CN109303921B

CN109303921B - Application of FXR agonist and SIRT1 agonist in preparation of anti-hepatic fibrosis drugs

Info

Publication number: CN109303921B
Application number: CN201811274825.7A
Authority: CN
Inventors: 郝海平; 王洪; 崔双; 王广基; 潘晓洁; 周济宇; 何青娴; 章鹏飞; 郭怡彤; 黄宁宁
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2018-10-30
Filing date: 2018-10-30
Publication date: 2021-02-09
Anticipated expiration: 2038-10-30
Also published as: CN109303921A

Abstract

The invention discloses an application of FXR agonist and SIRT1 agonist in preparation of anti-hepatic fibrosis drugs. Compared with the prior art, the invention discloses the application of the combination of the FXR agonist and the SIRT1 agonist in preparing anti-hepatic fibrosis drugs. Under the condition of liver injury, the expression level of FXR is obviously reduced, and the SIRT1 agonist can obviously inhibit the reduction of FXR protein, so that the FXR agonist and the SIRT1 agonist are jointly used for treating hepatic fibrosis, and the method has important significance.

Description

Application of FXR agonist and SIRT1 agonist in preparation of anti-hepatic fibrosis drugs

Technical Field

The invention relates to an application of FXR agonist and SIRT1 agonist in preparation of an anti-hepatic fibrosis drug, belonging to the technical field of new application of drugs.

Background

Liver diseases, particularly viral hepatitis, fatty liver disease, hepatic fibrosis, liver cancer and the like, have a high incidence rate in the global range. For example, the incidence rate of NAFLD in European and American common adults is 20-33%, and the incidence rate of NAFLD in obese people is 60-90%; the prevalence rate of common adults in China is about 15% (Fan J G, Farrell G C. epidemic of non-alcoholic facial liver disease in China. journal of hepatology,2009,50(1): 204-10.). Liver Fibrosis (Fibrosis) is a pathological condition, which refers to the excessive deposition of fibrous connective tissue in the liver. Hepatic fibrosis is an intermediate link of further development of chronic liver diseases such as chronic viral hepatitis, metabolic disorder, chronic alcoholic/non-alcoholic fatty liver and the like to liver cirrhosis, the hepatic fibrosis is a common important characteristic of chronic liver diseases, and 25-40% of patients with chronic liver diseases finally develop liver cirrhosis and even liver cancer. Recent studies have shown that liver fibrosis is a reversible lesion, while cirrhosis is an irreversible lesion. Therefore, inhibiting, arresting and reversing liver fibrosis is a central link in the treatment of various chronic liver diseases.

The research and development of anti-hepatic fibrosis drugs are the research and development hotspots of the current liver drugs, and meanwhile, certain research progress is also made. The currently reported therapeutic drugs for preventing and treating hepatic fibrosis are mainly in the following categories: 1) chinese herbal medicine and its extract, such as Curcuma rhizome, resveratrol, silibinin, allocryptopine and its salt (grant No. CN 10132721B); 2) chemical agents and formulations thereof, such as pirfenidone and creatinine combinations (grant publication No. CN103550242B), Cleistanone derivatives (grant publication No. CN 104095857B); 3) biological agents including recombinant proteins, gene drugs and the like, the former including TGF beta 1-inhibitory peptide (grant No. CN1203091C), IL-4 (grant No. CN101318013B) and the like. Although many drugs are used for treatment, no specific drug for hepatic fibrosis is found at present. Therefore, the search for an exact effective anti-hepatic fibrosis drug is the direction of the future development.

