Drug application of FXR-targeted saRNA combined with FXR agonist

Abstract

The invention discloses an application of an FXR targeted saRNA combined with an FXR agonist in preparation of a medicine for liver protection. According to the combined application of the medicine, the on-target effect of an FXR agonist and the regulation effect on an FXR signal channel are enhanced by reversing the down-regulation of the FXR expression level in a liver disease state, so that cholestasis is remarkably improved. Through combined application of the medicine, the regulation function of FXR transcription / non-transcription dependence is enhanced, and an effective strategy of targeting FXR is provided in the aspect of liver protection.

Description

Technical Field

[0001] This invention relates to the pharmaceutical application of an FXR-targeting saRNA combined with an FXR agonist, and more particularly to the application of an FXR-targeting saRNA combined with an FXR agonist in the preparation of a hepatoprotective drug. Background Technology

[0002] Hepatobiliary diseases caused by liver lesions leading to cholestasis are collectively referred to as cholestatic liver diseases, including primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and drug-induced cholestasis. All types of viral hepatitis and alcoholic liver disease are accompanied by cholestasis. Some non-alcoholic fatty liver diseases also present with cholestasis and intrahepatic bile duct histological lesions. The presence of cholestasis indicates a poor prognosis. Cholestasis further aggravates liver damage, and long-term untreated cholestasis can lead to liver fibrosis, cirrhosis, liver failure, and even death.

[0003] Currently, the treatment strategy for cholestatic liver disease mainly focuses on addressing the underlying cause. For example, in cases of drug-induced hepatitis-induced cholestasis, the relevant medications are discontinued promptly; in cases of alcoholic liver disease-induced cholestasis, alcohol consumption is stopped immediately; in cases of viral hepatitis-induced cholestasis, antiviral therapy is administered; in cases of autoimmune hepatitis-induced cholestasis, hormone therapy is used; and ursodeoxycholic acid (UDCA) is used to treat PBC and PSC. However, some patients do not respond to UDCA treatment. Furthermore, due to the limited availability of drugs for treating PSC, liver transplantation remains the primary treatment for prolonging the survival of PSC patients. In addition, FXR agonists in clinical trials, such as Tropifexor / LJN452, Ciloxor, and EDP-305, all exhibit severe pruritus and potential hepatotoxicity risks, limiting their clinical application. In conclusion, there are currently no safe and effective drugs for treating cholestatic liver disease. Summary of the Invention

[0004] Purpose of the invention: The present invention aims to provide a drug combination of FXR-targeting saRNA and FXR agonist with synergistic effect.

[0005] Technical Solution: The pharmaceutical application of this invention involves the use of FXR-targeting saRNA in combination with an FXR agonist in the preparation of a hepatoprotective drug. The FXR-targeting saRNA can also be used in the preparation of a drug that enhances the targeting of an FXR agonist. Furthermore, pharmaceutical compositions with FXR-targeting saRNA and an FXR agonist as active ingredients can also be used in the preparation of hepatoprotective drugs.

[0006] In various liver injury states, the expression level of FXR in the liver is significantly downregulated. The loss of this target leads to reduced on-target efficacy and enhanced off-target effects of FXR agonists, resulting in severe toxic side effects in clinical practice. Therefore, blocking the downregulation of FXR expression in the liver under liver disease conditions is a crucial prerequisite for ensuring the full efficacy of FXR agonists and effectively reducing toxic side effects. Based on the previously successfully screened saRNA targeting the FXR gene, this invention combines it with an FXR agonist to upregulate FXR expression levels, thereby enhancing the on-target effect of the FXR agonist and significantly improving its agonistic effect on the FXR signaling pathway. This effectively improves cholestasis and provides an effective strategy for targeting FXR in liver protection.

[0007] Preferably, the FXR-targeted saRNA is complementary to the FXR promoter or enhancer region.

[0008] Further preferably, the FXR-targeted saRNA has a nucleotide sequence complementary to the -103 to -85 site region or the -319 to -301 site region of the NR1H4 gene promoter region.

[0009] Preferably, the FXR-targeted saRNA has a sense strand with a 5'-AAGGUUCCUUUCUAUGUUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAACAUAGAAAGGAACCUU[dT][dT]-3' sequence (hdsFXR-103saRNA), or has a sense strand with a 5'-GGGAAGUGAUAGAGCUAUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAUAGCUCUAUCACUUCCC[dT][dT]-3' sequence (hdsFXR-319saRNA).

