Application of golden amide alcohol ester in preparation of medicine for treating cholestasis

Abstract

The invention belongs to but is not limited to the technical field of biomedicine, and discloses application of golden amide alcohol ester to preparation of a medicine for treating cholestasis. The in-vitro cell level verifies that the golden amide alcohol ester has relatively strong activity of activating FXR and CAR. The golden amide alcohol ester can relieve the cholestasis symptom of a cholestasis model mouse. The golden amide alcohol ester can obviously reduce the content of total bile acid and bilirubin in cells, relieve cholestasis and relieve hepatocyte damage. The golden amide alcohol ester can regulate and control the protein level of transporters and metabolic enzymes related to bile acid and bilirubin through double excitation of FXR and CAR, relieve cholestasis and achieve the purposes of benefiting gallbladder and removing jaundice.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of biomedical technology, and in particular relates to the use of golden amide alcohol esters for preparing drugs for treating cholestasis. Background Art

[0002] Cholestasis is a type of liver disease. It occurs due to various reasons inside and outside the liver that cause bile formation, secretion or excretion disorders, which prevent bile from entering the duodenum and entering the blood smoothly, causing mechanical damage or changes in liver cell function. Patients with cholestasis may experience symptoms such as jaundice, itching, fatigue, anorexia, and fatty diarrhea. Cholestasis usually has no obvious symptoms in the early stages, but as the disease progresses, bile acid disorders and hyperbilirubinemia may occur. If not treated in time, it can lead to bile duct necrosis, fibrosis, and liver failure.

[0003] Bile acids (BAs) are produced by the liver through cholesterol metabolism. The production process occurs in multiple places in the cell, including the cytoplasm, endoplasmic reticulum and mitochondria. At the same time, more than ten enzymes in the peroxisome are also involved in the synthesis of bile acids. As an important component of bile, total bile acid (total BA, TBA) is a key substance that promotes intestinal digestion and absorption. It is not only an important indicator reflecting the biochemical synthesis function of the liver, but also a sensitive indicator for detecting the secretion and uptake function of hepatocytes. The main function of bile acid is to dissolve lipids, sterols and fat-soluble vitamins in the intestine. Its synthesis includes classical pathways and alternative pathways. Bile acids are endogenous ligands of farnesoid X receptor (FXR) and have a natural agonist effect on it. FXR can be activated by specific bile acids and their metabolites, such as cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid. FXR is related to multiple physiological functions such as the uptake, transport, synthesis and metabolism of bile acids. Therefore, FXR is an important regulatory factor for regulating the synthesis and transport of bile acids in the liver.

[0004] Jaundice is a phenomenon in which the blood bilirubin concentration increases and the skin and sclera become yellow. It is not completely equivalent to cholestasis. In the early stage of cholestasis, only alkaline phosphatase (ALP) and glutamyltransferase (GGT) increase, and symptoms of jaundice may not appear. Jaundice will only appear clinically when bilirubin exceeds 34.2μmol / L. The incidence of jaundice involves many factors such as abnormal bilirubin metabolism, liver disease, blood disease and drug effects. The most common clinical jaundice is neonatal jaundice and cholestatic jaundice, among which abnormal bilirubin metabolism is one of the main causes of cholestatic jaundice.

[0005] The main pigment in human bile is bilirubin, which is used clinically to determine the degree of jaundice and is also a key indicator for measuring liver function. Bilirubin includes indirect bilirubin (IBIL) and direct bilirubin (DBIL), which is the collective name for the total amount of the two. Indirect bilirubin, also known as unconjugated bilirubin, has not undergone glucuronidation in the liver and is fat-soluble; while direct bilirubin, also known as conjugated bilirubin, is metabolized by indirect bilirubin. After indirect bilirubin enters the liver, it is combined with glucuronic acid through the action of glucuronyl transferase in the liver and is water-soluble. Abnormal functional expression of bilirubin transporters and convertases in hepatocytes can cause bilirubin uptake, conversion, and secretion disorders. Studies have found that activation of the constitutive androstane receptor (CAR) can effectively upregulate the transcriptional expression of bilirubin metabolizing enzyme CYP2B, bilirubin converting enzyme UGT1A1, and bilirubin efflux transporter multidrug resistance associated protein (MRPs). Therefore, CAR is an important regulatory factor affecting the synthesis and transport of bilirubin in the liver.

[0006] Currently, the main method for treating cholestasis is choleretic therapy. Ursodeoxycholic acid (UDCA) is a drug that has been approved for the treatment of cholestasis. Long-term use of UDCA is the preferred treatment for primary biliary cholangitis. UDCA occupies a dominant position in the application of drugs to treat cholestatic diseases. It plays a variety of effects in the treatment of primary biliary cholangitis (PBC), including reducing the production of inflammatory cytokines and maintaining the stability of bile duct cells and mitochondria, thereby repairing liver function and curbing the progression of liver disease. However, UDCA has limitations. 40% of PBC patients are insensitive to UDCA treatment, and the use of UDCA is accompanied by side effects such as nausea, vomiting, sleep disorders, and diarrhea.

[0007] Therefore, a new drug for treating cholestasis is needed to achieve the purpose of "promoting bile secretion and relieving jaundice". Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides use of aurantiamide acetate (AA) for preparing a drug for treating cholestasis.

[0009] The present invention realizes an innovative drug use for treating cholestasis by using golden amide alcohol ester. Golden amide alcohol ester regulates the related transporters and metabolic enzymes of bile acid and bilirubin metabolism through a unique molecular mechanism of action, thereby effectively alleviating the pathological state caused by cholestasis. The invention provides a new treatment method and opens up a new direction for the intervention and treatment of cholestasis-related diseases.

[0010] Furthermore, golden amide alcohol ester can regulate the protein expression levels of key transporters and enzymes in bile acid and bilirubin metabolism by dual stimulating nuclear receptors FXR and CAR. This mechanism of action achieves precise regulation of bile acid and bilirubin homeostasis, providing a scientific basis for its treatment of cholestasis.