Farnesoid X Receptor (FXR), also known as NR1H4(Nuclear Receptor Subfamily 1, Group H, Member 4), is a Member of the Nuclear Receptor superfamily. Since its cloning in 1995, the receptor has become increasingly functionally recognized: FXR plays an important role not only in the physiological processes of bile acid, lipid and carbohydrate metabolism, but also in the regulation of various pathological processes: FXR mediates the liver regeneration promoting effect of bile acid through an FXR-FGF15/19 signaling pathway; FXR exerts an anti-inflammatory effect via the FXR/NF-. kappa.B negative feedback loop (Wang Y D, Chen W D, Wang M, et alXiaology, 2008,48(5): 1632-43.); FXR exerts an anti-autophagy effect by blocking CREB-CRTC2 complex formation and inhibiting downstream autophagy-related gene expression (Seok S, Fu T, Choi SE, et al. transcriptional regulation of autophagy by an FXR-CREB a. Nature 2014; 516: 108-U274.); FXR is also closely associated with liver tumor formation: FXR^-/-Mice were normally raised to 15 months and all spontaneously developed liver tumors, while the same changes were not seen in age-matched wild-type mice (Yang F, Huang X, Yi T, et al, nanoparticles level of liver tumors in the absence of the sensitivity of the double-acid-receptor-X-receptor, cancer Res 2007; 67: 863-867.). The research shows that FXR is closely related to the occurrence and development of liver diseases. FXR agonists are the main research strategies of liver-protecting and anti-fibrosis drugs, and a large number of related patent applications (Wang H, He Q, Wang, et al. FXR modulators for enterohepatic and metabolic diseases. expert Optin peptide Pat. doi:10.1080/13543776.2018.) such as CN201210482982.3 (application No.) provides the application of the microtubulin compounds and the pharmaceutically acceptable salts thereof in the preparation of drugs for treating FXR-mediated diseases, and CN201180067346.8 and CN201080043283.8 (application No.) disclose the application of FXR activity modulators in pharmaceutical compositions, which are both authorized. In addition, 6E-CDCA (also known as Obeticholic Acid) is a potent FXR agonist whose anti-NASH efficacy is being studied in clinical stage III, and has been approved by the U.S. FDA to market as an anti-Primary Biliary Cirrhosis (PBC) drug in 2016 (Markham A, Keam SJ.Obeticholic Acid: First Global application. drugs. 2016Aug; 76(12):1221-6.) as the First drug to be successfully marketed with FXR as the target.

The results of previous animal experiments show that FXR agonists such as OCA have significant therapeutic effects on hepatic fibrosis (Fiorucci S, Antonelli E, Rizzo G, et al. the nuclear receptor SHP media inhibition of hepatic fibrous cells by FXR and protection against infection liver. gateway science. 2004 Nov; 127(5) 1497-512.Fiorucci S1, Rizzo G, Antonelli E, et al. A facial receptor-small liver modifier reagent regulation of fibrous tissue inhibition reagent inhibition of hepatic fibrosis. 584-3. the FXR agonists such as OCA and OCA have significant therapeutic effects on hepatic fibrosis (2. the FXR antagonists of liver tissue inhibition J, 95. the FXR agonists such as OCA and OCA. the FXR antibodies of liver cells of liver fibrosis of liver tissue inhibition of liver fibrosis of mouse liver fibrosis of liver tissue inhibition of liver fibrosis of liver tissue of liver of; however, two recent clinical studies have shown that: compared with Placebo, OCA has no significant improvement in the patient's indices of liver fibrosis (Nevens F, Androne P, Mazzella G, et al. A Placebo-Controlled Trial of Objectic Acid in Primary Biliary Cholatentitis. N Engl J Med.2016Aug 18; 375(7): 631-43; Kowdley KV, Lukentic V, Chapman R, et al. A randomised Trial of Objectic Acid Mongolic Acid in Patients with Primary Biliary Cholatentitis. Hepatency. 67; 5): 1890-1902). One important reason for the clinical failure of OCA is that FXR, the target of OCA, is significantly down-regulated in liver of liver-injured patients, which in turn leads to the inability of OCA to significantly activate the FXR pathway and thus exert anti-fibrotic effects.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the technical problems, the invention provides the application of the FXR agonist and the SIRT1 agonist in the preparation of anti-hepatic fibrosis drugs, and tests prove that the FXR agonist and the SIRT1 agonist have obviously enhanced anti-fibrotic effects.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

the combined use of FXR agonist and SIRT1 agonist in preparing anti-hepatic fibrosis medicine.

Preferably:

the FXR agonists include agonists of various structural types, including agonists of natural origin (chenodeoxycholic acid, CDCA, etc.), semi-synthetic, and total chemical synthesis (GW4064, Fexaramine, etc.). More preferably, the FXR agonist is selected from obeticholic acid.

The FXR agonist can promote FXR transcriptional activation, promote transcription of downstream SHP, BSEP, OST beta and other target genes, and inhibit transcription of CYP7A 1and other target genes.

The SIRT1 agonists include agonists of various structural types, and include naturally derived, semi-synthetic, and chemically synthesized agonists, including resveratrol, SRT1720, and the like.

