Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (21): 4315-4326.doi: 10.3864/j.issn.0578-1752.2022.21.017

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Effect of Matrine on NLRP3 Inflammasome Signaling Pathway in H9N2 AIV Infected Mice

WEI JingJie(),JIANG NingBo(),LIANG Yan,ZHANG Qian,SUN YingJian,HU Ge()   

  1. College of Animal Science and Technology, Beijing University of Agriculture, Beijing 102206
  • Received:2021-08-17 Accepted:2022-02-18 Online:2022-11-01 Published:2022-11-09
  • Contact: Ge HU E-mail:329974543@qq.com;1176693232@qq.com;bnhuge@126.com

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Abstract:

【Objective】 Influenza virus infection causes inflammatory response and imbalance of immune homeostasis. The resulting cytokine storm (CS) is the main cause of death of infected hosts. The aim of this study was to explore the protective effect of Matrine on H9N2 AIV infected mice as well as the characteristics and preliminary mechanism of regulating NLRP3 inflammatory body signal pathway, so as to further improve the theoretical basis of antiviral of traditional Chinese medicine and lay a foundation for the development of new antiviral drugs.【Method】72 8-week-old BALB/c mice were randomly divided into blank control group (0.1 mL sterile chicken embryo allantoic fluid) and virus group (0.1 mL 4×105 PFU/mL H9N2 AIV), amantadine group (0.1 mL 4×105 PFU/mL H9N2 AIV + 100×10-6 99% amantadine), matrine high concentration treatment group (0.1 mL 4×105 PFU/mL H9N2 AIV + 40 mL·kg-1 matrine), medium concentration treatment group (0.1 mL 4×105PFU/mLH9N2 AIV + 20 mL·kg-1 matrine), and low concentration treatment group (0.1 mL 4×105PFU/mL H9N2 AIV + 10 mL·kg-1 matrine), and allantoic fluid nasal drops were used to construct the mouse model of viral pneumonia. The matrine was administered by gavage or drinking water for 5 consecutive days, and the test was conducted for 7 days. The weight changes of mice in different groups were observed. On the 1st, 3rd, 5th and 7th days, three mice were aseptically collected and killed, and the lung tissue was taken for histopathological observation, the expression of H9N2 and NLRP3 gene in mouse lung tissues was detected by RT-PCR, the expression of NLRP3 inflammatory body signal pathway related protein in lung tissue of H9N2 infected mice after matrine treatment was detected by Western blot, and the cytokine TNF in mouse serum was measured by ELISA- α and IL-1 β changes in the expression of IL-18 and IL-10.【Result】Compared with the virus group, the area of pulmonary edema and the number of inflammatory cells in the pathological section of H9N2 AIV infected mice in the high concentration matrine treatment group were significantly reduced, and the exudation of red blood cells decreased, while the effect was close to that in the amantadine group. At day 7, the alveolar wall of mice in the high concentration matrine treatment group was intact, the boundary between alveoli was clear, and the number of inflammatory cells and plasma cells in lung tissue decreased significantly, which was almost the same as that in the blank group; in the treatment group with medium concentration of matrine, there was a small amount of bleeding in the lung tissue, there was no swollen fluid in the alveoli, and the alveolar septum was intact; in the low concentration matrine treatment group, the red blood cells exuded from the alveoli, a large number of plasma cells were recruited, and there was fusion between the alveoli. The expression of H9N2 virus gene in lung tissue of mice treated with Matrine and amantadine decreased significantly on day 3, 5 and 7 (P<0.01). After 3 and 5 days of treatment, the expression of NLRP3 gene, protein and TNF- α, IL-1 β in matrine high concentration treatment group and amantadine group decreased significantly (P<0.01); on day 7, the expression of NLRP3 gene in lung tissue of mice in matrine high, medium and low treatment groups decreased significantly (P<0.01); the expression of NLRP3 protein, caspase-1 protein in low concentration group and caspase-1 protein in medium concentration group decreased significantly on day 5 (P<0.05); the expression of TNF- α and IL-1 β in matrine medium and low concentration groups on days 3 and 5 decreased significantly (P<0.01).The expression of IL-10 in Shegan medium concentration treatment group decreased significantly.【Conclusion】Matrine could inhibit the expression of H9N2 AIV in vivo, reduce the expression of TNF-α, IL-1β and IL-10 by down regulating NLRP3 inflammatory body signal pathway related proteins, and reduce the inflammatory response, which had good antiviral and anti-inflammatory effects.

Key words: matrine, H9N2 AIV, NLRP3 inflammasome signaling pathway

Table 1

Sequence and fragment size of primers for NA, NLR and reference GAPDH gene"

基因 Gene 引物序列 Primer sequence (5′ to 3′) 片段大小 Fragment size (bp)
NA Forward primer：TGTGGGGCATAAATCATCA
Forward primer：ATGAGGCGACAGTCGAATTAA		 224
GAPDH Forward primer：TTGGCTACAGCAACAGGGTG
Forward primer：GAGGAGATGCTCGGTGTGTT		 236
NLR Forward primer：TGAAGTGGATTGAAGTGAAAGCC
Forward primer：TGTGAAAAAAACCCAGGGAAAGC		 256

Fig. 1

HE staining results of mice in the experimental group after two drug treatments on the third day of virus infection A. Matrine high dose; B. Matrine medium dose; C. Matrine low dose; D. Amantadine; E. Virus positive; F. Blank control; ★ : Inflammatory cell recruitment; ▲ : Pulmonary edema"

Fig. 2

H.E staining results of mice in the experimental group after four times of drug treatment on the fifth day of virus infection A. Matrine high dose; B. Matrine medium dose; C. Matrine low dose; D. Amantadine; E. Virus positive; F. Blank control; ★ : Inflammatory cell recruitment"

Fig. 3

H.E staining results of experimental group mice after five times of drug treatment on the seventh day of virus infection A. Matrine high dose; B. Matrine medium dose; C. Matrine low dose; D. Amantadine; E. Virus positive; F. Blank control; ★: Inflammatory cell recruitment"

Fig. 4

Expression of mRNA in lung tissues of mice infected with H9N2 AIV Compared with virus group *P<0.05; **P<0.01"

Fig. 5

Expression of NLRP3mRNA in lung tissues of mice infected with H9N2 AIV Compared with virus group *P<0.05; **P<0.01"

Fig. 6

Relative expression of NF-κB, NLRP3, Caspase-1 protein after five drug treatments on day 7 of viral infection"

Fig. 7

Gray value analysis of NF-κB, NLRP3, Caspase-1 Compared with virus group *P<0.05; **P<0.01; Compared with control group #P<0.05; ##P<0.01"

Fig. 8

Expression of TNF-α in lung tissues of mice infected with H9N2 AIV Compared with virus group *P<0.05; **P<0.01; Compared with the control group #P<0.05; ##P<0.01. The same as below"

Fig. 9

Expression of IL-1β in H9N2 AIV infected mice treated with drugs Compared with virus group *P<0.05; **P<0.01; Compared with the control group #P<0.05; ##P<0.01"

Fig. 10

Expression of IL-10 in mice infected with H9N2 AIV after drug treatment Compared with virus group *P<0.05; **P<0.01; Compared with the control group #P<0.05; ##P<0.01"

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