Abstract
目的
肝纤维化是由慢性肝病导致的严重病理后果，并最终发展为肝硬化。吗替麦考酚酯(mycophenolate mofetil，MMF)是器官移植术后常用的免疫抑制剂，与肝纤维化的关系尚不明确。本研究旨在探讨MMF对小鼠肝纤维化的影响及其机制。
方法
选用雄性8周龄C57BL/6小鼠24只，将其随机分为空白对照组、MMF组、四氯化碳(carbon tetrachloride，CCl4)组、CCl4+MMF组(每组均n=6)。小鼠处死后，取血清检测丙氨酸转氨酶(alanine aminotransferase，ALT)及天冬氨酸转氨酶(aspartate aminotransferase，AST)；肝组织行Masson染色评估纤维化程度，采用免疫组织化学染色评估I型胶原蛋白(collagen I，COL1)表达水平；然后采用蛋白质印迹法检测转化生长因子-β1(transforming growth factor-β1，TGF-β1)和α-平滑肌肌动蛋白(alpha-smooth muscle actin，α-SMA)的蛋白质表达水平；最后利用real-time PCR检测TGF-β1、α-SMA及COL1的mRNA相对表达量。
结果
与CCl4组相比，CCl4+MMF组小鼠的ALT与AST均下降(均P<0.05)，肝纤维化程度明显减轻，肝组织中COL1的沉积明显减少(P<0.01)。与CCl4组相比，CCl4+MMF组小鼠肝组织中TGF-β1与α-SMA的蛋白质表达水平均明显降低(均P<0.05)；TGF-β1、α-SMA及COL1的mRNA相对表达水平均明显降低(均P<0.05)。
结论
MMF能减轻CCl4诱导的小鼠肝纤维化程度，其机制可能与抑制TGF-β1的基因表达及蛋白质合成有关，本研究有望为肝纤维化的治疗提供新方向。
Keywords: 肝纤维化, 吗替麦考酚酯, 转化生长因子-β1, α-平滑肌肌动蛋白, I型胶原蛋白
Abstract
Objective
Hepatic fibrosis is a serious pathological consequence of chronic liver disease. Mycophenolate mofetil (MMF) is a commonly used immunosuppressant after organ transplant. However, the relationship between MMF and hepatic fibrosis remains unclear. This study aims to explore the effect of MMF on hepatic fibrosis in mice and the potential mechanism.
Methods
A total of 24 mice (male, 8-week old, C57BL/6) were randomly divided into a control group, a MMF group, a carbon tetrachloride (CCl4) group and a CCl4+MMF group (n=6 in each group). After the mice were sacrificed, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected. The liver tissues were taken up for Masson staining and collagen I (COL1) immunohistochemistry. The levels of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were detected by Western blotting. Finally, the levels of mRNA for TGF-β1, α-SMA, and COL1 were detected using real-time PCR.
Results
Compared with the CCl4 group, the ALT and AST levels were lower (both P<0.05), the degree of liver fibrosis was alleviated, and the deposition of COL1 in the liver was significantly decreased (P<0.01) in the CCl4+MMF group. Compared with the CCl4 group, the protein expression levels of TGF-β1 and α-SMA were significantly decreased (both P<0.05) and the relative expression levels of TGF-β1, α-SMA and COL1 mRNA in the liver were significantly decreased (all P<0.05) in the CCl4+MMF.
Conclusion
MMF could reduce CCl4-induced hepatic fibrosis, which might be related to the inhibition of TGF-β1. This study is expected to provide a target for the treatment of hepatic fibrosis.