The SIRT1 agonist is capable of promoting the deacetylation of proteins.

The SIRT1 agonist is an agonist of any enzyme and any link of a protein acetylation process. More preferably, the SIRT1 agonist is selected from SRT 1720.

In the application, the FXR agonist and the SIRT1 agonist are combined in any dose/proportion, and the aim of resisting hepatic fibrosis can be fulfilled.

The medicament is prepared from FXR agonist and SIRT1 agonist as the only components.

Or the medicine is prepared by taking FXR agonist and SIRT1 agonist as active ingredients and adding pharmaceutically acceptable auxiliary materials.

The pharmaceutically acceptable auxiliary materials refer to various conventional auxiliary materials required when different dosage forms are prepared, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbing materials and the like, and the pharmaceutically acceptable auxiliary materials are prepared into any one of common oral preparations by a conventional preparation method, such as granules, powder, tablets, capsules, pills, oral liquid, decoction, dropping pills and the like.

One important reason why obeticholic acid (OCA) fails in clinic is that the expression level of FXR, which is the target of the role of OCA, is significantly down-regulated in the liver of liver-injured patients, which in turn leads to the inability of OCA to significantly activate the FXR pathway and thus exert an anti-fibrotic effect. Therefore, blocking the expression down-regulation of FXR in pathological states is a precondition and important guarantee that FXR agonists such as OCA and the like can fully exert the drug effect. Thus, the present study found that FXR protein degradation in disease states, whereas SIRT1 agonists can block FXR degradation and in CCl₄In induced liver fibrosis state, the combined use of SIRT1 agonist and FXR agonist can play a significant enhanced anti-fibrosis role, and is a new liver fibrosis treatment strategy targeting FXR.

Compared with the prior art, the invention discloses the application of the FXR agonist and the SIRT1 agonist in the preparation of anti-hepatic fibrosis drugs. Under the condition of liver injury, the expression level of FXR is obviously reduced, and the SIRT1 agonist can obviously inhibit the reduction of FXR protein, so that the FXR agonist and the SIRT1 agonist are jointly used for treating hepatic fibrosis, and the method has important significance.

Drawings

FIG. 1: FXR protein decreases under hepatocyte injury conditions.

FIG. 2: SRT1720 can upregulate FXR expression.

FIG. 3: OCA and SRT1720 used in combination for CCl₄The function of the expression of the FXR protein of the liver in the induced hepatic fibrosis state.

FIG. 4: OCA and SRT1720 used in combination for CCl₄The role of the liver FXR signaling pathway in induced hepatic fibrosis states. Wherein A is Fxr mRNA expression level analysis; b is Shp mRNA expression level analysis. P<0.05，**P<0.01，***P<0.001。

FIG. 5: OCA and SRT1720 used in combination for CCl₄Effect of hepatic fibrosis Gene in induced hepatic fibrosis status (analysis of mRNA expression level of. alpha. SMA).

Detailed Description

In the research of the invention, a scheme for maintaining FXR expression to block degradation of FXR is provided, and the pharmacological effect of OCA is more remarkable under the condition of maintaining FXR expression level, so that a better strategy is provided for treating hepatic fibers.

The invention firstly proves that the expression level of FXR protein is obviously reduced in the damaged state of the liver cells cultured in vitro. The SIRT1 agonist SRT1720 can obviously improve the expression level of FXR and inhibit the FXR from degrading. In CCl₄In an induced hepatic fibrosis model, the combination of OCA and SRT1720 can obviously enhance the regulation and control effect of OCA on a liver FXR signal passage, obviously reduce the level of serum transaminase and improve the hepatic fibrosis state. The results of in vitro and in vivo tests are integrated, which shows that the technical scheme of the invention has better effect on the treatment of hepatic fibrosis.

The invention will be further illustrated with reference to the following specific examples. The biological materials (experimental animals) and reagents used in the examples of the present invention are commercially available, unless otherwise specified.

Example 1 SRT1720 can significantly reverse down-regulation of FXR protein levels

1 materials of the experiment

The HepG2 cell line used in the invention was purchased from Shanghai cell bank;

SRT1720 for use in the present invention is available from MCE corporation.

