CN112107585A - Application of micromolecular compound disalicylate in preparation of medicine for treating non-alcoholic fatty liver disease - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to an application of a small molecular compound disalicylate in preparation of a medicine for treating non-alcoholic fatty liver disease. The invention discovers that the salicylic acid compound has the function of directly activating the activity of AMPK enzyme through in vitro enzyme activity test screening, including

Disalicylate (PubChem CID 5161) and pharmaceutically acceptable salts thereof. These compounds all increase AMPK enzyme activity. Further studies have found that these compounds can activate AMPK in animals. Can reverse the AMPK activity inhibition state in fatty liver, reduce liver fat accumulation, and can be used in preparation of medicine for treating non-alcoholic fatty liver disease.

Description

Application of micromolecular compound disalicylate in preparation of medicine for treating non-alcoholic fatty liver disease

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to an application of a small molecular compound disalicylate in preparation of a medicine for treating non-alcoholic fatty liver disease.

Background

As obesity, diabetes, continues to increase worldwide, nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adults. The data show that the prevalence rate of NAFLD of specific people in China is as high as 27%, liver fat is gradually deposited in the early stage of NAFLD, and the activity of a liver tissue NF-kB pathway of fat deposition can be continuously increased and the secretion of inflammatory factors at the downstream of the liver tissue NF-kB pathway can be increased due to saturated fatty acid, bacterial lipopolysaccharide, toxic lipid metabolites such as sphingomyelin and the like, so that chronic metabolic inflammation is caused. Chronic metabolic inflammation is not only a prominent feature of NAFLD, but also plays a key role in the malignant course of NAFLD and the development of the underlying insulin resistance that is a common disease of metabolic disease, severely threatening human health. NAFLD not only develops into liver cirrhosis, liver failure and liver cancer, but also has positive correlation with incidence rates of extrahepatic tumors, diabetes and cardiovascular diseases, and therapeutic drugs are urgently needed. Since persistent inflammation can cause glycolipid metabolic abnormalities that accelerate NAFLD, reducing or inhibiting chronic metabolic inflammation becomes a key strategy for the exploration of effective treatments for NAFLD and other metabolic diseases.

Under the metabolic disease states of NAFLD and the like, the activity of liver cell AMPK is generally reduced, and because AMPK is a key factor for regulating and controlling the energy metabolism of tissue cells, the reduction of the activity of liver tissue AMPK can cause liver fat deposition and inflammatory reaction, so that the inhibition of the activity of AMPK is positively correlated with the disease course. AMPK is a serine/threonine protein kinase that is widely present in eukaryotic cells and highly conserved, and is a major regulator of cellular energy metabolism balance. AMPK not only directly regulates the activity of multiple enzymes and nuclear transcription factors involved in anabolism of lipids and the like, but also regulates cellular metabolism and proliferation through interaction with signaling pathways mediated by other molecules such as PGC-1 alpha, mTORC 1. AMPK is composed of three subunits, α, β and γ, where the α subunit is the unit of enzymatic activity and the β and γ subunits regulate the α unit of activity. The binding of the β and γ subunits to substances or molecules such as AMP results in structural changes in the AMPK protein that allosterically activate the α activity unit while also protecting the α activity unit from dephosphorylation. Therefore, changing the response state of AMPK activity of cells has the potential of reversing the metabolic abnormality under the pathological condition of NAFLD and treating the metabolic diseases such as NAFLD and the like.

Hawley et al reported that salicylic acid (SA, salicylic acid) binds to the β 1 subunit of AMPK, causing it to allosterically and activate AMPK. Aspirin (AS, aspirin), a commonly used drug, is acetylsalicylic acid, and does not have the ability to activate AMPK in vitro enzyme activity experiments, considering that aspirin can be rapidly hydrolyzed to salicylic acid in vivo, and thus AMPK can be activated at the cellular level. The salsalate is formed by bonding 2 salicylic acid molecules by ester bonds, has the effects of relieving fever, easing pain and resisting inflammation, is clinically used for treating moderate pains such as acute and chronic rheumatic arthritis, neuralgia and the like, and also has better curative effect on gout. Since the medicine is not decomposed in stomach, has less irritation to gastrointestinal tract, and can be used at higher dose (3-4.5 g per day for adult). It is believed that it gradually decomposes into 2 molecules of salicylic acid in alkaline intestinal fluid to act, so that it can reach higher blood concentration and maintain for a longer time.

