Discovery And Mechanism Of Novel Efficacy Of Emodin

ObjectiveRhubarb is a classic Chinese medicinal material with thousands of years’usage records,and its efficacy is precise,with many aspects of clinical efficacy including laxative heat and stool,detoxification of herbal blood,and strengthening the meridians.Emodin is a representative anthraquinone like active component of rhubarb and has a wide range of biological activities.Emodin has been found to exhibit cholesterol lowering function with external radiosensitizing efficacy,but studies on the mechanism of its cholesterol lowering function and whether emodin has brachyradiosensitizing efficacy are lacking.Therefore,this study is based on evidence-based evidence of rhubarb to explore the novel efficacy of emodin in both directions of cholesterol absorption inhibition as well as brachytherapy sensitization,as well as the underlying mechanism of action,and to explore the new druggable direction and potential value of emodin.MethodsIn the first part of this study,NBD cholesterol was firstly utilized to determine the cholesterol absorption inhibition ability of emodin;The mechanism of emodin inhibition of cellular cholesterol uptake was secondarily explored using Philippine;Again,the mode of action of emodin in inhibiting NPC1L1 mediated cholesterol uptake was determined by enzyme kinetic experiments;Finally,the effect of emodin on NPC1L1 protein expression in Hep G2 cells was explored by Western immunoblotting experiment.In the second part of the study,the brachyradiosensitizing effect of emodin on ¹²⁵I particles was preliminarily verified by CCK-8 experiments;Analysis of the intracellular transcriptome after ¹²⁵I particle irradiation;Measurement of intracellular peroxide（ROS）content,lipid peroxide content as well as ROS content after treatment with emodin and/or¹²⁵I irradiationγ-H2AX protein phosphorylation profile.ResultEfficacy and mechanism of emodin on cholesterol absorption inhibition:（1）After a 100μM,75μM,50μM,25μM,12.5μM The cholesterol absorption rates of Hep G2 versus Caco-2 cells after concentration of emodin treatment were 27%,31.3%,33.6%,41.6%,and 52.6%versus 29%,28%,32.3%,39.6%,44.3%for the blank control.The cholesterol absorption inhibition capacity of emodin was significantly better than that of ezetimibe in both cell lines;（2）CCK-8 results showed at 6.25μM,12μM,25μM,50μM,100μM Both emodin at concentrations significantly enhanced the killing of murine 4T1 cells by ¹²⁵I intraluminal particles;（3）The type of inhibitory effect of emodin on NPC1L1 mediated cholesterol uptake was determined as a anticompetitive inhibition in Hep G2 cells;（4）The results of Western blot experiments showed that emodin did not significantly affect the expression of NPC1L1 in Hep G2 cells.Studies on the efficacy and mechanism of emodin for intraparticle radiosensitization by ¹²⁵I:（1）Transcriptome profiling identified 3015 genes whose expression changed after125I particle irradiation in 4T1 cells,of which 982 were up-regulated and 2033 were down regulated;（2）CCK-8 results showed at 6.25μM,12.5μM,25μM,50μM,100μM.Both concentration of emodin significantly enhanced the killing of mouse 4T1 cells by ¹²⁵I intraluminal particles;（3）Ferrostatin-1 inhibited the radiosensitizing effect of emodin,and the combination of emodin and ¹²⁵I resulted in a significant increase in intracellular lipid peroxides compared with either emodin or ¹²⁵I alone;（4）Compared with treatment with emodin alone or ¹²⁵I irradiation,combined treatment of emodin and ¹²⁵I caused a significant increase in intracellular ROS;（5）Emodin combined with ¹²⁵I treatment significantly elevated the intracellularγ-H2AX protein phosphorylation levels.ConclusionOur study is the first to identify the inhibitory effect of emodin on cholesterol absorption and reveals that the mechanism of action is related to the inhibition of NPC1L1protein mediated cholesterol transport.Meanwhile,emodin was found to have sensitizing efficacy against ¹²⁵I particle based brachytherapy,and preliminary mechanistic studies revealed that ROS,Ferroptosis,as well as DNA damage played an important role in sensitization.The above studies add to the accumulation of knowledge about the new efficacy value of emodin,and at the same time provide new ideas and methods for the development of cholesterol absorption inhibitors as well as inner radiosensitizers.