宽体金线蛭提取物对HEK293细胞维甲酸诱导基因蛋白样受体(RLRs)通路的影响

doi:10.3969/j.issn.1004-1524.20221666

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (12): 2830-2843.DOI: 10.3969/j.issn.1004-1524.20221666

宽体金线蛭提取物对HEK293细胞维甲酸诱导基因蛋白样受体(RLRs)通路的影响

陈姿亦(), 何盛盛, 闫晶男, 吴怡蓉, 张雨婷, 高有领()

浙江万里学院生物与环境学院,浙江宁波 315100

收稿日期:2022-11-22 出版日期:2023-12-25 发布日期:2023-12-27
作者简介:陈姿亦(1995—),女,浙江舟山人,硕士研究生,研究方向为生物活性物质的利用。E-mail: ziyichenn@hotmail.com
通讯作者: *高有领,E-mail: gaoyl@zwu.edu.cn
基金资助:
浙江省科技厅研究项目(LGN22C190028);宁波市科技局项目(202002N3048)

Extract of Whitmania pigra on the retinoic acid-inducible gene I-like receptors (RLRs) signaling pathway of HEK293 cell

CHEN Ziyi(), HE Shengsheng, YAN Jingnan, WU Yirong, ZHANG Yuting, GAO Youling()

College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, Zhejiang, China

Received:2022-11-22 Online:2023-12-25 Published:2023-12-27

摘要/Abstract

摘要：

维甲酸诱导基因蛋白样受体(RLRs)信号通路是机体重要的抗病毒作用途径。宽体金线蛭体内含有抗凝血的活性物质,主要用于治疗血栓等疾病,但对于该信号通路的影响尚无任何报道。本试验目的是采用聚肌苷酸-聚胞苷酸(Poly I∶C)建立HEK293细胞RLRs信号通路激活模型,在此基础上揭示宽体金线蛭提取物(LE)对HEK293细胞RLRs信号通路的影响。试验首先转染3个不同质量浓度(1、2、4 μg·mL^-1)的Poly I∶C至HEK293细胞,并分别处理12 h、24 h和48 h,以维甲酸诱导基因I(RIG-I)的蛋白表达水平和mRNA转录水平为RLRs通路激活的指标。RLRs信号通路激活之后,采用LE对HEK293细胞进行处理,共设置4组,每组3个重复,分别为:对照组、2 μg·mL^-1 Poly I∶C转染组、2 μg·mL^-1 Poly I∶C转染且添加150 μg·mL^-1的水蛭提取物、2 μg·mL^-1 Poly I∶C转染且添加300 μg·mL^-1的水蛭提取物。处理时长分别为24 h和48 h。试验结果表明,转染3个剂量的Poly I∶C在3个处理时长均引起细胞活力降低,2 μg·mL^-1和4 μg·mL^-1 Poly I∶C转染HEK293细胞12 h、24 h和48 h均显著提高了RIG-I蛋白的表达量,4 μg·mL^-1的Poly I∶C转染组RIG-I mRNA转录水平在24 h和48 h显著提高。选择Poly I∶C质量浓度2 μg·mL^-1,处理24 h和48 h作为后续试验激活RLRs的处理条件。质量浓度为150 μg·mL^-1 LE可以显著抑制Poly I∶C介导的细胞活力降低;300 μg·mL^-1的LE显著降低了RIG-I的蛋白水平;150 μg·mL^-1和300 μg·mL^-1 LE处理24 h和48 h后均显著抑制了β干扰素的mRNA转录和生成。据此得出如下结论:Poly I∶C转染HEK293细胞成功地激活了RLRs信号通路,宽体金线蛭提取物具有促进HEK293细胞活力和抑制β干扰素生成的作用。

关键词: 维甲酸诱导基因蛋白样受体(RLRs), 宽体金线蛭, 聚肌苷酸-聚胞苷酸(Poly I∶C), HEK293细胞, 维甲酸诱导基因I, β干扰素

Abstract:

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) signaling pathway plays pivotal roles in antiviral innate immune response. Whitmania pigra contains the bioactive substances with the function of anticoagulation, and mainly uses for thrombosis treatment. However, there was no research concerning the effect of substance origin from W. pigra on RLRs signaling pathway. The aims of the present study were to activate RLRs signaling pathway by using polyinosinic: polycytidylic acid (Poly I∶C), and to determine the influence of extract from W.pigra(LE) on activated RLRs signaling pathway. 3 doses (1, 2, 4 μg·mL^-1) of Poly I∶C were transfected to HEK293 cell and lasted for 12, 24 and 48 h. The protein expression and mRNA transcription level of RIG-I were the indicators of activated RLRs signaling pathway. After activating RLRs signaling pathway, LE was used to treat HEK293 cell. 4 groups with triplicate were set. There were control, transfection with 2 μg·mL^-1 Poly I∶C, transfection with 2 μg·mL^-1 Poly I∶C and treatment with 150 μg·mL^-1 LE, transfection with 2 μg·mL^-1 Poly I∶C and treatment with 300 μg·mL^-1 LE. The treatment lasted for 24 and 48 h. The results showed that Poly I∶C transfection with 3 doses and durations reduced cell viability, and Poly I∶C transfection with 2 and 4 μg·mL^-1 Poly I∶C increased RIG-I protein expression significantly with 3 durations. At 24 and 48 h, RIG-I mRNA transcription level in HEK293 cell transfected with 4 μg·mL^-1 Poly I∶C was significantly increased. The dose of 2 μg·mL^-1 and durations of 24 h and 48 h were chosen to activate RLRs pathway in the following experiment. LE with 150 μg·mL^-1 counteracted the inhibited effect of Poly I∶C on cell viability. LE with 300 μg·mL^-1 reduced the protein expression level of RIG-I. Plus, Both 150 and 300 μg·mL^-1 LE inhibited interferon β mRNA transcription level and generation. In conclusion, Poly I∶C transfection succeed activating RLRs signaling pathway, as well as the extract from W. pigra promoted cell viability and inhibited interferon β generation of HEK293 cell.

