RNAi for reducing hepatotoxicity of triptolide and application of RNAi

Abstract

The invention discloses RNAi for reducing the hepatotoxicity of triptolide and application of the RNAi, and belongs to the technical field of biological medicine. According to the invention, siRNA of a targeted Cyp2e1 gene is subjected to 2 '-O methyl modification and dTsdT 3' phosphorothioate connection modification, and then is delivered through lipid nanoparticles (LNPs). After intravenous injection administration, mouse liver Cyp2e1 protein expression can be reduced by 70% or above within 24 h, and subacute liver injury induced by triptolide can be remarkably relieved by reducing the level of serum glutamic-pyruvic transaminase / aspartate transaminase, inhibiting liver oxidative stress and reducing release of inflammatory factors such as TNF-alpha / IL-1beta / IL-6. According to the invention, the defect that the drug effect is reduced or the targeting property is poor in the existing toxicity attenuation scheme is overcome, a clinical auxiliary toxicity attenuation technical scheme with remarkable targeting regulation effect and high safety is provided, and a new path is provided for safe clinical application of triptolide.

Description

Technical Field

[0001] This invention relates to RNAi for reducing the hepatotoxicity of triptolide and its applications, belonging to the field of biomedical technology. Background Technology

[0002] Tripterygium wilfordii Hook.f., a traditional Chinese medicine, is the root of the plant Tripterygium wilfordii, belonging to the Celastraceae family. It was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It is bitter and pungent in taste, highly toxic, and enters the liver and kidney meridians. It possesses significant anti-inflammatory and immunomodulatory functions and is widely used clinically to treat autoimmune and inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. With the widespread clinical application of Tripterygium wilfordii and its preparations, its toxic side effects have received increasing attention. Studies on the toxicity of Tripterygium wilfordii have found that hepatotoxicity is the most common cause of multi-organ damage induced by it. Tripterygium wilfordii has a complex chemical composition, mainly consisting of sesquiterpenes, diterpenes, triterpenes, and alkaloids. Among these, the diterpenoid component triptolide (TP) is its main active ingredient; and the quality control component of currently marketed Tripterygium wilfordii tablets and double-layer tablets is TP. Therefore, the treatment of TP-induced hepatotoxicity is extremely important.

[0003] It has been reported that the mechanism of TP-induced hepatotoxicity is complex, with oxidative stress being the main mechanism of TP-induced liver injury. Numerous in vitro and in vivo studies have shown that TP can significantly increase the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in HepG2, L-02 cells, and in the liver tissues of rats and mice, while significantly downregulating the activities of antioxidant enzymes such as glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), thus inducing oxidative stress and mediating liver injury.

[0004] Currently, treatment options for hepatotoxicity caused by phlebitis (TP) are extremely limited. Clinically, TP is often used in combination with the glucocorticoid dexamethasone to prevent hepatotoxicity. However, dexamethasone is a commonly used hepatic enzyme inducer that can significantly induce the expression of CYP3A4 in the human body. TP is metabolized by CYP3A4 in the human body; therefore, while using it in combination with dexamethasone reduces the incidence of hepatotoxicity, it also shortens the duration of TP's effects. Therefore, finding new methods to treat TP-induced hepatotoxicity is extremely important.

[0005] CYP2E1 is one of the main metabolic enzymes in the liver, significantly influencing oxidative stress and metabolic transformation. Studies have shown that troponin (TP) can induce CYP2E1 expression, thereby causing oxidative stress and inflammatory responses in vivo. In mice, administration of 500 μg / kg TP via gavage for two weeks, combined with treatment with the CYP2E1-specific inhibitor Clomethiazole (CMZ) for two weeks, significantly reduced TP-induced hepatotoxicity. Therefore, CYP2E1 holds promise as a therapeutic target for TP-induced hepatotoxicity. While CMZ offers some relief for TP-induced hepatotoxicity, as a small molecule inhibitor, it also inhibits CYP2A6 along with CYP2E1, posing risks of drug interactions and CYP2A6-mediated endogenous substance disturbances in clinical applications. Therefore, finding new CYP2E1-specific inhibitors holds promise for alleviating TP-induced hepatotoxicity.

