A model for evaluating idiosyncratic liver injury and its application

Abstract

The present application discloses an evaluation model for idiosyncratic liver injury. The evaluation model includes the AIM2 inflammasome and components that promote the activation of the AIM2 inflammasome. Among them, the component that promotes bakuchiol as an immune promoter to induce the release of mitochondrial DNA, thereby leading to the activation of the AIM2 inflammasome is bakuchiol. The present application also provides a method for constructing the evaluation model. The construction method includes: preparing a solution of bakuchiol monomer samples; pretreating primary macrophages derived from mouse bone marrow with LPS and adding LPS for induction for 4 hours; adding the bakuchiol monomer solution to primary macrophages derived from mouse bone marrow for stimulation; performing Western blot to detect proteins related to the activation of the AIM2 inflammasome, ELISA to detect the release of inflammatory factors IL-1β and TNF-α, and performing statistical analysis.

Description

Technical Field

[0001] This article relates to the field of drug screening, and in particular but not limited to, relates to a model for screening and evaluating idiosyncratic liver injury drugs and the application of this model in screening the components causing idiosyncratic liver injury in Psoralea corylifolia and screening drugs that can be used to treat idiosyncratic liver injury caused by Psoralea corylifolia. Background Art

[0002] Drug-induced liver injury is a serious drug adverse reaction and the main reason for drug R & D failure or withdrawal from the market. As a type of liver injury that is difficult to predict, treat, and prone to recurrence, idiosyncratic drug-induced liver injury threatens the lives of patients and increases the economic burden on families. Therefore, it is urgent to clarify the relevant scientific mechanisms of idiosyncratic liver injury to provide a scientific basis for rational clinical drug use and avoiding harm while seeking benefits. Currently, some studies have shown that idiosyncratic liver injury has the characteristic of rapid onset upon re-challenge, is a typical immune-mediated reaction, and histological examinations indicate a strong immune-mediated reaction.

[0003] The melanoma absent factor 2 (AIM2)-like receptor (ALR) family, and the AIM2 inflammasome containing a HIN domain and a pyrin domain (PYD) is the first protein member in the ALRs family with characteristics in innate immune sensing. AIM2 is expressed in the cytoplasm, can bind to foreign double-stranded DNA (dsDNA) and self dsDNA to form a DNA-AIM2 complex, recruit ASC and procaspase-1 precursor, and induce the activation of procaspase-1. Activated Caspase-1 can, on the one hand, induce pyroptosis, and on the other hand, induce the cleavage and activation of pro-IL-1β and pro-IL-18 to form IL-1β and IL-18, recruiting immune cells such as neutrophils, thus participating in the occurrence and development of many human diseases. However, whether drugs causing idiosyncratic liver injury can use the AIM2 inflammasome activation model (i.e., the effect of drugs on the AIM2 inflammasome under LPS stimulation) to evaluate idiosyncratic liver injury has not been reported.

[0004] Idiosyncratic liver injury caused by traditional Chinese medicine is one of the common and serious adverse reactions in the clinical application of traditional Chinese medicine. Although its incidence is usually low, severe cases can lead to acute liver failure and even death, which has attracted great attention from the medical community, pharmaceutical industry, regulatory authorities, and the public. Due to the public's misunderstanding that traditional Chinese medicine is "naturally safe and has no toxic side effects", as well as factors such as its complex composition, weak research foundation, common combined drug use, etc., its liver injury is often more concealed, making the evaluation of idiosyncratic liver injury caused by traditional Chinese medicine more difficult than that of chemical drugs.

