CN113101295A

CN113101295A - Use of stilbene analogues in the treatment of diabetic renal disease

Info

Publication number: CN113101295A
Application number: CN202010202539.0A
Authority: CN
Inventors: 梁威
Original assignee: Shanghai Jiangyun Medical Health Technology Co ltd
Current assignee: Shanghai Jiangyun Medical Health Technology Co ltd
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2021-07-13

Abstract

Disclosed is the use of a compound of formula (I), formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating or inhibiting diabetic kidney disease:

Description

Use of stilbene analogues in the treatment of diabetic renal disease

FIELD

The present disclosure relates generally to the field of pharmaceutical chemistry. More specifically, the present disclosure relates to stilbene analogs.

Background

Approximately 4.25 million diabetics worldwide by 2017, and approximately 6.29 million people are predicted to have diabetes by 2045 (international diabetes union, IDF). About 1.14 hundred million diabetics exist in 2017, namely about every 10 adults in China have one diabetic, and the total number of the diabetics is the first in the world.

Diabetic Kidney Disease (DKD), which is known as Diabetic Nephropathy (DN), is a Chronic Kidney Disease (CKD) caused by diabetes. One of the major microvascular complications of diabetes is the common cause of End-stage renal disease (ESRD). About 20-40% of diabetic patients will suffer from diabetic kidney disease, eventually worsening to end-stage renal disease, requiring dialysis treatment, even kidney transplantation. Up to 10% of diabetic patients die from diabetic kidney disease.

Diabetic renal disease patients suffer from renal tissue damage and functional impairment due to chronic hyperglycemia. The pathological changes can affect the whole kidney (including glomerulus, renal tubule, renal interstitium, renal blood vessel, etc.), and can be manifested by thickening of glomerular basement membrane, widening of mesangial matrix, glomerular sclerosis and loss of podocyte; thickening of renal tubule basement membrane, renal tubular atrophy and increase of apoptosis, interstitial inflammatory infiltration of kidney, interstitial fibrosis of kidney, and sparse peritubular capillary; the appearance of the wall of the arteriole changes. Clinical manifestations are mainly characterized by persistent albuminuria and/or progressive decline in Glomerular Filtration Rate (GFR).

Currently, only angiotensin II-type 1 receptor (AT1R) antagonists (blood pressure lowering: losartan and irbesartan) and sodium-glucose cotransporter-2 (SGLT2) inhibitors (urine glucose excretion: canagliflozin) are clinically approved drugs for treating diabetic renal diseases, and specific drugs for treating diabetic renal diseases are lacked. Thus, there is a great unmet clinical need in this field.

Diabetes is primarily characterized by persistent, chronically abnormally elevated blood glucose levels. Under hyperglycemic conditions, a series of highly active stable end products, collectively referred to as advanced glycation end products (AGEs), are formed between glucose and free amino groups of proteins, fatty acids or nucleic acids through non-enzymatic glycosylation reactions (Maillard reactions) via the aldehyde groups of the glucose, which are in a range higher than normal. High glucose also causes the body cells to block glycolytic metabolic pathways, which in turn generates active dihydroxy groups, which in turn are converted to AGEs.

In the process of conversion of diabetes to diabetic kidney disease, high AGEs lead to progressive changes in the kidney, such as glomerulosclerosis, interstitial fibrosis, tubular atrophy, and diminished kidney function. Renal function impairment, whose ability to eliminate AGEs is correspondingly reduced, leads in the opposite direction to an increase in the concentration of circulating AGEs in the body, further leading to structural and functional impairment of the kidney.

There are many studies in developing drugs that can inhibit the formation of AGEs and slow down their induced damage to the terminal organs. At present, no relevant medicine is on the market.

Researches on molecular pathogenesis and treatment mechanism of type 2 diabetes (more than 95% of diabetes subtypes) show that adenylate activated protein kinase (AMPK) and sirtuin 2-related enzyme 1(SIRT1, sirtuin 1, silent information regulator 2homolog 1) play important roles.

SUMMARY

Certain aspects of the present disclosure relate to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for reducing the inhibitory effect of advanced glycation end products (AGEs) on AMPK:

certain aspects of the present disclosure relate to the use of a compound represented by formula (II), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for reducing the inhibitory effect of high concentrations of glucose on AMPK:

certain aspects of the present disclosure relate to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for reducing the inhibitory effect of advanced glycation end products (AGEs) on SIRT 1:

certain aspects of the present disclosure relate to the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for inhibiting the increase of Reactive Oxygen Species (ROS) induced by advanced glycation end products (AGEs):

certain aspects of the present disclosure relate to the use of a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing diabetic kidney disease:

certain aspects of the present disclosure relate to the use of a compound represented by formula (II), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing diabetic kidney disease:

certain aspects of the present disclosure relate to compounds of formula (I) or pharmaceutically acceptable salts thereof for reducing the AMPK inhibitory effect of advanced glycation end products (AGEs):

certain aspects of the present disclosure relate to compounds represented by formula (II) or pharmaceutically acceptable salts thereof for reducing AMPK inhibition by high concentrations of glucose:

certain aspects of the present disclosure relate to compounds of formula (I) or pharmaceutically acceptable salts thereof for reducing the inhibitory effect of advanced glycation end products (AGEs) on SIRT 1:

certain aspects of the present disclosure relate to compounds of formula (I) or pharmaceutically acceptable salts thereof for inhibiting increased Reactive Oxygen Species (ROS) induced by advanced glycation end products (AGEs):

certain aspects of the present disclosure relate to compounds of formula (I) or a pharmaceutically acceptable salt thereof, for use in treating or preventing diabetic kidney disease:

certain aspects of the present disclosure relate to compounds represented by formula (II) or pharmaceutically acceptable salts thereof for treating or preventing diabetic kidney disease:

certain aspects of the present disclosure relate to methods of reducing the inhibition of advanced glycation end products (AGEs) on AMPK, comprising administering to a subject in need thereof an inhibitory effective amount of a compound of formula (I), formula (II), or a pharmaceutically acceptable salt thereof:

certain aspects of the present disclosure relate to methods of reducing the inhibition of SIRT1 by advanced glycation end products (AGEs) comprising administering to a subject in need thereof an inhibitory effective amount of a compound of formula (I):

certain aspects of the present disclosure relate to methods of reducing the AMPK inhibitory effect of high concentrations of glucose, comprising administering to a subject in need thereof an inhibitory effective amount of a compound represented by formula (II):

certain aspects of the present disclosure relate to methods of inhibiting advanced glycation end products (AGEs) induced increases in Reactive Oxygen Species (ROS) comprising administering to a subject in need thereof an inhibitory effective amount of a compound of formula (I):

certain aspects of the present disclosure relate to methods of treating or preventing diabetic kidney disease comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a compound of formula (I), formula (II), or a pharmaceutically acceptable salt thereof:

certain aspects of the present disclosure relate to pharmaceutical compositions for reducing the AMPK inhibitory effect of advanced glycation end products (AGEs) comprising an inhibitory effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a combination thereof and a pharmaceutically acceptable excipient:

certain aspects of the present disclosure relate to pharmaceutical compositions for reducing the inhibitory effect of advanced glycation end products (AGEs) on SIRT1, comprising an inhibitory effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or a combination thereof, and a pharmaceutically acceptable excipient:

certain aspects of the present disclosure relate to a pharmaceutical composition for reducing AMPK inhibition by high concentrations of glucose, comprising an inhibitory effective amount of a compound represented by formula (II):

certain aspects of the present disclosure relate to a pharmaceutical composition for inhibiting advanced glycation end products (AGEs) induced increase in Reactive Oxygen Species (ROS), comprising an inhibitory effective amount of a compound represented by formula (I):

certain aspects of the present disclosure relate to pharmaceutical compositions for treating or preventing diabetic kidney disease comprising a therapeutically or prophylactically effective amount of a compound represented by formula (I), formula (II), or a pharmaceutically acceptable salt thereof, or a combination thereof, and a pharmaceutically acceptable adjuvant:

brief description of the drawings

FIG. 1 shows that MBA inhibits AGEs-induced reduction of AMPK in HUVEC cells.

FIG. 2 shows that MBA inhibits AGEs-induced reduction of SIRT1 in HUVEC cells.

FIG. 3 shows that MBA inhibits AGEs-induced ROS production in HUVEC cells.

Figure 4 shows the activation of cellular AMPK activity by RES and OXY.

Figure 5 shows MBA reduces mouse Triglycerides (TG).

Figure 6 shows that MBA reduces mouse serum Creatinine (CREA) levels.

Figure 7 shows that MBA reduces mouse blood urea nitrogen levels.

Figure 8 shows MBA reduces total urine volume in mice for 24 hours.

Figure 9 shows MBA reduced mouse urine microalbumin (marlb).

Figure 10 shows MBA reduces total urinary Creatinine (CREA) in mice.

Figure 11 shows MBA inhibits glomerular sclerosis lesions in mice.

Figure 12 shows MBA inhibits mouse glomerulosclerosis lesions.

Detailed description of the invention

In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments. One skilled in the relevant art will recognize, however, that the embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth.

Throughout this specification and the claims which follow, unless the context requires otherwise, the words "comprise", "comprising", and "have" are to be construed in an open, inclusive sense, i.e., "including but not limited to".

Reference throughout the specification to "one embodiment," "an embodiment," "in another embodiment," or "in certain embodiments" means that a particular reference element, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Definition of

In the present disclosure, the term "mulberroside A" refers to 3- { (E) -2- [4- (β -D-glucopyranosyloxy) -2-hydroxyphenyl ] vinyl } -5-hydroxyphenyl- β -D-glucopyranoside.

In the present disclosure, the term "oxyresveratrol" refers to 4- [2- (3, 5-dihydroxyphenyl) ethenyl ] benzene-1, 3-diol, which includes both cis and trans configurations.

In the present disclosure, the term "Diabetic Kidney Disease (DKD)" refers to Chronic Kidney Disease (CKD) caused by diabetes, which is one of the major microvascular complications of diabetes.