[0010] Preferably, the FXR-targeting saRNA is a drug that upregulates FXR expression levels.

[0011] Preferably, the FXR-targeting FXR agonist is selected from FXR agonists of natural, semi-synthetic, or fully synthetic origin.

[0012] Further preferably, the FXR agonist is selected from chenodeoxycholic acid (CDCA), obeticholic acid (OCA), or LJN452.

[0013] More preferably, the FXR agonist is as follows:

[0014]

[0015] Preferably, the drug is a drug for treating cholestatic liver disease, drug-induced liver injury, viral hepatitis, non-alcoholic fatty liver disease, alcoholic liver disease, cirrhosis, or liver cancer.

[0016] Preferably, the drug is a drug for treating liver damage accompanied by bile acid accumulation.

[0017] Preferably, the drug is a drug for treating diseases with a cholestatic phenotype accompanied by downregulation of FXR expression.

[0018] Preferably, the drug is a drug that upregulates the expression level of FXR.

[0019] Preferably, the drug is a drug that activates the FXR signaling pathway.

[0020] Preferably, the mass concentration ratio of the FXR-targeted saRNA to the FXR agonist is 1000:0.60 to 1:60.36.

[0021] Preferably, the dosage ratio of the FXR-targeted saRNA to the FXR agonist is 5:1.

[0022] More preferably, the dosage of hdsFXR-103saRNA or hdsFXR-319saRNA is 1.5 mg / kg, and the dosage of LJN452 is 0.3 mg / kg.

[0023] More preferably, the drug is administered as a combination of single-ingredient drugs containing a single active ingredient or as a compound preparation containing two active ingredients.

[0024] The pharmaceutical composition of this invention further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be an excipient widely used in the pharmaceutical manufacturing field. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipient may be an inert filler, or may provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. The pharmaceutical excipient may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0025] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. For example, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0026] The pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0027] Furthermore, the drug described in this invention can also be used in combination with other drugs that improve bile acid stasis, including but not limited to ursodeoxycholic acid (UDCA), obeticholic acid (OCA), S-adenosylmethionine (SAMe), phenobarbital, and silymarin.

[0028] Technical advantages: Compared with the prior art, the present invention has the following significant advantages:

[0029] Combining FXR-targeting saRNA with an FXR agonist enhances the target effect of the FXR agonist by reversing the downregulation of FXR expression levels in liver disease, effectively restoring the regulatory role of the FXR agonist in downstream signaling pathways, thereby improving cholestasis and other conditions. The composition of this invention enhances the transcriptional / transcriptionally independent regulation of FXR, providing an effective strategy for targeting FXR in liver protection. Attached Figure Description

[0030] Figure 1 The effects of the FXR agonist LJN452 on serum biochemical parameters in BDL mice were: A. ALT level, B. AST level, C. ALP level, and D. TBIL level. *** P<0.001, no statistically significant difference in ns;

[0031] Figure 2Effects of the FXR agonist LJN452 on liver tissue pathology in BDL mice;

[0032] Figure 3 To investigate the effects of the FXR agonist LJN452 on the hepatic FXR signaling pathway in BDL mice, the following parameters were considered: A. Bsep level; B. Shp level; C. Mdr2 level. *** P<0.001, no statistically significant difference in ns;

[0033] Figure 4 The effect of combining hdsFXR saRNA with the FXR agonist LJN452 on the expression of BSEP, a downstream target gene of FXR, in L02 cells; among which... * P<0.05, ** P<0.01;

[0034] Figure 5 The effect of combining FXR saRNA with the FXR agonist LJN452 on the expression of the FXR downstream target gene BSEP in L02 cells; among which... * P<0.05, ** P<0.01, *** P<0.001, no statistically significant difference in ns, compared with the NC group;

[0035] Figure 6 The effect of hdsFXR saRNA on FXR expression induced by DCA / TNFα in L02 cells; among which, * P<0.05, *** P<0.001;

[0036] Figure 7 The effect of combined hdsFXR saRNA and the FXR agonist LJN452 on bile acids in DCA / TNFα-stimulated L02 cells; among which, *** P<0.001, no statistically significant difference in ns;