[0011] In addition, golden amide alcohol ester can significantly reduce the content of total bile acid and bilirubin in cells, effectively improving cell damage and functional abnormalities caused by bile acid accumulation and bilirubin metabolism disorders. This feature further verifies the outstanding role and practical application value of golden amide alcohol ester in the treatment of cholestasis-related diseases.

[0012] Another object of the present invention is to provide a method for verifying the golden amide alcohol ester stimulating FXR and CAR based on molecular docking technology, comprising the following steps:

[0013] a) Using the hFXR (PDB code: 5Z12) and hCAR (PDB code: 1XVP) crystal structures in the PDB database for preprocessing, setting grid points and verifying the binding potential of gold amide alcohol esters to hFXR and hCAR;

[0014] b) Using molecular docking technology, the structure of the gold amide alcohol ester compound was docked into the active sites of hFXR and hCAR, and the docking score and binding mode were analyzed and compared with the binding modes of known agonists WAY-362450 and CITCO to determine its agonistic potential.

[0015] Another object of the present invention is to provide a cell verification method for in vitro stimulating FXR and CAR by golden amide alcohol ester, comprising the following steps:

[0016] a) examining the cytotoxicity of golden amide alcohol ester to HepG2 cells by MTT method to determine the applicable dosage range;

[0017] b) Dual luciferase reporter gene assay was used to verify that auramide ester can stimulate the activity of FXR and CAR in HepG2 cells, and the activity was concentration-dependent.

[0018] Another object of the present invention is to provide an animal experimental method for alleviating cholestasis with golden amide alcohol ester, comprising the following steps:

[0019] a) Male C57BL / 6J mice were used for modeling and randomly divided into groups;

[0020] b) The mice in each experimental group were intraperitoneally injected with auramide alcohol ester and intragastrically administered with ANIT on the 5th day;

[0021] c) After the experiment, blood, liver and gallbladder were collected, serum ALT, AST, ALP, GGT, TBIL and TBA levels were measured, and bile acid and liver tissue pathology were examined.

[0022] Another object of the present invention is to provide a cell experiment method for explaining the molecular mechanism of golden amide alcohol ester in relieving cholestasis, comprising the following steps:

[0023] a) FXR and CAR in HepG2 cells were silenced by lentiviral technology to construct siFXR-HepG2 and siCAR-HepG2 cell models;

[0024] b) Using α-naphthyl isothiocyanate, bilirubin and probenecid to establish a high bile salt and high bilirubin cell model;

[0025] c) After intervention with aureamide, the levels of ALT, AST, ALP, GGT, TBIL, DBIL, and TBA were detected, and the expressions of FXR, CAR, and their downstream proteins were detected by Western Blot to analyze their molecular mechanisms.

[0026] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0027] First, the present invention provides the use of golden amide alcohol ester for preparing drugs for treating cholestasis; golden amide alcohol ester can relieve cholestasis symptoms in cholestasis model mice. Golden amide alcohol ester can significantly reduce the content of total bile acid and bilirubin in cells, relieve cholestasis, and reduce liver cell damage. Golden amide alcohol ester can regulate the protein levels of bile acid and bilirubin-related transporters and metabolic enzymes by dual stimulating FXR and CAR, relieve cholestasis, and achieve the purpose of "choleretic and jaundice-removing".

[0028] This invention uses golden amide alcohol ester as the research object, and based on the molecular docking technology combined with experimental technology, verifies the mechanism of golden amide alcohol ester in alleviating cholestasis by dual stimulating FXR+CAR, clarifies the stimulation of dual targets to achieve the purpose of "promoting bile secretion and relieving jaundice", and provides new ideas for multi-target relief of cholestasis.

[0029] The present invention focuses on nuclear receptors and demonstrates the feasibility of alleviating cholestasis by stimulating FXR and CAR dual targets.

[0030] The present invention uses molecular docking technology to discover that golden amide alcohol ester can be well bound to the active pockets of hFXR and hCAR, and has a similar binding mode with the FXR agonist WAY-362450 and the CAR agonist CITCO, proving that golden amide alcohol ester has the potential to dual-agonize FXR and CAR.

[0031] The present invention uses a dual luciferase reporter gene experiment to verify at the in vitro cell level that golden amide alcohol ester can stimulate the expression of FXR and CAR in HepG2 cells, proving that golden amide alcohol ester has biological activity and is a strong dual agonist of FXR and CAR.

[0032] The present invention establishes a cholestasis model mouse, confirms that golden amide alcohol ester can alleviate intrahepatic cholestasis of the cholestasis model mouse, reduces liver damage in the mouse, and proves that golden amide alcohol ester has the pharmacological effect of relieving cholestasis.

[0033] The present invention uses HepG2 cells and silences intracellular FXR and CAR respectively. On this basis, a high bile salt and high bilirubin cell model is constructed. By comparing the biochemical levels and protein expression of normal cells and FXR / CAR silenced cells, it is proved that golden amide alcohol ester can relieve cholestasis by dual stimulating FXR and CAR.

[0034] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0035] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation

[0036] Cholestasis is a common liver disease that is usually manifested by obstruction of bile flow, which leads to bile accumulation in the liver and liver damage. With the increasing number of patients with liver diseases, especially those with chronic cholestasis, there is a huge demand for new drugs to treat such diseases. TMAccording to research, the global liver disease drug market is growing at a relatively fast pace, and is expected to grow from approximately US$25 billion to more than US$35 billion between 2023 and 2028, with an average annual growth rate of approximately 6%-7%. Although cholestasis is a relatively small subset of liver diseases, the related drug market still has great development potential. According to reports from Grand View Research and Evaluate Pharma, drugs for the treatment of cholestasis (such as detoxification drugs, bile acid regulators, immunosuppressants, etc.) are currently in a growth stage, especially in the treatment of chronic biliary diseases such as primary biliary cholangitis and primary sclerosing cholangitis. Focusing on cholestasis drugs, the global market is expected to reach approximately more than US$1 billion around 2025, and with the launch of new drugs and technological advances, this figure is expected to continue to grow in the next few years.