[0006] It is noteworthy that although CYP2E1 plays a role in various types of liver poisoning, such as alcoholic liver injury and acetaminophen-induced liver injury, the molecular mechanisms of TP-induced hepatotoxicity differ significantly from those of these liver injuries. Specifically, the activation pathways of CYP2E1 differ: TP, as a diterpenoid active ingredient unique to Tripterygium wilfordii, can mediate the transcriptional activation of CYP2E1 by directly binding to hepatocyte nuclear receptors (such as CAR and PXR), without relying on indirect induction by metabolic intermediates (such as acetaldehyde produced from alcohol metabolism) or toxic intermediates (such as NAPQI produced from acetaminophen metabolism); while in other CYP2E1-related liver injuries, CYP2E1 is mostly "metabolic-dependently activated," and its upregulation is a secondary effect of the metabolic process of toxic substances. Summary of the Invention

[0007] Technical issues The technical problem to be solved by the present invention is to provide a drug that reduces the hepatotoxicity of triptolide, so as to solve the problems of high incidence of hepatotoxicity in the current clinical application of triptolide, and the reduced efficacy and short duration of efficacy of existing attenuation regimens (such as dexamethasone combination and CYP2E1 small molecule inhibitors).

[0008] Technical solution This invention provides a siRNA that targets and inhibits CYP2E1, which is formed by annealing two single-stranded RNAs to create a double-stranded siRNA: ccAuGuAcAcAAuGGAAAA, UUUUCcAUUGUGuAcAUGG.

[0009] The present invention also provides a derivative of the siRNA that targets and inhibits CYP2E1. Two single-stranded RNAs are modified with 2′-O methyl groups and dTsdT 3′ thiophosphate linkages. The resulting single-stranded RNA sequences are: si-P450 2e1-F:ccAuGuAcAcAAuGGAAAAdTsdT; si-P450 2e1-R: UUUUCcAUUGUGuAcAUGGdTsdT.

[0010] This invention provides a drug delivery system for siRNA or its derivatives that target and inhibit CYP2E1. The system can be lipid nanoparticles comprising an oil phase and an aqueous phase. The oil phase uses anhydrous ethanol as a solvent, and the solute is a mixture of D-Lin-MC3-DMA: distearate lecithin choline (DSPC): high-purity cholesterol (CHO-HP): 14:0 PEG2000 PE in a molar ratio of 50 / 10 / 38.5 / 1.5. The aqueous phase uses 10 mM sodium acetate buffer as a solvent, and the solute is the siRNA that targets and inhibits CYP2E1. The siRNA is modified with a 2′-O methyl group and linked to dTsdT 3′ thiophosphate.

[0011] The present invention provides a drug for reducing the hepatotoxicity of triptolide, which contains the lipid nanoparticles and can be administered by intravenous injection at a dose of 0.5 mg / kg (calculated as siRNA).

[0012] Beneficial effects This study investigated the therapeutic effect and related mechanisms of siRNA delivery via lipid nanoparticles to target and regulate the expression of Cyp2e1 in the liver of mice, aiming to explore its efficacy against TP-induced hepatotoxicity. No existing techniques utilize RNAi technology to target... Cyp2e1 There are related reports on genes, and on the reduction of triptolide hepatotoxicity through lipid nanoparticle delivery. The present invention's si- Cyp2e1 LNPs exhibit significantly superior inhibitory effects on Cyp2e1 protein: within 24 hours of administration, Cyp2e1 protein expression in mouse liver was knocked down by more than 70%, and the duration of action (inhibition rate of more than 50%) lasted for up to one week. Furthermore, the siRNA of this invention, through artificial synthesis and 2′-O methyl modification and dTsdT 3′ thiophosphate linkage modification, greatly enhances its in vivo stability. This invention provides a more efficient targeted regulatory tool for specific intervention in TP hepatotoxicity. Attached Figure Description

[0013] Figure 1 Construction of CYP2E1 overexpression cell lines.

[0014] Figure 2In vitro screening of siRNAs targeting CYP2E1.

[0015] Figure 3 Representative images of lipid nanoparticles under a transmission electron microscope (Note: A: si-Control LNPs; B: si- Cyp2e1 LNPs).

[0016] Figure 4 For si- Cyp2e1 Western blotting image of changes in Cyp2e1 protein expression in mouse liver 24 hours after LNP injection.