[0005] Psoralea corylifolia, as a traditional non-toxic and tonifying Chinese medicine, was first recorded in *Leigong's Treatise on Processing of Medicinal Herbs*, and has the effects of tonifying kidney yang, strengthening bones and muscles, and dispelling wind and dampness. However, in recent years, there have been frequent reports of liver injury caused by Psoralea corylifolia and related preparations. Among them, Zhuanggu Guanjie Pills and Xianling Gubao Capsules have been repeatedly reported by the National Adverse Reaction Center. At present, there have been many studies on the hepatotoxicity of Psoralea corylifolia and its related preparations. One study found that fatty degeneration of the liver was visible after continuously intragastric administrating mice with Psoralea corylifolia for 6 months. The results showed that Psoralea corylifolia has potential hepatotoxicity, which is closely related to the extraction process, dosage, and treatment course. Another study showed that high-dose bakuchiol can significantly cause elevated alanine aminotransferase and aspartate aminotransferase, as well as liver tissue lesions. How to objectively understand the problem of liver injury caused by Psoralea corylifolia is a difficult point in the current research on the safety of traditional Chinese medicine. Summary of the Invention

[0006] The following is an overview of the subject matter described in detail in this document. This overview is not intended to limit the scope of protection of the claims.

[0007] Existing studies on idiosyncratic liver injury are mostly based on the traditional toxicological evaluation model, and the body state has not been taken as the core consideration factor. Based on the immune response characteristics of idiosyncratic liver injury and the important role of AIM2 inflammasome in idiosyncratic liver injury, this application established a drug idiosyncratic liver injury model and explored its application.

[0008] Specifically, this application provides an evaluation model for immune idiosyncratic liver injury, and the evaluation model includes AIM2 inflammasome and components that promote the activation of AIM2 inflammasome. Among them, the component that promotes the activation of AIM2 inflammasome is bakuchiol.

[0009] This application also provides a method for constructing an in vitro evaluation model for immune idiosyncratic liver injury, and the construction method includes:

[0010] S100. Evaluation at the cellular level;

[0011] S101. Preparation of a sample solution of the monomer component of Psoralea corylifolia;

[0012] S102. Prepare primary bone marrow macrophages of mice and stimulate them with lipopolysaccharide (LPS);

[0013] S103. Add the solution prepared in step S101 to the primary bone marrow macrophages of mice for stimulation;

[0014] S104. Detect the expression of AIM2 inflammasome-related proteins by Western blot, detect the contents of IL-1β and TNF-α in the cell supernatant by ELISA, and perform statistical analysis.

[0015] In this application, the psoralen monomer components may be selected from any one or more of angelicin, bakuchiol, bavachin, bavachinin, corylin, daidzin, isobavachin, isobavachalcone, neobavaisoflavone, psoralidin, psoralen, 5-methoxypsoralen, and 8-methoxypsoralen.

[0016] In this application, step S100 may further include: screening the components in the psoralen monomer components that promote the activation of the AIM2 inflammasome, and further verifying the activation effect of this component on the AIM2 inflammasome at a concentration of 40 μM.

[0017] In this application, in step S101, take the psoralen monomer component, dissolve it in dimethyl sulfoxide to 40 mM, store it at -20 °C for later use, and dilute it to 40 μM with Opti-MEM before use.

[0018] This application also provides a method for constructing an in vivo evaluation model of immune idiosyncratic liver injury, and the construction method includes:

[0019] S100. Evaluation at the cellular level;

[0020] S101. Preparation of the psoralen monomer component sample solution;

[0021] S102. Prepare primary mouse bone marrow macrophages and stimulate them with LPS;

[0022] S103. Add the solution prepared in step S101 to primary mouse bone marrow macrophages for stimulation;

[0023] S104. Detect the expression of AIM2 inflammasome-related proteins by Western blot, detect the contents of IL-1β and TNF-α in the cell supernatant by ELISA, and perform statistical analysis;

[0024] S200. Evaluation at the animal level;

[0025] S201. Inject LPS into the tail vein of mice;

[0026] S202. Inject a solution of the component that promotes the activation of the AIM2 inflammasome intraperitoneally;

[0027] S203. Determine the contents of ALT, AST, IL-1β, and TNF-α in the serum, determine the contents of IL-1β, IL-18, and TNF-α in the liver tissue, and perform H&E staining on the liver.