2 method of experiment

2.1 cell culture

Cells were grown in T25 cell culture flasks (Costar) and cultured at 37 deg.C, 5% CO2 and 90% relative humidity. The medium was DMEM medium containing 10% Fetal Bovine Serum (FBS), and penicillin 1unit/ml and streptomycin 1. mu.g/ml were added thereto. All the cells are adherent cells, the liquid is changed every other day, and the cells are divided into bottles for passage in three days.

2.2 Western blot

1) The cells were collected and placed on ice, 100. mu.l of RIPA cell lysate, 1% Protease Inhibitor Cocktail (Sigma), 1% phosphatase Inhibitor were added to each 20. mu.l of the cell volume, the pellet was blown out thoroughly, and after 30min on ice, the pellet was sonicated 5 times for 2s each, with a few seconds pause to allow the lysate to cool.

2)13000g, centrifuging for 10min, transferring the obtained supernatant as the extracted total protein sample into a new centrifuge tube, measuring the protein content by using a BCA method, adding 4 × loading buffer, mixing uniformly, and boiling for 10min to obtain the sample.

3) SDS-PAGE of 3% -5% concentrated gel and 6% -12% separation gel is prepared, and protein samples are separated by electrophoresis. The samples were thoroughly shaken and loaded with 50-100. mu.g of the same amount of total protein per sample. The electrophoresis is carried out in a gradient manner by using a concentration gel constant pressure of 75V 40min and a separation gel constant pressure of 115V 60 min.

4) Protein transfer was performed using semi-dry transfer conditions, the filter paper, the excised gel, and the PVDF membrane (Bio-Rad) were equilibrated in a semi-dry transfer buffer, the filter paper was placed flat in a semi-dry transfer bath, the PVDF membrane, the gel, and another portion of the filter paper were placed in this order, and the air bubbles between all layers were carefully purged. And (5) buckling the half-dried cover on the groove, setting a constant pressure of 25V, and performing transfer printing for 30 min.

5) And sealing the transferred PVDF membrane in 5 percent skimmed milk powder sealing solution prepared from TBST solution, and sealing for 1h at 37 ℃. An appropriate amount of primary antibody prepared in blocking solution was added in a relative amount of 100. mu.l/cm 2PVDF and incubated overnight at 4 ℃.

6) The PVDF membrane was rinsed 4 times 5min each with TBST solution.

7) The membrane was incubated with HRP-conjugated secondary antibody dilutions for 60min at 37 ℃ and the PVDF membrane was rinsed 4 times 5min each with TBST solution.

8) Exposure was performed using a ChemiDocTM XRS + gel imaging system (Bio-Rad) and densitometric analysis and semi-quantitative comparison were performed using an Image Lab TM software.

3 results of the experiment

3.1 expression levels of FXR in the TNF α -induced apoptosis model of HepG2

The results of western blot showed that the expression level of FXR decreased with the increase of the modeling time in the TNF α -induced hepatocyte apoptosis model (fig. 1).

3.2 Effect of SIRT1 agonist SRT1720 on FXR expression

The results from western blot showed (figure 2) that the SIRT 1-specific agonist SRT1720 was able to significantly increase the expression of FXR.

Example 2 Effect of combination of SRT1720 and OCA on hepatic fibrosis

1 materials of the experiment

Experimental mice (C57BL/6) were purchased from Experimental animals technology, Inc. of Wei Tong Li Hua, Beijing;

CCl₄purchased from Shanghai Lingfeng Chemicals, mineral oil from Sigma-Aldrich, Trizol RNAisso plus from Takara, reverse transcription kit and SYBR Green from Applied Biosystems, the primers used were synthesized by Life.

The remaining biological materials/reagents are available from commercial sources.

The remaining experimental materials were the same as in example 1.

2 method of experiment

2.1 SRT1720 in combination with OCA on CCl₄Induced liver fibrosis in mice.

Experiment ofThe first C57BL/6 mouse is bred adaptively for one week at room temperature of 22-26 ℃, relative humidity of 40-60% and alternating light and shade for 12 hours. All mice were housed in cages with free access to water and weight maintained at 20-22 g. The experiment was divided into a blank control group, a model group, an SRT1720 administration group, an OCA administration group, and an SRT1720 combined OCA administration group. The SRT1720 group and the combined group C57BL/6 mice were pre-dosed by intraperitoneal injection of 10mg/kg/day SRT1720, and the remaining groups were given the same dose of physiological saline. CCl was performed after 3 days₄The model was made and the blank group was given the same dose of mineral oil. After 24h of molding, the mice in the administration group were given the corresponding drug treatment, wherein the dose of OCA was 5mg/kg/day, and the mice were gavaged. Mice were euthanized after 48 h. Mouse blood and liver were collected.

