田晓玲,张彧,杜珊,伍梦思,谭年花,陈斌
TIAN Xiao-ling,ZHANG Yu,DU Shan,WU Meng-si,TAN Nian-hua,CHEN Bin

• 1:湖南中医药大学
• 2:湖南中医药大学第一附属医院

摘要(Abstract)：

基于肝细胞生长因子(hepatocyte growth factor, HGF)/磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)/蛋白激酶B(protein kinase B, Akt)信号轴研究赤芍-附子药对对慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)大鼠的治疗作用及对肝细胞再生的影响。采用牛血清白蛋白皮下及尾静脉注射联合脂多糖(lipopolysaccharides, LPS)+D-氨基半乳糖(D-galactosamine, D-GalN)腹腔注射构建ACLF大鼠模型，实验设置正常对照(NC)组、模型(vehicle)组、赤芍-附子药对(SFYD,5.85 g·kg~(-1))组、促肝细胞生长颗粒(HGFG,4.05 g·kg~(-1))组。苏木素-伊红(hematoxylin-eosin staining, HE)染色观察大鼠肝组织病理变化。全自动生化分析仪检测血清谷丙转氨酶(alanineamino transferase, ALT)、谷草转氨酶(aspartate transa-minase, AST)、总胆红素(total bilirubin, TBIL)。ELISA法检测白细胞介素-1β(interleukin-1β, IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)炎症因子水平。免疫荧光染色检测增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)、细胞增殖相关抗原(antigen identified by monoclonal antibody, Ki67)及细胞周期蛋白(cell cycle protein B1, CyclinB1)阳性表达。实时荧光定量聚合酶链式反应(RT-qPCR)法、蛋白免疫印迹(Western blot)法检测HGF、结合蛋白Gab1(grb2 associated binding protein 1, Gab1)、PI3K、Akt、磷酸化PI3K(phosphorylation PI3K, p-PI3K)、磷酸化Akt(phosphorylation Akt, p-Akt)mRNA及蛋白表达水平。结果显示，与vehicle组比较，SFYD组大鼠肝组织病理评分降低，肝功能及炎症反应改善，PCNA、Ki67、CyclinB1荧光表达增强。同时与模型组相比，SFYD组HGF、Gab1蛋白表达上调，PI3K、Akt磷酸化激活。以上研究表明赤芍-附子药对通过减轻肝细胞炎症损伤以及促进肝细胞再生来达到抗肝衰竭的效应，其具体调控机制可能与激活HGF/PI3K/Akt信号通路有关。
Based on the hepatocyte growth factor(HGF)/phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt) signaling axis, this study investigated the therapeutic effect of Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata(PRR-ALRP) her-bal pair on acute-on-chronic liver failure(ACLF) rats and its impact on hepatocellular regeneration. The rat model of ACLF was constructed by subcutaneous and tail vein injection of bovine serum albumin combined with intraperitoneal injection of lipopolysaccharides(LPS)+D-galactosamine(D-GalN). The rats were divided into normal control(NC) group, model(vehicle) group, PRR-ALRP(5.85 g·kg~(-1)) group, and hepatocyte growth factor granules(HGFG, 4.05 g·kg~(-1)) group. Hematoxylin-eosin(HE) staining was used to observe pathological changes in rat liver tissues. Serum alanine aminotransferase(ALT), aspartate transaminase(AST), and total bilirubin(TBIL) were detected using an automatic biochemical analyzer. The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) inflammatory factors were detected by ELISA. Immunofluorescence staining was used to detect the positive expression of proliferating cell nuclear antigen(PCNA), antigen identified by monoclonal antibody(Ki67), and cell cycle protein B1(CyclinB1). Real-time fluorescence-based quantitative polymerase chain reaction(RT-qPCR) and Western blot were used to detect the mRNA and protein expression levels of HGF, growth factor receptor-bound protein 1(Gab1), PI3K, Akt, phosphorylated PI3K(p-PI3K), and phosphorylated Akt(p-Akt). The results showed that compared with the vehicle group, the PRR-ALRP group had reduced liver tissue pathological scores, improved liver function, and reduced inflammatory response, with enhanced PCNA, Ki67, and CyclinB1 fluorescence expression. Furthermore, compared with the model group, the PRR-ALRP group showed upregulated expression of HGF and Gab1 proteins, as well as activation of PI3K and Akt phosphorylation. These findings suggest that PRR-ALRP herbal pair exerts anti-liver failure effects by alleviating hepatocyte inflammatory damage and promoting hepatocellular regeneration, and its specific regulatory mechanism may be related to the activation of the HGF/PI3K/Akt signaling pathway.