[0028] In this application, the administration dose of the monomer component of Psoralea corylifolia can be 15 mg / kg - 30 mg / kg. Preferably, the monomer component of Psoralea corylifolia is bakuchiol.

[0029] In this application, mg / kg refers to the dosage in mg per kg of the animal body weight.

[0030] In this application, the administration time of the monomer component of Psoralea corylifolia is 2 hours after lipopolysaccharide injection.

[0031] In this application, the administration dose of lipopolysaccharide is 2 mg / kg.

[0032] This application also provides the use of bakuchiol as an AIM2 inflammasome activator, such as the use of bakuchiol as an immune adjuvant.

[0033] This application also provides the use of bakuchiol in the evaluation of drug idiosyncratic liver injury.

[0034] The advantage of this application is that in this model method, under the stimulation of LPS, the AIM2 inflammasome is not activated. When the target component causing idiosyncratic hepatotoxicity is administered, it induces the activation of the AIM2 inflammasome, and the pro-inflammatory factor IL-1β increases significantly, thereby mediating a severe immune inflammatory response and further inducing liver injury.

[0035] This model method has not been used for the prediction, screening, and evaluation of drug immune idiosyncratic liver injury before. This method can be used for the prediction, screening, and evaluation of a series of drugs causing immune idiosyncratic liver injury.

[0036] This application combines the AIM2 inflammasome activation model and the LPS-induced idiosyncratic liver injury model for the screening and evaluation of drugs with immune idiosyncratic liver injury. Under the guidance of this method, the chemical component in Psoralea corylifolia with immune idiosyncratic liver injury was screened out as bakuchiol.

[0037] The screening and evaluation method of this application is simple and easy to perform, and it consumes less manpower and financial resources, has a short experimental period, and a high model formation rate. It is applicable to the study of the pathogenesis of drug idiosyncratic liver injury and the evaluation of drugs with idiosyncratic liver injury, providing a stable and reliable animal model for the expansion of drug idiosyncratic liver injury treatment strategies and new drug research and development.

[0038] The AIM2 inflammasome activation model and the idiosyncratic liver injury model induced by LPS provided in this application can rapidly evaluate the immune idiosyncratic hepatotoxicity of drugs, and provide references for drug hepatotoxicity evaluation models, research on scientific mechanisms, and clinical applications.

[0039] Other features and advantages of this application will be described in the following specification. Moreover, some of them will become obvious from the specification, or be understood by implementing this application. Other advantages of this application can be realized and obtained through the solutions described in the specification and the accompanying drawings. Brief Description of the Drawings

[0040] The drawings are used to provide an understanding of the technical solutions of this application, and constitute a part of the specification. Together with the embodiments of this application, they are used to explain the technical solutions of this application, and do not constitute a limitation to the technical solutions of this application.

[0041] Figure 1 : In primary mouse bone marrow macrophages, after pretreatment with lipopolysaccharide, the medium was replaced with the monomer components of psoralen (40 μM) prepared with serum-free opti-MEM, and then the cell supernatant and cell lysate were collected for detection of relevant indicators. The protein expression of caspase-1 p20 was detected in the cell supernatant, and commassie was used as the loading control. The protein expressions of pro-IL-1β, caspase-1 p45, NLRP3, and ASC were detected in the cell lysate, and GAPDH was used as the loading control. The bar graphs are the contents of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the cell supernatant, respectively.

[0042] Figure 2 : In mouse bone marrow-derived macrophages, after pretreatment with lipopolysaccharide, the medium was replaced with bakuchiol (10, 20, 40 μM) prepared with serum-free opti-MEM. The protein expression of caspase-1 p20 was detected in the cell supernatant, and commassie was used as the loading control. The protein expressions of pro-IL-1β, caspase-1 p45, and ASC were detected in the cell lysate, and LaminB was used as the loading control. The bar graphs are the contents of interleukin 1β and tumor necrosis factor α in the cell supernatant, respectively.

