NF-κB抑制剂PDTC诱导人肝星状细胞LX-2凋亡的药理机制

doi:10.5246/jcps.2022.09.056

中国药学(英文版) ›› 2022, Vol. 31 ›› Issue (9): 665-676.DOI: 10.5246/jcps.2022.09.056

NF-κB抑制剂PDTC诱导人肝星状细胞LX-2凋亡的药理机制

黄津¹, 邓凯翔³, 黄美珍¹, 林高敏¹, 林媚¹, 练水梅³, 张美泉²^,^*()

1. 福建中医药大学附属第二人民医院,　福建福州 350003
2. 福建省老年医院,　福建福州 350009
3. 南平市第一医院中医康复科, 福建南平 353000

收稿日期:2022-03-18 修回日期:2022-05-12 接受日期:2022-06-19 出版日期:2022-09-30 发布日期:2022-09-30
通讯作者: 张美泉
作者简介:
+ Tel.: +86-13859369873, E-mail: yisheng0620@163.com
基金资助:
The Fujian Provincial Natural Science Foundation, China (Grant No. 2019J01611), and Youth Project of Fujian Provincial Health Commission, Fujian Province, China (Grant No. 2017-1-94).

The pharmacological mechanism underlying the apoptosis of human hepatic stellate cells LX-2 induced by NF-κB inhibitor PDTC

Jin Huang¹, Kaixiang Deng³, Meizhen Huang¹, Gaomin Lin¹, Mei Lin¹, Shuimei Lian³, Meiquan Zhang²^,^*()

1 Department of Infection, Second affiliated Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, China
2 Department of Respiratory and Critical Care Medicine, Fujian Geriatric Hospital, Fuzhou 350009, China
3 Department of Traditional Chinese Medicine, First Hospital of Nanping City, Nanping 353000, China

Received:2022-03-18 Revised:2022-05-12 Accepted:2022-06-19 Online:2022-09-30 Published:2022-09-30
Contact: Meiquan Zhang

摘要/Abstract

摘要：

本研究旨在探讨NF-κB抑制剂吡咯烷二硫代氨基甲酸酯(PDTC)是否可以诱导人肝星状细胞LX-2凋亡, 并分析其潜在药理机制。通过体外培养人肝星状细胞系LX-2细胞, 将实验分为空白对照组和PDTC干预组。将LX-2细胞暴露于PDTC后, 使用CCK8检测法评估LX-2细胞活力; 采用AO/EB双染试剂盒检测PDTC的抗凋亡作用。此外, 分别采用Western blotting法和免疫荧光染色法测定NF-κB、Fas/FasL、凋亡相关蛋白的活性和AIF的细胞定位。PDTC处理12和24小时后, AO/EB双染呈现典型的凋亡变化, 比如细胞体积缩小、细胞核固缩、核碎裂等。PDTC干预浓度在60 μmol/L时可显著提高细胞增殖抑制率, 减少LX-2细胞中的胶原蛋白I、胶原蛋白III和α-SMA的分泌。Western blotting和RT-PCR结果显示, 对照组与PDTC干预组的AIF表达水平无统计学意义, 且各组均未观察到Fas和FasL的表达(P > 0.05)。PDTC还可以促进AIF从线粒体向细胞核的移位, 激活细胞核内的凋亡信号, 并可能参与LX-2细胞的凋亡过程。综上所述, PDTC抗肝纤维化的药理机制可能是促进AIF从线粒体向细胞核移位, 进而激活细胞核内的凋亡信号, 最终诱导LX-2细胞凋亡。同时, 也揭示了Fas/FasL介导的细胞凋亡通路不参与PDTC诱导的LX-2细胞凋亡过程。

关键词: 肝纤维化, 人肝星状细胞, Fas/FasL凋亡通路, 凋亡诱导因子, 吡咯烷二硫代氨基甲酸酯

Abstract:

In the present study, we aimed to confirm whether NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could induce apoptosis of human hepatic stellate cells (HSCs) LX-2 and explore the potential pharmacological mechanism underlying these effects. In this study, LX-2 cells were cultured in vitro, and the experiment was divided into two groups, including the control and PDTC groups. The viability of LX-2 cells was measured by CCK8 assay after the cells were exposed to PDTC. The anti-apoptotic effect of PDTC was detected by AO/EB double assay staining kit. Additionally, the activities of NF-κB, Fas/FasL, apoptosis-related proteins, as well as the cellular localization of AIF, were determined by Western blotting analysis and immunofluorescence staining respectively. After PDTC treatment for 12 and 24 h, AO/EB dual staining showed typical apoptotic changes, such as cell volume reduction, cell shrinkage, nuclear fragmentation, and so on. PDTC at 60 μmol/L significantly increased the proliferation inhibition rate and decreased the secretion of collagen I, collagen III, and α-SMA in LX-2 cells. The Western blotting analysis and RT-PCR showed no significant difference in the expression of AIF between the control group and PDTC group, and the expressions of Fas and FasL were not observed in all groups (P > 0.05). Further results showed that PDTC could promote the displacement of AIF from mitochondria to the nucleus, activate the apoptotic signaling in the cell nucleus, and possibly participate in the apoptosis process of LX-2 cells. In conclusion, the pharmacological mechanism of PDTC against hepatic fibrosis might be to promote the displacement of AIF from mitochondria to the nucleus, then activate the apoptotic signaling in the cell nucleus, and finally induce the apoptosis of LX-2 cells. Meanwhile, these results also revealed that the Fas/FasL-mediated apoptosis pathway was not involved in the PDTC-induced apoptosis process of LX-2 cells.

