岩生忍冬黄酮提取工艺优化及其抗炎镇痛、免疫增强和抑制细胞焦亡作用

Optimization of extraction of flavonoids from Lonicera rupicola and analysis of its effects in resisting inflammation,relieving pain,enhancing immunity,and inhibiting pyroptosis

优化岩生忍冬黄酮提取工艺并初步探究其抗炎镇痛、免疫增强和抑制细胞焦亡等药理学作用,为岩生忍冬的开发和利用提供数据支持和科学依据。基于单因素实验结果,采用响应面分析法(RSM)优化岩生忍冬黄酮提取工艺;分别建立二甲苯耳肿胀、角叉菜胶足跖肿胀、醋酸扭体反应和舔足反应等小鼠抗炎镇痛模型并评价其抗炎镇痛作用;建立小鼠环磷酰胺免疫抑制模型分析岩生忍冬黄酮对单核细胞碳廓清指数和血清免疫球蛋白IgA、IgM含量的影响;复制D-半乳糖小鼠慢性/亚急性衰老模型,通过检测肝脏超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和丙二醛(MDA)含量以评价岩生忍冬黄酮抗体内抗氧化作用;检测J774A.1小鼠单核巨噬细胞上清液中caspase-1、IL-1β和IL-18的水平,评价岩生忍冬黄酮对脂多糖+三磷酸腺苷(LPS+ATP)联合诱导小鼠单核巨噬细胞焦亡的影响,同时探讨其对cAMP-PKA信号通路的影响。岩生忍冬黄酮最佳提取工艺如下,提取温度65℃、乙醇浓度50%、提取时间60 min、料液比1∶25,黄酮实际得率为0.553%。响应面分析确定响应值与变异因素的多元二次回归方程模型:黄酮得率=0.61-0.48A+0.1B+0.029C-0.014D+0.32AB+0.04AC-0.012AD-0.02BC+0.037BD-0.031CD-0.058A^(2)-0.068B^(2)-0.069C^(2)-0.057D^(2)。岩生忍冬黄酮有效地抑制小鼠耳朵肿胀和足跖肿胀、减少小鼠醋酸扭体次数并延长小鼠热板舔足反应时间;岩生忍冬黄酮提升免疫抑制小鼠碳廓清指数,增强D-半乳糖衰老小鼠肝脏SOD和CAT抗氧化酶活性和降低MDA水平,同时有效降低巨噬细胞caspase-1、IL-1β和IL-18细胞因子水平并抑制巨噬细胞焦亡。上述结果表明,岩生忍冬黄酮具有良好的抗炎镇痛、免疫增强作用和抗氧化能力并通过增强cAMP-PKA信号途径抑制巨噬细胞焦亡,该研究结果能够为岩生忍冬的药理学研究及其开发利用奠定基础。

The present study optimized the extraction of flavonoids from Lonicera rupicola Hook.f.et Thoms(LRH)and explored its pharmacological effects,such as resisting inflammation,relieving pain,enhancing immunity,and inhibiting pyroptosis,aiming to provide data support and scientific basis for the development and utilization of LRH.Response surface methodology(RSM)was applied to optimize the extraction of flavonoids from LRH based on the results of single-factor experiments.Anti-inflammatory and analgesic effects of LRH flavonoids were evaluated via inflammation and pain models in mice,such as xylene-induced ear swelling,carrageenaninduced footpad swelling,writhing caused by acetic acid,and paw licking.The effect of LRH flavonoids on the carbon clearance index of monocytes and serum immunoglobulin A(IgA)and IgM levels was analyzed on the immunosuppression model induced by cyclophosphamide in mice.The anti-oxidative effect in vivo of LRH flavonoids on liver superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA)levels was determined based on the chronic/subacute aging model in mice induced by D-galactose.The levels of cysteinyl aspartate specific proteinase-1(caspase-1),interleukin-1β(IL-1β),and IL-18 in the supernatant of J774A.1 mononuclear phagocytes were detected to evaluate the effect of LRH flavonoids on the pyroptosis of mononuclear phagocytes in mice induced by the combination of lipopolysaccharide(LPS)and adenosine triphosphate(ATP).Meanwhile,the effect of LRH flavonoids on the cAMPPKA signaling pathway was also explored.The optimum conditions for the extraction of LRH flavonoids are listed below:extraction temperature of 65℃,the ethanol concentration of 50%,extraction time of 60 min,a material-liquid ratio at 1∶25,and the yield of LRH flavonoids of 0.553%.RSM determined the multiple quadratic regression equation model of response value and variables as follows:the yield of LRH flavonoids=0.61-0.48A+0.1B+0.029C-0.014D+0.32AB+0.04AC-0.012AD-0.02BC+0.037BD-0.031CD-0.058A^(2)-0.068B^(2)-0.069C2-0.057D^(2).LRH flavonoids could effectively inhibit ear swelling and footpad swelling,reduced acetic acid-induced writhing,and delayed the paw licking response time in mice.Additionally,LRH flavonoids could improve the carbon clearance index in immunosuppressed mice,potentiate the activities of SOD and CAT and reduce MDA levels in the liver of aging mice induced by D-galactose,and effectively inhibit macrophage pyroptosis by decreasing the levels of caspase-1,IL-1β,and IL-18.The results reveal that LRH flavonoids possess excellent pharmacological activities such as resisting inflammation and oxidation,relieving pain,and enhancing immunity.They can inhibit pyroptosis by enhancing the cAMP-PKA signaling pathway.The results of this study can underpin the pharmacological research,development,and utilization of LRH.