苦参碱对H9N2 AIV感染小鼠NLRP3炎性体信号通路的影响

doi:10.3864/j.issn.0578-1752.2022.21.017

摘要/Abstract

摘要：

【目的】流感病毒感染引发机体的炎症反应及免疫稳态失衡，由此产生的细胞因子风暴（CS）是引起感染宿主死亡的主要原因。通过探究苦参碱对H9N2 AIV感染小鼠的保护作用及其调节NLRP3炎性体信号通路的特点及作用机制，可进一步完善中药抗病毒的理论依据，为新型抗病毒药物的研发奠定基础。【方法】72只8周龄BALB/c小鼠随机分为空白组（0.1 mL无菌鸡胚尿囊液）、病毒组（0.1 mL 4×10⁵PFU/mLH9N2 AIV）、金刚烷胺组（0.1 mL 4×10⁵PFU/mLH9N2 AIV+100×10^-6 99%金刚烷胺）、苦参碱高浓度治疗组（0.1 mL 4×10⁵PFU/mLH9N2 AIV+40 mL·kg^-1苦参碱）、中浓度治疗组（0.1 mL 4×10⁵PFU/mLH9N2 AIV+20 mL·kg^-1苦参碱）和低浓度治疗组（0.1 mL 4×10⁵PFU/mLH9N2 AIV+10 mL·kg^-1苦参碱），每组12只，含病毒鸡胚尿囊液滴鼻构建小鼠病毒性肺炎模型，灌服或饮水给药治疗连续5 d，试验共进行7 d，观察不同组小鼠的体重变化。在第1、3、5、7天分别取3只小鼠无菌采血并处死，取小鼠肺组织进行病理组织学观察，RT-PCR检测小鼠肺组织中H9N2 NA、NLRP3 NLR基因表达情况，Western Blot检测苦参碱治疗后H9N2感染小鼠肺组织NLRP3炎性体信号通路相关蛋白表达量的变化，小鼠血清用ELISA法测定细胞因子TNF-α、IL-1β和IL-10表达量的变化。【结果】与病毒组相比，苦参碱高浓度治疗组病理组织学观察中H9N2 AIV感染小鼠肺部水肿的区域明显减少，红细胞渗出减少，炎性细胞数量减少，效果接近金刚烷胺组。7 d时苦参碱高浓度治疗组小鼠肺泡壁完好，肺泡之间分界清楚，肺组织内炎性细胞、浆细胞数量大量减少，与空白组无异;苦参碱中浓度治疗组肺组织少量出血，肺泡内无渗出液，肺泡间隔完好;苦参碱低浓度治疗组可见肺泡内红细胞渗出，大量浆细胞募集，肺泡之间出现融合现象。经过苦参碱和金刚烷胺治疗的各组小鼠肺组织中H9N2病毒基因表达量在3、5和7 d极显著降低（P<0.01）。经治疗3、5 d时，苦参碱高浓度治疗组和金刚烷胺组的NLRP3基因和蛋白表达量、TNF-α、IL-1β蛋白表达量极显著降低（P<0.01）;7 d时，苦参碱高、中、低治疗组小鼠肺组织中NLRP3基因表达量极显著降低（P<0.01）;5 d时苦参碱低浓度治疗组NLRP3蛋白、Caspase-1蛋白和中浓度组Caspase-1蛋白表达量显著降低（P<0.05）;3 d、5 d时苦参碱中、低浓度治疗组TNF-α和IL-1β蛋白表达量极显著降低（P<0.01），苦参碱中浓度治疗组IL-10表达量极显著降低。【结论】苦参碱可在体内抑制H9N2 AIV的表达，通过下调NLRP3炎性体信号通路相关蛋白，下调TNF-α、IL-1β和IL-10的表达，减轻炎症反应，有良好的抗病毒、抗炎作用。

关键词: 苦参碱, H9N2 AIV, NLRP3炎性体信号通路

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 4×10⁵PFU/mL H9N2 AIV), amantadine group (0.1 mL 4×10⁵PFU/mL H9N2 AIV + 100×10^-6 99% amantadine), matrine high concentration treatment group (0.1 mL 4×10⁵PFU/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

魏婧洁,蒋宁波,梁燕,张倩,孙英健,胡格. 苦参碱对H9N2 AIV感染小鼠NLRP3炎性体信号通路的影响[J]. 中国农业科学, 2022, 55(21): 4315-4326.

WEI JingJie,JIANG NingBo,LIANG Yan,ZHANG Qian,SUN YingJian,HU Ge. Effect of Matrine on NLRP3 Inflammasome Signaling Pathway in H9N2 AIV Infected Mice[J]. Scientia Agricultura Sinica, 2022, 55(21): 4315-4326.

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https://www.chinaagrisci.com/CN/Y2022/V55/I21/4315

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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