[0037] Figure 8 The effects of combined hdsFXR-103saRNA and the FXR agonist LJN452 on the hepatic FXR signaling pathway in BDL mice were investigated, including A. Bsep level, B. Shp level, and C. Mdr2 level. ** P<0.01, *** P<0.001, no statistically significant difference in ns;

[0038] Figure 9 The effects of combined hdsFXR-319saRNA and the FXR agonist LJN452 on the hepatic FXR signaling pathway in BDL mice were investigated, including A. Bsep level, B. Shp level, and C. Mdr2 level.* P<0.05, ** P<0.01, *** P<0.001;

[0039] Figure 10 The effects of combined use of hdsFXR-103saRNA and the FXR agonist LJN452 on serum biochemical parameters in BDL mice were investigated, including: A. ALT level; B. AST level; C. ALP level; D. TBIL level. * P<0.05, *** P<0.001;

[0040] Figure 11 The effects of combined use of hdsFXR-319saRNA and the FXR agonist LJN452 on serum biochemical parameters in BDL mice were investigated, including: A. ALT level, B. AST level, C. ALP level, and D. TBIL level. * P<0.05, ** P<0.01, *** P<0.001;

[0041] Figure 12 The effect of combined use of hdsFXR-103saRNA and FXR agonist LJN452 on liver tissue pathology in BDL mice;

[0042] Figure 13 The effects of the combination of hdsFXR-319saRNA and the FXR agonist LJN452 on liver histopathology in BDL mice. Detailed Implementation

[0043] The technical solution of the present invention will be further described below with reference to the embodiments.

[0044] Example 1: Evaluation of the effect of FXR agonists on bile duct ligation (BDL)-induced cholestatic liver injury in mice.

[0045] 1. Experimental Materials

[0046] The experimental mice (C57BL / 6J) were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0047] saRNA was synthesized by Genemax Biotechnology Co., Ltd. (Shanghai, China). LJN452 was purchased from MCE. TrizolRNAiso plus was purchased from Takara, the reverse transcription kit and SYBR Green were purchased from Vazyme, and the primers used were synthesized by Life.

[0048] All other biological materials / reagents can be obtained from commercial sources.

[0049] 2. Experimental Methods

[0050] (1) Animal drug administration

[0051] Male C57BL / 6J mice, weighing 20–22 g, were housed in an environment with a temperature of 25±2℃ and a relative humidity of 50%±10%, simulating a 12-hour day / night cycle. They were given free access to food and water for one week prior to the experiment. All mice were housed separately and randomly divided into three groups of six mice per cage, with free access to food and water. This experiment included a blank control group, a BDL group, and an LJN452 administration group. Starting on day 5 of BDL-induced cholestasis, LJN452 was administered by gavage at a dose of 0.3 mg / kg once daily for three consecutive days. Mice were euthanized 24 hours after the last administration. Blood and liver were collected from the mice.

[0052] (2) Serum biochemical factor detection

[0053] The biochemical parameters of mouse serum, including ALT, AST, ALP and TBIL levels, were measured according to the kit instructions.

[0054] (3) Pathological tissue

[0055] Liver tissue specimens from mice with liver injury were fixed, embedded in paraffin, sectioned, and stained with H&E before microscopic observation of hepatocyte damage.

[0056] (4) PCR

[0057] ① Collect liver tissue, wash with pre-cooled PBS, add 1.0 ml of RNAiso Plus and homogenize, centrifuge at 12000g for 5 min, then transfer 800 μl of supernatant. Add 160 μl of chloroform, vortex vigorously for 15 sec, incubate at room temperature for 5 min, then centrifuge at 12000g for 15 min.

[0058] ② Carefully transfer 300 μl of the upper aqueous phase into a new tube, add 300 μl of isopropanol, invert and mix well, let stand at room temperature for 10 min, then centrifuge at 12000g for 10 min, and discard the supernatant.

[0059] ③ Wash the RNA precipitate with 1.0 ml of pre-cooled 75% ethanol, then centrifuge at 12000 g for 5 min, discard the supernatant to obtain total RNA, and reconstitute with 20 μl of DEPC water. After quantification, dilute to 0.5 μg / μl.

[0060] ④ Reverse transcription, the specific ratio requirements are as follows:

[0061]

[0062] ⑤ Real-time quantitative PCR, the specific ratio requirements are as follows:

[0063]

[0064] The conditions for using PCR are as follows:

[0065] Pre-denaturation: 95℃ for 1 min;

[0066] PCR reaction: 95℃ for 15 seconds, 60℃ for 15 seconds, 72℃ for 30 seconds, 40 cycles;

[0067] Melting curve analysis: 65~95℃.