[0037] The golden amide alcohol ester described in the present invention is a dipeptide derivative, which is commonly found in many natural medicinal plants, such as Radix Isatidis, Portulaca Oleracea and Clematis. The reported pharmacological effects include antiviral, antibacterial, antiparasitic, anti-inflammatory and antioxidant, but there are no reports that golden amide alcohol ester can relieve cholestasis and protect liver function. The golden amide alcohol ester described in the present invention is a new compound molecule for treating cholestasis that dually excites FXR and CAR, which can more effectively reduce cholestasis and improve liver function, has good clinical application prospects, is expected to fill the market gap, and meet the growing treatment needs. Its market application will also have good market prospects and commercial value.

[0038] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0039] The clinical manifestations of cholestasis are often itching, fatigue and jaundice, etc. There are no obvious symptoms in the early stage. Hyperbilirubinemia may occur as the disease progresses. In severe cases, liver failure or even death may occur. The treatment methods for cholestasis are limited. The main methods are currently removing the cause and choleretic treatment. Among them, the commonly used therapeutic drugs for cholestasis are ursodeoxycholic acid, and the therapeutic drugs for jaundice are glucuronyl transferase inducers, activated carbon and albumin. However, the clinical performance of existing drugs for the treatment of cholestasis and jaundice is not satisfactory, and there are shortcomings such as poor efficacy and more side effects, and the target of the above-mentioned therapeutic drugs is a single target. The golden amide alcohol ester described in the present invention can dually excite the nuclear receptors FXR and CAR, and by regulating the protein levels of bile acid and bilirubin-related transporters and metabolic enzymes, it can significantly reduce the content of total bile acid and bilirubin in cells, relieve cholestasis, achieve the purpose of "choleretic jaundice", and also reduce liver cell damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1The effect of the golden amide alcohol ester provided in the embodiment of the present invention on the viability of HepG2 cells. A: 24h; B: 72h, (n=6). **P<0.01, ***P<0.001 indicate that the difference is statistically significant compared with the blank group.

[0041] Figure 2 The dual luciferase reporter gene experiment provided in the embodiment of the present invention detects the agonist activity of golden amide alcohol ester on FXR and CAR. The results are expressed as relative luciferase activity, and the luciferase activity measured by the compound is compared with the luciferase activity of the solvent group. A: agonist activity of golden amide alcohol ester on FXR; B: agonist activity of golden amide alcohol ester on CAR. *P<0.05, **P<0.01, ***P<0.001 indicate that the difference is statistically significant compared with the control group.

[0042] Figure 3 It is a schematic diagram of the weight changes of each group of mice within seven days provided in the embodiments of the present invention.

[0043] Figure 4 The effect of the golden amide alcohol ester provided in the embodiment of the present invention on the serum biochemical index levels of cholestasis model mice (n=8). A: ALT; B: AST; C: ALP; D: GGT; E: TBIL; F: TBA. *P<0.05, ***P<0.001 indicate that the difference is statistically significant compared with the control group; #P<0.05, ##P<0.01, ###P<0.001 indicate that the difference is statistically significant compared with the ANIT group.

[0044] Figure 5 The effect of the golden amide alcohol ester provided in the embodiment of the present invention on the total bile acid content in the bile of cholestasis model mice (n=8). *P<0.05, ***P<0.001 indicate that the difference is statistically significant compared with the control group; ##P<0.01, ###P<0.001 indicate that the difference is statistically significant compared with the ANIT group.

[0045] Figure 6 The effect of aureamide alcohol ester provided in the embodiment of the present invention on liver pathology of cholestatic mice (20×). A: Control group; B: ANIT group; C: OCA group; D: aureamide alcohol low-dose group AA-L (1 mg / Kg); E: aureamide alcohol ester medium-dose group AA-M (5 mg / Kg); F: aureamide alcohol ester high-dose group AA-H (10 mg / Kg).

[0046] Figure 7The FXR and CAR silencing results provided in the examples of the present invention (mean±SD, n=3). A: protein expression level after FXR silencing; B: protein expression level after CAR silencing. *P<0.05, ***P<0.001 indicate that the difference is statistically significant compared with the control group.

[0047] Figure 8 The effect of the golden amide alcohol ester provided in the embodiment of the present invention on the biochemical indicators after FXR silencing in the high bile salt high bilirubin model cells (mean ± SD, n = 4). A: cell viability; B: ALT; C: AST; D: GGT; E: ALP; F: TBA; G: TBIL; H: DBIL. *P<0.05, **P<0.01, ***P<0.001 indicate that the difference is statistically significant compared with the normal cell group (control); +P<0.05, ++P<0.01, +++P<0.001 compared with the normal cell high bile salt high bilirubin model group (model), the difference is statistically significant; #P<0.05, ##P<0.01, ###P<0.001 compared with the normal cell high bile salt high bilirubin model golden amide alcohol ester intervention group (model+AA), the difference is statistically significant.

[0048] Fig. 9 The effect of the golden amide alcohol ester provided in the embodiment of the present invention on the expression of FXR and its downstream proteins in HepG2 cells after FXR silencing (mean±SD, n=3). A: FXR; B: SHP; C: BSEP; D: NTCP; E: MRP2; F: CYP8B1; G: CYP7A1. *P<0.05, **P<0.01, ***P<0.001 indicate that compared with the normal cell control group, +P<0.05, ++P<0.01, +++P<0.001 indicate that compared with the normal cell model group, #P<0.05, ##P<0.01, ###P<0.001 indicate that compared with the normal cell model+AA group, the difference is statistically significant.