Key words: retinoic acid-inducible gene I-like receptors (RLRs), Whitmania pigra, polyinosinic:polycytidylic acid (Poly I∶C), HEK293 cell, retinoic acid-inducible gene I, interferon β

中图分类号:

S937.3

陈姿亦, 何盛盛, 闫晶男, 吴怡蓉, 张雨婷, 高有领. 宽体金线蛭提取物对HEK293细胞维甲酸诱导基因蛋白样受体(RLRs)通路的影响[J]. 浙江农业学报, 2023, 35(12): 2830-2843.

CHEN Ziyi, HE Shengsheng, YAN Jingnan, WU Yirong, ZHANG Yuting, GAO Youling. Extract of Whitmania pigra on the retinoic acid-inducible gene I-like receptors (RLRs) signaling pathway of HEK293 cell[J]. Acta Agriculturae Zhejiangensis, 2023, 35(12): 2830-2843.

图/表 11

表1 qRT-PCR PCR引物

Table 1 Primers for qRT-PCR

基因Gene	引物名称Primer name	引物序列(5’→3’)Primer sequence (5’→3’)	产物长度Product length/bp
GAPDH	hGAPDHf-249	GCACCGTCAAGGCTGAGAAC	119
	hGAPDHr-368	TGGTGAAGACGCCAGTGGA
RIG-Ⅰ	hRIG-f496	GTGGAATGCCTTCTCAGATCAGAC	271
	hRIG-r767	CAGGCAAAGCAAGCTCTAATTGGT
MAVS	hMAVS-f9678	TCGAATACTACTGCACAGCAACGAA	177
	hMAVS-r9855	CTGTTCCCCTGGCTTCTAACACC
MDA5	hMDA5-f601	ACAATTGAAGACAGAAACCGGAT	272
	hMDA5-r873	TCCATGCCCCAGACCTCC
TRAF3	hTRAF3-f642	AAATGTACAGCGTGTCAAGAGAGCATC	243
	hTRAF3-r885	GCTTCCCGGTATTTACACGCCTT
IRF3	hIRF3-f439	GGATAAGCCAGACCTGCCAACCTG	183
	hIRF3-r622	TCCTGGGTATCAGAAGTACTGCCT
IFN-β1	hIFNB1-f101	GCTCTCCTGTTGTGCTTC	287
	hIFNB1-r388	CATTAGCCAGGAGGTTCTCA
TRAF2	hTRAF2-f405	CCTGAAAGAATACGAGAGCTGCCACGAA	323
	hTRAF2-r728	GCTGTTTCTCACCCTCTACCGTCT
NF-κB	hNFKB-f1150	ACGCCATCTATGACAGTAAAGCC	287
	hNFKB-r1437	TTTCCAAGTCAGATTTCCTCCGAAG

图1 Poly I∶C转染HEK293细胞后细胞形态变化

Fig.1 Morphology change of HEK293 cell transfected with Poly I∶C

图2 Poly I∶C转染后HEK293的细胞活力 *, P<0.05。下同。

Fig.2 Cell viability of HEK293 transfected with Poly I∶C *, P<0.05. The same as below.

图3 poly I∶C转染后HEK293细胞中RIG-Ⅰ蛋白的表达量 A,Western blot;B,条带灰度值;*,P<0.05。

Fig.3 RIG-Ⅰ protein expression in HEK293 cell transfected with poly I∶C A, Western blot; B, Gray density of bands; *, P<0.05.

图4 poly I∶C转染后HEK293细胞中RIG-Ⅰ基因的转录水平

Fig.4 mRNA transcription level of RIG-Ⅰ in HEK293 cell transfected with poly I∶C

图5 宽体金线蛭提取物处理HEK293细胞后细胞形态的变化

Fig.5 Morphology change of HEK293 cell treated with Whitmania pigra extract

图6 宽体金线蛭提取物和poly I∶C共同作用对HEK293细胞活力的影响

Fig.6 Combined effect of Whitmania pigra extract and poly I∶C on HEK293 cell viability

图7 宽体金线蛭提取物处理后HEK293细胞中RIG-Ⅰ蛋白的表达量 A,Western blot;B,条带灰度值。

Fig.7 RIG-Ⅰ protein expression in HEK293 cell treated with Whitmania pigra extract A, Western blot; B, Gray density of bands.

图8 宽体金线蛭提取物处理后HEK293细胞中RIG-Ⅰ mRNA的转录水平

Fig.8 mRNA transcription level of RIG-Ⅰ in HEK293 cell treated with Whitmania pigra extract

图9 宽体金线蛭提取物处理后HEK293细胞中IFN-β的mRNA转录水平和IFN-β生成量 *,P<0.05;N.D表示未检出。

Fig.9 mRNA transcription level and production of IFN-β in HEK293 cell treated with Whitmania pigra extract *P<0.05; N.D represents non-detected.

图10 宽体金线蛭提取物处理48 h后HEK293细胞中RLRs信号通路因子的mRNA转录水平

Fig.10 mRNA transcription level of the factors involved in RLRs signaling pathway in HEK293 cell treated with Whitmania pigra extract for 48 h

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宽体金线蛭提取物对HEK293细胞维甲酸诱导基因蛋白样受体(RLRs)通路的影响

Extract of Whitmania pigra on the retinoic acid-inducible gene I-like receptors (RLRs) signaling pathway of HEK293 cell

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