[0068] 3. Experimental Results

[0069] (1) Effects of the FXR agonist LJN452 on serum biochemical parameters in BDL mice

[0070] Serum transaminase results showed that, compared with the control group, the serum ALT and AST levels in the BDL model group were significantly increased; compared with the model group, LJN452 administration had no effect on improving ALT and AST levels in BDL mice. Meanwhile, the serum ALP and TBIL levels in BDL mice were significantly increased compared with the sham-operated group, and LJN452 administration showed no significant improvement in BDL mice compared with the model group. Figure 1 ).

[0071] (2) Effects of FXR agonist LJN452 on liver histopathology in BDL mice

[0072] In the sham-operated group, the liver plates of mice were neatly arranged and the hepatocytes were intact and plump. In the BDL group, the portal area of ​​the liver showed infiltration of inflammatory cells such as neutrophils and lymphocytes, and the hepatocytes began to show degeneration and necrosis, mostly punctate and focal necrosis. Dilation of small bile ducts and a few newly formed bile ducts and small bile ducts without lumens were also observed. LJN452 administration did not significantly improve this condition. Figure 2 ).

[0073] (3) Effects of FXR agonist LJN452 on the liver FXR signaling pathway in BDL mice

[0074] RNA was extracted from mouse liver tissue samples to examine the expression of genes related to bile acid cycling in downstream target genes of FXR. The results showed that, compared with sham-operated mice, the expression of Bsep, Shp, and Mdr2 genes in the liver of mice with BDL-induced cholestasis was significantly reduced; compared with BDL mice, administration of LJN452 to BDL mice did not significantly improve these phenomena. Figure 3 ).

[0075] Example 2: Evaluation of the effect of combined use of FXR-targeting saRNA and FXR agonists on the bile acid signaling pathway

[0076] 1. Experimental Materials

[0077] Lipofectamine RNAiMAX was purchased from Thermo Fisher (CA, USA). Opti-MEM medium was purchased from GIBCO (Grand Island, New York, USA).

[0078] For the design and validation of FXR-targeting saRNA, please refer to CN112941076B.

[0079] The remaining experimental materials are the same as in Example 1.

[0080] 2. Experimental Methods

[0081] (1) Effects and synergistic effects of combined use of FXR-targeting saRNA and FXR agonists on the FXR signaling pathway

[0082] ① Cell culture and drug administration stimulation

[0083] The L02 human hepatocyte cell line was cultured in T25 cell culture flasks at 37°C, 5% CO2, and 90% relative humidity. The culture medium was DMEM containing 10% fetal bovine serum, with 1 unit / ml of penicillin and 1 μg / ml of streptomycin added. The medium was changed every other day, and the cells were passaged in separate flasks after 3 days. Cells in the logarithmic growth phase were harvested at a rate of 2 × 10⁻⁶ cells / flask. 5 Cells were seeded per well in 12-well plates. After 24 hours of cell adhesion, 50 nM saRNA was transfected into the cells, and after 48 hours of transfection, the cells were stimulated with 2 μM LJN452 for another 8 hours.

[0084] ②PCR

[0085] After stimulation with the drug, the cell plate was removed from the cell culture incubator. After washing with pre-cooled PBS, 1.0 ml of RNAiso Plus was added. After the cells were completely lysed, 200 μl of chloroform was added, the cells were vigorously shaken for 15 seconds, incubated at room temperature for 5 min, and then centrifuged at 12000g for 15 min.

[0086] Subsequent experimental methods are the same as in Example 1.

[0087] (2) Effects of FXR-targeted saRNA combined with FXR agonists on total bile acids in DCA / TNFα-induced cholestatic cells

[0088] ① Cell culture and drug administration stimulation

[0089] The L02 human hepatocyte cell line was cultured in the same way as in (1). After the cells adhered for 24 hours, 50 nM saRNA was transfected into the cells. After 48 hours of transfection, the cells were pre-stimulated with 2 μM LJN452 for 2 hours, and then the cells were further stimulated with DCA / TNFα for 12 hours.

