Susceptible Components And Mechanism Studies Of Epimedii Folium-Induced Immunological Idiosyncratic Liver Injury Based On NLRP3 Inflammasome

Traditional Chinese medicine Epimedii Folium(EF)is the dried leaves of Berberidaceae plants Epimedium brevicomu Maxim.,E.sagittatum(Sieb.Et Zucc.)Maxim.,E.pubescens Maxim or E.koreanum Nakai.The name of EF is first found in the Shennong Materia Medica Classic,which has the effects of tonifying kidney and strengthening Yang,dispelling wind and dehumidifying dampness.In recent years,however,liver injury caused by EF and its related preparations has been reported frequently,and the adverse reaction(ADR)Detection Center of China Food and Drug Administration has also repeatedly reported to be alert to liver injury caused by EF related preparations,including Zhuanggu Guanjie Pill and Xianling Gubao Capsule.Previous studies have confirmed the objectivity of Zhuanggu Guanjie Pill and Xianling Gubao Capsule in liver injury.Moreover,the composition of the two formulations is analyzed,and it is found that there are many traditional"non-toxic"Chinese herbal medicines(EF,Psoraleae Fructus(PF),Rehmanniae Radix,Dipsaci Radix)in the formulations.Combined with the clinical symptoms and detection data,it is found that the liver injury caused by EF related preparations has immunological idiosyncratic characteristics.To further clarify whether drugs in Zhuanggu Guanjie Pill could induce liver injury,the effects of suspected drugs EF and PF on liver injury in rats were studied.The results showed that Zhuanggu Guanjie Pill and its components,EF and PF,could induce liver injury in the LPS model,among which EF has the strongest effect on liver injury.Which components in EF are susceptible to liver damage?Do these components have the effect of synergistically inducing liver injury?Moreover,what is the specific mechanism by which these susceptible components induce liver injury?Previous studies have shown that EF can cause liver injury in an LPS-induced immune stress model,accompanied by the release of the inflammatory cytokine IL-1?.The large release of IL-1?is closely related to the abnormal activation of NLRP3 inflammasome.Abnormal activation of NLRP3 inflammasome plays an important role in the development of alcoholic/non-alcoholic hepatitis,drug-induced liver injury and other liver diseases.Is EF-induced liver injury related to the activation of NLRP3inflammasome in this model?Therefore,an in vitro NLRP3 inflammasome activation screening model,the in vivo LPS-induced idiosyncratic liver injury model and the Nlrp3-knockout(Nlrp3^-/-)model were established based on the toxicology of diseases and syndromes.The main active ingredients in the EF were used as the research object.The methods of cell biology,cytotoxicology and immunohistochemistry were used to screen and study the susceptible components,synergistic effects and the mechanism of hepatotoxicity of EF.Moreover,we proposed and constructed the precise prevention strategies and methods of traditional Chinese medicines-induced liver injury based on the"human-drug-use"three-dimensional warning technology system,providing reference for the rational clinical use of traditional non-toxic Chinese medicines such as EF.The main research contents and conclusions are as follows:1.A screening and evaluation model for liver injury in vitro based on activation of NLRP3 inflammasome is established,and the susceptible component of EF regulating NLRP3 inflammasome activation and its liver injury effect is identified.This project first established an in vitro idiosyncratic liver injury evaluation model based on the activation of NLRP3 inflammasome.The main active components of EF were used as research object to evaluate the regulatory effects of these components on NLRP3 inflammasome activation.The results showed that ICS?,icariside?