[0043] Figure 3: In mouse bone marrow-derived macrophages, after pretreatment with lipopolysaccharide, the medium was replaced with psoralen (40 μM) prepared with serum-free opti-MEM and incubated for 0.25 h, 0.5 h, 1 h, and 2 h respectively. The protein expression of caspase-1 p20 was detected in the cell supernatant, and commassie was used as the loading control. The protein expressions of pro-IL-1β, caspase-1 p45, and ASC were detected in the cell lysate, and Lamin B was used as the loading control. The bar graphs represent the contents of interleukin 1β and tumor necrosis factor α in the cell supernatant respectively.

[0044] Figure 4 : Psoralen activates the AIM2 inflammasome rather than the NLRP3 inflammasome. Wild-type and Nlrp3 - / - knockout BMDMs were primed with LPS for 4 hours and then treated with psoralen for 1 hour; BMDMs were induced with LPS for 4 hours and then treated with inhibitors of various inflammasomes (MCC950, echinatin, ODN) for 1 hour and then treated with psoralen for 1 hour. Western blot analysis of pro-caspase-1 (p45), pro-IL-1β, and ASC in whole cell lysates (WCL); secretion of activated caspase-1 (p20) in the BMDM culture supernatant was shown. Coomassie blue staining was used as the loading control for the supernatant, while GAPDH was used as the loading control for the lysate. iBMDMs were transfected with four siRNAs targeting AIM2 (siRNA#1, #2, #3, #4) or control RNA for 24 h, induced with LPS for 4 hours, and then treated with BAK or poly(dA:dT) for 1 hour. Western blot analysis of pro-caspase-1 (p45), pro-IL-1β, and ASC in whole cell lysates (WCL); secretion of activated caspase-1 (p20) in the BMDM culture supernatant was shown. The bar graphs represent the inhibitory effects of each siRNA on AIM2 determined by RT-PCR, and interleukin 1β in the cell supernatant determined by Elisa kit.

[0045] Figure 5 : ASC was detected in the cell lysate, and GAPDH was used for loading quantification; the bar graphs represent the ratios of ROS release and JC-1 green fluorescence respectively; the structural integrity of mitochondria and the localization of AIM2 (red fluorescence) and mtDNA (green fluorescence) were detected by Zeiss LSM800.

[0046] Figure 6:After mice were injected with lipopolysaccharide (2 mg / kg) via the tail vein, they were treated with different concentrations of bakuchiol (15, 30 mg / kg), and then the contents of alanine aminotransferase, aspartate aminotransferase, interleukin-1β, interleukin-18, and tumor necrosis factor-α in the serum were measured. The large liver lobes were used for H&E staining. After protein extraction from the liver lobules, the activation of caspase-1 in the liver tissue was determined by immunoblotting. Detailed implementation manners

[0047] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of protection of the claims.

[0048] Most of the existing studies on idiosyncratic liver injury are based on traditional toxicology evaluation models and do not take the body state as the core consideration factor. Based on the immune response characteristics of idiosyncratic liver injury and the important role of AIM2 inflammasome in idiosyncratic liver injury, this research method establishes a drug idiosyncratic liver injury model and explores its application. The technical problem to be solved by the present invention is to provide an animal model induced by a liver toxicant formula, which is applicable to conventional experimental animals, is simple and easy to perform, can activate AIM2, and has low labor and financial costs, a short experimental period, and a high model formation rate. It is applicable to the study of the pathogenesis of drug idiosyncratic liver injury and the evaluation of drugs for idiosyncratic liver injury, providing a stable and reliable animal model for the expansion of treatment strategies for drug idiosyncratic liver injury and the research and development of new drugs.

[0049] Specifically, the present application provides an evaluation model for immune idiosyncratic liver injury, and the evaluation model includes AIM2 inflammasome and components that promote the activation of AIM2 inflammasome. Among them, the component that promotes the activation of AIM2 inflammasome is bakuchiol.