[0090] ② Determination of total bile acids in cells

[0091] After stimulation with the drug, the cell plate was removed from the cell culture incubator. After washing with pre-cooled PBS, an appropriate amount of 75% ethanol solution was added, and the cells were sonicated. After incubation with the lysis buffer at 50°C for 2 hours, the cells were centrifuged at 6000g for 10 minutes, and the supernatant was collected for assay using the TBA kit.

[0092] 3. Experimental Results

[0093] (1) Effects of the combination of FXR-targeting saRNA and FXR agonist on the FXR signaling pathway in L02 cells

[0094] Compared with the blank control group, L02 cells treated with the FXR agonist LJN452 alone significantly upregulated the mRNA level of BSEP, a downstream target gene of FXR. Compared with LJN452 alone, administration of LJN452 to the FXR-targeting saRNA (hdsFXR-103 / 319) significantly increased the agonistic effect on the downstream target gene BSEP. Figure 4 ).

[0095] The synergistic effect of FXR saRNA and FXR agonists was further investigated. Results showed that, compared to the blank control group, LJN452 alone at concentrations below 15 μg / mL did not activate the BSEP mRNA level, a downstream target gene of FXR. Cell transfection with hdsFXR-319saRNA (1 μg / mL) followed by different doses of LJN452 (0.06 ng / mL–60.36 μg / mL) showed no significant difference in BSEP mRNA levels compared to the blank control transfection group; however, when combined with LJN452, BSEP mRNA levels were significantly upregulated within a concentration range of 0.60 ng / mL–60.36 μg / mL (FXR saRNA to LJN452 dose ratio of 1000:0.6–1:60.36). Figure 5 ).

[0096] (2) Effect of FXR-targeting saRNA on FXR expression in DCA / TNFα-induced L02 cells

[0097] Compared with the blank control group, DCA / TNFα stimulation of L02 cells significantly downregulated FXR mRNA levels, and subsequent administration of FXR-targeting saRNA (hdsFXR-103 / 319) significantly reversed the FXR mRNA level. Figure 6 ).

[0098] (3) Effects of the combination of FXR-targeting saRNA and FXR agonist on DCA / TNFα-induced total bile acids in L02 cells

[0099] Compared with the blank control group, DCA / TNFα significantly upregulated the total bile acid level in L02 cells, and the FXR agonist LJN452 had no significant effect on this. The combined application of hdsFXR-103 / 319 and LJN452 significantly inhibited the DCA / TNFα-induced increase in total bile acid levels in cells. Figure 7 ).

[0100] Example 3: Evaluation of the effect of FXR-targeting saRNA combined with FXR agonist on improving bile duct ligation (BDL)-induced cholestasis in mice.

[0101] 1. Experimental Materials

[0102] Human FXR transgenic mice with a C57BL / 6J background were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0103] The remaining experimental materials are the same as in Example 1.

[0104] 2. Experimental Methods

[0105] (1) Animal drug administration

[0106] Male human FXR transgenic mice with a C57BL / 6J background, weighing 20–22 g, were housed in an environment with a temperature of 25±2℃ and a relative humidity of 50%±10%, simulating a 12-hour day / night cycle, with free access to food and water. They were acclimatized for one week before the experiment. All mice were housed separately and randomly divided into 4 groups of 5 mice per cage, with free access to food and water. This experiment included a blank control group, a BDL group, an FXR saRNA administration group, and an FXR saRNA combined with LJN452 administration group. FXR saRNA (1.5 mg / kg) was administered to mice via tail vein injection once daily for 5 consecutive days. BDL surgery was performed 8 hours after the last FXR saRNA administration. LJN452 (0.3 mg / kg) was administered by gavage to mice starting on day 5 of BDL-induced cholestasis, once daily for 3 consecutive days. Mice were euthanized 24 hours after the last administration. Blood and liver were collected from the mice.

[0107] (2) Serum biochemical factor detection

[0108] The biochemical parameters of mouse serum, including ALT, AST, ALP and TBIL levels, were measured according to the kit instructions.

[0109] (3) Pathological tissue

[0110] Liver tissue specimens from mice with liver injury were fixed, embedded in paraffin, sectioned, and stained with H&E before microscopic observation of hepatocyte damage.

[0111] (4) PCR

[0112] The experimental method is the same as in Example 1.

