肝纤维化是指各种慢性肝病向肝硬化发展时肝脏损伤修复愈合反应的一种病理生理过程[1]，其中肝细胞凋亡在肝纤维化的形成过程中具有重要作用[2]。关于肝细胞凋亡与肝纤维化的联系，目前主要有2种机制[3]：(1)肝细胞凋亡后产生的凋亡小体被肝脏中的Kupffer细胞、肝星状细胞、肝窦周细胞吞噬，促进纤维化进展；(2)凋亡的肝细胞会释放核苷酸腺苷三磷酸、脂质溶血磷脂胆碱等促纤维化介质从而促进肝纤维化。
目前国内已有多种中成药应用于肝纤维化的治疗，如扶正化瘀胶囊、鳖甲软肝片等。这些方药中大多含有虫草菌丝或冬虫夏草。笔者在既往针对扶正化瘀胶囊有效组分的研究[4-6]中发现，虫草多糖具有显著的抗肝纤维化作用，其可通过抑制肝星状细胞活化和抑制TGFβ/Smads信号通路而发挥抗肝纤维化作用[7]，同时体内研究[8]也发现，虫草多糖有抗肝细胞凋亡的作用，因此，本文拟运用体外TNFα诱导的L02细胞凋亡模型，进一步探讨虫草多糖对肝细胞凋亡的影响及可能的机制。
人正常肝细胞株L02，购自中国科学院上海细胞生物学研究所细胞库。
虫草多糖(批号180316，纯度≥45%UV)购自上海融禾医药科技发展有限公司，称取0.028 g虫草多糖，加入1 ml细胞全培养基后进行冰水浴超声5 min混匀，配置为12.8 mg/ml的虫草多糖母液储存在超低温冰箱备用，后续根据所需实验药物浓度进行配置。
细胞培养基DMEM(1X，货号11965-092，购自GibcoTM)；胎牛血清(货号10099141C，购自GibcoTM)；青霉素和链霉素(10 000 U/ml，货号15140122，购自GibcoTM)；PBS(货号21-040-CV，购自Corning公司)；TNFα(货号210-TA，购自美国R & D公司)；CCK8试剂盒(货号CCK02-181102，购自上海博谷生物科技有限公司)；Annexin V/PI凋亡检测试剂盒[货号E606336-0100，购自生工生物工程(上海)股份有限公司]；兔cleaved-caspase3单克隆抗体(货号9664)和兔cleaved-caspase8单克隆抗体(货号9496)均购自cell signaling technology公司；兔GAPDH多克隆抗体(货号10494-1-AP，购自Proteintech公司)；UNIQ-10柱式Trizol总RNA抽提试剂盒(货号B511321-0100，购自生工生物工程上海股份有限公司)；TB Green Rremix Ex Taq(货号PK0445，购自TaKaRa Bio公司)；iScriptTM cDNA Synthesis Kit(货号1708891，购自TaKaRa Bio公司)；RT-PCR使用引物以及片段长度详见表 1。
| 引物名称 | 引物序列 | 产物长度(bp) |
| Bax | 5′TCG CCC TTT TCT ACT TTG CC 3′ | 97 |
| caspase3 | 5′CTG GTA TGT GTG GAT GAT GCT 3′ | 100 |
| caspase8 | 5′CTG GGA GAA GGA AAG TTG GAC 3′ | 68.5 |
| caspase9 | 5′TGC CTC AAT GCC AGT AAC 3′ | 129 |
| Fas | 5′ATT TTT GCC TTG GTG CTC A 3′ | 94 |
| GAPDH | 5′GGG AAG GTG AAG GTC GGA GT 3′ | 105 |
| ACTB | 5′AAG GTG ACA GCA GTC GGT T 3′ | 195 |
细胞孵育箱(型号Galasxy170s，购于美国NewBrunswick公司)；细胞超净操作台(型号HR50-IIA2，购自中国海尔公司)；PCR机(型号Applied Biosystems ViiA7，购自美国赛默飞公司)；Odyssey双色红外激光成像系统(购自Li-COR公司)；高内涵系统(购自美国赛默飞公司)。
L02正常肝细胞在提前配置好的完全DMEM培养基中培养，其中添加1%青霉素-链霉素和10%胎牛血清。于37 ℃、5%CO2的恒温细胞培养箱中培养，取对数生长期，生长状态良好的细胞进行后续实验。
将细胞按照所需组数接种于96孔板或6孔板，每孔细胞密度根据后续具体实验方法进行选择，TNFα造模浓度为0、5、10、20、40 ng/ml，干预时间为24 h，使用CCK8细胞增殖毒性检测法和Annexin V/PI双染法以确定最佳造模浓度。
细胞按照所需组数接种于96孔板或者6孔板，虫草多糖设置浓度为50、100、200 μg/ml共3组，在造模前预作用时间为12 h。
不经任何药物和TNFα处理的正常培养细胞为正常组(N)，通过实验筛选确定TNFα造模浓度并作用24 h的细胞为模型组(M)，根据实验设计，50、100、200 μg/ml虫草多糖预作用12 h并给予TNFα造模浓度作用24 h为实验组。