[0050] In the present application, the evaluation model is constructed using AIM2 inflammasome and components that promote the activation of AIM2 inflammasome.

[0051] In the present application, the evaluation model can also be constructed using AIM2 inflammasome and components that promote the activation of AIM2 inflammasome as markers.

[0052] The present application also provides a method for constructing an in vitro evaluation model for immune idiosyncratic liver injury, and the construction method includes:

[0053] S100. Evaluation at the cellular level;

[0054] S101. Preparation of a solution of bakuchiol monomer sample;

[0055] S102. Preparation of primary mouse bone marrow macrophages and stimulation with lipopolysaccharide (LPS).

[0056] S103. Add the solution prepared in step S101 to primary murine bone marrow macrophages for stimulation;

[0057] S104. Detect the expression of AIM2 inflammasome-related proteins by Western blot, detect the contents of IL-1β and TNF-α in the cell supernatant by ELISA, and perform statistical analysis.

[0058] In the present application, the psoralen monomer components may be selected from any one or more of angelicin, bakuchiol, bavachin, bavachinin, corylin, daidzin, isobavachin, isobavachalcone, neobavaisoflavone, psoralidin, psoralen, 5-methoxypsoralen, and 8-methoxypsoralen.

[0059] In the present application, step S100 may further include: screening the components in psoralen that promote the activation of the AIM2 inflammasome, and further verifying the activation effect of this component on the AIM2 inflammasome at a concentration of 40 μM.

[0060] In the present application, in step S101, take the psoralen monomer component, dissolve it in dimethyl sulfoxide to 40 mM, store it at -20 °C for standby, and dilute it to 40 μM with Opti-MEM before use.

[0061] The present application also provides a method for constructing an in vivo evaluation model of immune idiosyncratic liver injury, and the construction method includes:

[0062] S100. Evaluation at the cellular level;

[0063] S101. Preparation of a psoralen monomer sample solution;

[0064] S102. Prepare primary murine bone marrow macrophages and add LPS for stimulation;

[0065] S103. Add the solution prepared in step S101 to primary murine bone marrow macrophages for stimulation;

[0066] S104. Detect the expression of AIM2 inflammasome-related proteins by Western blot, detect the contents of IL-1β and TNF-α in the cell supernatant by ELISA, and perform statistical analysis;

[0067] S200. Animal-level evaluation;

[0068] S201. Inject LPS into the tail vein of mice;

[0069] S202. Intraperitoneally inject a solution of a component that promotes the activation of the AIM2 inflammasome;

[0070] S203. Measure the contents of ALT, AST, IL-1β, and TNF-α in the serum, measure the contents of IL-1β, IL-18, and TNF-α in the liver tissue, and perform H&E staining of the liver.

[0071] In this application, the administration dose of bakuchiol can be 15 mg / kg - 30 mg / k.

[0072] In this application, mg / kg refers to the administration amount in mg per kg of animal body weight.

[0073] In this application, the administration time of bakuchiol is 2 hours after lipopolysaccharide injection.

[0074] In this application, the administration dose of lipopolysaccharide is 2 mg / kg.

[0075] This application also provides the use of bakuchiol as an AIM2 inflammasome activator, such as the use of bakuchiol as an immune adjuvant.

[0076] This application also provides the use of bakuchiol in evaluating drug-induced idiosyncratic liver injury.

[0077] To make the purpose, technical solution, and advantages of this application clearer, the embodiments of this application will be described in detail below with reference to the accompanying drawings. It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other arbitrarily.

[0078] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0079] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0080] Materials and Methods

[0081] This study mainly evaluated the activation effect of bakuchiol on the AIM2 inflammasome and explored the mechanism of its activation of the AIM2 inflammasome. The effect of bakuchiol on the activation of the AIM2 inflammasome in BMDM and its potential mechanism of action. Its activation effect was evaluated by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence.