[0113] 3. Experimental Results

[0114] (1) Effects of FXR-targeting saRNA combined with FXR agonists on the hepatic FXR signaling pathway in BDL mice

[0115] Following bile duct ligation (BDL) surgery, mice showed significantly reduced expression of FXR downstream target genes Bsep, Shp, and Mdr2 mRNA in the liver compared to control mice. Compared to BDL mice, administration of hdsFXR-103 / 319 partially alleviated the downregulation of these genes caused by bile duct ligation. Furthermore, the combination of hdsFXR-103 / 319 and an FXR agonist significantly restored the levels of these FXR target genes, and the upregulated levels were significantly better than those in the hdsFXR-103 / 319-only group. Figure 8 and Figure 9 ).

[0116] (2) Effects of FXR-targeted saRNA combined with FXR agonists on serum biochemical indicators in BDL mice

[0117] Serum transaminase results showed that, compared with the control group, BDL mice had significantly elevated serum ALT, AST, ALP, and TBIL levels. Compared with the model group, BDL mice administered hdsFXR-103 / 319 showed significantly improved ALT, AST, ALP, and TBIL levels caused by bile duct ligation, and the combination of hdsFXR-103 / 319 and LJN452 further reduced the levels of serum biochemical factors in BDL mice. Figure 10 and Figure 11 ).

[0118] (3) Effects of FXR-targeted saRNA combined with FXR agonists on liver histopathology in BDL mice

[0119] H&E staining of mouse liver tissue showed that the liver cells in the sham-operated group were morphologically intact, clearly demarcated, and had round and distinct nuclei. BDL mice showed obvious necrotic lesions in their hepatocytes, with some cells exhibiting swelling, accompanied by extensive inflammatory cell infiltration. Tail vein injection of hdsFXR-103 / 319 into BDL mice significantly improved liver tissue necrosis and inflammatory cell infiltration caused by bile duct ligation. Combination of hdsFXR-103 / 319 with an FXR agonist further improved the liver lesions in BDL mice. Figure 12 and 13 ).

Claims

1. The application of an FXR-targeting saRNA in combination with an FXR agonist in the preparation of a hepatoprotective drug; wherein the saRNA has a sense strand with a 5'-AAGGUUCCUUUCUAUGUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAACAUAGAAAGGAACCUU[dT][dT]-3' sequence, or has a sense strand with a 5'-GGGAAGUGAUAGAGCUAUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAUAGCUCUAUCACUUCCC[dT][dT]-3' sequence.

2. The application of an FXR-targeting saRNA in the preparation of a drug for targeting an FXR agonist; wherein the saRNA has a sense strand with a 5'-AAGGUUCCUUUCUAUGUUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAACAUAGAAAGGAACCUU[dT][dT]-3' sequence, or has a sense strand with a 5'-GGGAAGUGAUAGAGCUAUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAUAGCUCUAUCACUUCCC[dT][dT]-3' sequence.

3. The use of a pharmaceutical composition comprising an FXR-targeting saRNA and an FXR agonist as active ingredients in the preparation of a hepatoprotective drug; wherein the saRNA has a sense strand with a 5'-AAGGUUCCUUUCUAUGUUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAACAUAGAAAGGAACCUU[dT][dT]-3' sequence, or a sense strand with a 5'-GGGAAGUGAUAGAGCUAUU[dT][dT]-3' sequence and an antisense strand with a 5'-AAUAGCUCUAUCACUUCCC[dT][dT]-3' sequence.

4. The application according to any one of claims 1 to 3, characterized in that, The FXR-targeted saRNA is complementary to the FXR promoter or enhancer region.

5. The application according to any one of claims 1 to 3, characterized in that, The saRNA targeted by FXR is a drug that upregulates the expression level of FXR.

6. The application according to any one of claims 1 to 3, characterized in that, The FXR agonist is selected from CDCA, OCA or LJN452.

7. The application according to any one of claims 1 to 3, characterized in that, The medications mentioned are for treating cholestatic liver disease, drug-induced liver injury, viral hepatitis, non-alcoholic fatty liver disease, alcoholic liver disease, cirrhosis, or liver cancer.

8. The application according to any one of claims 1 to 3, characterized in that, The drug described is for treating liver damage accompanied by bile acid accumulation.

9. The application according to any one of claims 1 to 3, characterized in that, The drug described is for treating diseases with a cholestatic phenotype accompanied by downregulation of FXR expression.

10. The application according to claim 1 or 3, characterized in that, The mass concentration ratio of the FXR-targeted saRNA to the FXR agonist is 1000:0.60 to 1:60.36.

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