[0049] Fig.10 It is the effect of the golden amide alcohol ester provided in the embodiment of the present invention on the expression of CAR and its downstream proteins in HepG2 cells after FXR silencing (mean ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 indicate that compared with the normal cell control group, +P < 0.05, ++P < 0.01, +++P < 0.001 indicate that compared with the normal cell model group, #P < 0.05, ##P < 0.01, ###P < 0.001 indicate that compared with the normal cell model + AA group, the difference is statistically significant.

[0050] Fig.11 This is the effect of the golden amide alcohol ester provided in the embodiment of the present invention on the biochemical level of HepG2 cells after CAR silencing (mean±SD, n=4). A: cell viability; B: ALT; C: AST; D: GGT; E: ALP; F: TBA; G: TBIL; H: DBIL. *P<0.05, **P<0.01, ***P<0.001 indicate that compared with the normal cell control group, +P<0.05, ++P<0.01, +++P<0.001 indicate that compared with the normal cell model group, #P<0.05, ##P<0.01, ###P<0.001 indicate that compared with the normal cell model+AA group, the difference is statistically significant.

[0051] Fig.12 The effect of the golden amide alcohol ester provided in the embodiment of the present invention on the expression of FXR and its downstream proteins in HepG2 cells after CAR silencing (mean±SD, n=3). A: FXR; B: SHP; C: BSEP; D: NTCP; E: MRP2; F: CYP8B1; G: CYP7A1. *P<0.05, **P<0.01, ***P<0.001 indicate that compared with the normal cell control group, +P<0.05, ++P<0.01, +++P<0.001 indicate that compared with the normal cell model group, #P<0.05, ##P<0.01, ###P<0.001 indicate that compared with the normal cell model+AA group, the difference is statistically significant.

[0052] Fig.13 This is the effect of the golden amide alcohol ester provided in the embodiment of the present invention on the expression of CAR and its downstream proteins in HepG2 cells after CAR silencing (mean±SD, n=3). A: CAR; B: UGT1A1; C: CYP2B6; D: CYP2C9. *P<0.05, **P<0.01, ***P<0.001 indicate that compared with the normal cell control group, +P<0.05, ++P<0.01, +++P<0.001 indicate that compared with the normal cell model group, #P<0.05, ##P<0.01, ###P<0.001 indicate that compared with the normal cell model+AA group, the difference is statistically significant.

[0053] Fig.14 This is a schematic diagram of the mechanism by which golden amide alcohol esters dually stimulate FXR and CAR to relieve cholestasis provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] The embodiment of the present invention provides the use of golden amide alcohol ester for preparing a drug for treating cholestasis.

[0056] Golden amide alcohol ester can significantly reduce the content of total bile acid and bilirubin in cells, relieve cholestasis, and reduce liver cell damage.

[0057] Golden amide alcohol ester can regulate the protein levels of related transporters and metabolic enzymes of bile acid and bilirubin through dual stimuli of FXR and CAR, relieve cholestasis, and achieve the purpose of "promoting choleresis and relieving jaundice".

[0058] This invention uses golden amide alcohol ester as the research object, and based on the molecular docking technology combined with the macroscopic experimental technology, verifies the mechanism of golden amide alcohol ester in alleviating cholestasis by dual stimulating FXR+CAR, clarifies the stimulation of dual targets to achieve the purpose of "promoting bile secretion and relieving jaundice", and provides new ideas for multi-target relief of cholestasis.

[0059] (1) Molecular docking technology was used to verify that golden amide alcohol ester has the potential activity of stimulating hFXR hCAR

[0060] The crystal structures of human FXR (hFXR) and human CAR (hCAR) from the PDB database (hFXRPDB code: 5Z12, hCARPDB code: 1XVP) were used to preprocess the structures of proteins and chemical molecules. The ligand small molecule WAY-362450 in the crystal structure of hFXR and the ligand small molecule CITCO in the crystal structure of hCAR were used to set the position of the docking point, and the confirmed FXR agonist WAY-362450 and CAR agonist CITCO were docked into the active sites of hFXR and hCAR, respectively, to verify the accuracy of the docking method. Then the processed golden amide alcohol ester compound structure was docked into the active sites of hFXR and hCAR, and the docking score and binding mode were analyzed and compared with the binding mode of the known agonists WAY-362450 and CITCO to determine the potential of golden amide alcohol ester to agonize FXR and CAR.

[0061] Molecular docking technology was used to verify whether golden amide alcohol ester has the potential to stimulate hFXR and hCAR:

[0062] After verification of the docking method, it was found that in hFXR, the root mean square deviation (RMSD) value of the docked conformation of WAY-362450 and the crystal structure conformation of its complex was In hCAR, the RMSD value of the docked conformation of CITCO and its complex crystal structure conformation is These data show that the docking method can effectively reproduce the interaction between agonist molecules and hFXR and hCAR, and has a certain degree of credibility. The conformation of the golden amide alcohol ester after docking is compared with the binding mode of hFXR-WAY-362450 and hCAR-CITCO, and the hydrophobic and electrostatic forces are the same, and the golden amide alcohol ester has a stronger bonding affinity. The golden amide alcohol ester can be well combined in the binding pockets of hFXR and hCAR, and has a potential agonist effect.

[0063] (2) Verification of the application of golden amide alcohol esters on FXR and CAR agonism at the cell level

[0064] The MTT method was used to investigate the cytotoxicity of golden amide alcohol esters in HepG2 cells at the in vitro cell level for 24h and 72h to determine the application dose. The dual luciferase reporter gene experiment was used to verify the agonist activity of golden amide alcohol esters on FXR and CAR in HepG2 cells.