[0017] Figure 5 The activity levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum (Note: n=6, A: Control group; B: TP model group; C: Pre-si-Control group; D: Pre-si-Control group). Cyp2e1 Group; E: si-Control group; F: si- Cyp2e1 Group; G: NAC group, *P<0.05, **P<0.01 vs Control; # P<0.05, ## P<0.01 vs. model group; & P<0.05, && P<0.01 vs si-Control).

[0018] Figure 6 HE staining images of mouse liver tissue (Note: A: Control group; B: TP model group; C: Pre-si-Control group; D: Pre-si- Cyp2e1 Group; E: si-Control group; F: si- Cyp2e1 Group; G: NAC group).

[0019] Figure 7 The levels of reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) in mouse liver tissue were measured (Note: n=6, A: Control group; B: TP model group; C: Pre-si-Control group; D: Pre-si-Control group). Cyp2e1 Group; E: si-Control group; F: si- Cyp2e1 Group; G: NAC group, *P<0.05, **P<0.01 vsControl; # P<0.05, ## P<0.01 vs. model group; & P<0.05, &&P<0.01 vs si-Control).

[0020] Figure 8 Relative table of mRNA levels of TNF-α, IL-1β, and IL-6 inflammatory factors in mouse liver tissue (Note: n=6, A: Control group; B: TP model group; C: Pre-si-Control group; D: Pre-si- Cyp2e1 Group; E: si-Control group; F: si- Cyp2e1 Group; G: NAC group, *P<0.05, **P<0.01 vs Control; # P<0.05, ## P<0.01 vs. model group; & P<0.05, && P<0.01 vs si-Control). Detailed Implementation

[0021] Example 1: Preparation and characterization of si-Cyp2e1 LNPs (1) Targeting Cyp2e1 Preparation, screening, modification and optimization of gene siRNA Four siRNA sequences and their derivatives targeting mouse Cyp2e1 were designed. si-Control is a siRNA sequence that does not target any gene product. The relevant sequences are shown in Table 1. The single-stranded RNAs were produced by Jiangsu GenScript Biotech Co., Ltd.

[0022] The single-stranded siRNA used in the following experiments was a derivative modified with 2′-O methyl groups and linked with dTsdT 3′ thiophosphate (as shown in Table 1). The crude oligonucleotides were deprotected and purified by anion-exchange HPLC. Equal volumes of complementary strands were mixed in annealing buffer (50 mM Tri-HCl buffer, 100 mM NaCl, pH=8.0). The mixture was then heated at 95°C for 5 min and slowly cooled to 25°C at a rate of 0.1°C / s using PCR to synthesize double-stranded siRNA.

[0023] The cDNA sequence of mouse Cyp2e1 was obtained from NCBI and synthesized by Jiangsu GenScript.

[0024] Hepa 1-6 cells were cultured in DMEM supplemented with 10% FBS in a humidified incubator at 37°C and 5% CO2. Stable cell lines overexpressing CYP2E1 protein were obtained by transfection with a lentiviral vector for 48 hours. After selection with puromycin, Hepa 1-6 cells overexpressing Cyp2e1 (Cyp2e1 OE cells) were finally constructed. Figure 1 As shown.

[0025] Cyp2e1 OE cells were planted at 1.0 × 10⁶ cells per well. 5 Cells were seeded at a density of 10 nM in 24-well plates. Simultaneously, Cyp2e1 siRNA was transfected using lipofectamine™ RNAiMAX transfection reagent for 24 hours. Double-stranded siRNA sequences were transfected at concentrations of 10 nM and gradient concentrations (10 nM, 2 nM, 400 pM, 80 pM, 16 pM). The knockout effect of Cyp2e1 was assessed by qRT-PCR, and the IC50 value was calculated. 50 The optimal siRNA silencing sequence was determined, and the knockdown effect at 10 nM concentration after 24 and 48 hours of transfection was verified by qRT-PCR and Western blot, respectively. Specifically, a successfully constructed stable CYP2E1 overexpression cell line was used for siRNA silencing screening. First, four siRNAs were transfected at a concentration of 10 nM, and the knockdown effect was as follows: Figure 2 As shown in Table A, 24 hours after transfection, the knockdown rates of si-Cyp2e1-1, si-Cyp2e1-2, si-Cyp2e1-3, and si-Cyp2e1-4 were 56%, 86%, 88%, and 86%, respectively, with the latter three siRNA sequences showing better knockdown effects. The latter three siRNAs were transfected at gradient concentrations of 10 nM, 2 nM, 400 pM, 80 pM, and 16 pM, and the IC50 value of each sequence was calculated. The results are shown in Table 2, with the si-Cyp2e1-3 sequence having the lowest IC50 value. The knockdown effect was verified by transfecting si-Cyp2e1-3 at a concentration of 10 nM for 24 h and 48 h, with the results as follows. Figure 2 As shown in B and 2C, 82% mRNA inhibition was still achieved 48 hours after cell transfection, and protein expression was almost completely inhibited after 48 hours. Therefore, si-Cyp2e1-3 was identified as the target siRNA.