Keywords: hepatic fibrosis, mycophenolate mofetil, transforming growth factor-β1, α-smooth muscle actin, collagen I
器官纤维化是疾病长期进展的结果，肝纤维化是慢性肝病进展中由于肝内细胞外基质(extracellular matrix，ECM)的产生超过其降解速率，致使胶原、糖蛋白、蛋白多糖等过度沉积的病理学改变，常伴有炎症并可发展为肝硬化，当患者处于肝硬化失代偿期或出现并发症时其病死率较高[1]。
器官移植是目前治疗终末期器官衰竭最为理想的手段，为了减轻器官移植后的排斥反应，移植术后常需有规律地服用免疫抑制剂[2]。有研究[3-4]指出长期服用免疫抑制剂可导致肝损伤，甚至引起肝纤维化；但同时亦有研究[5-6]发现他克莫司(Tacrolimus，FK506)及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)抑制剂等免疫抑制剂能缓解肝纤维化，而吗替麦考酚酯(mycophenolate mofetil，MMF)作为器官移植后常用的免疫抑制剂，已有研究[7-8]报道其可减轻肺、肾等器官纤维化，然而其对肝纤维化的影响尚无文献报道。本实验基于国内外研究现状，通过使用C57BL/6小鼠腹腔注射四氯化碳(carbon tetrachloride，CCl4)模拟肝纤维化进程，同时给予MMF悬浮液灌胃，探讨MMF对小鼠肝纤维化的影响及其机制，以期为临床治疗肝纤维化提供新的思路。
1. 材料与方法
1.1. 实验动物
选取8周龄的健康C57BL/6小鼠24只，均购自中南大学湘雅三医院动物实验中心。本研究获得中南大学湘雅三医院伦理委员会批准[审批号：LLSC(LA)2018-042]。
1.2. 主要试剂与仪器
MMF胶囊购自上海罗氏制药有限公司；CCl4购自麦克林生物技术有限公司；4%中性甲醛购自兰杰柯科技有限公司；丙氨酸转氨酶(alanine amino-transferase，ALT)及天冬氨酸转氨酶(aspartate aminotransferase，AST)试剂盒购自南京建成科技有限公司；PCR引物由上海晶莱生物技术有限公司合成；转化生长因子-β1(transforming growth factor-β1)抗体、I型胶原蛋白(collagen I，COL1)抗体、α-SMA抗体购自北京博奥森生物技术有限公司；甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase，GAPDH)购自北京中杉金桥生物技术有限公司。
1.3. 实验分组及模型建立
按随机数字表法将24只小鼠平均分成空白对照组、CCl4组、MMF组、CCl4+MMF组(每组均n=6)。空白对照组给予橄榄油溶液腹腔注射+空白载体灌胃4周，MMF组给予橄榄油溶液腹腔注射+MMF灌胃4周，CCl4组给予20% CCl4橄榄油溶液腹腔注射+空白载体灌胃4周，CCl4+MMF组给予20% CCl4橄榄油溶液腹腔注射+MMF灌胃4周。20% CCl4橄榄油溶液配制方法为99.9% CCl4与橄榄油按体积比1꞉4配制，腹腔注射剂量为0.5 mL/kg，隔天1次；MMF配制方法为500 mg MMF溶于含体积分数0.4%吐温-80及0.9%苯甲醇溶液的生理盐水10 mL中，充分震荡均匀，灌胃剂量为500 mg/(kg·d)，每天1次。末次灌胃24 h后摘眼球取血，然后处死小鼠，收集血清及肝标本。
1.4. 相关检测指标及方法
1.4.1. 肝功能检测
用离心机分离血清(1 000 r/min，5 min)，用AST及ALT试剂盒检测血清中的AST及ALT。
1.4.2. 肝组织病理学检测
取肝左叶，用4%中性甲醛固定后行脱水、透明、浸蜡、石蜡包埋、切片、烤片处理，行Masson染色及免疫组织化学染色。
1.4.3. 蛋白质印迹法
将30 mg肝组织加入蛋白裂解液中，研磨均匀后于冰上裂解30 min，以12 000 r/min离心15 min，取出上清液，采用BCA蛋白浓度测定试剂盒进行蛋白质定量。加样后行SDS-PAGE电泳、转膜后封闭，加入一抗于4 ℃摇床上孵育过夜，用电泳缓冲液洗膜3次，每次10 min，加入二抗后孵育2 h，加入电化学发光试剂显影，以GADPH作为内参，检测组织中TGF-β1和α-SMA的蛋白相对水平。
1.4.4. Real-time PCR检测
称取50 mg肝组织，于液氮中研磨成粉状，采用TRIzol提取总RNA，紫外分光光度计测量浓度，使用PrimScript RT Master Mix试剂盒将RNA反转录成互补脱氧核糖核酸(complementary DNA，cDNA)，用real-time PCR系统进行实时扩增。选择GAPDH为内参，根据2-ΔΔCt的方法计算α-SMA、COL1、TGF-β1 mRNA的相对表达量，2-ΔΔCt法的计算方法如下：首先计算每个样本目标基因的Ct值和参考基因的Ct值；然后计算ΔCt，ΔCt(目标基因)=Ct(目标基因)- Ct(参考基因)；接着用校准样本的ΔCt值归一实验样本的ΔCt值[ΔΔCt值=ΔCt(实验组)-ΔCt(对照组)]；最后计算表达水平比率2-ΔΔCt。引物见表1。
表1.
