藏药秦艽花黄酮提取物对LPS/ATP复合诱导巨噬细胞焦亡的影响

Effect of Gentiana flavonoids extract from Tibetan medicine on LPS/ATP complex-induced pyrolysis of macrophages

目的:探讨藏药秦艽花黄酮提取物对LPS/ATP复合诱导小鼠单核巨噬细胞焦亡的影响,为民族药物深度开发利用提供科学依据。方法:实验采用倒置荧光显微镜观察小鼠单核巨噬细胞形态,首先测定藏药秦艽花黄酮提取物对J774A.1小鼠单核巨噬细胞的最大无毒浓度,采用ELISA法检测细菌脂多糖、脂多糖+三磷酸腺苷、LPS+藏药秦艽花黄酮提取物(TC_(-2)、TC_(-1)、TC_(0))+三磷酸腺苷分别与小鼠单核巨噬细胞共培养,测定培养上清液中caspase-1、IL-1β、IL-18的水平及LDH的含量,同时探讨对cAMP-PKA信号通路的影响。结果:藏药秦艽花黄酮提取物对小鼠单核巨噬细胞最大无毒浓度为5.53 mg/mL,LPS致敏巨噬细胞,在ATP作用下,细胞焦亡率48.76%;不同浓度的藏药秦艽花黄酮处理致敏巨噬细胞后,在ATP作用下,细胞焦亡率分别为4.55%(TC_(0))、7.21%(TC_(-1))、21.30%(TC_(-2))且呈剂量依赖性。同时,有效降低小鼠单核巨噬细胞Caspase-1、IL-1β、IL-18及LDH细胞因子水平;同时,LPS+MDL1233+ATP、LPS+H89+ATP诱导体系细胞焦亡率分别为62.11%和64.52%,经藏药秦艽花黄酮提取物处理后的小鼠单核巨噬细胞焦亡率分别为6.39%、8.84%。结论:藏药秦艽花黄酮提取物通过cAMP-PKA信号通路影响cAMP、PKA活性,剂量依赖性地抑制LPS/ATP诱导小鼠单核巨噬细胞焦亡,对炎症小体NLRP3异常活化和细胞焦亡相关疾病具有潜在治疗价值。

To explore the effect of the Tibetan medicine Gentiana flavonoids extract on the LPS/ATP compound-induced pyrolysis of mouse mononuclear macrophages,and to provide a scientific basis for the in-depth development and utilization of national drugs.In the experiment,an inverted fluorescence microscope was used to observe the morphology of mouse mononuclear macrophages.First,the maximum non-toxic concentration of the Tibetan medicine Gentiana flower flavonoids extract on J774A.1 mouse mononuclear macrophages was determined.The ELISA method was used to detect bacterial lipopolysaccharide,Lipopolysaccharide+adenosine triphosphate,LPS+Tibetan medicine Gentiana flavonoid extract(TC_(-2),TC_(-1),TC_(0))+adenosine triphosphate were co-cultured with mouse monocyte macrophages,and the caspase-1 and IL-in the culture supernatant were determined The level of 1β,IL-18 and the content of LDH,while discussing their influence on the cAMPPKA signaling pathway.The results showed that the maximum non-toxic concentration of Tibetan medicine Gentiana flower flavonoids extract on mouse monocyte macrophages was 5.53 mg/mL,LPS sensitized macrophages,under the action of ATP,the cell pyrolysis rate was 48.76%;different concentrations After the Tibetan medicine Gentiana flower flavonoids were treated with sensitized macrophages,under the action of ATP,the cell pyrosis rates were 4.55%(TC_(0)),7.21%(TC_(-1)),21.30%(TC_(-2))and were dose-dependent..At the same time,it effectively reduces the levels of Caspase-1,IL-1β,IL-18 and LDH cytokines in mouse monocytes and macrophages;at the same time,the LPS+MDL1233+ATP and LPS+H89+ATP induced cell pyrolysis rates were 62.11,respectively.%And 64.52%.The pyrolysis rates of mouse mononuclear macrophages treated with Tibetan medicine Gentiana flower flavonoid extract were 6.39%and 8.84%,respectively.Conclusion:Tibetan medicine Gentiana flower flavonoids extract affect cAMP and PKA activities through the cAMP-PKA signaling pathway,and dose-dependently inhibit LPS/ATP-induced pyroptosis of mouse monocyte macrophages,and abnormal activation of inflammasome NLRP3.Diseases related to pyrolysis have potential therapeutic value.