细胞按照5×103 /孔接种于96孔板，每组10孔，待细胞生长至80%以上时，弃上清，按照实验设计进行分组并待药物干预以及造模后，使用PBS缓冲液0.1 ml/孔清洗1次，加入CCK8工作液(CCK8试剂∶细胞全培养基=1∶ 10)0.1 ml/孔，然后于培养箱内继续培养，选取最佳时间放入多功能酶标仪内(450 nm和630 nm波长)读取各孔吸光度值。
细胞按照2×106/孔接种于6孔板，每组3孔，待细胞生长至80%以上时弃上清，按照实验设计进行分组并待药物干预以及造模后，使用PBS洗涤收集不同组细胞，将细胞悬液摇匀后用195 μl的1×Binding Buffer重悬细胞；使细胞密度为2×105细胞/ml，分别加入5 μl的Annexin V和FITC染液至195 μl细胞重悬液，避光，混匀，4 ℃孵育15 min后进行上机检测，为防止荧光衰变，在1 h内进行流式细胞检测。
取生长状态良好的细胞接种于6孔板，每组3孔，按照实验设计进行分组并待药物干预以及造模后，弃上清，使用PBS缓冲液清洗，加入Trizol裂解细胞，按照说明书使用总RNA抽提试剂盒进行细胞内总RNA提取，逆转录后进行RT-PCR检测。
将生长状态良好的细胞接种于6孔板中，每组3孔，待药物干预以及造模后，4 ℃预冷离心取细胞沉淀，加入60 μl RIPA细胞裂解液后置于冰上进行细胞总蛋白提取并使用100 ℃加热器致蛋白变性3次，每次10 min后取相应样本进行SDS-聚丙烯酰胺凝胶电泳，使用Odyssey双色红外激光成像系统(Li-COR)进行蛋白印迹显影并分析。
应用SPSS 20.0统计软件进行数据分析。正态分布的计量资料以x±s表示，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。非正态分布的计量资料M(P25~P75)表示，多组间比较采用Kruskal-Wallis H检验，进一步两两比较采用Dunnett-t检验。P＜0.05为差异有统计学意义。
以不同浓度(0、5、10、20、40 ng/ml)TNFα作用于L02细胞24 h，结果显示，随着浓度的增加，细胞增殖逐渐受到抑制，其中40 ng/ml浓度TNFα对L02细胞的增殖具有显著抑制作用(P＜0.01)(图 1)。
根据增殖实验结果，选用0、20、40 ng/ml的TNFα浓度，通过Annexin V/PI双染法检测其诱导L02肝细胞24 h的细胞凋亡作用。结果显示，细胞凋亡数量随TNFα浓度升高而增加，其中40 ng/ml组与正常组比较差异具有统计学意义(9.85%±0.87% vs 7.71%±1.20%，P＜0.05)(图 2)。因此，后续实验以40 ng/ml的TNFα浓度作为诱导L02肝细胞凋亡的造模浓度。
CCK8法检测结果显示，与正常组比较，50、100、200 μg/ml的虫草多糖均对L02肝细胞无明显细胞毒性，其中100、200 μg/ml虫草多糖显著促进L02肝细胞增殖(P值均＜0.05)(图 3)。
与正常组比较，模型组细胞增殖活力显著下降(P＜0.05)，而50、100、200 μg/ml的虫草多糖组增殖活力均显著高于正常组和模型组(P值均＜0.01)(图 4)。
Annexin V/PI双染法检测结果显示，与正常组比较，模型组凋亡细胞数量显著增加(P＜0.01)；与模型组比较，50、100、200 μg/ml虫草多糖组凋亡细胞数量均明显降低(P值均＜0.05)；100 μg/ml组作用效果最优，其与200 μg/ml组比较差异有统计学意义(P＜0.01)(图 5)。
RT-PCR结果显示，与正常组比较，模型组的内源性线粒体途径(Bax)、外源性死亡受体途径Fas的mRNA表达均显著升高(P值均＜0.05)；50、100、200 μg/ml的虫草多糖均可显著降低模型组内源性线粒体途径caspase9的mRNA表达和关键执行蛋白酶caspase3的mRNA表达(P值均＜0.05)；50、100 μg/ml的虫草多糖可显著降低Bax mRNA表达(P值均＜0.05)；100、200 μg/ml的虫草多糖可明显降低Fas、caspase8 mRNA表达(P值均＜0.05)(表 3)
| 组别 | Bax | caspase 9 | Fas | caspase 8 | caspase 3 |
| 正常组 | 0.92(0.63~1.90) | 1.00±0.05 | 0.97(0.85~1.14) | 1.01(0.83~1.15) | 0.94(0.87~1.20) |
| 模型组 | 2.38(1.46~4.14)1) | 1.11±0.32 | 1.66(1.20~3.87)1) | 1.33(0.92~4.44) | 1.41(1.03~2.80) |
| 50 μg/ml | 0.81(0.78~1.11)3) | 0.83±0.142) | 1.06(0.86~3.18) | 0.90(0.83~1.40) | 0.90(0.66~1.16)2) |