[0082] Mice (8-week-old C57BL / 6 mice) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). The researchers were blinded to the animal experiments. Mice were randomly selected and grouped and housed in a specific sterile facility (12-hour / 12-hour light / dark cycle; 20 ± 2 °C). All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of the Fifth Medical Center of the Chinese PLA General Hospital.

[0083] Reagents

[0084] Bakuchiol, MCC950, and protease inhibitors were obtained from Topscience (Shanghai, China). Ultra-pure LPS, DMSO, poly(dA:dT), and nigericin were produced by Sigma. Anti-mouse CASPASE-1 (AG-20B-0042), 24 anti-mouse ASC (sc-51441), and anti-GAPDH (60004-1-1g) were from Adipogen (USA); anti-mouse AIM2 (66066S), anti-mouse IL-1β (12507) were purchased from Bioss (China).

[0085] Cell culture

[0086] Mice were immersed in 75% alcohol after euthanasia. Bone marrow-derived macrophages (BMDMs) isolated from femurs were cultured in DMEM (Macgene, Beijing, China) containing FBS (10% [v / v]; Gibco, Rockford, IL, USA), penicillin / streptomycin (1%, 100 U / mL; Sigma, Darmstadt, Germany), and M-CSF (50 ng / ml; MedChemExpress, Monmouth, NJ, USA). Cells were used for experiments 2 days after the second replenishment of the culture medium. Immortalized wild-type mouse bone marrow macrophages (iBMDMs) were cultured in DMEM supplemented with FBS (10% [v / v]) and penicillin / streptomycin (1%, 100 μg / mL). All cells were cultured at 37 °C in 5% carbon dioxide.

[0087] Cell activation

[0088] BMDMs (1.0 × 10 6 cells / mL) and iBMDMs (2.0 × 10 6Cells / mL) were seeded in 24-well plates overnight, fresh medium was replaced, and the cells were stimulated with LPS (50 ng / mL) for 4 hours. Then, the medium was replaced with Opti-MEM containing bakuchiol for 1 hour. Lipofectamine 2000 (Sigma) was used to transfect poly(dA:dT) (2 μg / mL) into BMDMs to activate the AIM2 inflammasome.

[0089] Detection by microplate reader

[0090] The production of IL-1β and TNF-α in the cell supernatants was determined using mouse IL-1β and mouse TNF-α ELISA kits (Dakewe, Beijing, China).

[0091] Lactate dehydrogenase (LDH) assay

[0092] The release of LDH reflects the occurrence of cell pyroptosis. According to the manufacturer's instructions, the production of LDH was evaluated using a non-radioactive cytotoxicity assay (G1780, Progen).

[0093] Knockdown of AIM2 with siRNAs

[0094] iBMDMs were seeded in 24-well plates at a density of 1.0×106 cells per well. After 2 hours of culture, the cells were transfected with siRNAs against AIM2 (2 μg / mL) or control siRNA (Sigma) using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA). Twenty-four hours after transfection, the iBMDMs were stimulated with 50 ng / mL LPS. Four hours later, the cells were stimulated with Bak and poly(dA:dT) respectively.

[0095] The sequences of the siRNAs were as follows:

[0096] #1: AGUACUAAGAAAUCAGUGAdTdT, UCACUGAUUUCUUAGUACUdTdT;

[0097] #2: CAAAUGCUCUUGAAGAGAAdTdT, UUCUCUUCAAGAGCAUUUGdTdT;

[0098] #3: CAGUUGUGGUUGAUGUUGAdTdT, UCAACAUCAACCACAACUGdTdT;

[0099] #4: GUAUGGUAUACAUGAUAAAdTdT, UUUAUCAUGUAUACCAUACdTdT.