关键词(KeyWords)： 慢加急性肝衰竭;赤芍-附子药对;HGF/PI3K/Akt;肝再生
acute-on-chronic liver failure;Paeoniae Radix Rubra and Aconiti Lateralis Radix Praeparata herbal pair;HGF/PI3K/Akt;hepatocellular regeneration

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81673959);; 湖南省科技创新计划项目(2021SK51415);; 湖南省研究生科研创新项目(CX20230805);; 湖南省中医药科研计划项目(C2022005);; 湖南中医药大学校院联合基金项目重点课题(2022XYLH003)

作者(Author): 田晓玲,张彧,杜珊,伍梦思,谭年花,陈斌
TIAN Xiao-ling,ZHANG Yu,DU Shan,WU Meng-si,TAN Nian-hua,CHEN Bin

DOI: 10.19540/j.cnki.cjcmm.20240412.707

参考文献(References)：

• [1] SARIN S K,CHOUDHURY A,SHARMA M K,et al.Acute-on-chronic liver failure:consensus recommendations of the Asian Pacific association for the study of the liver (APASL):an update[J].Hepatol Int,2019,13(4):353.
• [2] HERNAEZ R,SOLà E,MOREAU R,et al.Acute-on-chronic li-ver failure:an update[J].Gut,2017,66(3):541.
• [3] TREBICKA J,SUNDARAM V,MOREAU R,et al.Liver transplantation for acute-on-chronic liver failure:science or fiction?[J].Liver Transpl,2020,26(7):906.
• [4] 李佳琪，李君.慢加急性肝衰竭：定义、诊断与发病机制[J].中华肝脏病杂志，2022,30(2):121.
• [5] HUANG W,HAN N,DU L,et al.A narrative review of liver regeneration-from models to molecular basis[J].Ann Transl Med,2021,9(22):1705.
• [6] 陈斌，丁秀丽，彭杰，等.清温并用法对慢加急性肝衰竭肠源性内毒素血症大鼠结肠组织FoxP3,ROR-γt表达的影响[J].中国实验方剂学杂志，2020,26(2):19.
• [7] 桑培敏，达呼巴雅尔，王乐三，等.阳黄-阴阳黄-阴黄辨证论治联合西药治疗乙型肝炎相关慢加急性肝衰竭患者生存分析[J].中医杂志，2019,60(7):582.
• [8] 熊焰.温阳解毒化瘀法对肝衰竭IETM非阳黄证的干预机制及临床疗效研究[D].长沙：湖南中医药大学，2014.
• [9] 谭年花，唐陈琴，曹钰楠，等.基于“清温并用”理论的赤芍-附子不同配伍治疗慢加急性肝衰竭大鼠的疗效比较[J].时珍国医国药，2022,33(8):1821.
• [10] 曹钰楠，谭年花，唐陈琴，等.赤芍-附子对慢加急性肝衰竭大鼠PI3K/AKT信号通路及相关炎症因子表达的影响[J].中医药导报，2023,29(10):12.
• [11] GUPTA S,KUMAR M,CHAUDHURI S,et al.The non-canonical nuclear functions of key players of the PI3K-AKT-MTOR pathway[J].J Cell Physiol,2022,237(8):3181.
• [12] ZHAO Y,YE W,WANG Y D,et al.HGF/c-Met:a key promoter in liver regeneration[J].Front Pharmacol,2022,13:808855.
• [13] TAN N H,JAN G H,PENG J,et al.Chishao-Fuzi herbal pair restore the macrophage M1/M2 balance in acute-on-chronic liver failure[J].J Ethnopharmacol,2024,doi:10.1016/j.jep.2024118010.
• [14] TAND D,WANG R Y,SUN K W,et al.Netwerk pharmaeelogybased prediction of active compounds in the Wenyang Jiedu Huayu-formula acting on acute-on-chronic liver failure with experimental support in vitro and in viro[J].Front Pharmacol,2022,13:1003479.
• [15] 魏伟.药理实验方法学[M].北京：人民卫生出版社，2010.
• [16] 谭年花，曹钰楠，唐陈琴，等.基于巨噬细胞极化研究“赤芍-附子”治疗慢加急性肝衰竭的作用机制[J].中国实验方剂学杂志，2022,28(15):35.
• [17] 中华医学会感染病学分会肝衰竭与人工肝学组，中华医学会肝病学分会重型肝病与人工肝学组.肝衰竭诊治指南(2018年版)[J].中华传染病杂志，2019,37(1):9.
• [18] HUANG R,ZHANG X,GRACIA-SANCHO J,et al.Liver rege-neration:cellular origin and molecular mechanisms[J].Liver Int,2022,42(7):1486.
• [19] MA J T,XIA S,ZHANG B K,et al.The pharmacology and mechanisms of traditional Chinese medicine in promoting liver regeneration:a new therapeutic option[J].Phytomedicine,2023,116:154893.
• [20] 彭杰，陈斌，孙克伟，等.慢性乙型重型肝炎“湿热-血瘀-脾虚”证候分布与演变特点的回顾性分析[J].中西医结合肝病杂志，2011,21(3):135.