| 100 μg/ml | 1.09(0.65~1.64)2) | 0.75±0.073) | 0.83(0.77~1.19)2) | 0.85(0.76~1.02) | 0.83(0.68~0.95)3) |
| 200 μg/ml | 1.88(0.90~3.69) | 0.77±0.103) | 0.77(0.72~0.88)3) | 0.82(0.75~0.84)2) | 0.79(0.65~0.95)3) |
| 统计值 | H=14.17 | F=5.298 | H=15.88 | H=10.45 | H=14.63 |
| P值 | 0.006 | ＜0.001 | 0.003 | 0.003 | 0.005 |
| 注：与正常组比较，1)P＜0.05；与模型组比较，2)P＜0.05，3)P＜0.01。 | |||||
与正常组比较，模型组cleaved-caspase3(P＜0.05)、cleaved-caspase8(P＜0.01)均显著升高，200 μg/ml虫草多糖组与模型组比较，cleaved-caspase3蛋白表达显著降低(P＜0.01)；与模型组比较，50、100、200 μg/ml虫草多糖组的cleaved-caspase8蛋白表达均明显降低(P值均＜0.01)(图 6)。
外源性死亡受体通路和内源性线粒体通路介导细胞凋亡的发生，外源性死亡受体途径主要由TNF超家族成员介导，涉及半胱氨酸蛋白酶的层联级激活，包括caspase8、caspase7、caspase10、caspase3。caspase3的激活导致肝细胞凋亡事件的发生，包括核DNA片段化，磷脂酰丝氨酸外露至细胞膜表面等。内源性线粒体途径中线粒体促凋亡蛋白Bax通过拮抗抗凋亡蛋白Bcl2导致线粒体膜通透性改变，从而介导细胞色素C的释放并激活caspase9和凋亡小体的形成，然后caspase9直接激活半胱氨酸蛋白酶3，后续步骤同外源性死亡受体途径[9]。越来越多的研究[10]表明细胞凋亡的失调广泛参与了各种疾病和病理过程，包括药物毒性损伤、免疫反应所导致的损伤、感染以及肿瘤的发生、代谢紊乱、各种肝脏疾病等。
虫草多糖是一种重要的机体稳态调节剂，基于肝细胞凋亡与肝纤维化的关系，本实验重点研究虫草多糖对TNFα诱导的肝细胞凋亡模型的具体影响以及作用机制。CCK8细胞增殖毒性实验和流式细胞检测结果显示：(1) 不同浓度的虫草多糖对L02正常肝细胞均无明显细胞增殖毒性，其中100、200 μg/ml虫草多糖对L02正常肝细胞具有显著的促增殖作用，且可以明显恢复TNFα抑制的L02正常肝细胞的细胞增殖毒性；(2)100、200 μg/ml虫草多糖能够显著减少TNFα诱导的细胞凋亡数量。此外，TNFα诱导L02正常肝细胞发生细胞凋亡的外源性途径中死亡受体Fas的mRNA以及激活形式cleaved-caspase8的蛋白表达均升高，Fas被激活后可激活caspase8和细胞凋亡执行关键酶caspase3从而导致细胞凋亡；同时，对于内源性线粒体途径，Bax作为重要的线粒体促凋亡蛋白，可导致线粒体功能障碍；关键酶caspase9功能与caspase8相近，同样可激活caspase3导致细胞凋亡。TNFα诱导L02正常肝细胞Bax、caspase9、caspase3的mRNA和激活形式cleaved-caspase3的蛋白表达均升高，而100、200 μg/ml虫草多糖可显著抑制外源性途径和内源性途径中的Fas、caspase8、caspase9的mRNA以及激活形式cleaved-caspase8的蛋白表达，200 μg/ml虫草多糖还可以显著抑制激活形式cleaved-caspase3的蛋白表达，虫草多糖对肝细胞凋亡的抑制作用可能随药物浓度升高而逐渐增强，因此，在安全药物浓度的前提下，100、200 μg/ml可能是虫草多糖抑制TNFα诱导肝细胞凋亡的最优浓度，未来仍需进一步实验证实。
综上所述，虫草多糖抑制肝细胞凋亡的具体机制可能涉及如下：(1)抑制内源性线粒体途径中的促凋亡相关蛋白功能，保护线粒体功能完整性；(2)抑制肝细胞凋亡相关的半胱氨酸蛋白酶激活；(3)抑制外源性死亡受体的表达。总之，该研究结果为虫草多糖及其复方制剂在抗肝纤维化中的临床应用提供了一定依据。
| [1] |
NAKATSUKA T, TATEISHI R, KOIKE K. Changing clinical management of NAFLD in Asia[J]. Liver Int, 2021. DOI: 10.1111/liv.15046.[Online ahead of print]
|
| [2] |
ZHOU F, ZHOU J, WANG W, et al. Unexpected rapid increase in the burden of NAFLD in China from 2008 to 2018: A systematic review and meta-analysis[J]. Hepatology, 2019, 70(4): 1119-1133. DOI: 10.1002/hep.30702.
