001）， the level of M1 hepatic macrophages （P<0.001）， and the ratio of M1 and M2 hepatic macrophages （P<0.001）， as well as a significant increase in the level of TNF-α （P<0.001） and a significant reduction in the level of IL-4 in liver tissue （P=0.025）. The 1，25（OH）2D3 group had significant reductions in the serum levels of ALT （P<0.001）， the SAF score of liver histopathology （P<0.001）， the level of M1 hepatic macrophages （P<0.001）， and the ratio of M1 and M2 hepatic macrophages （P=0.001）， the level of IL-1β （P<0.001） and a significant increase in the level of M2 hepatic macrophages （P=0.017）， the level of IL-10 （P=0.039）， the level of IL-4 （P<0.001）， the level of PPAR-γ （P=0.016）. There were significant interactions between CDAA diet-induced NASH model and 1，25（OH）2D3 in serum the levels of AST and ALT （P=0.007 and P=0.008）， the SAF scores of liver histopathology （P<0.001）， the level of M1 hepatic macrophages （P<0.001）， the level of M2 hepatic macrophages （P=0.008）， the ratio of M1 and M2 of hepatic macrophages （P=0.005）， the level of TNF-α （P<0.001）， the level of IL-10 （P=0.038）， the level of IL-4 （P<0.001） and the level of PPAR-γ （P=0.009）. The correlation analysis showed that PPAR-γ was negatively correlated with the ratio of M1 and M2 hepatic macrophages （r=-0.415， P=0.044） and was positively correlated with M2 hepatic macrophages （r=0.435， P=0.033）， IL-10 （r=0.433， P=0.035）， and IL-4 （r=0.532， P=0.007）. Conclusion This study shows that 1，25（OH）2D3 improves liver inflammation in NASH by activating PPAR-γ to regulate the phenotypic transformation of hepatic macrophages. -
Key words:
- Non-alcoholic Steatohepatitis /
- Calcitriol /
- Inflammation /
- PPAR gamma /
- Rats， Wistar
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图 1 1，25（OH）2D3对NASH大鼠血清生化指标的影响
Figure 1. Effects of 1，25（OH）2D3 on serum biochemical levels in NASH rats
注： a，正常组；b，正常+1，25（OH）2D3组；c，模型组；d，模型+1，25（OH）2D3组。
图 2 1，25（OH）2D3对NASH大鼠肝组织病理学的影响（HE染色，×200）
Figure 2. Effect of 1，25（OH）2D3 on liver histopathology in NASH rats（HE staining，×200）
注： a，1，25（OH）2D3对NASH大鼠肝组织CD68、CD11c以及双染细胞的影响；b，1，25（OH）2D3对NASH大鼠肝组织CD68、CD163以及双染细胞的影响；c，1，25（OH）2D3对NASH大鼠肝组织M1型、M2型肝巨噬细胞及M1/M2表型比值的影响。绿色箭头指CD68细胞；红色箭头指CD11c或CD163细胞；黄色箭头指双染细胞。
图 3 1，25（OH）2D3对NASH大鼠肝组织巨噬细胞的影响
Figure 3. Effect of 1，25（OH）2D3 on macrophages in liver tissue of NASH rats
图 4 1，25（OH）2D3对NASH大鼠肝组织促炎因子水平的影响
Figure 4. Effect of 1，25（OH）2D3 on the level of pro-inflammatory factors in liver tissue of NASH rats
图 5 1，25（OH）2D3对NASH大鼠肝组织抗炎因子水平的影响
Figure 5. Effect of 1，25（OH）2D3 on the level of anti-inflammatory factors in liver tissue of NASH rats
图 6 1，25（OH）2D3对NASH大鼠肝组织PPAR-γ水平的影响
Figure 6. Effect of 1，25（OH）2D3 on PPAR-γ level in liver tissue of NASH rats
表 1 引物序列
Table 1. Primer sequence
基因 序列（5'-3'） 长度 参考序列号 PPAR-γ F：CCTTTACCACGGTTGATTTCTC 141 bp NM_013124.3 R：CAGGCTCTACTTTGATCGCACT GAPDH F：TTCAACGGCACAGTCAAGG 114 bp NM_017008.4 R：CTCAGCACCAGCATCACC 
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表 2 1，25（OH）2D3对NASH大鼠肝组织病理学以及SAF评分的影响
Table 2. Effects of 1，25（OH）2D3 on hepatic histology and scores
分组 脂肪变性（分） 气球样变（分） 小叶炎症（分） 活动度（分） SAF评分（分） 正常组 0 0 0 0 0 正常+1，25（OH）2D3组 0 0 0 0 0 模型组 2.67±0.521） 1.09±0.091） 1.33±0.261） 2.42±0.331） 5.09±0.841） 模型+1，25（OH）2D3组 1.00±0.022） 0.23±0.232） 0.48±0.122） 0.71±0.312） 1.71±0.312） 注：与正常组相比，1）P<0.001；与模型组相比，2）P<0.001。
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