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下瘀血汤对非酒精性脂肪性肝病小鼠模型肾损伤的干预作用及其机制
DOI: 10.12449/JCH241114
伦理学声明：本研究的临床试验方案于2020年8月25日经上海中医药大学附属普陀医院伦理委员会审批，批号：PTEC-2020-32（Y）-1，所纳入患者均签署知情同意书；动物实验方案于2020年9月11日经由上海中医药大学附属普陀医院实验动物伦理委员会审批，批号：PTEC-A-2020-31-1，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：刘成负责设计论文框架，起草论文；赵鑫、王知意、陶乐、杨广越、张玮负责实验操作，研究过程的实施；马文婷、吴柳、陈蒨负责数据收集，统计学分析，绘制图表；刘成负责修改论文；刘成、刘旭凌负责拟定写作思路，指导撰写文章并最后定稿。
Effect of Xiayuxue Decoction against renal injury in mice with non-alcoholic fatty liver disease and its mechanism
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摘要:
目的 探讨高脂饮食（HFD）诱导非酒精性脂肪性肝病（NAFLD）对小鼠肾脏的影响，以及下瘀血汤对NAFLD小鼠模型的保护作用及其机制。 方法 选取2020年9月—2021年9月在上海中医药大学附属普陀医院健康对照及NAFLD患者各25例，检测总胆固醇（TC）、甘油三酯（TG）、尿素氮（BUN）、肌酐（Cr）及尿酸（UA）水平；将24只雄性C57BL/6小鼠随机分为对照组（LFD组）、高脂组（HFD组）及高脂+下瘀血汤组（XYXD组），每组8只，第13周开始给予下瘀血汤每日1次灌胃至18周末，维持6周。检测各组TC、TG、BUN及Cr水平。HE、油红O染色观察肝脏和肾脏病理变化，免疫组化双染技术检测CD68和α-SMA表达。实时荧光定量PCR检测肾组织固醇调节元件结合蛋白1（SREBP1）、脂肪酸合酶（FASN）、IL-6、TNF-α、结蛋白（Desmin）和α-平滑肌肌动蛋白（α-SMA）的mRNA表达。Western Blot检测SREBP1和TNF-α蛋白水平。计量资料多组间比较采用单因素方差分析，两两比较采用LSD-t检验；两组间比较采用成组t检验。 结果 与健康对照组患者相比，NAFLD患者TC、TG、BUN、Cr、UA水平均升高（P值均<0.05）。与LFD组小鼠相比，HFD组小鼠体质量、TC、TG、BUN、Cr水平均升高（P值均<0.001）；与HFD组相比，XYXD显著抑制NAFLD小鼠TC、TG、BUN、CR表达（P值均<0.001）。肝脏病理学显示：HFD组小鼠肝脂肪变及炎症浸润较LFD组显著增加，而XYXD组病变显著减轻。肾脏病理学显示：与LFD组相比，HFD组肾组织炎性浸润明显，脂肪变显著，肾脏胶原形成明显；与HFD组相比，XYXD显著减轻炎症浸润、抑制脂肪变及肾脏胶原形成。实时荧光定量PCR结果显示：与LFD组相比，HFD组肾组织中SREBP1、FASN、IL-6、TNF-α、Desmin和α-SMA的mRNA相对表达水平均显著升高（P值均<0.001），XYXD组较HFD组均显著降低（P值均<0.001）。Western Blot结果显示：与LFD组比较，HFD组SREBP1及TNF-α蛋白表达水平均明显上调（P值均<0.05）；与HFD组比较，XYXD组SREBP1及TNF-α蛋白表达水平均明显下调（P值均<0.05）。免疫组化染色结果显示：HFD组α-SMA和CD68阳性染色及双阳性染色较LFD组均显著升高（P值均<0.05），XYXD组较HFD组均显著下降（P值均<0.05）。 结论 HFD可诱导肾脏脂肪变、炎症浸润及胶原形成，下瘀血汤可能通过抑制肾组织巨噬细胞与肌成纤维细胞表达发挥肾脏保护作用。 -
关键词:
- 非酒精性脂肪性肝病 /
- 膳食， 高脂 /
- 肾损伤 /
- 下瘀血汤 /
- 小鼠， 近交C57BL
Abstract:Objective To investigate the effect of non-alcoholic fatty liver disease （NAFLD） induced by high-fat diet （HFD） on the kidneys of mice and the protective effect and mechanism of Xiayuxue Decoction. Methods A total of 25 healthy controls and 25 NAFLD patients who attended Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from September 2020 to September 2021 were enrolled， and the levels of total cholesterol （TC）， triglyceride （TG）， blood urea nitrogen （BUN）， creatinine （Cr）， and uric acid （UA） were measured. A total of 24 male C57BL/6 mice were randomly divided into low-fat diet （LFD） group， HFD group， and HFD+Xiayuxue Decoction group （XYXD group）， with 8 mice in each group， and since week 13， XYXD was administered by gavage once a day for 6 weeks till the end of week 18. The level of TC， TG， BUN， and Cr were measured for each group. HE staining and oil red staining were used to observe the