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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1. 病例资料
病例1:患者女性,58岁,以“体检发现胰腺尾部占位3天”于2019年3月4日入本院,患者2年来体质量下降5 kg,增强CT检查示:胰腺尾部动脉期以及门静脉期显著强化结节,动脉期CT值为198 HU,考虑神经内分泌肿瘤(图1)。超声胃镜示:胰腺尾部低回声病灶(0.6 cm×0.8 cm),为富血供结节。患者癌胚抗原、糖类抗原(CA)19-9、CA125等肿瘤标志物指标正常,既往无糖耐量异常病史,口服葡萄糖耐量试验结果正常。术前诊断为胰腺神经内分泌肿瘤(pancreatic neuroendocrine tumor,pNET),患者在全麻下行腹腔镜下胰腺体尾切除术,术后标本可见胰腺内副脾(intrapancreatic accessory spleen,IPAS)组织,切面红褐色、质韧(图2);术后病理示:胰腺体尾部副脾组织,胰腺组织切缘未见癌(图3)。
病例1误诊原因分析:(1)CT影像学表现是主要原因之一。胰腺尾部动脉早期和门静脉期均呈显著强化结节,边缘规则,未见腹膜后和周围肿大淋巴结,这些影像学特点与pNET的特点相符。其中,非功能性pNET患者的症状隐蔽,激素水平正常,缺乏特异性肿瘤标志物,因其血供丰富且内部可有变性坏死,当病灶较大时,增强CT可见不均匀强化,但当pNET病灶较小也可表现为均匀强化,此时较难与IPAS区分;(2)该患者虽行超声胃镜检查,但患者家属拒绝行穿刺,遂未操作;(3)未能将病灶强化程度与脾脏强化程度相比,对IPAS的诊断缺乏经验。
病例2:患者男性,60岁,因“间断性腹痛10余天”于2022年9月15日入本院。既往病史:30年前因外伤行脾切除。CT平扫+增强检查示:胰腺钩突部占位,考虑恶性肿瘤可能(图4);胰尾部见类圆形低密度影,较大直径为3 cm,边缘见钙化,增强扫描轻度不均匀强化,实性假乳头状瘤待排除(图5)。术前诊断:胰头钩突肿瘤;胰尾部占位,考虑胰腺实性假乳头状瘤;脾切除术后。肿瘤标志物:CA19-9 1 537.07 U/mL,CA125 125.68 U/mL。术中行胰十二指肠联合胰尾部分切除术;术后病理示:钩突部胰腺导管腺癌(中低分化);胰体尾副脾组织(图6),胰腺组织切缘未见癌。
病例2误诊原因分析:(1)该患者有脾切除病史,术前影像学检查提示脾脏缺如,主观上忽视了副脾存在的可能,客观上影像学检查时缺少副脾组织与正常脾脏组织的影像学特点比较。(2)该患者CT特征为胰尾部见类圆形稍低密度影,增强扫描示轻度不均匀强化,边缘可见钙化,可能为胰腺实性假乳头状瘤,或者其他低度恶性肿瘤。而脾脏组织较少发生钙化,该患者胰尾部分占位的钙化表现进一步干扰了临床判断。而胰腺实性假乳头状瘤实性部分和囊壁常呈轻、中度强化,病灶内可见钙化灶,往往位于病灶实性部分、分隔处或其周边包膜。该患者的影像学表现与胰腺实性假乳头状瘤极为相似,对明确鉴别诊断造成严重影响[1]。
2. 讨论
副脾与胚胎阶段脾脏的胚芽形成密切相关,正常情况下,在胚胎发育第5周,来自间充质细胞的胃背系膜分化为脾脏,但如果脾脏胚芽未完全融合,或者分离出单个细胞,则发育形成副脾[2]。80%的副脾发生在脾门附近,17%发生在胰腺尾部,还可见于胃壁、大网膜、脾结肠韧带、睾丸、肾上腺等部位。副脾的发生率约为10%,其中完全包裹在胰腺内的副脾发生率仅为2%[3],即IPAS。IPAS最常被误诊为pNET,pNET分为功能性pNET和非功能性pNET,功能性pNET易通过患者激素水平及其他临床症状与IPAS鉴别诊断,而IPAS最易被误诊为非功能性pNET。pNET在所有胰腺肿瘤中占比不超过2%,非功能性pNET仅占全部pNET的15%~41%,其中大多数为恶性,肿瘤根治性切除是实现患者长期生存最重要的手段,因此对肿瘤最大径>2 cm的非功能性pNET必须手术切除,此外,除了肿瘤最大径<1 cm或手术风险较大者,其余≤2 cm的非功能性pNET也均应行手术切除。但IPAS无需外科干预,故对二者的明确鉴别诊断具有重要的临床意义[4]。
