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基于PI3K/AKT/mTOR信号通路探讨大黄煎剂对轻微型肝性脑病大鼠脑组织炎症损伤的保护机制
DOI: 10.12449/JCH240215
Protective mechanism of rhubarb decoction against inflammatory damage of brain tissue in rats with mild hepatic encephalopathy： A study based on the PI3K/AKT/mTOR signaling pathway
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摘要:
目的 探讨大黄煎剂保留灌肠对轻微型肝性脑病（MHE）大鼠模型脑组织炎症损伤的改善作用及可能机制。 方法 60只SD雄性大鼠按完全随机方法分为空白组（CON组，n=6）和慢性肝硬化造模组（n=54）。12周后慢性肝硬化造模成功并经Morris水迷宫测试确认符合MHE模型大鼠40只，采用完全随机方法分为模型组（MOD组，n=8）、乳果糖组（LT组，n=8）、大黄煎剂低剂量组（RD1组，n=8）、大黄煎剂中剂量组（RD2组，n=8）和大黄煎剂高剂量组（RD3组，n=8）。其中CON组和MOD组大鼠用生理盐水保留灌肠，2 mL/只，1次/d；LT组大鼠用乳果糖按22.5%剂量保留灌肠，2 mL/只，1次/d；RD1组、RD2组、RD3组大鼠分别用大黄煎剂按2.5、5.0、7.5 g/kg三种剂量保留灌肠，2 mL/只，1次/d。所有大鼠治疗10 d后，进行Morris水迷宫测试，分析大鼠的空间学习记忆能力。分析大鼠行为学状态；检测大鼠血清ALT、AST、IL-1β、IL-6、TNF-α和血氨水平；观察大鼠肝组织和脑组织病理学变化；检测大鼠脑组织磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（AKT）、雷帕霉素靶蛋白（mTOR）的mRNA和蛋白表达水平。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。 结果 与MOD组比较，RD1组、RD2组和RD3组大鼠逃避潜伏期时间显著缩短（P值均<0.01），ALT、AST、IL-1β、IL-6、TNF-α和血氨水平明显降低（P值均<0.05），肝细胞、脑细胞变性、坏死和炎症程度减轻，脑组织PI3K、AKT、mTOR的mRNA和蛋白表达水平均降低（P值均<0.05），且RD3组治疗效果优于RD1组和RD2组。 结论 大黄煎剂保留灌肠能够改善MHE大鼠认知功能及脑组织炎症损伤，其作用机制可能与调控PI3K/AKT/mTOR信号通路有关。 -
关键词:
- 肝性脑病 /
- 大黄煎剂 /
- 治疗学 /
- 大鼠， Sprague-Dawley
Abstract:Objective To investigate the role and possible mechanism of action of rhubarb decoction （RD） retention enema in improving inflammatory damage of brain tissue in a rat model of mild hepatic encephalopathy （MHE）. Methods A total of 60 male Sprague-Dawley rats were divided into blank group （CON group with 6 rats） and chronic liver cirrhosis modeling group with 54 rats using the complete randomization method. After 12 weeks， 40 rats with successful modeling which were confirmed to meet the requirements for MHE model by the Morris water maze test were randomly divided into model group （MOD group）， lactulose group （LT group）， low-dose RD group （RD1 group）， middle-dose RD group （RD2 group）， and high-dose RD group （RD3 group）， with 8 rats in each group. The rats in the CON group and the MOD group were given retention enema with 2 mL of normal saline once a day； the rats in the LT group were given retention enema with 2 mL of lactulose at a dose of 22.5% once a day； the rats in the RD1， RD2， and RD3 groups were given retention enema with 2 mL RD at a dose of 2.5， 5.0， and 7.5 g/kg， respectively， once a day. After 10 days of treatment， the Morris water maze test was performed to analyze the spatial learning and memory abilities of rats. The rats were analyzed from the following aspects： behavioral status； the serum levels of alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） and the level of blood ammonia； pathological changes of liver tissue and brain tissue； the mRNA and protein expression levels of phosphatidylinositol 3-kinase （PI3K）， protein kinase B （AKT）， and mammalian target of rapamycin （mTOR） in brain tissue. 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 further comparison between two groups. Results Compared with the MOD group， the RD1， RD2， and RD3 groups had a significantly shorter escape latency （all P<0.01）， significant reductions in the levels of ALT， AST， IL-1β， IL-6， TNF-α， and blood ammonia （all P<0.05）， significant alleviation of the degeneration， necrosis， and inflammation of hepatocytes and brain cells， and significant reductions in the mRNA and protein expression levels of PI3K， AKT， and mTOR in brain tissue （all P<0.05）， and the RD3 group had a better treatment outcome than the RD1 and RD2 groups. Conclusion Retention enema with RD can improve cognitive function and inflammatory damage of brain tissue in MHE rats， possibly by regulating the PI3K/AKT/mTOR signaling pathway. -
Key words:
- Hepatic Encephalopathy /
- Rhubarb Decoction /
- Therapeutics /
- Rats， Sprague-Dawley
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图 1 各组大鼠肝功能和血氨水平比较
Figure 1. Comparison of liver function and blood ammonia levels among groups of rats
图 2 各组大鼠药物干预后的逃避潜伏期比较
Figure 2. Comparison of escape latency after drug intervention in each group of rats
图 3 光镜下各组大鼠肝组织HE染色结果（×200）
注： a，CON组；b，MOD组；c，LT组；d，RD1组；e，RD2组；f，RD3组。
Figure 3. Light microscopy results of HE staining of rat liver tissue in each group （×200）
图 4 光镜下各组大鼠脑组织HE染色结果（×200）
注： a，CON组；b，MOD组；c，LT组；d，RD1组；e，RD2组；f，RD3组。
Figure 4. Light microscopy results of HE staining of rat brain tissue in each group （×200）
图 5 各组大鼠相关炎性细胞因子水平比较
Figure 5. Comparison of levels of relevant inflammatory cytokines among groups of rats
图 6 各组大鼠脑组织中PI3K、AKT、mTOR的mRNA相对表达量比较
Figure 6. Relative mRNA expression of PI3K， AKT and mTOR in the brain tissue of rats in each group
图 7 各组大鼠脑组织中PI3K、AKT、mTOR的蛋白免疫印迹结果及蛋白表达量比较
Figure 7. Comparison of protein expression of PI3K， AKT， and mTOR in brain tissues of rats from various groups
表 1 引物序列
Table 1. Primer sequences
引物名称 引物序列（5′-3′） β-actin F：CTAAGGCCAACCGTGAAAAG R：ACCAGAGGCATACAGGGACA PI3K F：TGCACACATGCTTGCTACCTC R：TTGCTTTGACCTGACCCATCT AKT F：ATGGACTTCCGGTCAGGTTCA R：GCCCTTGCCCAGTAGCTTCA mTOR F：GCTTATCAAGCAAGCGACATCTCA R：TCCACTGGAAGCACAGACCAAG 
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