pathological changes of the liver and the kidneys； immunohistochemical double staining was used to measure the expression levels of CD68 and alpha-smooth muscle actin （α-SMA）； quantitative real-time PCR was used to measure the expression levels of sterol regulatory element binding protein 1 （SREBP1）， fatty acid synthase （FASN）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， Desmin， and α-SMA in renal tissue； Western blot was used to measure the protein expression levels of SREBP1 and TNF-α. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for pairwise comparison； the independent-samples t-test was used for comparison between two groups. Results Compared with the healthy controls， NAFLD patients showed significant increases in the levels of TC， TG， BUN， Cr， and UA （all P<0.05）. Compared with the LFD group， the HFD group had significant increases in body weight， TC， TG， BUN， and Cr （all P<0.001）， and compared with the HFD group， the XYXD group showed significant inhibition of the expression of TC， TG， BUN， and Cr （all P<0.001）. Liver pathological examination showed that compared with the LFD group， the HFD group showed significant increases in hepatic steatosis and inflammatory infiltration， while the XYXD group showed significant alleviation of lesions. Renal pathological examination showed that compared with the LFD group， the HFD group had significant inflammatory infiltration， steatosis， and collagen formation in renal tissue， and compared with the HFD group， XYXD significantly alleviated inflammatory infiltration and inhibited steatosis and collagen formation. Quantitative real-time PCR showed that compared with the LFD group， the HFD group had significant increases in the relative mRNA expression levels of SREBP1， FASN， IL-6， TNF-α， Desmin， and α-SMA in renal tissue （all P<0.001）， and compared with the HFD group， the XYXD group had significant reductions in the relative expression levels of these indicators （all P<0.001）. Western blot showed that compared with the LFD group， the HFD group had significant increases in the protein expression levels of SREBP1 and TNF-α （P<0.05）， and compared with the HFD group， the XYXD group had significant reductions in the protein expression levels of SREBP1 and TNF-α （P<0.05）. Immunohistochemical staining showed that compared with the LFD group， the HFD group had significant increases in the positive staining or the double positive staining of α- SMA and CD68 （P<0.05）， and compared with the HFD group， the XYXD group showed significant reductions （P<0.05）. Conclusion HFD can induce renal steatosis， inflammatory infiltration， and collagen formation， and XYXD might exert a protective effect on the kidneys by inhibiting the expression of macrophages and myofibroblasts in renal tissue. -
