Susceptibility Mechanism And Detoxification Studies Of Psoralea Corylifolia-induced Immunological Idiosyncratic Liver Injury

Research background:Psoralea corylifolia Linn.(PF)is a clinically used tonic Chinese medicine,which has the effects of tonifying the kidney and strengthening yang,warming the spleen and stopping diarrhea,etc.It is mostly used to treat various diseases such as kidney deficiency and impotence,lumbago and osteoporosis,etc.However,liver injury caused by PF and its preparations,have occurred frequently in recent years.Our previous studies have demonstrated that PF could induce immunological idiosyncratic liver injury through a large number of clinical cases and basic research.However,the susceptible substances and mechanisms of PF-induced idiosyncratic liver injury are still unclear,which makes it difficult to form and formulate targeted prevention and control strategies for the safe use of PF.Therefore,this paper intends to conduct an exploratory study on the substance and mechanism of PF-induced idiosyncratic liver injury,order to provide a reference basis for the scientific formulation of hepatic injury prevention and control strategies for PF.Research methods:1.Based on mouse PF-derived macrophages(BMDMs),an LPS-induced in vitro inflammasome activation model was constructed to screen for PF-induced specific liver injury susceptibility components.Western Blot(WB)technique was used to detect the expression of caspase-1 p20,and enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of IL-1β and TNF-α.2.To establish an LPS-induced immune stress model in mice and compare the susceptible components leading to liver injury under normal and immune stress conditions.AST,ALT,IL-1β,TNF-α in serum and IL-1β,IL-18,TNF-α in the liver were measured by Lai’s method,ELISA,and QRT-PCR,and caspase-1 p20 protein expression in mouse liver tissues was detected by WB technique,furthermore,the pathological changes in liver of each group of mice were observed by H&E staining method.3.The specificity of Bak-induced inflammasome activation was investigated based on an in vitro model of LPS-induced inflammasome activation using multiple inflammasomes inhibitors(MCC950,echinatin,ODN),Nlrp3 gene knockdown and targeted silencing of Aim2 gene expression.Changes in cell supernatant caspase-1 p20 protein expression were detected by WB method,and changes in cell supernatant IL-1β and TNF-α levels were measured by ELISA assay,respectively.4.Based on the activation of the inflammasome by susceptible components,the effects of susceptible components on upstream targets related to inflammasome activation were detected to clarify their activation mechanism.The effects of susceptible components on ASC oligomerization associated with inflammasome activation were analyzed by WB method;the effects of susceptible components on mitochondrial membrane potential and ROS production were measured by flow cytometry;the effects of susceptible components on mitochondrial morphology and protein binding associated with intracellular inflammasome assembly were observed by immunofluorescence assay.5.Based on the guideline of target identification and detoxification,we proposed a strategy for the prevention and control of hepatotoxicity of PF based on the interplay of effector targets.The polysaccharide of glycyrrhiza and echinatin,components of glycyrrhiza that inhibit the activation of the inflammasome,were combined with the components of PF that activate inflammasome,and the changes of caspase-1 p20 protein and ASC oligomerization before and after the combination were detected by WB method,and the changes of IL-1β and TNF-α content before and after the combination were measured by ELISA method.Results:1.Based on the in vitro liver injury drug screening and evaluation system established in the previous studies,the regulatory effects of various active components in PF on inflammasome were investigated.The results showed that the monoterpene phenolic component of Bakuchiol(Bak)and the coumarin component psoralidin both induced caspase-1 p20 protein expression and significantly increased the levels of IL-1β and TNF-α in cell supernatants,indicating that both could activate inflammasome,and the activation effect of Bak was stronger,suggesting that Bak may be a major susceptible component of PF-induced idiosyncratic liver injury.2.Based on the LPS-induced immune stress mouse model,we investigated the role of Bak in causing hepatic injury in a heterogeneous manner.The results showed that Bak(15,30 mg/ kg)did not induce an increase in serum levels of ALT,AST,IL-1β,and TNF-α in normal mice,nor did it increase the expression levels of IL-1β,IL-18,and TNF-α in the liver,and there was no inflammatory infiltration of liver tissue and intact hepatocytes,indicating that the administered dose did not cause liver injury;Bak(15,30 mg/ kg)significantly increased ALT,AST,IL-1β,TNF-α expression in serum and IL-1β,IL-18,TNF-α expression in liver,and dose-dependently increased hepatic caspase-1 p20 protein expression in the LPS-induced immune stress model,and also aggravated pathological changes such as inflammatory infiltration and hepatocyte injury in liver tissues,indicating that Bak caused idiosyncratic liver injury by activating inflammasome.3.Based on an in vitro model of LPS-induced inflammasome activation,we investigated the specificity of Bak-induced inflammasome activation by inhibiting Nlrp3,Nlrc4,and Aim2 expression using various inflammasomes inhibitors and knockdown /knockdown techniques.The results showed that MCC950,echinatin,and knockdown of Nlrp3 gene did not affect Bak-induced caspase-1 p20 protein expression and IL-1β and TNF-α release;AIM2 inhibitor and knockdown of Aim2 gene significantly inhibited Bak-induced inflammasome activation,indicating that Bak mainly induces AIM2 inflammasome activation.4.Based on the activation of inflammasomes by Bak,the regulatory targets related to the activation of AIM2 inflammasome assembly were investigated.The results showed that Bak could induce ASC oligomerization associated with AIM2 inflammasome activation;observing and measuring cellular mitochondrial morphology,membrane potential,and ROS,we found that Bak could damage mitochondrial morphology,decrease mitochondrial membrane potential,and increase mitochondrial ROS production,and it was hypothesized that Bak could be induced by inducing mitochondrial damage to cause the abnormal release of mitochondrial DNA into the cytoplasm,and the mitochondrial DNA was recognized by AIM2 receptors and activated AIM2 inflammasomes.Further,Bak was observed to promote the polymerization of DNA with AIM2 in the cytoplasm by immunofluorescence,suggesting that Bak activates AIM2 inflammasomes mainly by inducing mitochondrial damage and releasing mitochondrial DNA.5.The combination of glycyrrhiza polysaccharide and echinatin with Bak and psoralidin,respectively,revealed that the component pairing significantly inhibited Bak and psoralidin-induced caspase-1 p20 protein expression,ASC oligomerization,and the production of inflammatory factors IL-1β and TNF-α.The above results suggest that the components in glycyrrhiza,such as glycyrrhiza polysaccharide and echinatin,can inhibit Bak-and psoralidin-induced inflammasome activation,respectively,and act as an effector target-based modulation of the antitoxic effect of the pairing.Study conclusion:In this study,we investigated the susceptibility factors and potential mechanisms leading to PF-induced idiosyncratic liver injury.The in vitro inflammasome activation model was used to screen and identify Bak as the main chemical component in PF that activates inflammasomes,and in combination with the in vivo immune stress model,Bak was clearly identified as the main susceptible component of heterogeneous liver injury in PF.It was also clear that the combination of PF and glycyrrhiza could inhibit the excessive inflammatory response induced by multiple components of PF.This study revealed the susceptibility factors and potential mechanisms of hepatic injury induced by PF,and proposed a prevention and control strategy for PF hepatotoxicity based on the interaction of effector targets,which provided a theoretical basis and treatment plan for the safe and rational use of PF in clinical practice.