[0026] Table 1 siRNA primer sequences

[0027] Table 2 Gradient Concentration Screening and IC50 50 value

[0028] (2) Preparation of lipid nanoparticles The lipid nanoparticles consist of an oil phase and an aqueous phase. The oil phase solution is anhydrous ethanol, and the solutes are D-Lin-MC3-DMA: distearate lecithin choline (DSPC): high-purity cholesterol (CHO-HP): 14:0 PEG2000 PE in a molar ratio of 50 / 10 / 38.5 / 1.5. The aqueous phase is a 10 mM sodium acetate buffer solution containing dissolved siRNA.

[0029] A threaded syringe was used to draw up solutions of oil and water phases in a 1:3 volume ratio and connected to the two inlets of a microfluidic device. The solutions were mixed spontaneously in the volume mixing chip of the LNP at a flow rate of 1:3 to form lipid nanoparticles. The products were collected and quickly added to a 20KD dialysis bag. The solution was then subjected to rotary dialysis for 2 hours in 2 liters of phosphate buffer solution prepared with enzyme-free water to remove residual ethanol.

[0030] (3) Measurement of lipid nanoparticle size, polydispersity index (PDI) and zeta potential A small amount of the prepared lipid nanoparticle solution was taken, diluted 100-fold with DPBS, and sonicated. The Si- in the diluted solution was analyzed using a NanoBrook 90Plus PALS device. Cyp2e1 The particle size, polydispersity index (PDI), and zeta potential of LNPs and si-Control LNPs were determined.

[0031] (4) Determination of lipid nanoparticle encapsulation efficiency and drug loading Triton-100 was selected as the demulsifier and diluted to 2% with enzyme-free water as a surfactant to release the siRNA encapsulated in lipid nanoparticles. The fluorescence absorbance of free nucleic acids before demulsification and the fluorescence absorbance of total nucleic acids after demulsification were measured using the Quant-iT™ RiboGreen® RNA Kit (Thermo Fisher, catalog number R11490). The encapsulation was calculated based on the obtained fluorescence absorbance values ​​using the following formula: Encapsulation efficiency (%) = [(Total nucleic acid fluorescence value – Unbroken nucleic acid fluorescence value) / Total nucleic acid fluorescence value] × 100 (5) Observation of the morphology of the prepared lipid nanoparticles by transmission electron microscopy After drying, negative staining, and dilution with distilled water, the lipid nanoparticle solution was placed on a copper grid for detection.

[0032] (6) si-Control LNPs and si- Cyp2e1 Verification of the in vivo knockdown effect of LNPs Nine healthy female C75BL / 6J mice aged 6-8 weeks were randomly divided into three groups: a control group, a si-control group, and a si- Cyp2e1 In the LNPs group, three mice were included in each group, and the experiment was repeated three times. Data are expressed as mean ± standard deviation. Independent samples t-tests were performed using SPSS 22.0 software, and P < 0.05 was considered statistically significant. Lipid nanoparticles were diluted to a target dosage of 0.5 mg / kg using DPBS and injected into mice via the tail vein. Mice in the control group were injected with DPBS. Mice were anesthetized and sacrificed 24 hours after injection, and their livers were harvested for Western blotting analysis of Cyp2e1 protein expression.