(ICS?)and epimedin B could enhance NLRP3 inflammasome activation.ICS?,as an important active and metabolic component in EF,had the strongest effect in promoting NLRP3 inflammasome activation,suggesting that ICS?may be the main susceptibility component of EF-induced idiosyncratic liver injury.To further verify whether ICS?causes idiosyncratic liver injury,the LPS model was used to evaluate its hepatotoxicity.The results showed that ICS?significantly increased the alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities in the model,accompanied by the increase in the levels of inflammatory cytokines IL-1?and TNF-?,indicating that ICS?can cause liver damage and is the susceptible component of EF-induced liver injury.2.The susceptible component ICS?promotes the NLRP3 inflammasome activation stimulated by ATP and nigericin.The established model was used to evaluate ICS?on the activation of inflammasomes induced by multiple stimuli.The results showed that ICS?can promote NLRP3 inflammasome activation stimulated by ATP and nigericin specifically.The activation of NLRP3 inflammasome induced by other NLRP3 agonists,such as MSU and Si O₂,was not affected by ICS?;in addition,ICS II had no effect on the activation of non-canonical NLRP3 inflammasome,AIM2 and NLRC4 inflammasomes.NLRP3 inflammasome activation can also lead to a kind of programmed cell death-pyroptosis.Pyroptosis is widely involved in the occurrence and development of a variety of diseases.Similarly,ICS?also significantly promoted the NLRP3inflammasome-induced pyroptosis.Therefore,ICS?-induced the increase of inflammatory cytokine IL-1?stimulated by ATP and nigericin,the recruitment of inflammatory cells,and the occurrence of pyroptosis may play an important role in the development of EF-induced liver injury.3.Clarifying the target mechanism of ICS?regulating NLRP3inflammasome activation.Further research on the target mechanism of ICS?in promoting the activation of NLRP3 inflammasome.Mitochondrial reactive oxygen species(ROS)play a vital role in the NLRP3 inflammasome activation.The results showed that mitochondrial ROS were significantly enhanced under ATP or nigericin stimulation.ICS?further promoted the production of mitochondrial ROS under the stimulation of ATP or nigericin,indicating that the production of mitochondrial ROS is a possible mechanism for its enhancement of NLRP3 inflammasome activation.Furthermore,ROS inducer H₂O₂ can promote the activation of NLRP3 inflammasome stimulated by ATP and nigericin;while ROS scavenger N-Acetylcysteine amide(NAC)can significantly inhibit the promotion effect of ICS?on NLRP3 inflammasome stimulated by ATP and nigericin.The above research results indicate that ICS?mainly promotes the production of mitochondrial ROS to enhance NLRP3 inflammasome activation triggered by ATP and nigericin.4.LPS-induced idiosyncratic liver injury model combined with Nlrp3knockout(Nlrp3^-/-)model to evaluate the correlation between ICS?-induced liver injury and NLRP3 inflammasome.The LPS model and Nlrp3^-/-mouse were established to evaluate the correlation between the liver injury induced by ICS?and NLRP3 inflammasome activation.The results showed that in the LPS-induced idiosyncratic liver injury model,ICS?significantly increased the levels of inflammatory cytokines IL-1?and TNF-?,the activity levels of ALT and AST.The activity of caspase-1 related to inflammasome activation was significantly enhanced;HE and Tunel staining of liver tissue showed that ICS?can cause hepatocyte death and inflammatory cell infiltration in mice in the LPS model,and the positive rate of Tunel was significantly increased.On the contrary,in Nlrp3^-/-mice,the same treatment was given.In the LPS model,ICS?could not cause changes in the levels of IL-1?,TNF-?,ALT,and AST.And there was no effect on caspase-1 activity and liver damage.The above results further indicate that NLRP3inflammasome is indeed involved in ICS?-induced idiosyncratic liver injury.Activating NLRP3 inflammasome-mediated IL-1?release and subsequent TNF-?release,pyroptosis and oxidative stress synergistically induces the occurrence of liver injury caused by ICS?.5.The multi-component synergistic effect of EF on enhancing NLRP3inflammasome activation is explained preliminarily.The in vitro screening model has shown that,ICS?,ICS?and epimedin B can significantly promote the activation of NLRP3 inflammasome.ICS?and ICS?coordination enhancement effect on NLRP3 inflammasome activation was carried out to evaluate EF multicomponent synergies on liver injury.The results showed that at a non-toxic dose of ICS?cannot directly activate inflammasome but could only promote the activation of NLRP3 inflammasome induced by ATP and nigericin;while ICS?not only directly induces inflammasome activation,but also promotes the activation of