IPAS无明显特异性临床表现,其诊断主要依靠影像学检查。在CT平扫中,IPAS表现的密度与脾脏相似,增强扫描强化程度高于胰腺组织,与脾脏一致,动脉期表现为“花斑样”不均匀强化,门静脉期为均匀强化,但当IPAS直径较小时,动脉期也表现为均匀强化[5]。MRI对IPAS的诊断也有重要价值,IPAS的T1WI信号低于胰腺,T1WI可显示肿块的位置、边缘、形态,T2WI信号表现为与脾脏等信号,MRI强化特点与CT强化类似。由于IPAS的白髓与红髓比例较高,有时在T2加权像上IPAS的信号强度略高于脾脏[6]。此外,弥散加权成像对IPAS的诊断也有积极意义,尤其在鉴别IPAS与胰腺实体肿瘤方面[7]。比IPAS更罕见的是IPAS伴上皮样囊肿(epidermoid cyst in intrapancreatic accessory spleen,ECIPAS)[8]。ECIPAS直径通常为2~4 cm;病理学镜下通常表现为囊肿内衬复层鳞状上皮,囊外可见脾脏组织;CT和MRI检查的主要着眼点为识别囊肿周围的脾脏组织,ECIPAS的CT表现为实性部分强化,强化程度与脾脏类似,囊性部分CT平扫呈低密度,增强后不强化[9]。明确的术前影像学诊断和术中冰冻病理切片对于避免过度治疗至关重要。
此外,由于副脾和脾脏具有相同的生理功能,因此具有脾脏功能显示作用的放射性核素扫描在鉴别IPAS和pNET也有重要价值。脾脏组织利用网状内皮细胞可拦截并破坏衰老的红细胞,99mTc标记的热变性红细胞(99mTc-heat denaturation RBC,99mTc-HDRBC)可以在脾脏和IPAS处浓聚,并在核素检查中显影。然而,当脾脏包裹胰尾或覆盖IPAS时,IPAS处的显影被正常脾脏组织掩盖,易造成误诊[10]。非功能性pNET因其含有生长抑素受体,故可通过68Ga生长抑素类似物(68Ga somatostatin analogue imaging,68Ga-SSA)核素扫描进行鉴别,其在诊断pNET方面比MRI更敏感,但假阳性率较高,只有当68Ga-SSA图像中摄取显著高于脾脏才具有诊断价值,可能与脾脏组织中淋巴细胞同样含有生长抑素受体有关[11]。常规超声内镜(endoscopic ultrasonography,EUS)对IPAS的诊断价值有限,但可利用Levovist或Sonazid作为静脉造影剂的对比增强EUS来提高诊断IPAS的能力。Makino等[12]研究发现,以Sonazoid作为造影剂的增强EUS对IPAS中存在的丰富血管和网状内皮细胞系统较为敏感。此外,IPAS与pNET在EUS定性定量弹性成像下表现也有差异,IPAS由于病灶质地松软呈现绿色为主的图像,pNET则由于病灶质地较硬呈现质地均匀的蓝色图像[13]。进一步将弹性成像结果量化为弹性应变率比值可以提高诊断效率[14]。
除了影像学检查以外,EUS引导下细针穿刺(EUS-FNA)检查是鉴别IPAS的高特异性手段。Tatsas等[15]研究发现,脾窦薄层血管的内皮细胞中的CD8经免疫细胞化学染色可检出,只要取得脾窦的内皮细胞即可诊断为副脾。但由于肿块较小、位置较深等不利因素,往往无法准确取得病理标本,且细针穿刺可能导致胰漏、出血等不良事件。当影像学检查无法确诊且胰腺内肿块较大时,EUS-FNA仍值得考虑。近年来,基于探针的激光共聚焦显微内镜检查(confocal laser endomicroscopy,CLE)在诊断胰腺占位肿块的应用成为研究热点,CLE可以通过获取放大1 000倍后的黏膜图像来识别细胞和亚细胞的微结构,并进行活体组织学诊断(光学虚拟活检)。CLE既可以避免EUS-FNA的副损伤,又可与EUS-FNA联合应用提高胰腺肿块的诊断准确度[16]。
避免副脾的误诊,首先需要临床医师对IPAS具备充分的认识,对位于胰腺尾部、直径1~3 cm、边界清、质地均匀的富血管病灶,应考虑IPAS的可能性。其次,在鉴别诊断过程中需要综合利用CT、MRI、PET-CT等影像学检查,尤其是99mTc-HDRBC等敏感性较高的检查。必要时可采用EUS-FNA检查,但要提高操作技巧,避免发生不良事件。确诊IPAS的患者无需手术治疗,随访观察即可。总之,临床上应进一步提升对IPAS的认识与鉴别,避免IPAS患者被误诊并接受非必要手术,增加医疗负担。
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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 -