[0033] As shown in Table 3, the lipid nanoparticles had a particle size of about 72 nm, and both the PDI and Zeta potentials met the standards. The encapsulation efficiency was about 75%, and the drug loading was 0.46 mg / kg. Figure 3 Displaying si-Control LNPs (A) and si- under a transmission electron microscope. Cyp2e1 The two types of lipid nanoparticles in LNPs (B) are approximately spherical in shape with a particle size of about 70 nm, which is consistent with the measured particle size results. Figure 4 This indicates that si- Cyp2e1 LNPs can significantly reduce the expression of Cyp2e1 protein in mouse liver.

[0034] Table 3. Relevant characterization data of lipid nanoparticles

[0035] Example 2: (1) Establishment of animal models Forty-two healthy female C75BL / 6J mice aged 6-8 weeks were randomly divided into 7 groups of 6 mice each. The groups were: saline control group, 800 μg / kg TP model group, Pre-si-Control LNPs group, Pre-si- Cyp2e1 LNPs group, si-Control group, si- Cyp2e1 LNPs group and N-acetylcysteine ​​(NAC) positive drug group. A dose of 800 μg / kg TP was pre-validated to stably induce subacute liver injury in mice (serum ALT / AST levels increased more than 2-fold compared to the control group, P<0.01). Pre-si-Control LNPs group and Pre-si- Cyp2e1 The LNPs group received a tail vein injection of si-Control LNPs and si-control LNPs respectively on the third day prior to oral TP administration. Cyp2e1 LNPs, si-Control group and si- Cyp2e1 The LNPs group received tail vein injections of si-Control LNPs and si- during TP administration via gavage. Cyp2e1The LNPs and NAC groups were administered 500 mg / kg NAC via gavage simultaneously with TP. Except for the saline Control group, the other groups of mice were administered 800 μg / kg TP via gavage daily for 7 consecutive days. Twenty-four hours after the last administration, the mice were anesthetized, weighed, and their eyeballs were enucleated to collect blood and liver samples for subsequent biochemical analysis.

[0036] (2) Subacute toxicity evaluation The blood collected in step (1) was allowed to stand, and then centrifuged at 4°C and 3500g for 15 minutes to collect the supernatant. The serum ALT and AST levels were detected using an ALT and AST kit developed in Nanjing. Mouse liver tissue was fixed with 4% paraformaldehyde and then sectioned and stained with hematoxylin and eosin (HE) by Wuhan Sewell Biotechnology Co., Ltd. to observe cell morphology and pathological damage.

[0037] The results are as follows Figure 5 As shown, administration of 800 μg / kg TP for one week significantly induced an increase in ALT and AST levels in mice, and si- Cyp2e1 LNP pretreatment and simultaneous treatment can significantly reduce serum ALT and AST levels.

[0038] HE staining results of mouse liver tissue are as follows Figure 6 As shown, the results indicated that administration of 800 μg / kg TP for one week significantly affected liver tissue damage in mice, manifested as cellular edema, inflammatory infiltration, nuclear pyknosis and lysis, and si- Cyp2e1 LNP treatment significantly improved the pathological state of mouse liver, demonstrating that si- Cyp2e1 The therapeutic effect of LNPs on TP-induced subacute liver injury.

[0039] (3) Evaluation of oxidative stress levels in mice Weigh 50 mg of each liver sample obtained in step (1) and perform ROS assay using the ROS detection kit from Bebo Biotechnology; perform assays using the GSH, MDA, and SOD kits from Nanjing Jiancheng. Quantify BCA protein using the BCA protein concentration assay kit from GlpBio (USA). Measure absorbance using an ELISA reader and calculate data according to the instructions.

[0040] The results are as follows Figure 7 As shown, administration of 800 μg / kg TP for one week significantly increased ROS and MDA levels in mouse liver, depleted GSH, and decreased SOD levels, thereby increasing hepatotoxicity caused by tissue oxidative stress. Cyp2e1 LNP treatment can significantly reduce ROS and MDA levels, upregulate GSH and SOD, and combat oxidative stress to reduce liver damage.