|
| [3] |
BESSONE F, RAZORI MV, ROMA MG. Molecular pathways of nonalcoholic fatty liver disease development and progression[J]. Cell Mol Life Sci, 2019, 76(1): 99-128. DOI: 10.1007/s00018-018-2947-0.
|
| [4] |
EL-AGROUDY NN, KURZBACH A, RODIONOV RN, et al. Are lifestyle therapies effective for NAFLD treatment?[J]. Trends Endocrinol Metab, 2019, 30(10): 701-709. DOI: 10.1016/j.tem.2019.07.013.
|
| [5] |
ARMSTRONG MJ, HULL D, GUO K, et al. Glucagon-like peptide 1 decreases lipotoxicity in non-alcoholic steatohepatitis[J]. J Hepatol, 2016, 64(2): 399-408. DOI: 10.1016/j.jhep.2015.08.038.
|
| [6] |
ANDERSEN A, LUND A, KNOP FK, et al. Glucagon-like peptide 1 in health and disease[J]. Nat Rev Endocrinol, 2018, 14(7): 390-403. DOI: 10.1038/s41574-018-0016-2.
|
| [7] |
FUJIWARA Y, EGUCHI S, MURAYAMA H, et al. Relationship between diet/exercise and pharmacotherapy to enhance the GLP-1 levels in type 2 diabetes[J]. Endocrinol Diabetes Metab, 2019, 2(3): e00068. DOI: 10.1002/edm2.68.
|
| [8] |
SMITH NK, HACKETT TA, GALLI A, et al. GLP-1: Molecular mechanisms and outcomes of a complex signaling system[J]. Neurochem Int, 2019, 128: 94-105. DOI: 10.1016/j.neuint.2019.04.010.
|
| [9] |
DEACON CF. Physiology and pharmacology of DPP-4 in glucose homeostasis and the treatment of type 2 diabetes[J]. Front Endocrinol (Lausanne), 2019, 10: 80. DOI: 10.3389/fendo.2019.00080.
|
| [10] |
KAMAKURA R, RAZA GS, PRASANNAN A, et al. Dipeptidyl peptidase-4 and GLP-1 interplay in STC-1 and GLUTag cell lines[J]. Peptides, 2020, 134: 170419. DOI: 10.1016/j.peptides.2020.170419.
|
| [11] |
VARIN EM, MULVIHILL EE, BAGGIO LL, et al. Distinct neural sites of GLP-1R expression mediate physiological versus pharmacological control of incretin action[J]. Cell Rep, 2019, 27(11): 3371-3384.e3. DOI: 10.1016/j.celrep.2019.05.055.
|
| [12] |
MA H, HUANG W, WANG X, et al. Structural insights into the activation of GLP-1R by a small molecule agonist[J]. Cell Res, 2020, 30(12): 1140-1142. DOI: 10.1038/s41422-020-0384-8.
|
| [13] |
MVLLER TD, FINAN B, BLOOM SR, et al. Glucagon-like peptide 1 (GLP-1)[J]. Mol Metab, 2019, 30: 72-130. DOI: 10.1016/j.molmet.2019.09.010.
|
| [14] |
LI S, WANG X, ZHANG J, et al. Exenatide ameliorates hepatic steatosis and attenuates fat mass and FTO gene expression through PI3K signaling pathway in nonalcoholic fatty liver disease[J]. Braz J Med Biol Res, 2018, 51(8): e7299. DOI: 10.1590/1414-431x20187299.