NLRP3 inflammasome induced by ATP and nigericin.Further studies showed that without stimuli such as ATP and nigericin,ICS?combined with ICS?can significantly enhance inflammasome activation;and the combination of the two can also promote NLRP3 inflammasome activation stimulated by ATP.The above results suggest that multiple components of EF,such as ICS?and ICS?,synergistically induce or enhance NLRP3 inflammasome activation,which inducing a strong and continuous immune inflammatory response,and maybe ultimately lead to immune idiosyncratic liver injury.6.Based on the objective recognition of EF-induced idiosyncratic liver injury,the prevention strategies of EF and its preparations hepatotoxicity and reasonable drug use recommendations are proposed.Our study has confirmed that ICS?is the main susceptible component of EF-induced idiosyncratic liver injury,and the liver injury induced by this component is closely related to the activation of NLRP3 inflammasome.To prevent and control the liver injury of EF from three aspects:body factors,drug quality and drug use,a three-dimensional warning technology system based on human-drug-use was proposed and established.In terms of body factors,the abnormal activation of NLRP3 inflammasome is the main susceptible population feature of EF-induced hepatotoxicity.In the presence of certain underlying diseases,the body releases a small amount of DAMPs,such as endotoxin LPS and ATP,nigericin.If any patients with these basic diseases taking EF related preparations,ICS?can amplify the inflammatory response and induce a more powerful abnormal activation of NLRP3 inflammasome,resulting in idiosyncratic property.However,not all abnormal activation of NLRP3 inflammasome is susceptible.In some basic diseases such as MSU-induced gout and Si O₂-induced silicosis patients,taking EF-containing preparations may not cause liver injury.In terms of drug quality,as a result of ICS?,ICS?,and epimedin B is effective components of EF,also they can also promote NLRP3 inflammasome activation.So,controlling the ingredients to make it within a certain range of content could reach the purpose to improve effect and decrease toxicity.In terms of drug use,the results of this study suggest that abnormal activation of NLRP3 inflammasome is an important mechanism of EF-induced liver injury.Therefore,targeting inhibition of NLRP3 inflammasome may be an effective strategy for the prevention and control of hepatotoxicity of EF.Cardamonin,a specific NLRP3 inflammasome inhibitor,can significantly inhibit ICS?-induced NLRP3inflammation activation,suggesting the possibility of preventing and treating ICS?-induced idiosyncratic liver injury.Furthermore,the recognized NLRP3 inflammasome-specific inhibitor MCC950 was selected to evaluate its prevention effects on ICS?-induced idiosyncratic liver injury.The results showed that the administration of MCC950 can effectively inhibit the abnormal increase of inflammatory cytokines IL-1?and TNF-?,liver function index ALT,AST in the LPS-induced idiosyncratic liver injury model,suggesting that NLRP3 inflammasome inhibitor may be a treatment strategy for EF(ICS?)-induced liver injury.The results provide candidate drugs for the treatment of liver injury caused by abnormal activation of NLRP3 inflammasome.In summary,our study aimed at the problem of EF-induced hepatotoxicity,using the in vitro screening evaluation model based on NLRP3 inflammasome and the in vivo LPS-induced idiosyncratic liver injury evaluation model to screen and evaluate the active components in EF.ICS?is a susceptible component of EF-induced idiosyncratic liver injury.We explain the characteristics of ICS?in promoting NLRP3inflammasome,the target mechanism and the objective truthfulness of the occurrence of liver injury.The multi-component synergistic effect of EF on regulating NLRP3inflammasome activation is preliminarily clarified.Accordingly,a three-dimensional warning technology system and risk prevention and control strategy of EF-induced liver injury based on human-drug-use were proposed and established.This study provides a reference for the drug screening and evaluation of the abnormal activation of NLRP3inflammasome,and also provides research basis and treatment plan for the prevention of liver injury caused by abnormal activation of NLRP3 inflammasome.