Key words: Hepatic fibrosis, LX-2, Fas/FasL, AIF, Pyrrolidine dithiocarbamate

Supporting:

黄津, 邓凯翔, 黄美珍, 林高敏, 林媚, 练水梅, 张美泉. NF-κB抑制剂PDTC诱导人肝星状细胞LX-2凋亡的药理机制[J]. 中国药学(英文版), 2022, 31(9): 665-676.

Jin Huang, Kaixiang Deng, Meizhen Huang, Gaomin Lin, Mei Lin, Shuimei Lian, Meiquan Zhang. The pharmacological mechanism underlying the apoptosis of human hepatic stellate cells LX-2 induced by NF-κB inhibitor PDTC[J]. Journal of Chinese Pharmaceutical Sciences, 2022, 31(9): 665-676.

图/表 7

Table 1. Primer sequences used for RT-PCR.

Figure 1. The expressions of collagen I, collagen III, and α-SMA in LX-2 cells. Each data point is a mean ± SD (n = 5). Collagen I (A), collagen III (B), and α-SMA (C) levels in LX-2 cells. All the data were analyzed using an ANOVA.

Figure 2. Proliferation inhibition rate of LX-2 cells exposed to PDTC. The proliferation inhibition rate was tested using CCK-8 assay by absorbance at 490 nm. (A) OD value of cells exposed to different concentrations of PDTC. (B) Proliferation inhibition rate of LX-2 cells exposed to different concentrations of PDTC. Each value indicates the mean ± SD and is representative of results obtained from five wells in all experiments. *P < 0.05, **P < 0.01 as compared with control group (0 μmol/L PDTC).

Figure 3. AO/EB staining for the apoptosis rate of LX-2 cells. The total number of stained cells was 100, and the percentage of apoptotic cells was further calculated. (A) LX-2 cells were cultured for 1 h (1 h control group). (B) LX-2 cells were cultured for 6 h (6 h control group). (C) LX-2 cells were cultured for 12 h (12 h control group). (D) LX-2 cells were cultured for 24 h (24 h control group). (E) LX-2 cells were treated with PDTC for 1 h (1 h PDTC group). (F) LX-2 cells were treated with PDTC for 6 h (6 h PDTC group). (G) LX-2 cells were treated with PDTC for 12 h (12 h PDTC group). (H) LX-2 cells were treated with PDTC for 24 h (24 h PDTC group). (I) The apoptosis rate of LX-2 cells was compared between the control group and PDTC group. Each value indicates as mean ± SD and is representative of results obtained from five wells (n = 5). ΔΔP < 0.01 as compared with 12 h PDTC group; **P < 0.01 as compared with 24 h PDTC group. The data between groups were analyzed using an ANOVA.

Figure 4. The expressions of AIF, Fas, and FasL at the mRNA level in LX-2 cells. Each data point is a mean ± SD (n = 5). AIF (A), FasL (C), and Fas (D) levels in LX-2 cells. β-Actin was used as the loading control. (1–4): 1, 6, 12, and 24 h for the control group, respectively; (5–8): PDTC treatment for 1, 6, 12, and 24 h, respectively. (M): Relative molecular weight standard. (E) The expression of AIF at the mRNA level was compared between the control and PDTC groups. ΔP > 0.05 as compared with 24 h PDTC group; *P > 0.05 as compared with 24 h control group. The data between groups were analyzed using an ANOVA.

Figure 5. The expressions of NF-κB, AIF, Fas, and FasL at the protein level in LX-2 cells. Each data point is a mean ± SD (n = 5). AIF (B) and NF-κB (C) are protein levels in LX-2 cells. β-Actin was used as the loading control. (1–4): 1, 6, 12, and 24 h for the control group, respectively; (5–8): PDTC treatment for 1, 6, 12, and 24 h, respectively. All the data were analyzed using an ANOVA.