[1] Chinese Society of Hepatology, Chinese Medical Association. Guidelines on the management of hepatic encephalopathy in cirrhosis[J]. J Clin Hepatol, 2018, 34( 10): 2076- 2089. DOI: 10.3969/j.issn.1001-5256.2018.10.007.中华医学会肝病学分会. 肝硬化肝性脑病诊疗指南[J]. 临床肝胆病杂志, 2018, 34( 10): 2076- 2089. DOI: 10.3969/j.issn.1001-5256.2018.10.007. [2] HÄUSSINGER D, SCHLIESS F. Pathogenetic mechanisms of hepatic encephalopathy[J]. Gut, 2008, 57( 8): 1156- 1165. DOI: 10.1136/gut.2007.122176. [3] STARK AK, SRISKANTHARAJAH S, HESSEL EM, et al. PI3K inhibitors in inflammation, autoimmunity and cancer[J]. Curr Opin Pharmacol, 2015, 23: 82- 91. DOI: 10.1016/j.coph.2015.05.017. [4] NIU YB. Effect of metformin on acute liver injury induced by carbon tetrachloride in mice by inhibiting PI3K-AKT-mTOR signaling pathway[D]. Taiyuan: Shanxi Medical University, 2020.牛艳邦. 二甲双胍通过抑制PI3K-AKT-mTOR信号通路对四氯化碳诱导的小鼠急性肝损伤的影响[D]. 太原: 山西医科大学, 2020. [5] MAO DW, WU JH, QIU H, et al. Theoretical discussion on the treatment of hepatic encephalopathy with the method of”dredging fu organs and inducing resuscitation”[J]. J Sichuan Tradit Chin Med, 2008, 26( 10): 22- 24.毛德文, 武建华, 邱华, 等.” 通腑开窍”法治疗肝性脑病的理论探讨[J]. 四川中医, 2008, 26( 10): 22- 24. [6] YAO C, YAO F, XIE W, et al. Clinical observation on rhubarb decoction retention Enema in treatment of minimal hepatic encephalopathy[J]. Liaoning J Tradit Chin Med, 2013, 40( 3): 474- 476. DOI: 10.13192/j.ljtcm.2013.03.96.yaoch.089.姚春, 姚凡, 谢武, 等. 大黄煎剂保留灌肠治疗轻微肝性脑病临床研究[J]. 辽宁中医杂志, 2013, 40( 3): 474- 476. DOI: 10.13192/j.ljtcm.2013.03.96.yaoch.089. [7] ZHU RH, HUANG JJ, HUANG HN, et al. Mechanism of action of Rheum officinale decoction in treatment of minimal hepatic encephalopathy based on network pharmacology[J]. Hunan J Tradit Chin Med, 2021, 37( 11): 172- 177, 214. DOI: 10.16808/j.cnki.issn1003-7705.2021.11.057.朱荣火, 黄晶晶, 黄鸿娜, 等. 基于网络药理学探究大黄煎剂治疗轻微型肝性脑病的作用机制[J]. 湖南中医杂志, 2021, 37( 11): 172- 177, 214. DOI: 10.16808/j.cnki.issn1003-7705.2021.11.057. [8] ZHOU XX, ZENG SL, YAN HP, et al. Retention enema therapy with rhubarb decoction in the adjuvant treatment of hepatic encephalopathy[J]. J Guangxi Univ Chin Med, 2022, 25( 1): 12- 16. DOI: 10.3969/j.issn.2095-4441.2022.01.003.