[0065] Verification of the activity of golden amide alcohol esters in stimulating FXR and CAR at the cellular level: The results of the MTT experiment showed that when golden amide alcohol esters with a concentration of 0-100 μmol / L were used to intervene in HepG2 cells for 24 hours, there was no significant change in cell viability; after 72 hours of intervention, cell viability decreased significantly with the increase in dose. Therefore, the present invention uses golden amide alcohol esters in a concentration range of 0-100 μmol / L to intervene in HepG2 cells for 24 hours. The results of the dual luciferase reporter gene experiment showed that golden amide alcohol esters can stimulate the activity of FXR and CAR in HepG2 cells, and are concentration-dependent.

[0066] (3) Animal studies on the pharmacological effects of golden amide alcohol esters in relieving cholestasis

[0067] Male C57BL / 6J mice were used for modeling and randomly divided into control group, model group, positive drug OCA group, golden amide alcohol low-dose group (AA-L1mg / Kg), golden amide alcohol ester medium-dose group AA-M (5mg / Kg), golden amide alcohol ester high-dose group AA-H (10mg / Kg), 8 mice in each group, intraperitoneal injection once a day for seven days. On the fifth day, except for the control group, mice in other experimental groups were gavaged with ANIT (75mg / Kg). After the experiment, the mice were anesthetized and killed, and blood, liver tissue and gallbladder were collected. The levels of ALT, AST, ALP, GGT, TBIL and TBA in serum were detected using an automatic biochemical analyzer, the total bile acid content in mouse bile was detected using a total bile acid kit, and liver tissue pathology was examined using hematoxylin-eosin staining.

[0068] Animal experiments investigated the pharmacological effects of golden amide alcohol ester in relieving cholestasis: the experimental results showed that the weight of mice did not change in the first 5 days. After oral administration of ANIT on the 5th day, the weight of mice in the model group decreased significantly. After intervention with the positive drug group and golden amide alcohol ester, the weight of mice did not change significantly. Serum biochemistry results showed that the levels of ALT, AST, ALP, GGT, TBIL and TBA in the model group mice increased significantly, indicating that the mice had severe cholestatic liver injury after ANIT intervention; compared with the model group, the liver enzyme activity and TBIL and TBA levels of mice in the positive drug OCA and golden amide alcohol ester groups were significantly reduced. Liver pathology results showed that the mice in the model group showed obvious liver necrosis, inflammatory factor infiltration and edema, accompanied by gallbladder filling and blackening; after intervention with positive drugs, the size of mouse liver cells was uniform and the nucleus was clearly visible; after intervention with golden amide alcohol ester, the inflammation of mouse liver cells was alleviated, and the morphology and structure were close to those of the normal group. The above results show that golden amide alcohol ester has the pharmacological effect of relieving cholestasis.

[0069] (4) Elucidating the molecular mechanism of golden amide alcohol esters in relieving cholestasis at the cellular level

[0070] Lentivirus was used to silence FXR and CAR in HepG2 cells, respectively. Normal HepG2 cells, siFXR-HepG2 cells and siCAR-HepG2 cells were used as research objects, and α-naphthyl isothiocyanate, bilirubin and probenecid were used to establish a high bile salt and high bilirubin cell model. After 24 hours of intervention with 100 μmol / L auramide alcohol ester, the levels of ALT, AST, ALP, GGT, TBIL, DBIL and TBA in cells and culture medium were detected. Western Blot technology was used to detect the expression of FXR and CAR and their downstream related proteins in cells, and the molecular mechanism of auramide alcohol ester stimulating dual targets to relieve cholestasis was explained.

[0071] The molecular mechanism of golden amide alcohol ester in relieving cholestasis was explained at the cellular level: the results of lentiviral silencing showed that after the intervention of normal HepG2 cells with FXR and CAR silencing lentiviruses, respectively, the expression levels of intracellular FXR and CAR proteins decreased significantly, indicating that FXR-silenced HepG2 cells (siFXR-HepG2) and CAR-silenced HepG2 cells (siCAR-HepG2) were successfully constructed. The results of biochemical tests showed that after the establishment of high bile salt and high bilirubin model cells, the levels of ALT, AST, ALP, GGT, TBIL, DBIL and TBA in the normal HepG2 cell model group were significantly increased; after the intervention of the model cells with golden amide alcohol ester, the levels of ALT, AST, ALP, GGT, TBIL, DBIL and TBA in the cells were significantly decreased; the levels of ALT, AST, ALP, GGT, TBIL, DBIL and TBA in the siFXR-HepG2 cell and siCAR-HepG2 cell model groups were consistent with those in the normal HepG2 cell model group, and after the intervention with golden amide alcohol ester, the biochemical indicators of the two silenced cells showed a downward trend, but there were significant differences compared with the normal HepG2 cell drug group, which indicates that when FXR or CAR is silenced, the effect of golden amide alcohol ester in alleviating high bile salt and high bilirubin model cells is reduced.

[0072] Western Blot results showed that after intervention with aureoacetate, FXR and its downstream proteins small heterodimer partner (SHP), bile salt export pump (BSEP), MRP2 and sodium-taurocholate cotransporter (Na-TAC) in normal HepG2 cells were upregulated. +The expression of CAR and its downstream related proteins UDP-glucuronosyltransferase1A1 (UGT1A1), CYP2B6 and CYP2C9 were significantly increased; there were significant differences in CAR and FXR and their downstream related proteins in siFXR-HepG2 cells and siCAR-HepG2 cells compared with normal HepG2 cells. Compared with the normal cell administration group, the siFXR-HepG2 cell FXR protein expression was significantly decreased and the CAR protein expression was significantly increased in the silencing cell administration group; there was no significant difference in the expression level of FXR protein in siCAR-HepG2 cells, and the CAR protein expression was significantly decreased. This indicates that golden amide alcohol ester can accelerate the excretion of bile acid and bilirubin and relieve cholestasis by stimulating FXR and CAR.

[0073] Relevant evidence of the technical effects achieved by the embodiments of the present invention.