引物序列
Table 1 Primer sequences
| 目的基因 | 引物序列 | |
|---|---|---|
| GAPDH | F: | 5'-AGAAGGTGGTGAAGCAGGCATCT-3' |
| R: | 5'-CGGCATCGAAGGTGGAAGAGTG-3' | |
| COL1 | F: | 5'-AGACAGGCGAACAAGGTGACAGA-3' |
| R: | 5'-CAGGAGAACCAGGAGAACCAGGAG-3' | |
| α-SMA | F: | 5'-GTACCACCATGTACCCAGGC-3' |
| R: | 5'-GCTGGAAGGTAGACAGCGAA-3' | |
| TGF-β1 | F: | 5'-CCCGAAGCGGACTACTATGC-3' |
| R: | 5'-CATAGATGGCGTTGTTGCGG-3' | |
F：正向；R：反向。
1.5. 统计学处理
应用SPSS 19.0软件学软件进行统计学分析，计量资料采用均数±标准差( ±s)表示，组间比较采用方差分析，P<0.05表示差异有统计学意义。
2. 结 果
2.1. 小鼠血清ALT和AST水平
CCl4组血清ALT和AST水平较空白对照组明显升高，CCl4+MMF组较CCl4组降低，差异均有统计学意义(均P<0.05，图1)。
图1.
各组小鼠血清AST和ALT水平
Figure 1 Serum AST and ALT levels in the different groups
A: Serum AST levels; B: Serum ALT levels. **P<0.01, ***P<0.001. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; MMF: Mycophenolate mofetil; CCl4: Carbon tetrachloride.
2.2. 小鼠肝组织的纤维化程度
小鼠肝组织Masson染色结果表明：与空白对照组相比，CCl4组中肝小叶汇管区有大量蓝色的胶原纤维包裹肝小叶，提示明显肝纤维化；但在CCl4+MMF组中，蓝色胶原纤维明显减少，并主要聚集于肝小叶汇管区，肝纤维化程度较CCl4组轻(图2)。
图2.
各组小鼠肝Masson染色结果
Figure 2 Result of Masson staining of mouse liver in the different groups
A: No collagen fibers in the blank control group; B: No collagen fibers in the MMF group; C: Abundant collagen fibers surrounding the hepatic lobules in the CCl4 group; D: Lower collagen fibers in the CCl4+MMF group compared with the CCl4 group; E: Statistic results of collagen fibers in the 4 groups. ** P<0.01. MMF: Mycophenolate mofetil; CCl4: Carbon tetrachloride.
2.3. 小鼠肝组织中COL1沉积情况
免疫组织化学染色显示：与空白对照组相比，CCl4组肝内大量表达COL1(P<0.001)，而CCl4+MMF组表达的COL1明显减少(P<0.01，图3)。
图3.
各组小鼠肝COL1免疫组织化学染色结果
Figure 3 Collagen I in mouse liver by immunohistochemistry staining in the different groups
A: Only little COL1 expression in the control group; B: Only little COL1 expression in the MMF group; C: A larger number of COL1 expression in the CCl4 group; D: Little COL1 expression in the CCl4+MMF group; E. Statistic results of COL1 expression in the 4 groups. **P<0.01, ***P<0.001. MMF: Mycophenolate mofetil; CCl4: Carbon tetrachloride; COL1: Collagen I.
2.4. 小鼠肝组织中TGF-β1和α-SMA的蛋白质表达水平
蛋白质印迹法结果显示：与空白对照组比较，CCl4组的TGF-β1表达升高(P<0.01)，α-SMA蛋白表达升高，但差异无统计学意义(P>0.05)；与CCl4组相比，CCl4+MMF组TGF-β1和α-SMA蛋白表达明显降低(分别P<0.001，P<0.05；图4)；空白对照组与MMF组的TGF-β1和α-SMA蛋白质表达水平差异均无统计学意义(均P>0.05)。
图4.