[0041] (4) Evaluation of mouse inflammatory factor levels Approximately 50 mg of liver tissue from each mouse was collected in a 5 mL centrifuge tube. 1 mL of Beyotime Trizol reagent (catalog number 15596-026) was added to the tube while on ice. Note that the sample volume should not exceed 10% of the Trizol volume. Homogenize using a micro-electric homogenizer for 10 seconds, pause for 30 seconds, and repeat 5-6 times to prevent overheating from prolonged homogenization. After homogenization, the sample was incubated on ice for 3 minutes. Centrifuged at 12000 g for 10 minutes at 4 °C. The supernatant was transferred to a 1.5 mL enzyme-free centrifuge tube, and the precipitate was discarded. 200 μL of chloroform was added to the tube and vortexed with the supernatant for 15 seconds. The mixture was then incubated on ice for 5 minutes. The mixture was centrifuged again using the same procedure. 400 μL of the upper aqueous phase was transferred to a 1.5 mL enzyme-free centrifuge tube, mixed with 400 μL of isopropanol, and incubated for 10 minutes. The mixture was then centrifuged once more using the same low-temperature centrifugation procedure, retaining the bottom precipitate. After pipetting 100 μl of pre-cooled anhydrous ethanol, centrifuge according to the above procedure, repeating the washing 2-3 times to remove as much residual ethanol as possible, leaving the RNA at the bottom. Add 40 μL of enzyme-free water to dissolve the extracted total RNA, aliquot into 2 μL portions, and check the concentration using Nanodrop before use.

[0042] Prepare a 20 μl reverse transcription system according to the Beyotime cDNA synthesis kit instructions, set the reverse transcription program (42°C, 60 min; 80°C, 10 min; 4°C, ∞), and store the synthesized cDNA product in a -20°C freezer for later use.

[0043] by Gapdh The gene was used as an internal control gene, and the procedure was performed according to the Beyotime SYBR Green qPCR Mix (2×) kit instructions. The qPCR reaction volume per well was 20 μl, as shown in Table 3. The qPCR program was set as follows (95°C, 120 s; 95°C, 30 s; 72°C, 30 s; 45 cycles), with three replicates per well. Finally, 2... -△△Ct The method is to conduct data analysis.

[0044] The results are as follows Figure 8 As shown, administration of 800 μg / kg TP for one week significantly increased the levels of inflammatory factors such as Tnf-α, IL-1β, and IL-6 in the liver of mice, and si- Cyp2e1 LNP treatment can significantly reduce the level of inflammatory factors and alleviate liver damage.

[0045] Table 4. qPCR reaction system

[0046] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An siRNA targeting the inhibition of CYP2E1, characterized in that, The double-stranded siRNA is formed by annealing the following two single-stranded RNAs: ccAuGuAcAcAAuGGAAAA, UUUUCcAUUGUGuAcAUGG.

2. The derivative of the siRNA targeting and inhibiting CYP2E1 according to claim 1.

3. The derivative according to claim 2, characterized in that, The two single-stranded RNAs are modified by 2'-O-methyl and dTsdT 3'-thiophosphate linkage, and the obtained single-stranded RNA sequences are: ccAuGuAcAcAAuGGAAAAdTsdT, UUUUCcAUUGUGuAcAUGGdTsdT.

4. A drug delivery system containing and used for delivering the siRNA targeting and inhibiting CYP2E1 according to claim 1, or the derivative of the siRNA targeting and inhibiting CYP2E1 according to claim 2 or 3.

5. The drug delivery system of claim 4, wherein, The lipid nanoparticle is a lipid nanoparticle.

6. The drug delivery system of claim 5, wherein, The raw materials of the lipid nanoparticle include D-Lin-MC3-DMA, DSPC, cholesterol, and 14:0 PEG2000 PE.

7. The drug delivery system of claim 6, wherein, The lipid nanoparticle includes an oil phase and an aqueous phase, the oil phase has anhydrous ethanol as a solvent, and the solute is D-Lin-MC3-DMA: distearoylphosphatidylcholine: high-purity cholesterol: 14:0 PEG2000 PE with a molar ratio of 50 / 10 / 38.5 / 1.5; the aqueous phase has a 10 mM sodium acetate buffer solution as a solvent, and the solute is the siRNA targeting and inhibiting CYP2E1 or the derivative thereof.

8. The siRNA targeting and inhibiting CYP2E1 according to claim 1, or the derivative according to claim 2, or the derivative according to claim 3, for use in the development or preparation of a medicament for treating or alleviating the hepatotoxicity of triptolide.

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