2.2 Western blot analysis

The experimental procedure was as in example 1.

2.3 RT-PCR

2.3.1 Total RNA extraction from animal tissue samples

1) Separately weighing 20mg of liver tissue sample of each mouse, adding 1.0ml of RNAasso Plus, homogenizing by a homogenizer, transferring the homogenate to a centrifuge tube, standing at room temperature for 5min, and centrifuging at 12000g for 5min at 4 ℃.

2) The supernatant was transferred to 800. mu.l, 160. mu.l of chloroform was added thereto, the mixture was vigorously shaken for 15sec, and then centrifuged at 12000g for 15min after leaving at room temperature for 5 min. The sample was divided into three layers: the bottom layer is a yellow organic phase, and the upper layer is a colorless aqueous phase and an intermediate layer.

3) Carefully transferring 300 mu l of the upper-layer water phase into a new tube, adding 300 mu l of isopropanol, reversing, uniformly mixing, standing at room temperature for 10min, then centrifuging at 12000g for 10min, and discarding the supernatant;

4) the RNA pellet was washed with 1.0ml of pre-cooled 75% ethanol, then centrifuged at 12000g for 5min, the supernatant was discarded to give total RNA, which was redissolved with 20. mu.l of DEPC water, quantified and diluted to 0.5. mu.g/. mu.l.

2.3.2 reverse transcription

Preparing an RNA solution and kit components into a system with the total volume of 20 mul according to the system proportion required by the specification, setting a program temperature for reverse transcription, and specifically, the proportion is as follows:

2.3.3 PCR

the PCR system was as follows:

the PCR conditions were as follows:

stage 1: pre-denaturation at 95 ℃ for 1min

Stage 2: PCR reaction at 95 ℃ for 15 sec; such as 30sec for 40Cycles at 60 ℃; 72 ℃ 30sec

Stage 3: melting curve analysis at 65-95 deg.C, 0.5 deg.C/5

3 results of the experiment

3.1 Effect of SRT1720 in combination with OCA on mouse liver FXR protein levels

Results from western blot (FIG. 3) are shown by CCl₄The expression level of FXR in liver tissues of the modeled mice is obviously reduced compared with that of a normal group. In CCl₄In a mouse acute liver injury induction model, FXR agonist OCA cannot improve the expression level of FXR, while the expression level of FXR in a combination group of SIRT1 agonist SRT1720 and OCA is obviously improved.

3.2 Effect of combination of SRT1720 and OCA on mouse liver FXR Signal pathway

Results from real-time RT-PCR are shown (FIG. 4), in CCl₄In the induced mouse acute liver injury model, the Fxr mRNA level is obviously reduced (P)<0.001), the FXR agonist OCA, the SIRT1 agonist SRT1720, and combinations thereof, are unable to reverse the level of FXR mRNA. However, the combination of FXR agonist OCA and SIRT1 agonist SRT1720 significantly improves the mRNA level of FXR downstream target gene Shp, and compared with the model group, P<0.05, P compared with the group administered with FXR agonist OCA<0.01, P compared to the SIRT1 agonist SRT1720 administered group<0.01。

3.3 improvement of fibrosis in mice by combination of SRT1720 and OCA

The results from real-time RT-PCR are shown (FIG. 5), in CCl₄In the induced mouse acute liver injury model, the mRNA level of the liver fibrosis gene alpha-SMA is obviously increased (P)<0.001), the FXR agonist OCA, the SIRT1 agonist SRT1720 failed to reduce alpha-SMA mRNA levels. However, the combination of the FXR agonist OCA and the SIRT1 agonist SRT1720 significantly reduced the alpha-SMA mRNA level, and P compared with the model group<0.001, P compared with the group administered with FXR agonist OCA<0.001。

Claims

The application of FXR agonist and SIRT1 agonist in preparing anti-hepatic fibrosis drugs; wherein, the FXR agonist is obeticholic acid, and the SIRT1 agonist is SRT 1720.
2. The use of claim 1, wherein the medicament is prepared from an FXR agonist and a SIRT1 agonist as active ingredients, together with pharmaceutically acceptable excipients.