Figure 6. The nuclear localization of AIF in LX-2 cells × 40. (A) Control group for 48 h; (B) PDTC treatment group for 6 h; (C) PDTC treatment group for 12 h; (D) PDTC treatment group for 24 h. Red fluorescence: Cy3-labeled immunofluorescence; Blue fluorescence: DAPI immunofluorescence.

参考文献 25

[1]	Lan, Y.T.; Wang, Z.L.; Tian, P.; Gong, X.N.; Fan, Y.C.; Wang, K. Treg/Th17 imbalance and its clinical significance in patients with hepatitis B-associated liver cirrhosis. Diagn. Pathol. 2019, 14, 114.
[2]	Anbar, A.; Piran, T.; Farhadi, M.; Karimi, P. Iranian crack induces hepatic injury through mitogen-activated protein kinase pathway in the liver of Wistar rat. Iran. J. Basic Med. Sci. 2018, 21, 1179–1185.
[3]	Du, G.; Wang, J.; Zhang, T.; Ding, Q.; Jia, X.; Zhao, X.; Dong, J.; Yang, X.; Lu, S.; Zhang, C.; Liu, Z.; Zeng, Z.; Safadi, R.; Qi, R.; Zhao, X.; Hong, Z.; Lu, Y. Targeting Src family kinase member Fyn by Saracatinib attenuated liver fibrosis in vitro and in vivo. Cell Death Dis. 2020, 11, 118.
[4]	He, Z.; Yang, D.Y.; Fan, X.L.; Zhang, M.W.; Li, Y.; Gu, X.B.; Yang, M.Y. The roles and mechanisms of lncRNAs in liver fibrosis. Int. J. Mol. Sci. 2020, 21, 1482.
[5]	Park, Y.J.; Lee, K.H.; Jeon, M.S.; Lee, Y.H.; Ko, Y.J.; Pang, C.; Kim, B.; Chung, K.H.; Kim, K.H. Hepatoprotective potency of chrysophanol 8-O-glucoside from rheum palmatum L. against hepatic fibrosis via regulation of the STAT3 signaling pathway. Int. J. Mol. Sci. 2020, 21, 9044.
[6]	Tan, Z.; Sun, H.B.; Xue, T.X.; Gan, C.L.; Liu, H.Y.; Xie, Y.T.; Yao, Y.Q.; Ye, T.H. Liver fibrosis: therapeutic targets and advances in drug therapy. Front. Cell Dev. Biol. 2021, 9, 730176.
[7]	Li, R.; Guo, Y.J.; Zhang, Y.M.; Zhang, X.; Zhu, L.P.; Yan, T.H. Salidroside ameliorates renal interstitial fibrosis by inhibiting the TLR4/NF-κB and MAPK signaling pathways. Int. J. Mol. Sci. 2019, 20, 1103.
[8]	Kapusta, P.; Dulińska-Litewka, J.; Totoń-Żurańska, J.; Borys, A.; Konieczny, P.S.; Wołkow, P.P.; Seweryn, M.T. Dysregulation of transcription factor activity during formation of cancer-associated fibroblasts. Int. J. Mol. Sci. 2020, 21, 8749.
[9]	Zhou, L.; Jiang, H.; Du, J.; Li, L.; Li, R.; Lu, J.; Fu, W.; Hou, J. USP15 inhibits multiple myeloma cell apoptosis through activating a feedback loop with the transcription factor NF-κBp65. Exp. Mol. Med. 2018, 50, 1–12.
[10]	Zhao, J.Q.; He, B.; Zhang, S.J.; Huang, W.X.; Li, X.S. Ginsenoside Rg1 alleviates acute liver injury through the induction of autophagy and suppressing NF-κB/NLRP3 inflammasome signaling pathway. Int. J. Med. Sci. 2021, 18, 1382–1389.
[11]	Kou, X.X.; Xu, X.T.; Chen, C.; Sanmillan, M.L.; Cai, T.; Zhou, Y.H.; Giraudo, C.; Le, A.; Shi, S.T. The Fas/Fap-1/Cav-1 complex regulates IL-1RA secretion in mesenchymal stem cells to accelerate wound healing. Sci. Transl. Med. 2018, 14, eaai8524.
[12]	Zhao, H.; Li, C.D.; Li, L.N.; Liu, J.Y.; Gao, Y.H.; Mu, K.; Chen, D.H.; Lu, A.P.; Ren, Y.Y.; Li, Z.H. Baicalin alleviates bleomycin‑induced pulmonary fibrosis and fibroblast proliferation in rats via the PI3K/AKT signaling pathway. Mol. Med. Rep. 2020, 21, 2321–2334.
[13]	Wang, C.H.; Luo, H.; Xu, Y.N.; Tao, L.; Chang, C.R.; Shen, X.C. Salvianolic acid B-alleviated angiotensin II induces cardiac fibrosis by suppressing NF-κB pathway in vitro. Med. Sci. Monit. 2018, 24, 7654–7664.