[0100] Western blot assay:

[0101] Detect the protein expression level of caspase-1 (P20) in the supernatant, and detect the protein levels of ASC, caspase-1 (P45), Pro-IL-1β, GAPDH, and LaminB in the cell lysate. The protein samples in the cell supernatant were subjected to SDS-PAGE gel electrophoresis with a 12% concentration, and the cell lysate samples were subjected to SDS-polyacrylamide gel electrophoresis with a 10% concentration. The power supply was turned on, and a constant voltage of 80V was set. After 1h, the voltage was changed to 135V. When the bromophenol blue approached the bottom of the separating gel, the electrophoresis was ended, and the transfer device was installed. After the electrophoresis of the cell supernatant was completed, the gel was cut along the 45kDa position of the marker. The part above 45kDa was stained with Coomassie Brilliant Blue for 45min, and the decolorizing solution (water: methanol: acetic acid (v:v:v) = 5:4:1) was used until the background blue became transparent; the remaining part was transferred to a PVDF membrane. After completion, it was blocked at room temperature in 5% skim milk for 1h. After incubating overnight on a shaker at 4°C with caspase-1 (P20), IL-1β, caspase-1 (P45), Pro-IL-1β, ASC, GAPDH, and LaminB antibodies, the antibodies were recovered. The PVDF membrane was washed 3 times with 1% TBST solution for 5min each time, and then incubated with the corresponding labeled mouse or rabbit secondary antibody (1:5000, according to the instructions) solution for 1h. The secondary antibody was discarded, and the membrane was washed 3 times with 1% TBST solution at 5min intervals. After mixing equal volumes of the two reagents of the developing solution A and B, they were immediately added to the membrane, and the membrane was developed by pressing an X-ray film in a darkroom for exposure.

[0102] Detection of RNA expression

[0103] According to the manufacturer's instructions, RNA was extracted from stimulated iBMDMs or liver tissues using Trizol reagent (Invitrogen), and then the concentration of RNA was detected using a nanodrop 2000. Subsequently, the charged RNA was transcribed into cDNA using the StarScript II First-strand cDNA Synthesis Kit (A212-02, Genstar, Beijing, China), and RT-PCR analysis was performed using SYBR Green qPCR Master Mix (HY-K0522, MCE) and primers.

[0104] Based on the LPS model, it was clarified that LPS / bakuchiol could cause severe idiosyncratic liver injury

[0105] C57BL / 6 female mice aged 6 - 8 weeks were injected with LPS (2 mg / kg) via the tail vein. After 2 h, bakuchiol (15 or 30 mg / kg) was injected intraperitoneally. After 6 h, blood was collected from the orbital cavity of the mice and centrifuged at 3500 rpm / min to measure the contents of ALT and AST in the serum. The mice were dissected, the large liver lobes were placed in 5% formalin, and the liver lobule tissues were homogenized to extract proteins. HE staining was used to detect hepatocyte injury.

[0106] Results:

[0107] 1. Effects of various monomeric components of Psoralea corylifolia on inflammasome activation

[0108] In BMDMs cells, the cells were first treated with LPS (50 ng / ml) for 4 h, and then the medium was replaced with various monomeric components of Psoralea corylifolia (40 μM) prepared with serum-free Opti-MEM and treated for 1 h. The cell supernatants and lysates were collected for detection of relevant indicators. It can be seen from Figure 1 this that under LPS stimulation, caspase-1 and IL-1β proteins were not expressed in the cell supernatant, indicating that the inflammasome was not activated; under the co-stimulation of LPS and monomeric components of Psoralea corylifolia, caspase-1 protein was expressed in the cell supernatant, and the measurement results of the content of IL-1β by ELISA kit showed that both bakuchiol and psoralidin promoted inflammasome activation. This indicates that this model can evaluate idiosyncratic liver injury drugs or components and can be used for the screening and evaluation of drugs or components related to idiosyncratic liver injury.

[0109] 2. Bakuchiol dose-dependently induced the maturation of caspase-1 and the production of IL-1β and TNF-α, confirming the up-regulatory effect of Bak-induced inflammasome activation.

[0110] 3. Bakuchiol time-dependently induced the maturation of caspase-1 and the production of IL-1β and TNF-α, confirming the up-regulatory effect of Bak-induced inflammasome activation.

