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

doi:10.3969/j.issn.1004-1524.20221666

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (12): 2830-2843.DOI: 10.3969/j.issn.1004-1524.20221666

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S937.3

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.

Figures/Tables 11

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

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

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

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.

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

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

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

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

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

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.

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

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