• [21] GUMURDULU D,ERDOGAN S,KAYASELUK F,et al.Expression of COX-2,PCNA,Ki-67 and p53 in gastrointestinal stromal tumors and its relationship with histopathological parameters[J].World J Gastroenterol,2007,13(3):426.
• [22] BEN YA′ACOV A,MEIR H,ZOLOTARYOVA L,et al.Impaired liver regeneration is associated with reduced cyclin B1 in natural killer T cell-deficient mice[J].BMC Gastroenterol,2017,17(1):44.
• [23] XIA J,ZHOU Y,JI H,et al.Loss of histone deacetylases 1 and 2 in hepatocytes impairs murine liver regeneration through Ki67 depletion[J].Hepatology,2013,58(6):2089.
• [24] MA Y J,LANG X M,YANG Q,et al.Paeoniflorin promotes intestinal stem cell-mediated epithelial regeneration and repair via PI3K-AKT-mTOR signalling in ulcerative colitis[J].Int Immunopharmacol,2023,119:110247.
• [25] 周昊言，孙若岚，季千惠，等.基于网络药理-分子对接解析川芎-赤芍药对干预脑缺血的作用机制[J].中国中药杂志，2021,46(12):3007.
• [26] SUN X H,ZHU K D,FENG C C,et al.Paeoniflorin ameliorates hyperprolactinemia-induced inhibition of osteoblastogenesis by suppressing the NF-κB signaling pathway[J].Int J Endocrinol,2022,2022:4572033.
• [27] WANG M,HU W J,ZHOU X,et al.Ethnopharmacological use,pharmacology,toxicology,phytochemistry,and progress in Chinese crude drug processing of the lateral root of Aconitum carmichaelii Debeaux.(Fuzi):a review[J].J Ethnopharmacol,2023,301:115838.
• [28] WANG Y T,ZHANG H X,WANG J,et al.Aconiti lateralis radix praeparata inhibits Alzheimer′s disease by regulating the complex regulation network with the core of GRIN1 and MAPK1[J].Pharm Biol,2021,59(1):311.
• [29] ITSUJI T,TONNMURA H,ISHIBASHI H,et al.Hepatocyte growth factor regulates HIF-1α-induced nucleus pulposus cell proliferation through MAPK-,PI3K/Akt-,and STAT3-mediated signaling[J].J Orthop Res,2021,39(6):1184.
• [30] STANCIU S,IONITA-RADU F,STEFANI C,et al.Targeting PI3K/AKT/mTOR signaling pathway in pancreatic cancer:from molecular to clinical aspects[J].Int J Mol Sci,2022,23(17):124.
• [31] ZHAO M,JUNG Y,JIANG Z,et al.Regulation of energy metabolism by receptor tyrosine kinase ligands[J].Front Physiol,2020,11:354.
• [32] HE Z Y,LOU K H,ZHAO J H,et al.Resina draconis reduces acute liver injury and promotes liver regeneration after 2/3 partial hepatectomy in mice[J].Evid Based Complement Alternat Med,2020,2020:2305784.
• [33] LI X,SUN J,FAN X,et al.Schisandrol B promotes liver regene-ration after partial hepatectomy in mice[J].Eur J Pharmacol,2018,818:96.
• [34] SALAMA S A,KABEL A M.Taxifolin ameliorates iron overload-induced hepatocellular injury:modulating PI3K/AKT and p38 MAPK signaling,inflammatory response,and hepatocellular regeneration[J].Chem Biol Interact,2020,330:109230.
• [35] GAO D D,FU J,QIN B,et al.Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats[J].World J Gastroenterol,2016,22(16):4136.
• [36] 修文丽，毛成刚，杨晨宇，等.肝细胞生长因子基因修饰的间充质干细胞治疗肝损伤的研究进展[J].中华细胞与干细胞杂志(电子版),2020,10(6):373.