|
| [15] |
LEE J, HONG SW, RHEE EJ, et al. GLP-1 receptor agonist and non-alcoholic fatty liver disease[J]. Diabetes Metab J, 2012, 36(4): 262-267. DOI: 10.4093/dmj.2012.36.4.262.
|
| [16] |
YU X, HAO M, LIU Y, et al. Liraglutide ameliorates non-alcoholic steatohepatitis by inhibiting NLRP3 inflammasome and pyroptosis activation via mitophagy[J]. Eur J Pharmacol, 2019, 864: 172715. DOI: 10.1016/j.ejphar.2019.172715.
|
| [17] |
GILBERT MP, PRATLEY RE. GLP-1 analogs and DPP-4 inhibitors in type 2 diabetes therapy: Review of head-to-head clinical trials[J]. Front Endocrinol (Lausanne), 2020, 11: 178. DOI: 10.3389/fendo.2020.00178.
|
| [18] |
TOBITA H, YAZAKI T, KATAOKA M, et al. Comparison of dapagliflozin and teneligliptin in nonalcoholic fatty liver disease patients without type 2 diabetes mellitus: A prospective randomized study[J]. J Clin Biochem Nutr, 2021, 68(2): 173-180. DOI: 10.3164/jcbn.20-129.
|
| [19] |
SHIRAKAWA J, FUJII H, OHNUMA K, et al. Diet-induced adipose tissue inflammation and liver steatosis are prevented by DPP-4 inhibition in diabetic mice[J]. Diabetes, 2011, 60(4): 1246-1257. DOI: 10.2337/db10-1338.
|
| [20] |
SHEN T, XU B, LEI T, et al. Sitagliptin reduces insulin resistance and improves rat liver steatosis via the SIRT1/AMPKα pathway[J]. Exp Ther Med, 2018, 16(4): 3121-3128. DOI: 10.3892/etm.2018.6554.
|
| [21] |
SANTOS FO, CORREIA B, MARINHO TS, et al. Anti-steatotic linagliptin pleiotropic effects encompasses suppression of de novo lipogenesis and ER stress in high-fat-fed mice[J]. Mol Cell Endocrinol, 2020, 509: 110804. DOI: 10.1016/j.mce.2020.110804.
|
| [22] |
OKUYAMA T, SHIRAKAWA J, TAJIMA K, et al. Linagliptin ameliorates hepatic steatosis via non-canonical mechanisms in mice treated with a dual inhibitor of insulin receptor and IGF-1 receptor[J]. Int J Mol Sci, 2020, 21(21): 7815. DOI: 10.3390/ijms21217815.
|
| [23] |
KAWAGUCHI T, NAKANO D, KOGA H, et al. Effects of a DPP4 inhibitor on progression of NASH-related HCC and the p62/ Keap1/Nrf2-pentose phosphate pathway in a mouse model[J]. Liver Cancer, 2019, 8(5): 359-372. DOI: 10.1159/000491763.
|
| [24] |
RANJBAR G, MIKHAILIDIS DP, SAHEBKAR A. Effects of newer antidiabetic drugs on nonalcoholic fatty liver and steatohepatitis: Think out of the box![J]. Metabolism, 2019, 101: 154001. DOI: 10.1016/j.metabol.2019.154001.
|
| [25] |
LEE M, SHIN E, BAE J, et al. Dipeptidyl peptidase-4 inhibitor protects against non-alcoholic steatohepatitis in mice by targeting TRAIL receptor-mediated lipoapoptosis via modulating hepatic dipeptidyl peptidase-4 expression[J]. Sci Rep, 2020, 10(1): 19429. DOI: 10.1038/s41598-020-75288-y.
|
| [26] |
ZHENG W, ZHOU J, SONG S, et al. Dipeptidyl-peptidase 4 inhibitor sitagliptin ameliorates hepatic insulin resistance by modulating inflammation and autophagy in ob/ob mice[J]. Int J Endocrinol, 2018, 2018: 8309723. DOI: 10.1155/2018/8309723.
|
| [27] |
WU C, CHEN W, DING H, et al. Salvianolic acid B exerts anti-liver fibrosis effects via inhibition of MAPK-mediated phospho-Smad2/3 at linker regions in vivo and in vitro[J]. Life Sci, 2019, 239: 116881. DOI: 10.1016/j.lfs.2019.116881.
|
| [28] |
KHALIL R, SHATA A, ABD EL-KADER EM, et al. Vildagliptin, a DPP-4 inhibitor, attenuates carbon tetrachloride-induced liver fibrosis by targeting ERK1/2, p38α, and NF-κB signaling[J]. Toxicol Appl Pharmacol, 2020, 407: 115246. DOI: 10.1016/j.taap.2020.115246.
