芍药苷调控JAK/STAT3通路干预HepG2细胞PD-L1表达的研究
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摘要
为探究芍药苷对程序性死亡受体-配体1(programmed cell death-Ligand 1,PD-L1)表达的影响及作用机制,研究采用干扰素γ(IFN-γ)诱导HepG2细胞建立体外PD-L1高表达细胞模型。采用MTT法检测芍药苷的细胞毒性,通过流式细胞术、ELISA、RT-PCR法检测其对PD-L1蛋白和mRNA表达的影响;建立HepG2细胞和Jurkat T细胞共培养体系,ELISA检测芍药苷干预后共培养24 h后的IL-2的表达,CCK-8法检测药物干预后T细胞增殖情况,Western blot检测芍药苷作用后HepG2细胞中PD-L1、Janus激酶(Janus kinase,JAK)和信号传导及转录激活因子(signal transducers and activators of transcription,STAT3)的蛋白表达。实验结果表明,芍药苷能够显著下调PD-L1蛋白和mRNA的表达水平,增加共培养体系中的IL-2的浓度,促进T细胞显著增殖,此外,芍药苷能显著抑制JAK和STAT3的蛋白磷酸化。实验结果表明,芍药苷能够下调PD-L1的表达,其机制可能与JAK/STAT3通路有关。
In order to explore the effect and its mechanism of paeoniflorin on PD-L1,a PD-L1 high expression cell model was established in interferon gamma(IFN-γ)-induced HepG2 cells.The cytotoxicity of paeoniflorin was detected by MTT assay.Flow cytometry,ELISA and RT-PCR were performed to detect protein and mRNA levels of PD-L1 regulated by paeoniflorin.In HepG2 cells and Jurkat T cell co-culture system,the expression of IL-2 was detected by ELISA.Besides,T cell proliferation was evaluated by CCK-8 method,and the protein expression levels of PD-L1,JAK and STAT3 after drug treatment were determined by Western blot.These results indicated that paeoniflorin could significantly down-regulate the levels of PD-L1 protein and mRNA.In addition,it increased the number of T cells and the concentration of IL-2 in the co-culture system.Furthermore,paeoniflorin could significantly inhibit the protein expression of JAK and STAT3.Au the above experimental data indicated that paeoniflorin could down-regulate the expression of PD-L1,and its mechanism might be related to the JAK/STAT3 pathway.
In order to explore the effect and its mechanism of paeoniflorin on PD-L1,a PD-L1 high expression cell model was established in interferon gamma(IFN-γ)-induced HepG2 cells.The cytotoxicity of paeoniflorin was detected by MTT assay.Flow cytometry,ELISA and RT-PCR were performed to detect protein and mRNA levels of PD-L1 regulated by paeoniflorin.In HepG2 cells and Jurkat T cell co-culture system,the expression of IL-2 was detected by ELISA.Besides,T cell proliferation was evaluated by CCK-8 method,and the protein expression levels of PD-L1,JAK and STAT3 after drug treatment were determined by Western blot.These results indicated that paeoniflorin could significantly down-regulate the levels of PD-L1 protein and mRNA.In addition,it increased the number of T cells and the concentration of IL-2 in the co-culture system.Furthermore,paeoniflorin could significantly inhibit the protein expression of JAK and STAT3.Au the above experimental data indicated that paeoniflorin could down-regulate the expression of PD-L1,and its mechanism might be related to the JAK/STAT3 pathway.
引文
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[30] Chen J,Jiang CC,Jin L,et al.Regulation of PD-L1:a novel role of pro-survival signalling in cancer[J].Ann Oncol,2015,27(3):409-416.
[2] Gao Q,Wang XY,Qiu SJ,et al.Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma[J].Clin Cancer Res,2009,15(3):971-979.
[3] Zhou J,Liu M,Sun H,et al.Hepatoma-intrinsic CCRK inhibition diminishes myeloid-derived suppressor cell immunosuppression and enhances immune-checkpoint blockade efficacy[J].Gut,2017,67(5):gutjnl-2017-314032.
[4] Dai X,Pi G,Yang SL,et al.Association of PD-L1 and HIF-1α coexpression with poor prognosis in hepatocellular carcinoma[J].Transl Oncol,2018,11(2):559.
[5] Tian JP,Zhang J,Zhou JP,et al.Advances in small molecule inhibitors of PD-1/PD-L1 immune checkpoint pathway[J].J China Pharm Univ(中国药科大学学报),2019,50(1):1-10.
[6] Sharma P,Allison JP.The future of immune checkpoint therapy[J].Science,2015,348(6230):56.
[7] Zou W,Wolchok JD,Chen L.PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy:mechanisms,response biomarkers,and combinations[J].Sci Transl Med,2016,8(328):328rv4.
[8] Greten TF,Xin WW,Korangy F.Current concepts of immune based treatments for patients with HCC:from basic science to novel treatment approaches[J].Gut,2015,64(5):842-848.