周学寻, 曾胜澜, 严惠萍, 等. 大黄煎剂保留灌肠辅助治疗肝性脑病的临床观察[J]. 广西中医药大学学报, 2022, 25( 1): 12- 16. DOI: 10.3969/j.issn.2095-4441.2022.01.003. [9] LUO S, KONG X, WU JR, et al. Neuroinflammation in acute hepatic encephalopathy rats: imaging and therapeutic effectiveness evaluation using 11C-PK11195 and 18F-DPA-714 micro-positron emission tomography[J]. Metab Brain Dis, 2018, 33( 5): 1733- 1742. DOI: 10.1007/s11011-018-0282-7. [10] WANG K, GUAN DS, ZHAO X, et al. Proteomics and metabolomics of raw rhubarb and wine-processed rhubarb in the treatment of rats with intracerebral hemorrhage[J]. Ann Transl Med, 2020, 8( 24): 1670. DOI: 10.21037/atm-20-7831. [11] ZHAO XL, ZHU SF, LI J, et al. Epigenetic changes in inflammatory genes and the protective effect of cooked rhubarb on pancreatic tissue of rats with chronic alcohol exposure[J]. Biomed Pharmacother, 2022, 146: 112587. DOI: 10.1016/j.biopha.2021.112587. [12] DONG LL, DU HL, ZHANG MY, et al. Anti-inflammatory effect of Rhein on ulcerative colitis via inhibiting PI3K/Akt/mTOR signaling pathway and regulating gut microbiota[J]. Phytother Res, 2022, 36( 5): 2081- 2094. DOI: 10.1002/ptr.7429. [13] ZHU D, XU JH, MO W. Effect of Luling Huoluo Granules on the inflammatory responses in rats with cervical spondylotic radiculopathy based on the PI3K/Akt signaling pathway[J]. J Changchun Univ Chin Med, 2022, 38( 5): 506- 509. DOI: 10.13463/j.cnki.cczyy.2022.05.010.朱栋, 许金海, 莫文. 基于PI3K/Akt信号通路探究鹿灵活络颗粒对神经根型颈椎病大鼠炎症反应的影响[J]. 长春中医药大学学报, 2022, 38( 5): 506- 509. DOI: 10.13463/j.cnki.cczyy.2022.05.010. [14] HUANG XJ, NI BW, MAO ZK, et al. NOV/CCN3 induces cartilage protection by inhibiting PI3K/AKT/mTOR pathway[J]. J Cell Mol Med, 2019, 23( 11): 7525- 7534. DOI: 10.1111/jcmm.14621. [15] LUO M, GUO JY, CAO WK. Inflammation: A novel target of current therapies for hepatic encephalopathy in liver cirrhosis[J]. World J Gastroenterol, 2015, 21( 41): 11815- 11824. DOI: 10.3748/wjg.v21.i41.11815. -