|
| [29] |
PATEL CHAVEZ C, CUSI K, KADIYALA S. The emerging role of glucagon-like peptide-1 receptor agonists for the management of NAFLD[J]. J Clin Endocrinol Metab, 2022, 107(1): 29-38. DOI: 10.1210/clinem/dgab578.
|
| [30] |
JIANPING W, XUELIAN Z, ANJIANG W, et al. Efficacy and safety of glucagon-like peptide-1 receptor agonists in the treatment of metabolic associated fatty liver disease: A systematic review and meta-analysis[J]. J Clin Gastroenterol, 2021, 55(7): 586-593. DOI: 10.1097/MCG.0000000000001556.
|
| [31] |
LV X, DONG Y, HU L, et al. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) for the management of nonalcoholic fatty liver disease (NAFLD): A systematic review[J]. Endocrinol Diabetes Metab, 2020, 3(3): e00163. DOI: 10.1002/edm2.163.
|
| [32] |
WANG XC, GUSDON AM, LIU H, et al. Effects of glucagon-like peptide-1 receptor agonists on non-alcoholic fatty liver disease and inflammation[J]. World J Gastroenterol, 2014, 20(40): 14821-14830. DOI: 10.3748/wjg.v20.i40.14821.
|
| [33] |
VENDRELL J, EL BEKAY R, PERAL B, et al. Study of the potential association of adipose tissue GLP-1 receptor with obesity and insulin resistance[J]. Endocrinology, 2011, 152(11): 4072-4079. DOI: 10.1210/en.2011-1070.
|
| [34] |
FANG QH, SHEN QL, LI JJ, et al. Inhibition of microRNA-124a attenuates non-alcoholic fatty liver disease through upregulation of adipose triglyceride lipase and the effect of liraglutide intervention[J]. Hepatol Res, 2019, 49(7): 743-757. DOI: 10.1111/hepr.13330.
|
| [35] |
FANG Y, JI L, ZHU C, et al. Liraglutide alleviates hepatic steatosis by activating the TFEB-regulated autophagy-lysosomal pathway[J]. Front Cell Dev Biol, 2020, 8: 602574. DOI: 10.3389/fcell.2020.602574.
|
| [36] |
YANG M, MA X, XUAN X, et al. Liraglutide attenuates non-alcoholic fatty liver disease in mice by regulating the local renin-angiotensin system[J]. Front Pharmacol, 2020, 11: 432. DOI: 10.3389/fphar.2020.00432.
|
| [37] |
KUCHAY MS, KRISHAN S, MISHRA SK, et al. Effect of dulaglutide on liver fat in patients with type 2 diabetes and NAFLD: Randomised controlled trial (D-LIFT trial)[J]. Diabetologia, 2020, 63(11): 2434-2445. DOI: 10.1007/s00125-020-05265-7.
|
| [38] |
FENG WH, BI Y, LI P, et al. Effects of liraglutide, metformin and gliclazide on body composition in patients with both type 2 diabetes and non-alcoholic fatty liver disease: A randomized trial[J]. J Diabetes Investig, 2019, 10(2): 399-407. DOI: 10.1111/jdi.12888.
|
| [39] |
CHRISTOU GA, KATSIKI N, BLUNDELL J, et al. Semaglutide as a promising antiobesity drug[J]. Obes Rev, 2019, 20(6): 805-815. DOI: 10.1111/obr.12839.
|
| [40] |
NEWSOME P, FRANCQUE S, HARRISON S, et al. Effect of semaglutide on liver enzymes and markers of inflammation in subjects with type 2 diabetes and/or obesity[J]. Aliment Pharmacol Ther, 2019, 50(2): 193-203. DOI: 10.1111/apt.15316.
|
| [41] |
SAAD ZA, KHODEER DM, ZAITONE SA, et al. Exenatide ameliorates experimental non-alcoholic fatty liver in rats via suppression of toll-like receptor 4/NFκB signaling: Comparison to metformin[J]. Life Sci, 2020, 253: 117725. DOI: 10.1016/j.lfs.2020.117725.
|
| [42] |
LUO Y, YANG P, LI Z, et al. Liraglutide improves non-alcoholic fatty liver disease in diabetic mice by modulating inflammatory signaling pathways[J]. Drug Des Devel Ther, 2019, 13: 4065-4074. DOI: 10.2147/DDDT.S224688.
