基于流式细胞术系统评估京尼平对线粒体功能和炎症的影响

A Flow Cytometry-based System to Assess the Effects of Genipin on Mitochondrial Function and Inflammation

摘要:

线粒体是细胞主要供能者,也是免疫调节中枢。本研究选择栀子苷、京尼平、西红花苷Ⅰ等9种栀子代表性成分,以HEK293T-mtKeima和 HeLa-mtKeima细胞为工具,以促线粒体自噬活性为切入点,对它们进行了初步筛选。结果表明,京尼平在两种细胞系中均具有最强的促进线粒体自噬活性,线粒体自噬激活比例分别达到了78.91±1.63%和18.96±3.86%,其可能是有前景的线粒体功能保护活性物质。进一步以京尼平为主要研究对象,探究了其对线粒体自噬的量效关系。结果表明,京尼平可以剂量依赖性地激活两种细胞的线粒体自噬且不会导致细胞损伤,在40 μM浓度下效果甚至显著强于阳性对照,超过80%的细胞线粒体自噬被激活。以THP1细胞为工具,系统评估了京尼平对线粒体功能的量效关系。50 μM京尼平孵育可以显著增加线粒体膜电位至对照组的1.15倍。此外,京尼平还剂量依赖性地增加线粒体活性氧,可能与其促进能量代谢有关。在THP1-ASC-GFP细胞中初步评估京尼平的抗炎能力,结果显示京尼平可以显著减少ASC炎症小体的数量,且效果具有浓度依赖性。以上结果表明京尼平可以调节线粒体功能,并具备抗炎功效。

Abstract:

Mitochondria are major cellular energy donors and immunoregulatory centers. This study selected nine representative components of Gardenia, including geniposide, genipin, crocetin I, etc., and used HEK293T-mtKeima and HeLa-mtKeima cells as tools to conduct a preliminary screening of these components, focusing on their pro-mitophagy activity. The results showed that genipin exhibited the strongest pro-mitophagy activity in both cell lines, with the proportion of activated mitophagy reaching 78.91±1.63% and 18.96±3.86%, respectively. It is therefore considered a promising active substance for protecting mitochondrial function. Further investigation focused on genipin to explore its dose-response relationship with respect to mitophagy. The results demonstrated that genipin could activate mitophagy in both cell lines in a dose-dependent manner without causing cellular damage. At a concentration of 40 μM, its effect was even significantly stronger than that of the positive control, activating mitophagy in over 80% of the cells. Using THP1 cells as a tool, the dose-response relationship of genipin on mitochondrial function was systematically evaluated. Incubation with 50 μM genipin significantly increased mitochondrial membrane potential to 1.15 times that of the control group. Additionally, genipin also dose-dependently increased mitochondrial reactive oxygen species (ROS), which may be related to its promotion of energy metabolism. A preliminary evaluation of genipin"s anti-inflammatory capacity was conducted in THP1-ASC-GFP cells. The results showed that genipin could significantly reduce the number of ASC specks, and this effect was concentration-dependent. These findings indicate that genipin can modulate mitochondrial function and possesses anti-inflammatory properties.