改良育阴方对非洲猪瘟病毒感染PAMs的cGAS-STING通路影响

doi:10.11843/j.issn.0366-6964.2024.12.045

Abstract

Abstract:

In order to evaluate the efficacy of Chinese traditional medicine against African swine fever virus (ASFV) in vitro, five experimental drugs were used in this study. The effects of different Chinese herbal medicines on ASFV encored capshell protein p72 in porcine alveolar macrophages (PAMs) infected with African swine fever virus were detected by qPCR. Modified Yuyin decoction (MYY) was selected as a Chinese medicine that could significantly reduce the expression of ASFV-p72 gene. The main components of anti-ASFV were detected by LC-MS analysis. CCK8 method was used to observe the effect of modified Yuyin decoction on the activity of PAMs. The expression of ASFV-p72 gene was detected by fluorescence quantitative PCR. Western blot and ELISA were used to detect CGAS-STING signaling pathway protein expression. RU.521 was used as a cGAS inhibitor to verify the anti-ASFV effect of MYY in the CGAS-STING pathway. The results showed that MYY can significantly reduce the expression of ASFV-p72 gene in PAMs, and the best effect is given at 2 h after ASFV infection, and it has a concentration-dependent improvement effect on the cell viability after ASFV infection of PAMs. The expression levels of interferon gene stimulating protein (STING), TANK binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3) and interferon induced transmembrane protein 3 (IFITM3) in the supernatant of PAMs cells were decreased by MYY 2 h after the challenge. The expression of cyclic GMP-AMP synthetase (cGAS) and interferon β (IFNβ) protein in PAMs cells increased, and MYY could activate the cGAS-STING-TBK1-IRF3-IFNβsignaling pathway and promote the expression of IFITM3. After treatment with RU.521, MYY could still increase cell viability and decrease the expression of ASFV-p72 gene. In summary, MYY has a potential anti-ASFV effect, which may be related to the activation of cGAS-STING signaling pathway to promote the expression of IFITM3.

Key words: modified Yuyin decoction, African swine fever virus, ASFV-p72 gene, CGAS-STING signaling pathway, LC-MS analysis

CLC Number:

CHEN Xiaoli, ZHOU Jiahao, ZHOU Jing, QU Qian, WANG Zhihua, XIONG Ying, ZHU Yongqi, JIA Weixin, LÜ Weijie, GUO Shining. Effect of Modified Yuyin Decoction on cGAS-STING Pathway of African Swine Fever Virus Infected PAMs[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5839-5853.

Figures/Tables 12

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试验用药 Experimental drug	组方 Composition	剂量/g Dosage	作用功效 Function and efficacy
连翘 Forsythia	单药	20	清热，解毒，散结，消肿
马勃Sphaerium	单药	20	清肺利咽，解毒，止血
改良育阴方(MYY)(复方1) Modified Yuyin decoction (Compound 1)	石膏	30	清热泻火
	知母	18	和石膏同用，可增强石膏的清热泻火作用
	玄参	18	清热凉血，滋阴降火，除烦，解毒
	蒲公英	18	清热解毒，利尿散结
	金银花	20	清热，解毒
	连翘	20	清热，解毒，散结，消肿
	桑叶	16	祛风清热，凉血明目
	生地黄	18	滋阴清热，凉血补血
	牡丹皮	18	清热凉血，和血消瘀
	大青叶	18	清热解毒，凉血止血
	紫苏叶	16	发散表寒，开宣肺气
	甘草	10	和中缓急，润肺，解毒，调和诸药
复方鱼腥草(复方2) Compound Houttuynia (Compound 2)	鱼腥草	16	清热解毒，利尿消肿
	黄芩	18	泻实火，除湿热，止血，安胎
	板蓝根	18	清热解毒，凉血
	连翘	20	清热，解毒，散结，消肿
	金银花	20	清热，解毒
银翘马勃散(复方3) Yinqiao Mabo powder (Compound 3)	连翘	30	清热，解毒，散结，消肿
	牛蒡子	18	疏散风热，宣肺透疹，消肿解毒
	金银花	15	清热，解毒
	射干	9	降火，解毒，散血，消痰
	马勃	6	清肺利咽，解毒，止血

目标 Target	方向 Orientation	序列(5′- 3′) Sequence
ASFV-p72	正向 Forward	ACGGCGCCCTCTAAAGGT
ASFV-p72	反向 Reverse	CATGGTCAGCTTCAAACGTTTC
Cyclophilin A	正向 Forward	AGACAAGGTTCCAAAGACAGCAG
Cyclophilin A	反向 Reverse	AGACTGAGTGGTTGGATGGCA

化学式 Formula	名称 Name	质量 Exact_mass	测量误差(ppm) Measurement error(ppm)	CAS	Library	化学结构 Structure
C₆H₄O₂	1, 2-Benzoquinone	108.021 1	2.971 703 44	583-63-1	MetaDNA
C₅H₁₀N₂O	L-Prolinamide	114.079 3	2.815 271 87	7531-52-4	MetaDNA
C₁₅H₁₈O₈	1-O-(4-coumaroyl)-β-D-glucose	326.100 2	14.021 680 47	7139-64-2	MetaDNA
C₉H₁₈N₄O₄	D-Octopine	246.132 8	29.529 685 51	34522-32-2	MetaDNA
C₁₁H₂₀N₄O₆	Nopaline	304.138 3	24.608 987 88	22350-70-5	MetaDNA
C₉H₁₃N₃O₄	Deoxycytidine	227.090 6	6.605 293 218	951-77-9	MetaDNA
C₁₉H₂₆O₂	androst-5-ene-3, 17-dione	286.193 3	23.064 020 6	571-36-8	MetaDNA
C₁₉H₂₈O₂	Androstanedione	288.208 9	1.645 827 722	846-46-8	MetaDNA
C₂₁H₂₈O₄	11-Dehydrocorticosterone	344.198 8	22.415 016 9	72-23-1	MetaDNA
C₂₇H₄₆O₄S	Cholesterol sulfate	466.311 7	7.934 606 831	1256-86-6	MetaDNA

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Effect of Modified Yuyin Decoction on cGAS-STING Pathway of African Swine Fever Virus Infected PAMs

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