[0074] 1. Verification of the activity of golden amide alcohol esters at the in vitro level and investigation of pharmacological effects at the in vivo level

[0075] 1.1 Evaluation of cytotoxicity of golden amide alcohol esters

[0076] like Figure 1 As shown in the figure, HepG2 cells were intervened with different concentrations of golden amide alcohol ester ranging from 0 to 100 μmol / L for 24 hours and 72 hours, and then the cell viability was detected. The results showed that after golden amide alcohol ester intervened HepG2 cells for 24 hours, there was no significant difference in cell viability under different concentrations; after golden amide alcohol ester intervened HepG2 cells for 72 hours, cell viability decreased significantly with increasing doses, and when the concentration of golden amide alcohol ester reached 80 μmol / L, cell viability dropped to 50%. Therefore, in subsequent cell experiments, the condition of intervention for 24 hours within a concentration of 100 μmol / L will be used.

[0077] 1.2 Agonist activity of auramide alcohol esters on FXR and CAR

[0078] like Figure 2As shown in A, the dual luciferase reporter gene assay was used to detect the FXR agonist activity of aureamide alcohol ester, and the FXR endogenous agonist CDCA was selected as the positive drug. The results showed that aureamide alcohol ester has the activity of FXR agonism, and this agonistic effect is concentration-dependent. For the agonistic activity of CAR, Figure 2 As shown in B, auramide alcohol ester can stimulate CAR in a concentration-dependent manner. The above results show that auramide alcohol ester has the activity of stimulating FXR and CAR.

[0079] 1.3 Effect of golden amide alcohol ester on body weight of ANIT-induced cholestatic mice

[0080] like Figure 3 As shown, the weight of mice in each group did not change significantly in the first five days and remained at about 20g. After ANIT intervention on the fifth day, the weight of mice in all groups except the Control group decreased. From the fifth to the seventh day, the weight of mice after intervention with medium and high doses of golden amide alcohol ester did not change significantly, while the weight of mice after intervention with low doses of golden amide alcohol ester and positive drug OCA (5mg / Kg) further decreased.

[0081] 1.4 Effects of golden amide alcohol ester on serum biochemical parameters in mice with ANIT-induced cholestasis

[0082] like Figure 4 As shown in the results, compared with the control group, the levels of ALT, AST, GGT, TBIL and TBA in the ANIT group were significantly increased (P<0.001), and ALP was increased (P<0.05), which was statistically significant, indicating that the cholestasis mouse model was successfully prepared; there was no significant difference in the levels of ALT, AST, ALP and GGT in the positive drug OCA group, and the levels of TBIL and TBA were significantly increased (P<0.001); the levels of ALT, AST, ALP, TBIL and TBA in the AA-L group and AA-M group were significantly increased (P<0.001), and there was no significant difference in the content of GGT; the ALP content in the AA-H group was decreased (P<0.05), which was statistically significant, and the TBIL

[0083] The levels of ALT, AST and GGT were not significantly different.

[0084] Compared with the ANIT group, the levels of ALT, ALP, GGT, TBIL and TBA in the positive drug OCA group were significantly decreased (P<0.001), and the level of AST was decreased (P<0.05), which were statistically significant; the levels of ALT, AST, ALP and TBIL in the AA-L group were significantly increased (P<0.001), and the levels of GGT and TBA were significantly decreased (P<0.001); the levels of ALT, ALP and TBIL in the AA-M group were significantly increased (P<0.001), and the level of AST was significantly increased (P<0.01), and the levels of GGT and TBA were significantly decreased (P<0.001); the levels of ALT, AST, ALP, GGT and TBA in the AA-H group were significantly decreased (P<0.001), and the level of TBIL was significantly decreased (P<0.01).

[0085] Effect of 1.5-goldenamide alcohol ester on total bile acid content in bile of ANIT-induced cholestatic mice

[0086] like Figure 5 As shown in the data, compared with the control group, the TBA content in the ANIT group and the AA-M group was significantly increased (P<0.001), and the TBA content in the AA-H group was decreased (P<0.05), and the difference was statistically significant. There was no significant difference in the TBA content between the positive drug OCA group and the AA-M group. Compared with the ANIT group, the TBA content in the AA-L group was significantly decreased (P<0.01), and the TBA content in the positive drug OCA group, the AA-M group, and the AA-H group were all significantly decreased (P<0.001).

[0087] 1.6 Pathological effects of auramide on liver tissue in mice with ANIT-induced cholestasis

[0088] like Figure 6 As shown, representative pathological sections from each group were selected for display. Compared with the normal group, the model group mice had more inflammatory infiltration in the liver tissue, and hepatocyte necrosis was clearly visible. The comprehensive biochemical index results showed that the model was successfully established using ANIT. After the positive drug OCA was administered, the inflammatory infiltration decreased and the hepatocyte size was consistent; after different concentrations of golden amide alcohol ester were administered, the inflammatory infiltration in the liver tissue of the low-dose group mice did not decrease, and a large number of hepatocyte necrosis still existed; the inflammatory infiltration in the medium-dose group decreased, but some vacuoles were still visible; the liver tissue morphology of the high-dose group was close to that of the normal group.

[0089] The results showed that golden amide alcohol ester could stimulate FXR and CAR at the in vitro level, and had dual agonist activity; at the same time, golden amide alcohol ester could relieve cholestasis in cholestasis model mice in vivo, and had a pharmacological effect of relieving cholestasis.

[0090] 2. Investigation of the molecular mechanism of golden amide alcohol ester in relieving cholestasis at the in vitro cell level

[0091] 2.1 Verification of FXR and CAR silencing results

[0092] like Figure 7 As shown in A, compared with HepG2 cells infected with empty lentivirus, the expression of FXR protein in the siFXR group was significantly reduced (P<0.001), indicating that FXR in HepG2 cells was silenced; Figure 7 As shown in B, compared with HepG2 cells infected with empty lentivirus, the expression of CAR protein in the siCAR group was decreased (P<0.05), which was statistically significant, indicating that CAR in HepG2 cells was silenced.