图 4 肾组织免疫组化染色结果（×600）
注： 细箭头，α-SMA（棕色）；粗箭头，CD68（蓝色）。
Figure 4. Renal tissue immunohistochemical results（×600）
表 1 引物序列
Table 1. Primer sequence
名称 序列 18S F：5'-GTAACCCGTTGAACCCCATT-3'
R：5'-CCATCCAATCGGTAGTAGCG-3'
TNF-α F：5'-ATGAGAGGGAGGCCATTTG-3'
R：5'-CAGCCTCTTCTCATTCCTGC-3'
IL-6 F：5'-ACCAGAGGAAATTTTCAATAGGC-3'
R：5'-TGATGCACTTGCAGAAAACA-3'
FASN F：5'-GGAGGTGGTGATAGCCGGTAT-3'
R：5'-TGGGTAATCCATAGAGCCCAG-3'
SREBP -1c F：5'-GCAGCCACCATCTAGCCTG-3' R 5'-CAGCAGTGAGTCTGCCTTGAT-3' α-SMA F 5'-GTTCAGTGGTGCCTCTGTCA-3' R 5'-ACTGGGACGACATGGAAAAG-3' Desmin F 5'-TCGGCTCTAAGGGCTCCTC-3' R 5'-CGTGGTCAGAAACTCCTGGTT-3' 
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表 2 研究对象血脂与肾功能参数的分析
Table 2. Analysis of blood lipids and renal function parameters in subjects
组别 例数 年龄（岁） BUN（mmol/L） Cr（μmol/L） UA（μmol/L） TC（mmol/L） TG（mmol/L） 对照组 25 41.88±12.12 4.33±1.02 53.52±3.06 265.00±9.43 4.14±0.78 1.15±0.53 NAFLD组 25 48.38±13.82 4.49±1.04 79.33±3.06 381.00±18.87 6.21±1.21 3.01±1.32 t值 1.73 2.05 5.97 5.50 15.00 13.23 P值 0.090 0.012 <0.001 <0.001 <0.001 <0.001
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表 3 各组小鼠体质量肾功能、血脂表达改变
Table 3. Changes in body weight， renal function， and lipid expression of mice in each group
组别 动物数（只） 体质量（g） BUN（mmol/L） Cr（μmol/L） TC（mmol/L） TG（mmol/L） LFD组 8 30.74±2.34 6.14±0.38 5.29±0.30 2.63±0.23 0.53±0.03 HFD组 8 46.34±2.271） 7.49±0.121） 8.63±0.221） 5.46±0.141） 0.83±0.031） XYXD组 8 37.98±2.842） 6.64±0.252） 7.53±0.142） 3.62±0.192） 0.71±0.022） F值 78.13 75.70 432.41 474.30 312.04 P值 <0.001 <0.001 <0.001 <0.001 <0.001 注：与LFD组比较，1）P<0.001；与HFD组比较，2）P<0.001。
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表 4 各组小鼠肾脏病理学、炎症纤维化指标变化
Table 4. Changes of pathological in each group
指标 LFD组 HFD组 XYXD组 F值 P值 NAS评分（分） 0.65±0.05 6.04±0.461） 3.61±0.192） 8.74 <0.001 天狼星红染色阳性面积百分比（%） 2.20±0.34 5.45±0.171） 3.26±0.122） 23.73 <0.001 油红O染色阳性面积百分比（%） 7.44±0.87 21.05±0.931） 14.88±0.152） 1.33 <0.001 免疫印迹 TNF-α 1.00±0.22 11.06±2.691） 8.49±2.672） 16.67 <0.001 SREBP-1 1.00±0.03 2.36±0.281） 1.16±0.272） 28.59 <0.001 免疫组化染色阳性面积百分比（%） CD68 0.25±0.06 1.77±0.201） 0.92±0.082） 4.94 <0.001 α-SMA 0.48±0.09 2.29±0.151） 1.49±0.152） 2.71 <0.001 CD68+α-SMA双染色 1.69±0.27 4.79±0.501） 3.40±0.492） 64.54 <0.001 RT-PCR TNF-α 1.00±0.21 2.14±0.491） 1.32±0.192） 26.12 <0.001 IL-6 1.00±0.27 2.34±0.971） 1.21±0.122） 12.92 <0.001 FASN 1.00±0.21 10.05±1.131） 7.30±0.842） 35.42 <0.001 SREBP-1c 1.00±0.35 11.76±1.251） 9.06±1.032） 25.82 <0.001 Desmin 1.00±0.18 2.15±0.871） 1.11±0.192） 11.86 <0.001 α-SMA 1.00±0.57 3.76±0.731） 1.70±0.262） 53.53 <0.001 注：与 LFD组比较，1）P<0.001；与 HFD 组比较：2）P<0.001。
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