现代中西医结合杂志2025，Vol.34Issue(22)：3094-3101,8.DOI:10.3969/j.issn.1008-8849.2025.22.005
重连口服液体外抗甲型H1N1 pdm09病毒活性和抗炎机制研究
Antiviral activity and anti-inflammatory mechanism of Chonglian oral solution against influenza A(H1N1)pdm09 virus in vitro
摘要
Abstract
Objective It is to investigate the antiviral activity and anti-inflammatory mechanism of Chonglian oral solu-tion(COS)against influenza A(H1N1)pdm09 virus in vitro.Methods①The cytotoxicities of different concentrations of COS and oseltamivir on MDCK(Madin-Darby canine kidney)cells and A549(human non-small cell lung cancer)cells were assessed by CCK-8 kit,the 50%cytotoxic concentration(CC50)was calculated to determine the effective and safe concentration ranges for COS and oseltamivir.②The experiment included 3 groups:virus group,COS group and oseltami-vir group.MDCK cells were implanted in a 96-well plate,after 24 hours of culture,50 μL of diluted virus solution was added and adsorbed for 1 hour,then the virus group,COS group and oseltamivir group were cultured with virus mainte-nance solution,20000 μg/mL COS and 12.5 μg/mL oseltamivir phosphate solution,respectively.After 72 hours of cul-ture,the cells were detected and the tissue culture infectious dose fifty(TCID50)was calculated.③The experiment includ-ed 8 groups:normal group,virus group,oseltamivir group,COS 2 μg/mL group,COS 20 μg/mL group,COS 200 μg/mL group,COS 2000 μg/mL group,and COS 20000 μg/mL group.The seasonal influenza A virus representative strains A/TJA/swl1962 were selected and sequentially inoculated into 12-well plates with direct action and preventive action.The os-eltamivir group was cultured with oseltamivir solution of a final concentration of 12.5 μg/mL,while the COS groups were cultured with COS of corresponding concentrations.After 24 hours of culture,the viral load was measured,and the half-maximal inhibitory concentration(IC50)and viral inhibition rate in each group were calculated.④The experiment included 2 groups:virus group and COS group.MDCK cells were implanted into 12-well plates.After 24 hours of culture,100 TCID50 of H1N1pdm09 virus solution was added.After 1 hour of culture,the solution was removed and the cells were washed,and virus maintenance medium was added for continued culture.At 2,4,and 6 hours after virus inoculation,the virus group was added with virus maintenance medium,while the COS group was added with virus maintenance medium containing 20000 μg/mL COS.At 8 hours after virus inoculation,the expression of influenza virus nucleoprotein(NP)was detected by immunofluorescence staining.⑤The experiment included 6 groups:blank group,model group,low-dose,medium-dose,high-dose groups of COS,and oseltamivir group.A549 cells in the logarithmic growth phase were implanted in 12-well plates and cultured for 48 hours.All groups except for the blank group were cultured with 200 μL of H1N1 pdm09 virus dilution(100 TCID50).After 1 hour of culture,the low-dose,medium-dose,and high-dose groups of COS were added with 5 mg/mL,10 mg/mL,and 15 mg/mL COS,respectively,while the oseltamivir group was added with 50 μg/mL oseltamivir phosphate.After 24 hours of culture,the mRNA expressions of monocyte chemoattractant protein-1(MCP-1)and influenza virus matrix protein 2(M2)were detected by real-time quantitative PCR,the protein expressions of M2 and cellular inflammatory factors retinoic acid-inducible gene I(RIG-I),NOD-like receptor thermal protein domain associated protein 3(NLRP3),apoptosis-associated speck-like protein containing a CARD(ASC),Caspase-1,and inter-leukin-1β(IL-1β)were detected by Western blot.Results ①The CC50 values of COS for MDCK and A549 cells were 28.56 mg/mL and 14.59 mg/mL,respectively.②The IC50 of COS against the virus was 9.10 mg/mL,and its inhibitory effect was dose-dependent.③Administration of COS at 2 and 4 hours after infecting MDCK cells significantly suppressed NP expression.④The relative mRNA expressions of M2 and MCP-1,and the relative protein expressions of M2,RIG-I,NLRP3,ASC,Caspase-1,and IL-1β in A549 cells of the model group were significantly higher than those in the blank group(all P<0.05),these expressions in all medicated groups were significantly lower than those in the model group(all P<0.05),furthermore,all the afore-mentioned indicators in the COS groups decreased in a dose-dependent manner(all P<0.05).Conclusion Chonglian oral solution can effectively inhibit the replication of influenza A(H1N1 pdm09)virus.Its anti-inflammatory effect may be mediated by reducing RIG-I expression in A549 cells,thereby decreasing inflammasome production.关键词
重连口服液/磷酸奥司他韦/H1N1pdm09病毒/NOD样受体热蛋白结构域相关蛋白3/凋亡相关斑点样蛋白/半胱氨酸蛋白酶1/白细胞介素-1βKey words
Chonglian oral solution/oseltamivir phosphate/H1N1pdm09 virus/NLRP3/ASC/Caspase-1/IL-1β分类
医药卫生引用本文复制引用
卫宇航,张琳煜,俞峻岭,孙永,王新汝,李泽庚,童佳兵..重连口服液体外抗甲型H1N1 pdm09病毒活性和抗炎机制研究[J].现代中西医结合杂志,2025,34(22):3094-3101,8.基金项目
2022年度安徽省卫生健康科研项目(AHWJ2022a025) （AHWJ2022a025）
新安医学与中医药现代化研究所"揭榜挂帅"项目(2023CXMMTCM009,2023CXMMTCM005) （2023CXMMTCM009,2023CXMMTCM005）