[9] Naidoo J,Page DB,Li BT,et al.Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies[J].Ann Oncol,2015,26(12):2375-2391.
[10] Zhu H,Bengsch F,Svoronos N,et al.BET bromodomain inhibition promotes anti-tumor immunity by suppressing PD-L1 expression[J].Cell Rep,2016,16(11):2829-2837.
[11] Jiang Z,Yang Y,Yang Y,et al.Ginsenoside Rg3 attenuates cisplatin resistance in lung cancer by downregulating PD-L1 and resuming immune[J].Biomed Pharmacother,2017,96:378-383.
[12] Kim ID,Ha BJ.Paeoniflorin protects RAW 264.7 macrophages from LPS-induced cytotoxicity and genotoxicity[J].Toxicol In Vitro,2009,23(6):1014-1019.
[13] Liu DF,Wei W,Song LH.Protective effect of paeoniflorin on immunological liver injury induced by bacillus Calmette-Guerin plus lipopolysaccharide:modulation of tumour necrosis factor-alpha and interleukin-6 MRNA[J].Clin Exp Pharmacol Physiol,2010,33(4):332-339.
[14] Lu JT,He W,Song SS,et al.Paeoniflorin inhibited the tumor invasion and metastasis in human hepatocellular carcinoma cells[J].Bratisl Lek Listy,2014,115(7):427-433.
[15] Hung JY,Yang CJ,Tsai YM,et al.Antiproliferative activity of paeoniflorin is through cell cycle arrest and the Fas/Fas ligand-mediated apoptotic pathway in human non-small cell lung cancer A549 cells[J].Clin Exp Pharmacol Physiol,2010,35(2):141-147.
[16] Xu RX,Nie XH,Jia OY,et al.Paeoniflorin inhibits human glioma cells via STAT3 degradation by the ubiquitin-proteasome pathway[J].Drug Des Devel Ther,2015,9(default):5611-5622.
[17] Zheng YB,Xiao GC,Tong SL,et al.Paeoniflorin inhibits human gastric carcinoma cell proliferation through up-regulation of microRNA-124 and suppression of PI3K/Akt and STAT3 signaling[J].World J Gastroenterol,2015,21(23):7197-7207.
[18] Zhao MG,Yang HM,Jiang CH,et al.Intervention effects of the triterpenoids from Cyclocarya paliurus on free fatty acids-induced steatosis in HepG2 cells[J].J China Pharm Univ(中国药科大学学报),2018,49(3):333-340.
[19] Wang YT,Zhao MG,Sheng XP,et al.Effect of triterpenic acid-enriched fraction from Cyclocarya paliurus on high glucose-induced pancreatic α cells insulin resistance[J].J China Pharm Univ(中国药科大学学报),2018,49(2):215-221.
[20] Ribas A.Adaptive immune resistance:how cancer protects from immune attack[J].Cancer Discov,2015,5(9):915-919.
[21] Elkhoueiry AB,Sangro B,Yau T,et al.Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040):an open-label,non-comparative,phase 1/2 dose escalation and expansion trial[J].Lancet,2017,389(10088):2492.
[22] Coombs MR,Harrison ME,Hoskin DW.Apigenin inhibits the inducible expression of programmed death ligand 1 by human and mouse mammary carcinoma cells[J].Cancer Lett,2016,380(2):424-433.
[23] Hato T,Goyal L,Greten TF,et al.Immune checkpoint blockade in hepatocellular carcinoma:current progress and future directions[J].Hepatology,2014,60(5):1776-1782.
[24] Abiko K,Matsumura N,Hamanishi J,et al.IFN-γ from lymphocytes induces PD-L1 expression and promotes progression of ovarian cancer[J].Br J Cancer,2015,112(9):1501-1509.
[25] Muhlbauer M,Fleck M,Schutz C,et al.PD-L1 is induced in hepatocytes by viral infection and by interferon-a and-g and mediates T cell apoptosis[J].J Hepatol,2006,45:520-528.
[26] Yang W,Chen PW,Li H,et al.PD-L1:PD-1 interaction contributes to the functional suppression of T-cell responses to human uveal melanoma cells in vitro[J].Invest Ophthalmol Vis Sci,2008,49(6):2518.
[27] Niwa Y,Kanda H,Shikauchi Y,et al.Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma[J].Oncogene,2005,24:116-119.
[28] Timofeeva OA,Tarasova NI,Zhang X,et al.STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain[J].Proc Natl Acad Sci U S A,2013,110(4):1267-1272.
[29] Choudhari S,Khan M,Harris G,et al.Deactivation of Akt and STAT-3 signaling promotes apoptosis,inhibits proliferation and enhances sensitivity of HCC cells to an anti-cancer agent,Atiprimod[J].Mol Cancer Ther,2007,67:2377-2377.
[30] Chen J,Jiang CC,Jin L,et al.Regulation of PD-L1:a novel role of pro-survival signalling in cancer[J].Ann Oncol,2015,27(3):409-416.