蛋白质印迹法检测小鼠肝组织TGF-β1和α-SMA的蛋白质表达水平
Figure 4 Protein levels of TGF-β1 and α-SMA in mouse liver detected by Western blotting
A: Protein levels of TGF-β1 and α-SMA in the control group and the MMF group; B: Protein levels of TGF-β1 and α-SMA in the blank control group and the CCl4 group; C: Protein levels of TGF-β1 and α-SMA in the CCl4 group and the CCl4+MMF group; D: Histogram of protein levels of TGF-β1 in the blank control group and the CCl4 group; E: Histogram of protein levels of α-SMA in the blank control group and the CCl4 group; F: Histogram of protein levels of TGF-β1 in the CCl4 group and the CCl4+MMF group; G: Histogram of protein levels of α-SMA in the CCl4 group and the CCl4+MMF group. *P<0.05, **P<0.01, ***P<0.001. MMF: Mycophenolate mofetil; CCl4: Carbon tetrachloride; TGF-β1: Transforming growth factor-β1; α-SMA: Alpha-smooth muscle actin.
2.5. 小鼠肝组织中 TGF-β1 、 α-SMA 和 COL1 的mRNA表达
CCl4组小鼠肝组织中TGF-β1、α-SMA和COL1的mRNA相对表达量均高于CCl4+MMF组，且差异均有统计学意义(均P<0.05，图5)。
图5.
CCl4 组和CCl4+MMF组小鼠肝组织 TGF-β1 、 α-SMA 和 COL1 的mRNA表达水平
Figure 5 Expression levels of TGF-β1, α-SMA, and COL1 mRNA in mouse liver in the CCl4 mouse group and the CCl4+MMF group
A: TGF-β1 mRNA levels; B: α-SMA mRNA levels; C: COL1 mRNA levels. *P<0.05. CCl4: Carbon tetrachloride; MMF: Mycophenolate mofetil; TGF-β1: Transforming growth factor-beta 1; α-SMA: Alpha-smooth muscle actin; COL1: Collagen I.
3. 讨 论
肝纤维化是慢性肝病的一种常见病理后果[9]，其机制主要为在慢性肝损伤过程中，ECM的产生速率超过其降解速率，其特点是基质蛋白的生成大于基质重塑，纤维间隔的逐渐增厚和胶原的化学交联最终导致纤维化[1, 10]。在肝纤维化的病理生理学中，其核心过程便是肝星状细胞的激活[11]，TGF-β1是肝星状细胞最重要的激活因子，TGF-β1主要由肝巨噬细胞分泌[12]。在炎症状态下，肝巨噬细胞会分泌大量的TGF-β1等促炎和成纤维介质，激活肝星状细胞，促进肝纤维化的发生和发展[13-14]。因此，肝巨噬细胞与慢性肝损伤的进展密切相关，是预防及治疗肝纤维化的重要环节[15]。所以，识别这些细胞中的特异性抗炎通路可作为设计治疗肝纤维化新策略的基础。 α-SMA是哺乳动物组织中表达的一种平滑肌肌动蛋白，在肝中除大血管周围的平滑肌细胞及激活的肝星状细胞外，其他肝细胞均不含α-SMA，所以 α-SMA是肝星状细胞激活最可靠的标志[16]。在肝纤维化进展中，活化的肝星状细胞分泌多种类型的胶原，其中COL1是肝纤维化临床表现中最丰富和最重要的一种因子，因此，COL1能作为肝纤维化指标[17]。
器官移植是终末期器官衰竭最有效的治疗方法，为减轻排斥反应，术后需要长期规律服用免疫抑制剂[2]。既往的研究[18-20]报道多表明免疫抑制剂可通过导致移植后代谢综合征、感染和药物性肝损伤等并发症促进肝纤维化的进展；与此相反的是，最近研究[21]指出某些免疫抑制剂可抑制肝纤维化，改善肝功能。Manojlovic等[5]发现FK506可预防乙醇/CCl4诱导的大鼠肝纤维化的发生，其机制为FK506通过影响胶原合成的La核糖核蛋白结构域家族成员6(La ribonucleoprotein domain family member 6，LAP6）依赖性通路直接靶向胶原合成，导致活化肝星状细胞缺乏而抑制肝纤维化。研究[22]发现mTOR抑制剂西罗莫司和依维莫司可减轻肝纤维化，降低门静脉压力，减少腹水，并强效下调促纤维化基因的表达；Biecker等[23]亦发现这一现象，认为此为雷帕霉素通过抑制mTOR来抑制细胞增殖，进而使促纤维细胞因子的产生减少，最终减轻肝纤维化。MMF在实体器官移植术后常联合钙调磷酸酶类免疫抑制剂预防或治疗排斥反应。虽然目前暂无研究发现其对肝损伤与肝纤维化的影响，但在平滑肌细胞、肾小管细胞、系膜细胞和成纤维细胞等细胞系体外研究[24]中发现MMF的主要活性成分霉酚酸(mycophenolic acid，MPA)可减少甚至抑制增殖刺激反应引起的增殖，起到抗纤维化作用。Chang等[8]发现在大鼠肾成纤维细胞中，MPA通过刺激肿瘤坏死因子-α抑制趋化因子CCL2的产生，从而抑制肾成纤维细胞的增殖，同时还诱导了这些细胞的凋亡，这表明MPA具有抗肾纤维化作用。此外，Guo等[7]研究发现MMF治疗可降低诱发性系统性红斑狼疮小鼠肺组织中TGF-β1基因的表达和蛋白质合成，提示MMF对治疗狼疮引起的肺纤维化有很大的潜力。
本研究探讨了MMF对肝功能的影响。近年有研究[25]报道：在使用MMF后，13.9%的肾移植受者AST和ALT水平会升高，而在减少MMF剂量或停用后，AST和ALT水平逐渐下降至正常水平。与上述观点相反的是，Ferjani等[26]发现MMF对FK506所致的大鼠肾毒性和肝毒性有保护作用。本实验发现CCl4+MMF组小鼠的ALT与AST较CCl4组下降，提示MMF可减轻慢性肝损伤；同时，CCl4+MMF组小鼠肝组织的肝纤维化程度较CCl4组减轻，说明MMF能缓解CCl4导致的小鼠肝纤维化。Djamali等[27]发现MPA可能通过核因子κB通路抑制TGF-β1引起的NADPH氧化酶-2活化来减轻移植肾纤维化。为了进一步研究MMF对肝纤维化影响的机制，本研究用蛋白质印迹法及real-time PCR法检测3组小鼠肝组织中α-SMA和TGF-β1蛋白质的表达及转录水平，结果显示：与CCl4组相比，CCl4+MMF组肝内TGF-β1蛋白和mRNA的表达水平明显降低，差异有统计学意义；与CCl4组相比，CCl4+MMF组肝内α-SMA蛋白变化有上升趋势，但差异无统计学意义，其mRNA的表达水平明显降低，差异有统计学意义。因此推测MMF可能通过抑制巨噬细胞中TGF-β1基因的表达和蛋白质合成来抑制肝星状细胞激活，从而降低肝纤维化。
综上所述，本研究证实MMF能减轻CCl4诱导的小鼠肝纤维化程度，其机制可能是通过抑制巨噬细胞中TGF-β1的基因表达及蛋白质合成来减缓肝纤维化的进程。本研究有望为肝纤维化的防治提供新的思路。同时MMF作为器官移植后的常用免疫抑制剂，其降低肝纤维化作用可为器官移植患者个体化使用免疫抑制剂提供参考。
基金资助
国家自然科学基金(81771722，82100695)；湖南省自然科学基金(2022JJ40759)。
This work was supported by the National Natural Science Foundation (81771722, 82100695) and the Natural Science Foundation of Hunan Province (2022JJ40759), China.
利益冲突声明
作者声称无任何利益冲突。
作者贡献
丁鹏 实验实施、数据整理、论文构想及撰写；张朋朋 数据整理、统计分析及论文修改；李皓 实验实施、论文修改；明英姿 论文修改及研究指导。所有作者阅读并同意最终的文本。
原文网址
http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202306821.pdf
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