[0111] 4. Bakuchiol in wild-type and AIM2 - / -All BMDMs can activate the activation of caspase-1. Neither echinatin nor MCC950 affects the maturation of caspase-1 and the secretion of IL-1β induced by bakuchiol. On the contrary, ODN (known as a specific AIM2 inflammasome inhibitor) can significantly inhibit the inflammasome activation induced by Bak. In addition, after iBMDMs were transfected with four different siRNAs (siRNA#1, #2, #3, #4) targeting AIM2 or control for 24 hours, induced with LPS for 4 hours, and then treated with bakuchiol or poly(dA:dT) for 30 minutes, the results showed that siRNAs (siRNA#2, #3, #4) had a significant inhibitory effect on AIM2. At the same time, the siRNA targeting AIM2, rather than the control RNA, significantly inhibited the maturation of caspase-1 and the production of IL-1β induced by bakuchiol and poly(dA:dT).

[0112] 5. Bakuchiol acts upstream of inflammasome activation, promotes ASC oligomerization, induces the release of mtDNA and its subsequent binding to the AIM2 receptor, thereby activating the AIM2 inflammasome.

[0113] Bakuchiol induces ASC oligomerization; bakuchiol can induce mitochondrial damage; when the mitochondrial membrane potential decreases, the JC-1 kit shows green fluorescence, and the green fluorescence increases with the increase in the concentration of bakuchiol, indicating that bakuchiol can reduce the mitochondrial membrane potential; in addition, bakuchiol can also induce the production of mitochondrial ROS in a concentration-dependent manner; after LPS-induced BMDMs were treated with bakuchiol and the positive drug poly(dA:dT) for half an hour, DNA antibody (green) and AIM2 antibody (red) were added respectively. In the case of bakuchiol and the positive drug, DNA and AIM2 formed co-localization, but not in the negative control group. It shows that bakuchiol induces the activation of the AIM2 inflammasome by promoting the release of mtDNA.

[0114] 6. The idiosyncratic liver injury model induced by LPS further confirmed the liver injury effect of bakuchiol, the biomarker of idiosyncratic liver injury in Psoralea corylifolia L.

[0115] Compared with the control group, the serum levels of ALT, AST, IL-1β and TNF-α in the LPS group were significantly increased (P < 0.05), the activation of caspase-1 in the liver was weak, and the liver tissue showed mild injury. There was no significant difference between the single-dose groups (15, 30 mg / kg) and the control group. Compared with the control group, there were no changes in serum AST, IL-1β and TNF-α in the single-dose group (100 mg / kg), caspase-1 in the liver was not activated, and no injury was observed in the liver tissue, indicating that bakuchiol at this concentration did not produce liver toxicity. Compared with the LPS group, the serum levels of ALT, AST, IL-1β and TNF-α in the LPS + single-dose groups (15, 30 mg / kg) were significantly increased (P < 0.001). With the increase of concentration, the expression of caspase-1 in the liver increased and was accompanied by aggravated liver tissue injury, indicating that in the idiosyncratic liver injury model induced by LPS, bakuchiol caused liver injury, and this injury was accompanied by the increase of inflammatory factors IL-1β and TNF-α, suggesting that bakuchiol is indeed the main chemical component leading to idiosyncratic liver injury of psoralea.

[0116] This application describes multiple embodiments, but the description is exemplary rather than restrictive, and it will be apparent to those of ordinary skill in the art that there can be more embodiments and implementation schemes within the scope of the embodiments described in this application. Although many possible combinations of features are shown in the drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically restricted, any feature or element of any embodiment can be combined with any other feature or element in any other embodiment, or can replace any other feature or element in any other embodiment.

Claims

1. Use of bakuchiol in the preparation of an AIM2 inflammasome activator, wherein the AIM2 inflammasome activator is used for mouse bone marrow macrophages treated with LPS to prepare an evaluation model for immune idiosyncratic liver injury.

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