[0093] 2.2 Effects of golden amide alcohol ester intervention on the levels of biochemical indicators of HepG2 cells after FXR silencing Figure 8 As shown in the figure, in normal HepG2 cells, compared with the normal group (control), the cell viability of the model group (model) was significantly decreased (P<0.001), and the levels of ALT, AST, ALP, GGT, TBIL, DBIL and TBA were significantly increased (P<0.001), indicating that the model was successfully established. Compared with the model group, the cell viability of the drug administration group (model+AA) was significantly increased (P<0.001), the levels of AST, ALP, GGT, TBIL, DBIL and TBA were significantly decreased (P<0.001), and the ALT level was significantly decreased (P<0.01). In siFXR-HepG2 cells, compared with the normal group, the cell viability and the change trends of ALT, AST, ALP, GGT, TBIL, DBIL and TBA levels of the model group (model-siFXR) were consistent with those of the model group of normal cells. Compared with the normal HepG2 cell group, the cell viability of the siFXR-HepG2 cell group was significantly decreased (P<0.001), the levels of ALT, AST, GGT, TBIL and TBA were significantly increased (P<0.001), the level of ALP was significantly increased (P<0.01), and there was no statistically significant change in the level of DBIL.

[0094] 2.3 Effects of auramide on the expression of FXR and its downstream proteins in HepG2 cells after FXR silencing Fig. 9As shown in the figure, for FXR and its downstream proteins, in normal HepG2 cells, compared with the normal group, the protein expression of NTCP (P<0.05) in the model group increased, and the difference was statistically significant. The protein expression of CYP7A1 (P<0.01) increased significantly, and the expression levels of other proteins did not change significantly; the expression of FXR (P<0.001) in the drug administration group increased significantly, the expression of SHP, BSEP and NTCP increased significantly (P<0.01), the expression of MRP2 and CYP7A1 also increased, and the difference was statistically significant (P<0.05), and the protein expression of CYP8B1 (P<0.05) decreased, and the difference was statistically significant. Compared with the model group, the protein expression of FXR and BSEP in the drug administration group increased significantly (P<0.001), the protein expression of SHP, NTCP and MRP2 increased significantly (P<0.01), the protein expression of CYP8B1 decreased significantly (P<0.001), and there was no significant difference in the protein expression of CYP7A1.

[0095] In siFXR-HepG2 cells, compared with the normal group, there were no significant differences in the protein expressions of FXR, SHP, BSEP, MRP2 and CYP7A1 in the model group, while the protein expression of NTCP (P<0.01) was significantly increased, and the protein expression of CYP8B1 (P<0.05) was increased, and the differences were statistically significant; there were no significant changes in the protein expressions of FXR, SHP, BSEP, NTCP, MRP2 and CYP7A1 in the drug group, while the protein expression of CYP8B1 (P<0.01) was significantly increased; compared with the model group, there were no significant differences in the protein expressions of FXR, SHP, BSEP, NTCP, CYP7A1 and CYP8B1 in the drug group, while the protein expression of MRP2 (P<0.05) was increased, and the difference was statistically significant.

[0096] Compared with the normal HepG2 cell treatment group, the protein expressions of FXR, SHP and BSEP in the siFXR-HepG2 cell treatment group were significantly decreased (P<0.001), the protein expression of MRP2 was significantly decreased (P<0.01), the protein expression of CYP8B1 was significantly increased (P<0.001), and there was no significant difference in the protein expressions of CYP7A1 and NTCP.

[0097] 2.4 Effects of golden amide alcohol esters on the expression of CAR and its downstream proteins in HepG2 cells after FXR silencing Fig.10As shown in the figure, after FXR silencing, for CAR and its downstream proteins, in normal HepG2 cells, compared with the normal group, there was no significant difference in the protein expression of CAR, CYP2B6, CYP2C9 and UGT1A1 in the model group; the protein expression of CAR (P<0.01) in the drug administration group was significantly increased, and the protein expression of UGT1A1 (P<0.05) was increased, and the difference was statistically significant; compared with the model group, the protein expression of CYP2B6, CYP2C9 and UGT1A1 in the drug administration group was increased (P<0.05), and the difference was statistically significant.

[0098] In siFXR-HepG2 cells, compared with the normal group, the protein expression of UGT1A1 (P<0.01) in the model group was significantly increased, and the protein expression of CYP2B6 (P<0.05) was increased, and the differences were statistically significant; the protein expression of CAR, UGT1A1 and CYP2C9 in the drug administration group was significantly increased (P<0.01), and the protein expression of CYP2B6 (P<0.001) was significantly increased.

[0099] Compared with the normal HepG2 cell treatment group, the protein expressions of CAR (P<0.01), CYP2B6 and CYP2C9 (P<0.001) in the siFXR-HepG2 cell treatment group were significantly increased, and the protein expression of UGT1A1 (P<0.05) was increased, and the differences were statistically significant.

[0100] 2.5 Effects of golden amide alcohol ester intervention on the levels of biochemical indicators of HepG2 cells after CAR silencing

[0101] like Fig.11As shown in the figure, in normal HepG2 cells, compared with the normal group (control), the cell viability of the model group (model) was significantly decreased (P<0.001), and the levels of ALT, AST, ALP, GGT, TBIL, DBIL and TBA were significantly increased (P<0.001). Compared with the model group, the cell viability of the drug administration group (model+AA) was significantly increased (P<0.001), the levels of ALT, AST, ALP, GGT, TBIL and DBIL were significantly decreased (P<0.001), and the level of TBA was decreased (P<0.05), and the differences were statistically significant. In siCAR-HepG2 cells, compared with the normal group, the cell viability and the change trends of ALT, AST, ALP, GGT, TBIL, DBIL and TBA levels of the model group (model-siFXR) were consistent with those of the model group of normal cells. Compared with the normal HepG2 cell administration group, the cell viability of the siCAR-HepG2 cell administration group was significantly decreased (P<0.001), and the levels of ALT, AST, ALP, TBIL and DBIL were significantly increased (P<0.001), while there was no statistically significant difference in the levels of GGT and TBA.