|
| [43] |
SHAO N, YU XY, MA XF, et al. Exenatide delays the progression of nonalcoholic fatty liver disease in C57BL/6 mice, which may involve inhibition of the NLRP3 inflammasome through the mitophagy pathway[J]. Gastroenterol Res Pract, 2018, 2018: 1864307. DOI: 10.1155/2018/1864307.
|
| [44] |
TESHOME G, AMBACHEW S, FASIL A, et al. Efficacy of glucagon-like peptide-1 analogs in nonalcoholic fatty liver disease: A systematic review[J]. Hepat Med, 2020, 12: 139-151. DOI: 10.2147/HMER.S265631.
|
| [45] |
WU L, LIU Y, SHI L, et al. Promotion of apoptosis in high glucose-activated hepatic stellate cells by GLP-1 receptor agonist and its potential mechanism[J]. Gene Mol Res, 2017, 16(4): gmr16039818. DOI: 10.4238/gmr16039818.
|
| [46] |
HAN X, DING C, ZHANG G, et al. Liraglutide ameliorates obesity-related nonalcoholic fatty liver disease by regulating Sestrin2-mediated Nrf2/HO-1 pathway[J]. Biochem Biophys Res Commun, 2020, 525(4): 895-901. DOI: 10.1016/j.bbrc.2020.03.032.
|
| [47] |
GHADIEH HE, MUTURI HT, NAJJAR SM. Exenatide prevents diet-induced hepatocellular injury in a CEACAM1-dependent mechanism[J]. J Diabetes Treat, 2017, 2017(4): 10.29011/2574.7568.000033. DOI: 10.29011/2574.7568.000033.
|
| [48] |
REED J, KANAMARLAPUDI V. GLP-1. [M]//CHOI S. Encyclopedia of Signaling Molecules. Cham; Springer International Publishing. 2018: 2098-2106.
|
| [49] |
DHIR G, CUSI K. Glucagon like peptide-1 receptor agonists for the management of obesity and non-alcoholic fatty liver disease: A novel therapeutic option[J]. J Investig Med, 2018, 66(1): 7-10. DOI: 10.1136/jim-2017-000554.
|
| [50] |
RICHARDS P, PARKER HE, ADRIAENSSENS AE, et al. Identification and characterization of GLP-1 receptor-expressing cells using a new transgenic mouse model[J]. Diabetes, 2014, 63(4): 1224-1233. DOI: 10.2337/db13-1440.
|
| [51] |
CANTINI G, MANNUCCI E, LUCONI M. Perspectives in GLP-1 research: New targets, new receptors[J]. Trends Endocrinol Metab, 2016, 27(6): 427-438. DOI: 10.1016/j.tem.2016.03.017.
|
| [52] |
SOFOGIANNI A, FILIPPIDIS A, CHRYSAVGIS L, et al. Glucagon-like peptide-1 receptor agonists in non-alcoholic fatty liver disease: An update[J]. World J Hepatol, 2020, 12(8): 493-505. DOI: 10.4254/wjh.v12.i8.493.
|
| 1. | 王雪莹，侯惠宁，安永升，卢佳，李日顺，张才. 甘草多糖对H_2O_2诱导肉鸡肝细胞氧化应激损伤的保护作用. 中国兽医学报. 2024(08): 1773-1781 .  | |
| 2. | 马传贵，张志秀，贺宗毅. 虫草属真菌多糖的分离提取、理化性质及生物活性研究进展. 食用菌. 2023(02): 1-3+7 . | |
| 3. | 李爽，韩淑贞，戴瑜婷，修明慧，杜籼芹，和建政，蔺兴遥. 基于多种化疗肠损伤发生机制的中医药防治进展. 中国临床药理学与治疗学. 2023(05): 583-593 . | |
| 4. | 韩思婕，潘翔，张梦美，魏琼，叶晓川. 白蔹乙酸乙酯部位体内抗肿瘤的药效作用机制及其成分分析研究. 中药新药与临床药理. 2022(12): 1623-1631 . | |
| 5. | 常久，张琪，郑宏，季巍巍，向宇雁. 药用真菌抗肿瘤作用机制研究进展. 中国中医药信息杂志. 2021(12): 140-144 . |
Figures(1)
The first journal specializing in hepatobiliary and pancreatic diseases in China
Supervisor：Ministry of Education of the People's Republic of China
Sponsor：Jilin University
Academic Support: Chinese Society of Hepatology，Chinese Medical Association
Address: 461 Xinjiang Road, Changchun
Submit：0431-88782044
Peer review：0431-88783542
Email：lcgdb@vip.163.com
Website Design © 2020 Editorial Board of Journal of Clinical Hepatology
吉ICP备10000617号-1
Supported by: Beijing Renhe Information Technology Co. Ltd
ZHAO JN, PENG JH, LIU P, et al. Effect of Cordyceps polysaccharides on hepatocyte apoptosis induced by tumor necrosis factor-α[J]. J Clin Hepatol, 2021, 37(6): 1368-1372. DOI: 10.3969/j.issn.1001-5256.2021.06.029.