Disclosure of Invention

The invention aims to solve the problem of providing a method for treating nonalcoholic fatty liver disease by using a small molecular compound, which is characterized in that the small molecular compound can directly activate AMPK, reverse the inhibitory action of AMPK activity in vivo, reduce liver fat accumulation and reverse the NAFLD process. The micromolecular compound described by the invention is disalicylate and pharmaceutically acceptable salt, and the in-vitro AMPK activating effect of the micromolecular compound is described, and the characteristics of obviously reversing AMPK activity inhibition, improving liver fat deposition and the like on a NAFLD mouse model are described. The invention researches the effect of the Sal on the fat deposition of mouse liver tissues on a mouse NAFLD model, and finds that the Sal can obviously reduce the fat deposition of the liver tissues and reduce inflammatory reaction. In response, Sal treatment reversed mouse liver AMPK activity inhibition. Therefore, the application mainly researches the influence of Sal on the activity of AMPK enzyme, and proves that Sal can directly activate AMPK and has the capability of reversing the inhibition of AMPK activity for the first time. Can be prepared into medicine for treating non-alcoholic fatty liver disease.

Compared with the prior art, the invention has the beneficial effects that:

1. the disalicylate can directly activate AMPK and serve as a candidate compound to carry out structure optimization;

2. the salsalate exists in a salt form under the physiological pH condition, and can be used as a pharmaceutically acceptable salt;

3. the compound is used for clinical medication, can expand the application range and prepare the medicine for treating the non-alcoholic fatty liver disease;

4. the invention provides a method for screening AMPK activators in vitro;

drawings

FIG. 1 shows the AMPK activation assay by salsalate. Schematic diagram (A) of enzyme activity determination and result (B) of AMPK (adenosine monophosphate activated protein kinase) experiment activated by Sal.

FIG. 2 shows that 10mM salsalate solution in physiological saline at pH7.5 is left at room temperature for 1, 2, 4 and 24 hours, and HPLC is used to detect the content of salsalate and the salicylic acid as hydrolysate. The left side SA and Sal are standard control images.

FIG. 3 HEK293T or hepatoma HepG2 cells were treated with different concentrations of salsalate (Sal) or Aspirin (AS) or Salicylic Acid (SA). AMPK and ACC phosphorylation were identified by immunoblotting.

FIG. 4. Disalicylate effects on mouse body weight and liver tissue fat deposition (A) model building and treatment schematic, (B) mouse body weight change, (C) mouse glucose tolerance assay, (D, E) mouse liver and tissue fat deposition assay. Data were mean +/-SD, # p <0.01, # p <0.05.

Figure 5 salsalate improves liver and adipose tissue AMPK and inflammatory processes. Sal treatment improves the AMPK inhibition state of liver caused by high-fat feeding, B, mice are perfused with Sal (200mg/kg), sacrificed after 2 hours, liver and thigh muscle tissues are taken, and AMPK and ACC phosphorylation conditions are detected by an immunoblotting method. n-3. shows that the disalicylate activates AMPK experiment in vivo. C. Sal reverses inflammation and metabolic gene expression in mice fed high-fat food. Data were mean +/-SD, # p <0.01, # p <0.05.

FIG. 6 Sal improves high fat diet-induced fatty liver (A) and quantification assay (B), reduces hepatic triglyceride accumulation (C), inhibits macrophage migration (D). Data were mean +/-SD, # p <0.01, # p <0.05.

Detailed Description

The test methods used in the following examples are all conventional methods unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

HEK293T and HepG2 cells were purchased from shanghai academy of sciences, mice were purchased from the model animal center of the university of Nanjing, chemicals were purchased from Sigma (Shanghai), fluorospar kits were purchased from Japanese Wako, and SAM polypeptides were synthesized by Kinsley.

The embodiment relates to an in-vitro screening method of an AMPK activator and a study on AMPK activation by salsalate.

The invention researches whether Sal can directly activate AMPK by an in vitro enzyme activity method. The following buffer, 75mM NaCl,2mM MgCl2, 0.02% BSA, 0.02% Triton-100, and Tris-HCl (50mM, pH 7.5) was prepared first, then recombinant AMPK (purchased from Forsythia, α 1 β 1 γ 1) was added at 2 μ g/ml in 1.5ml EP tubes in 15 μ l volumes per reaction, in duplicate, blank control, ADP positive control or test samples, including different concentrations of SA, Sal, AS (deionized water adjusted to pH7.5 with NaOH), were added to the tubes, after 20 minutes of incubation at room temperature, freshly prepared ATP buffer containing 10 μ M SAMS and 200 μ M was added to each tube and incubated for 60 minutes at 30 ℃. The reaction was inactivated at 65 ℃ for 5 minutes and the ADP content was determined using the Fluorosporick kit (Wako, Japan) using a SpectraMax M3(Molecular Devices) with emission and detection wavelengths of 540nm and 590nm, respectively, and a blank reading of 1, the effect of the compound on AMPK activity was converted and plotted (FIG. 1).

The stability of disalicylate was determined at physiological pH. Salicylic Acid (SA) has been reported in the literature to activate AMPK. Since disalicylate is derived from the esterification of 2 molecules of salicylate, it is possible to activate AMPK by hydrolysis products. To this end, we simulated the stability of Sal under enzyme activity experiments (about 60 minutes in duration), and found that salsalate was poorly hydrolyzed under physiological pH conditions, and that 96% of Sal was present as the original drug even after being left at room temperature for 24 hours (FIG. 2), and that the concentration of SA produced was extremely low, and did not reach the concentration that activated AMPK, suggesting that Sal directly activated AMPK, rather than by its hydrolysate, etc.

Disalicylate promotes the study of AMPK phosphorylation. We treated 293T cells and the like with salsalate (Sal), Salicylic Acid (SA) sodium salt and Aspirin (AS) to study the capacity of the cells to induce AMPK activation, and the Sal is found to induce AMPK and ACC phosphorylation of a substrate of the AMPK (figure 3), thereby proving that the cells have the capacity of activating AMPK.

In the embodiment, the disalicylate can reduce the expression of mouse adipose tissue inflammatory factors and metabolism related genes, reverse the mouse liver and muscle tissue AMPK inactivation and improve the condition of non-alcoholic fatty liver.

We validated the effect of Sal on AMPK on a NAFLD mouse model fed with high fat food (figure 4). The literature reports significant inhibition of AMPK activity in mice fed high-fat foods. We measured the activation of AMPK in mouse liver and muscle tissues by immunoblotting and found that the inhibition of AMPK activity was reversed in the Sal-treated group (fig. 5A). This observation was further confirmed in mice. We fed healthy mice with Sal, sacrificed for 2 hours, examined AMPK phosphorylation in liver and muscle tissues, and found that Sal could activate AMPK in vivo (fig. 5B).

The invention also detects the expression of inflammatory factors and the like in adipose tissues by RT-qPCR, and proves that the expression of the inflammatory factors can be obviously increased by high-fat feeding, and the expression of the inflammatory factors can be obviously inhibited by Sal treatment. Obesity not only causes insulin resistance, but also promotes the synthesis of a large amount of triglyceride using glucose and fatty acid as raw materials, and then endogenous hyperlipidemia and fatty liver occur. The Sal treated group reversed HFD-induced fatty liver symptoms and reduced triglyceride levels. We also isolated SVF cells from adipose tissue by F4/80⁺CD11b⁺Double staining marked, flow cytometric analysis of mouse adipose tissue for the infiltration of ATMs was significantly reduced (fig. 6), suggesting that Sal may inhibit the metabolic inflammatory response process. These data indicate that Sal can reverse inhibition of AMPK activity and improve NAFLD pathology.

Claims (3)

1. An application of a micromolecular compound disalicylate in preparing a medicine for treating non-alcoholic fatty liver disease.

2. The small molecule compound disalicylate of claim 1, having the structure

And pharmaceutically acceptable salts thereof.

3. The small molecule compound disalicylate according to claim 1, characterized by its use in activating AMPK enzyme.

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