[0102] 2.6 Effects of golden amide alcohol esters on the expression of FXR and its downstream proteins in HepG2 cells after CAR silencing Fig.12 As shown in the figure, for FXR and its downstream proteins, in siCAR-HepG2 cells, compared with the normal group, the protein expression of CYP8B1 in the model group was significantly increased (P<0.01), and the expressions of other proteins showed no significant changes; in the drug-treated group, the protein expression of FXR was significantly increased, the protein expression of BSEP (P<0.05) was increased, and the differences were statistically significant, the protein expression of SHP and MRP2 (P<0.01) was significantly increased, the protein expression of NTCP (P<0.5) was decreased, and the differences were statistically significant, and the protein expression of CYP7A1 and CYP8B1 showed no significant changes; compared with the model group, the protein expression of FXR (P<0.01) in the drug-treated group was significantly increased, the protein expression of SHP and BSEP was significantly increased, the protein expression of MRP2 (P<0.05) was increased, and the differences were statistically significant, the protein expression level of CYP8B1 was significantly decreased (P<0.01), and the protein expression levels of NTCP and CYP7A1 showed no significant differences.

[0103] Compared with the normal HepG2 cell administration group, the protein expression of NTCP (P<0.01) and CYP8B1 (P<0.05) in the siCAR-HepG2 cell administration group was significantly decreased, and the differences were statistically significant. There was no significant difference in the expression of other proteins.

[0104] 2.7 Effects of golden amide alcohol esters on the expression of CAR and its downstream proteins in HepG2 cells after CAR silencing Fig.13 As shown in the figure, for CAR and its downstream proteins, in siCAR-HepG2 cells, compared with the normal group, there was no significant difference in the protein expression of CAR, CYP2B6, CYP2C9 and UGT1A1 in the model group; the protein expression of CYP2B6 in the drug-treated group increased (P<0.05), and the difference was statistically significant, and there was no significant difference in the expression of other proteins; compared with the model group, the protein expression of CYP2B6 in the drug-treated group was significantly increased (P<0.01), and there was no significant difference in the expression of other proteins.

[0105] Compared with the normal HepG2 cell administration group, the protein expressions of CAR (P<0.001), CYP2B6 (P<0.01) and CYP2C9 (P<0.01) in the siCAR-HepG2 cell administration group were significantly decreased, and the protein expression of UGT1A1 (P<0.05) was decreased, and the differences were statistically significant.

[0106] The results showed that golden amide alcohol ester can effectively reduce the levels of relevant biochemical indicators in high bile salt and high bilirubin model cells, and this effect is achieved by dual stimulating FXR and CAR and regulating the expression of their downstream related proteins.

[0107] The mechanism of aureamide dual agonist FXR and CAR in relieving cholestasis Fig.14 shown.

[0108] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. Use of golden amide alcohol ester for preparing drugs for treating cholestasis.

2. The use of the golden amide alcohol ester according to claim 1 for preparing a drug for treating cholestasis, characterized in that: Golden amide alcohol esters regulate the protein levels of bile acid and bilirubin related transporters and metabolic enzymes through dual agonist nuclear receptors FXR and CAR.

3. The use of the golden amide alcohol ester according to claim 1 for preparing a drug for treating cholestasis, characterized in that: Aureoside reduces the intracellular levels of total bile acid and bilirubin.

4. A method for verifying the activation of FXR and CAR by gold amide alcohol esters based on molecular docking technology, characterized in that: The following steps are involved: a) Using the hFXR (PDB code: 5Z12) and hCAR (PDB code: 1XVP) crystal structures in the PDB database for preprocessing, setting grid points and verifying the agonist potential of gold amide alcohol esters on hFXR and hCAR; b) Using molecular docking technology, the structure of the gold amide alcohol ester compound was docked into the active sites of hFXR and hCAR, and the docking score and binding mode were analyzed and compared with the binding modes of known agonists WAY-362450 and CITCO to determine its agonistic potential.

5. A cell verification method for a golden amide alcohol ester to stimulate FXR and CAR in vitro, characterized in that: The following steps are involved: a) examining the cytotoxicity of golden amide alcohol ester to HepG2 cells by MTT method to determine the applicable dosage range; b) Dual luciferase reporter gene assay was used to verify that auramide ester can stimulate the activity of FXR and CAR in HepG2 cells, and the activity was concentration-dependent.

6. An animal experimental method for alleviating cholestasis with golden amide alcohol ester, characterized in that: The following steps are involved: a) Male C57BL / 6J mice were used for modeling and randomly divided into groups; b) The mice in each experimental group were intraperitoneally injected with auramide alcohol ester and intragastrically administered with ANIT on the 5th day; c) After the experiment, blood, liver and gallbladder were collected, serum ALT, AST, ALP, GGT, TBIL and TBA levels were measured, and bile acid and liver tissue pathology were examined.

7. A cell experiment method for explaining the molecular mechanism of golden amide alcohol ester in relieving cholestasis, characterized in that: The following steps are involved: a) FXR and CAR in HepG2 cells were silenced by lentiviral technology to construct siFXR-HepG2 and siCAR-HepG2 cell models; b) Using α-naphthyl isothiocyanate, bilirubin and probenecid to establish a high bile salt and high bilirubin cell model; c) After intervention with aureoquinol ester, the levels of ALT, AST, ALP, GGT, TBIL, DBIL and TBA were detected, and the expressions of FXR, CAR and their downstream proteins were detected by Western Blot to analyze their molecular mechanisms.

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