[14]	Wei, J.Y.; Deng, X.N.; Li, Y.; Li, R.M.; Yang, Z.H.; Li, X.Y.; Song, S.; Shi, Y.H.; Duan, H.J.; Wu, H.J. PP₂ ameliorates renal fibrosis by regulating the NF-κB/COX-2 and PPARγ/UCP₂ pathway in diabetic mice. Oxidative Med. Cell Longev. 2021, 2021, 7394344.
[15]	Yang, X.X; Ma, L.P.; Wei, R.; Ye, T.H.; Zhou, J.K.; Wen, M.Y.; Men, R.T.; Aqeilan, R.I.; Peng, Y.; Yang, L. Twist1-induced miR-199a-3p promotes liver fibrosis by suppressing caveolin-2 and activating TGF-β pathway. Signal Transduct. Target. Ther. 2020, 5, 75.
[16]	Yan, J.; Tung, H.C.; Li, S.H.; Niu, Y.D.; Garbacz, W.G.; Lu, P.P.; Bi, Y.H.; Li, Y.P.; He, J.H.; Xu, M.S.; Ren, S.R.; Monga, S.P.; Schwabe, R.F.; da Yang, Xie, W. Aryl hydrocarbon receptor signaling prevents activation of hepatic stellate cells and liver fibrogenesis in mice. Gastroenterology. 2019, 157, 793–806.e14.
[17]	Jafari, O.; Babaei, H.; Kheirandish, R.; Samimi, A.; Zahmatkesh, A. Histomorphometric evaluation of mice testicular tissue following short- and long-term effects of lipopolysaccharide-induced endotoxemia. Iran. J. Basic Med. Sci. 2018, 21, 47–52.
[18]	Hanley, N.A. Monoclonal Antibodies for the Treatment of Multiple Myeloma: An Update. Int. J. Mol. Sci. 2018, 19, 3924.
[19]	Tan, S.; Liu, X.; Chen, L.; Wu, X.; Tao, L.; Pan, X.; Tan, S.; Liu, H.; Jiang, J.; Wu, B. Fas/FasL mediates NF-κBp65/PUMA-modulated hepatocytes apoptosis via autophagy to drive liver fibrosis. Cell Death Dis. 2021, 12, 474.
[20]	Shi, J.; Zhao, J.; Zhang, X.; Cheng, Y.; Hu, J.; Li, Y.; Zhao, X.; Shang, Q.; Sun, Y.; Tu, B.; Shi, L.; Gao, B.; Wang, F.S.; Zhang, Z. Activated hepatic stellate cells impair NK cell anti-fibrosis capacity through a TGF-β-dependent emperipolesis in HBV cirrhotic patients. Sci. Reports. 2017, 7, 44544.
[21]	Novo, N.; Ferreira, P.; Medina, M. The apoptosis-inducing factor family: Moonlighting proteins in the crosstalk between mitochondria and nuclei. IUBMB Life. 2021, 73, 568–581.
[22]	Daniel, J.K.; Amal, K.A.; Sara, A.; Mahmoud, A.; Asghar, A.; Steffen, A.; Hagai, A. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy. 2021, 17, 1–382.
[23]	Ye, J.; Wang, Y.; Wang, Z.; Liu, L.; Yang, Z.C.; Ye, D.; Wang, M.L.; Xu, Y.; Zhang, J.S.; Zhao, M.M.; Liu, J.F.; Lin, Y.Z.; Ji, Q.W.; Wan, J. Interleukin-12p35 deficiency enhances mitochondrial dysfunction and aggravates cardiac remodeling in aging mice. Aging. 2020, 12, 193–203.
[24]	Ghanbarinejad, V.; Jamshidzadeh, A.; Khalvati, B.; Farshad, O.; Li, H.F.; Shi, X.; Chen, Y.Y.; Ommati, M.M.; Heidari, R. Apoptosis-inducing factor plays a role in the pathogenesis of hepatic and renal injury during cholestasis. Naunyn Schmiedeberg’s Arch. Pharmacol. 2021, 394, 1191–1203.
[25]	Luo, Q.Y.; Wu, X.W.; Zhao, P.F.; Nan, Y.B.; Chang, W.; Zhu, X.L.; Su, D.; Liu, Z.H. OTUD1 activates caspase-independent and caspase-dependent apoptosis by promoting AIF nuclear translocation and MCL1 degradation. Adv. Sci. 2021, 8, 2002874.

NF-κB抑制剂PDTC诱导人肝星状细胞LX-2凋亡的药理机制

The pharmacological mechanism underlying the apoptosis of human hepatic stellate cells LX-2 induced by NF-κB inhibitor PDTC

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