Mechanism And Significance Of The Active Components Of Sophora Flavescens Targeted Regulating NLRP3 Inflammasome Pathway

Sophora flavescens is the dry root of Sophora flavescens Ait,a plant of the genus Sophora,with cold nature and bitter taste.It has the effects of clearing away heat and detoxifying,dispelling wind and drying dampness,killing insects and diuretic.Clinically,it is used for diseases such as dysentery,blood in stool,jaundice and closure of urine,eczema and itching of skin.It has various pharmacological activities such as enhancing immunity,anti-virus and anti-tumor.However,in recent years,adverse reactions of Sophora flavescens and its related preparations are frequently reported,there are also reports of liver injury caused by it.The inflammasome is an important part of the natural immune system and plays an important role in the occurrence and development of various diseases.Appropriate activation of inflammasomes can enhance immunity and resist the invasion of pathogens effectively,but excessive activation can also cause immune inflammatory damage and promote the development of various liver diseases.Objective:This project intends to explore the active components of Sophora flavescens that target and regulate the NLRP3 inflammasome and its mechanism through the NLRP3 inflammasome activation model in vitro.Combined with the LPS-mediated immune activation model in vivo,to further evaluate the role in the liver.In order to screen the active components of Sophora flavescens that regulate immunity,provide a basis for the development of anti-inflammatory and anti-tumor drugs.We will clarify the causes of liver damage induced by Sophora flavescens and its related preparations,promote the rational use of drugs,and ensure the safety of medications.Methods:（1）Construct an NLRP3 inflammasome activation model in vitro:LPS-pretreated BMDMs were treated with the main components of Sophora flavescens,and then stimulated with ATP to induce the activation of NLRP3 inflammasome.Western blot was used to detect the expressions of NLRP3 inflammasome-related proteins in the cell supernatant and lysate;ELISA kit was used to detect the changes of IL-1βcontent in cell supernatants;Caspase-Glo^?1 Inflammasome Assay detection system was used to detect the activity of caspase-1 in cell supernatants;（2）To construct a variety of agonist-induced inflammasome activation,Western blot,Caspase-Glo^?1 Inflammasome Assay and ELISA kits were used to clarify the effect of Isoxanthohumol（IXN）on Salmonella-induced NLRC4 inflammasome activation and Poly（d A:d T）-induced AIM2 inflammasome activation.（3）The effect of IXN on NF-κB signaling pathway was preliminarily explored by comparing the treatment with IXN first and then LPS and the treatment with LPS first and then IXN;while the inflammasome was activated,The effect of IXN on the ASC oligomerization during the activation of various inflammasomes was detected by Western blot;the effect of IXN on intracellular calcium ion concentration was detected by FLIPRT Tetra high-throughput real-time fluorescence detection system;Flow cytometry was used to detect the effect of IXN on mitochondrial reactive oxygen species（ROS）production.（4）Using LPS-induced mouse immune activation model to evaluate the effect of IXN on liver function indexes ALT,AST,inflammatory factors IL-1β,TNF-αand liver histopathology.Result:（1）IXN,oxysophocarpine and sophororicoside in Sophora flavescens can significantly enhance the activity of caspase-1,but only IXN can significantly promote the expression of NLRP3 inflammasome-related protein and the secretion of IL-1βin the supernatant,it is suggested that IXN can promote the activity of NLRP3 inflammasome.（2）IXN can dose-dependently promote ATP-induced NLRP3 inflammasome activation.In the constructed activation models of NLRP3,NLRC4 and AIM2inflammasome,IXN only specifically promotes ATP and Nigericin-induced the NLRP3inflammasome activation,but had no effect on the the activation of NLRC4inflammasome induced by Salmonella or the activation of AIM2 inflammasome induced by Poly（d A:d T）.（3）Mechanistic results showed that IXN promoted ASC oligomerization and mitochondrial ROS production during ATP-induced NLRP3 inflammasome activation,but had no effect on NF-κB signaling pathway and calcium influx.（4）In vivo data show that non-hepatotoxic dose of IXN can lead to the elevation of ALT and AST and the massive release of inflammatory factors TNF-αand IL-1βin the LPS-induced mouse immune activation model,and can lead to liver pathological changes and hepatocyte apoptosis.Conclusions:This study shows that IXN can enhance the activation of the NLRP3inflammasome and also lead to idiosyncratic liver injury.The mechanism may be to promote ATP,Nigericin-induced ASC oligomerization and mitochondrial ROS production,resulting in the massive release of inflammatory factors and triggering liver inflammation,which in turn leads to idiosyncratic liver injury.This study clarified the mechanism of the active ingredient IXN in Sophora flavescens targeting the regulation of the NLRP3 inflammasome pathway,provided new insights into the mechanism of action of Sophora flavescens,and suggested that IXN may be an important risk factor for liver injury caused by the clinical use of Sophora flavescens.It provides a reference for the clinical safety and rational drug use and the risk prevention and control of related adverse drug reactions.