| 引物名称 | 引物序列 | 产物长度(bp) |
| Bax | 5′TCG CCC TTT TCT ACT TTG CC 3′ | 97 |
| caspase3 | 5′CTG GTA TGT GTG GAT GAT GCT 3′ | 100 |
| caspase8 | 5′CTG GGA GAA GGA AAG TTG GAC 3′ | 68.5 |
| caspase9 | 5′TGC CTC AAT GCC AGT AAC 3′ | 129 |
| Fas | 5′ATT TTT GCC TTG GTG CTC A 3′ | 94 |
| GAPDH | 5′GGG AAG GTG AAG GTC GGA GT 3′ | 105 |
| ACTB | 5′AAG GTG ACA GCA GTC GGT T 3′ | 195 |
| 组别 | Bax | caspase 9 | Fas | caspase 8 | caspase 3 |
| 正常组 | 0.92(0.63~1.90) | 1.00±0.05 | 0.97(0.85~1.14) | 1.01(0.83~1.15) | 0.94(0.87~1.20) |
| 模型组 | 2.38(1.46~4.14)1) | 1.11±0.32 | 1.66(1.20~3.87)1) | 1.33(0.92~4.44) | 1.41(1.03~2.80) |
| 50 μg/ml | 0.81(0.78~1.11)3) | 0.83±0.142) | 1.06(0.86~3.18) | 0.90(0.83~1.40) | 0.90(0.66~1.16)2) |
| 100 μg/ml | 1.09(0.65~1.64)2) | 0.75±0.073) | 0.83(0.77~1.19)2) | 0.85(0.76~1.02) | 0.83(0.68~0.95)3) |
| 200 μg/ml | 1.88(0.90~3.69) | 0.77±0.103) | 0.77(0.72~0.88)3) | 0.82(0.75~0.84)2) | 0.79(0.65~0.95)3) |
| 统计值 | H=14.17 | F=5.298 | H=15.88 | H=10.45 | H=14.63 |
| P值 | 0.006 | ＜0.001 | 0.003 | 0.003 | 0.005 |
| 注：与正常组比较，1)P＜0.05；与模型组比较，2)P＜0.05，3)P＜0.01。 | |||||
| 引物名称 | 引物序列 | 产物长度(bp) |
| Bax | 5′TCG CCC TTT TCT ACT TTG CC 3′ | 97 |
| caspase3 | 5′CTG GTA TGT GTG GAT GAT GCT 3′ | 100 |
| caspase8 | 5′CTG GGA GAA GGA AAG TTG GAC 3′ | 68.5 |
| caspase9 | 5′TGC CTC AAT GCC AGT AAC 3′ | 129 |
| Fas | 5′ATT TTT GCC TTG GTG CTC A 3′ | 94 |
| GAPDH | 5′GGG AAG GTG AAG GTC GGA GT 3′ | 105 |
| ACTB | 5′AAG GTG ACA GCA GTC GGT T 3′ | 195 |
| 组别 | Bax | caspase 9 | Fas | caspase 8 | caspase 3 |
| 正常组 | 0.92(0.63~1.90) | 1.00±0.05 | 0.97(0.85~1.14) | 1.01(0.83~1.15) | 0.94(0.87~1.20) |
| 模型组 | 2.38(1.46~4.14)1) | 1.11±0.32 | 1.66(1.20~3.87)1) | 1.33(0.92~4.44) | 1.41(1.03~2.80) |
| 50 μg/ml | 0.81(0.78~1.11)3) | 0.83±0.142) | 1.06(0.86~3.18) | 0.90(0.83~1.40) | 0.90(0.66~1.16)2) |
| 100 μg/ml | 1.09(0.65~1.64)2) | 0.75±0.073) | 0.83(0.77~1.19)2) | 0.85(0.76~1.02) | 0.83(0.68~0.95)3) |
| 200 μg/ml | 1.88(0.90~3.69) | 0.77±0.103) | 0.77(0.72~0.88)3) | 0.82(0.75~0.84)2) | 0.79(0.65~0.95)3) |
| 统计值 | H=14.17 | F=5.298 | H=15.88 | H=10.45 | H=14.63 |
| P值 | 0.006 | ＜0.001 | 0.003 | 0.003 | 0.005 |
| 注：与正常组比较，1)P＜0.05；与模型组比较，2)P＜0.05，3)P＜0.01。 | |||||
下载:
DownLoad: