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粪菌移植与中药大黄治疗高脂血症性急性胰腺炎大鼠模型的效果比较
DOI: 10.3969/j.issn.1001-5256.2022.12.016
伦理学声明：本研究方案于2021年3月27日经由川北医学院实验动物伦理委员会审批，批号：NSMC伦理动物审[2021]105号，符合实验室动物管理与使用准则。
利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。
作者贡献声明：罗旭娟负责课题设计；罗旭娟、白雪负责资料分析，撰写论文并最后定稿；白雪、李增晖、唐浩、李若欣负责实验实施，分析数据；刘帆负责数据整理；杨国栋负责实验指导。
Clinical effect of fecal microbiota transplantation versus the traditional Chinese medicine Rheum officinale in a rat model of hyperlipidemic acute pancreatitis
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摘要:
目的 探索粪菌移植对高脂血症急性胰腺炎大鼠模型的影响。 方法 将72只雄性SD大鼠随机分为假手术组、模型组、大黄组、粪菌组，每组各18只。高脂饲料喂养8周，假手术组行假手术，其余3组采用5%牛黄胆酸钠逆行胰胆管注射诱导急性胰腺炎模型，大黄组在造模后予以大黄灌肠，粪菌组在造模后予以新鲜粪菌液灌肠。分别于术后6 h、24 h和36 h采集各组大鼠血液及胰腺、末端回肠组织标本。采用HE染色观察胰腺及肠道组织病理学改变，全自动生化分析仪检测血清淀粉酶、ALT、AST、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HLD-C)，ELISA检测血清IL-6、TNFα及肠道通透性指标内毒素。正态分布的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检验或Tamhane T2检验；非正态分布的计量资料多组间比较采用Kruskal-Wallis H检验，进一步两两比较采用Bonferroni法。 结果 大黄组与粪菌组6 h、24 h回肠末端病理评分均较假手术组无显著升高，且粪菌组在36 h时仍与假手术组差异无统计学意义(P值均＞0.05)；血清淀粉酶水平在36 h时，大黄组与粪菌组均较模型组显著降低(P值均＜0.05)；大黄组治疗36 h时血清ALT水平较模型组显著降低(P＜0.05)，血清AST水平在治疗24 h时明显低于模型组(P＜0.05)，而粪菌组ALT水平在各时间段均低于模型组(P值均＜0.05)，血清AST水平在24 h、36 h较模型组显著降低(P值均＜0.05)；大黄组与粪菌组均能显著降低血清TC、TG水平(P值均＜0.05)；粪菌组在治疗24 h、36 h时血清HDL-C较大黄组显著增高(P值均＜0.05)，且粪菌组在各时间段血清LDL-C均较模型组显著降低(P值均＜0.05)；炎症指标IL-6、TNFα，粪菌组在治疗各时间段与假手术组相比差异均无统计学意义(P值均＞0.05)，而大黄组均显著高于假手术组(P值均＜0.05)；大黄组与粪菌组血清内毒素水平均显著低于模型组(P值均＜0.05)，且粪菌组在治疗6 h时内毒素水平显著低于大黄组(P＜0.05)。 结论 大黄与粪菌移植均能改善高脂血症急性胰腺炎大鼠组织炎症及肠道通透性，可在一定程度上改善血脂代谢，缓解大鼠胰腺炎进展，且粪菌移植疗效优于单独使用中药大黄，但尚需更多随机对照试验加以研究。 -
关键词:
- 高脂血症 /
- 胰腺炎 /
- 大黄属 /
- 粪菌移植 /
- 大鼠, Sprague-Dawley
Abstract:Objective To investigate the effect of fecal microbiota transplantation (FMT) on a rat model of hypertriglyceridemic acute pancreatitis (HLAP). Methods A total of 72 male Sprague-Dawley rats were randomly divided into sham-operation group, model group, Rheum officinale group, and fecal microbiota group, with 18 rats in each group. After 8 weeks of feeding with high-fat diet, the rats in the sham-operation group were given sham operation, and those in the other three groups were given retrograde pancreaticobiliary injection of 5% sodium taurocholate to induce acute pancreatitis; after modeling, the rats in the Rheum officinale group were given enema with Rheum officinale, and those in the fecal microbiota group were given enema with fresh fecal microbiota solution. Blood, pancreatic, and terminal ileal tissue samples were collected at 6, 24, and 36 hours after surgery. HE staining was used to observe histopathological changes of the pancreas and the intestine; an automatic biochemical analyzer was used to measure the serum levels of amylase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HLD-C); ELISA was used to measure the serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and endotoxin as an index for intestinal permeability. A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test or the Tamhane T2 test was used for further comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups, and the Bonferroni method was used for further comparison between two groups. Results Compared with the sham-operation group, the Rheum officinale group and the fecal microbiota group had no significant increase in the pathological score of the terminal ileum at 6 and 24 hours, and there was no significant difference between the fecal microbiota group and the sham-operation group at 36 hours (all P > 0.05). At 36 hours, the Rheum officinale group and the fecal microbiota group had a significantly lower serum level of amylase than the model group (all P < 0.05). Compared with the model group, the Rheum officinale group had a significantly lower serum level of ALT at 36 hours (P < 0.05) and a significantly lower serum level of AST at 24 hours (P < 0.05), while the fecal microbiota group had a significantly lower level of ALT at each time point (P < 0.05) and a significantly lower serum level of AST at 24 and 36 hours (all P < 0.05). The Rheum officinale group and the fecal microbiota group had significant reductions in the serum levels of TC and TG (all P < 0.05); compared with the Rheum officinale group, the fecal microbiota group had a significantly higher serum level of HDL-C at 24 and 36 hours (all P < 0.05), and compared with the model group, the fecal microbiota group had a significantly lower serum level of HDL-C at each time period (all P < 0.05). There were no significant differences in the inflammatory indices IL-6 and TNF-α between the fecal microbiota group and the sham-operation group at each time point (all P > 0.05), and the Rheum officinale group had significantly higher levels than the sham-operation group (all P < 0.05); both the Rheum officinale group and the fecal microbiota group had a significantly lower serum level of endotoxin than the model group (all P < 0.05), and the fecal microbiota group had a significantly lower level of endotoxin than the Rheum officinale group within 6 hours of treatment (P < 0.05). Conclusion Both Rheum officinale and fecal microbiota transplantation can improve tissue inflammation and intestinal permeability in HLAP rats and can improve lipid metabolism and alleviate the progression of pancreatitis to a certain extent, and fecal microbiota transplantation shows a better clinical effect than Rheum officinale alone, but more randomized controlled trials are needed for further investigation. -
Key words:
- Hyperlipidemias /
- Pancreatitis /
- Rheum /
- Fecal Bacteria Transplantation /
- Rats, Sprague-Dawley
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图 1 各组大鼠术后36 h的胰腺组织病理学改变(HE染色，×200)
注：a，假手术组；b，模型组；c，大黄组；d，粪菌组。
Figure 1. Comparison of pancreatic histopathology at 36 h (HE, ×200)
图 2 各组大鼠术后36 h的回肠末端组织病理学改变(HE染色，×200)
注：a，假手术组；b，模型组；c，大黄组；d，粪菌组。
Figure 2. Histological comparison of terminal ileum at 36 h (HE, ×200)
表 1 各组不同时间段的大鼠胰腺组织病理评分
Table 1. Comparison of pathological scores of pancreatic tissue
组别 动物数(只) 6 h 24 h 36 h 假手术组 6 0.25(0~0.56) 0.15(0~0.29) 0(0~0.38) 模型组 6 6.25(5.69~8.06)1) 7.00(6.00~7.56)1) 6.50(5.19~8.19)1) 大黄组 6 3.75(3.18~4.19) 4.75(4.25~5.63)1) 4.75(3.88~6.00)1) 粪菌组 6 3.25(2.50~4.44) 4.00(3.63~4.09)2) 2.88(2.38~4.06) H值 19.123 20.108 18.416 P值 ＜0.001 ＜0.001 ＜0.001 注：与假手术组比较，1)P＜0.05；与模型组比较，2)P＜0.05。 
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表 2 各组不同时间段的大鼠回肠末端组织病理评分
Table 2. Comparison of pathological scores of terminal ileum tissue
组别 动物数(只) 6 h 24 h 36 h 假手术组 6 0.38(0~1.45) 0.35(0~0.50) 0.30(0.19~0.50) 模型组 6 4.05(4.00~4.53)1) 4.38(3.75~4.63)1) 4.08(3.64~4.28)1) 大黄组 6 2.55(2.08~3.41) 2.55(2.25~3.13) 3.00(2.46~3.50)1) 粪菌组 6 2.80(1.53~3.28) 2.50(1.90~3.00) 2.50(1.75~2.55) H值 14.973 18.864 19.903 P值 0.002 ＜0.001 ＜0.001 注：与假手术组比较，1)P＜0.05。
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表 3 各组不同时间段的大鼠血清淀粉酶
Table 3. Comparison of serum amylase
组别 动物数(只) 6 h 24 h 36 h 假手术组 6 1 732.48±628.60 1 717.65±271.91 2 071.55±320.06 模型组 6 4 625.58±1 073.431) 4 143.68±889.561) 6 377.90±2 694.431) 大黄组 6 3 512.38±892.281) 4 442.57±2 548.961) 2 648.47±1 241.502) 粪菌组 6 4 052.03±2 492.741) 3 749.08±1 703.071) 3 228.03±2 787.592) F值 4.389 3.540 6.325 P值 0.016 0.033 0.007 注：与假手术组比较，1)P＜0.05；与模型组比较，2)P＜0.05。
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表 4 各组不同时间段的大鼠生化相关指标比较
Table 4. Comparison of biochemical indexes in different time
组别 动物数(只) ALT(U/L) AST(U/L) 6 h 24 h 36 h 6 h 24 h 36 h 假手术组 6 116.13±20.43 125.50±41.22 113.75±23.32 349.92±169.32 292.73±112.75 301.07±44.67 模型组 6 238.17±70.501) 225.00±62.481) 214.63±25.811) 819.67±419.13 840.98±217.781) 769.35±212.741) 大黄组 6 183.10±52.771) 175.37±33.15 155.92±38.721)2) 480.38±243.31 515.28±151.421)2) 614.98±117.251) 粪菌组 6 140.47±47.922) 136.02±26.952) 136.07±42.102) 464.27±249.99 483.22±126.231)2) 402.33±82.032)3) F值 6.565 6.577 10.027 3.021 12.555 15.705 P值 0.003 0.003 ＜0.001 0.054 ＜0.001 ＜0.001 组别 动物数(只) TC(mmol/L) TG(mmol/L) 6 h 24 h 36 h 6 h 24 h 36 h 假手术组 6 2.30±0.30 2.52±0.33 2.37±0.37 1.21±0.32 1.38±0.31 1.17±0.28 模型组 6 3.76±0.721) 3.66±0.481) 3.78±0.511) 3.57±0.641) 3.58±0.701) 3.48±0.441) 大黄组 6 3.32±0.571) 3.10±0.371)2) 3.12±0.351)2) 3.04±0.481) 3.10±0.201) 3.15±0.521) 粪菌组 6 3.12±0.411)2) 3.19±0.381) 3.08±0.641)2) 2.98±0.451)2) 3.05±0.371)2) 3.09±0.301) F值 8.096 8.407 11.754 26.935 29.028 4.751 P值 0.001 0.001 ＜0.001 ＜0.001 ＜0.001 0.012 组别 动物数(只) HDL-C(mmol/L) LDL-C(mmol/L) 6 h 24 h 36 h 6 h 24 h 36 h 假手术组 6 0.54±0.10 0.76±0.34 0.64±0.32 0.38±0.29 0.64±0.30 0.51±0.25 模型组 6 0.70±0.26 0.92±0.29 0.98±0.40 1.33±0.231) 1.30±0.411) 1.23±0.421) 大黄组 6 0.83±0.21 0.87±0.18 0.97±0.33 0.97±0.311)2) 1.04±0.291) 0.95±0.161) 粪菌组 6 1.09±0.351)2) 1.22±0.251)2)3) 1.50±0.531)2)3) 0.63±0.352) 0.74±0.212) 0.80±0.332) F值 5.162 4.536 4.790 11.712 5.517 5.773 P值 0.008 0.014 0.011 ＜0.001 0.006 0.005 注：与假手术组比较，1)P＜0.05；与模型组比较，2)P＜0.05；与大黄组比较，3)P＜0.05。
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表 5 各组不同时间段的大鼠血清炎症因子水平比较
Table 5. Comparison of serum inflammatory factors in different time
组别 动物数(只) IL-6(pg/mL) TNFα(pg/mL) 6 h 24 h 36 h 6 h 24 h 36 h 假手术组 6 104.15±14.84 105.57±18.52 100.09±9.35 135.39±20.19 164.96±35.79 161.58±26.49 模型组 6 146.33±21.731) 140.94±14.081) 148.99±11.461) 241.30±27.901) 230.31±32.281) 253.34±25.521) 大黄组 6 128.64±5.751)2) 140.55±15.311) 127.42±20.611)2) 213.41±40.661) 224.20±27.581) 216.96±31.531)2) 粪菌组 6 109.28±10.042)3) 119.63±14.602)3) 108.12±14.542)3) 173.03±36.562)3) 190.45±32.032) 193.77±34.122) F值 18.102 7.211 13.535 12.350 5.448 10.213 P值 ＜0.001 0.002 ＜0.001 ＜0.001 0.007 ＜0.001 注：与假手术组比较，1)P＜0.05；与模型组比较，2)P＜0.05；与大黄组比较，3)P＜0.05。
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表 6 各组不同时间段的大鼠血清内毒素水平比较
Table 6. Comparison of serum endotoxin
组别 动物数(只) 6 h 24 h 36 h 假手术组 6 35.52±7.61 30.34±13.00 28.53±10.51 模型组 6 67.93±13.811) 74.29±12.711) 69.46±12.331) 大黄组 6 54.70±10.551)2) 57.53±11.981)2) 53.68±12.651)2) 粪菌组 6 37.62±9.542)3) 43.96±8.552) 42.63±9.951)2) F值 12.458 15.499 13.757 P值 ＜0.001 ＜0.001 ＜0.001 注：与假手术组比较，1)P＜0.05；与模型组比较，2)P＜0.05；与大黄组比较，3)P＜0.05。
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