In the present disclosure, the term "AMPK (5' -adenosine monophosphate-activated protein kinase)" refers to AMP-activated protein kinase, which is a kind of serine/threonine protein kinase and is an important energy receptor in cells.

In the present disclosure, the term "SIRT 1 (silence information regulator 2homolog 1)" refers to Sirtuin 2-related enzyme 1, a highly conserved nicotinamide adenine dinucleotide-dependent class III histone deacetylase, the first discovered Sirtuin family member in mammals, which is the homolog with the highest homology to saccharomyces cerevisiae Sirtuin 2 (Sir 2).

In the present disclosure, the term "ROS (reactive oxygen species)" refers to a byproduct of aerobic metabolism of organisms, including oxygen ions, peroxides, and oxygen-containing radicals. Excessive levels of reactive oxygen species can cause damage to cells and genetic structures.

In the present disclosure, the term "pharmaceutically acceptable salts" includes "acceptable acid addition salts" and "acceptable base addition salts".

In the present disclosure, the term "acceptable acid addition salts" refers to those salts that retain the biological effectiveness and properties of the free base, which are biologically or otherwise suitable and are formed using inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or organic acids such as, but not limited to, acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzenecarboxylic acid, 4-acetamidobenzenecarboxylic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, and the like, Mucic acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

In the present disclosure, the term "acceptable base addition salts" refers to those salts that retain the biological effectiveness and properties of the free acid, which are biologically or otherwise suitable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. In certain embodiments, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, salts of cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benzylamine, phenylenediamine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. In certain embodiments, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

In the present disclosure, the term "adjuvant" refers to a general term for other ingredients in the composition besides the drug having the active ingredient.

In the present disclosure, the term "pharmaceutically acceptable excipient" refers to an excipient and additive used in the production of a pharmaceutical product and the formulation of a prescription, which are substances that have been reasonably evaluated in terms of safety in addition to active ingredients and are included in a pharmaceutical composition.

In the present disclosure, the term "therapeutically effective amount" refers to an amount of a compound represented by formula (I), formula (II), or a pharmaceutically acceptable salt thereof that ameliorates, attenuates, or eliminates a particular disease or condition and symptoms of a particular disease or condition, or delays the onset of a particular disease or condition or symptoms of a particular disease or condition. The amount of the compound represented by formula (I), formula (II) or a pharmaceutically acceptable salt thereof described in the present disclosure constituting the "therapeutically effective amount" will vary depending on the compound represented by formula (I), formula (II) or a pharmaceutically acceptable salt thereof, the disease state and its severity, and the age, body weight, etc. of the subject to be treated, but the amount of the compound represented by formula (I), formula (II) or a pharmaceutically acceptable salt thereof described in the present disclosure can be conventionally determined by one skilled in the art based on its own knowledge and the present disclosure.

As used in this disclosure, "treating" or "treatment" encompasses treating a related disease or disease state in a subject, e.g., a mammal, e.g., a human, suffering from the related disease or disorder and includes:

(i) preventing the occurrence of a disease or condition in an individual, particularly when the individual is susceptible to said disease condition but has not yet been diagnosed as having such a disease condition;

(ii) inhibiting a disease or disease state, i.e., preventing its occurrence; or

(iii) Alleviating the disease or condition, even if the disease or condition regresses or does not progress.

As used in this disclosure, the terms "disease" and "disease state" may be used interchangeably, or may be different, in that a particular disease or disease state may not have a known causative agent (and therefore cannot be explained by etiology), and thus is not recognized as a disease, but rather is considered an undesirable disease state or condition, in which a clinician has identified a more or less specific series of symptoms.

In the present disclosure, the term "prevention" refers to an action taken to inhibit or reduce the severity or symptoms of a particular disease or condition prior to the onset of the development of the disease or condition in an individual.

In the present disclosure, the term "prophylactically effective amount" refers to an amount sufficient to prevent a disease or condition or to prevent recurrence thereof. A prophylactically effective amount of a compound of formula (I), formula (II) refers to an amount that alone or in combination with other drugs provides a prophylactic benefit in preventing disease. The term "prophylactically effective amount" can include an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

In the present disclosure, the term "mammal" refers to animals including, for example, dogs, cats, cattle, sheep, horses, and humans.

Detailed Description

certain aspects of the present disclosure relate to compounds of formula (II) or pharmaceutically acceptable thereof for use in reducing the AMPK inhibitory effect of high concentrations of glucose:

certain aspects of the present disclosure relate to methods of reducing the inhibition of advanced glycation end products (AGEs) on AMPK, comprising administering to a subject in need thereof an inhibitory effective amount of a compound of formula (I):

in certain embodiments, illustrative examples of individuals that can be used in the methods of the present disclosure include, but are not limited to, mammals.

In certain embodiments, illustrative examples of mammals that can be used in the methods of the present disclosure include, but are not limited to, humans.

in certain embodiments, illustrative examples of pharmaceutically acceptable excipients that can be used in the pharmaceutical compositions of the present disclosure for reducing the inhibitory effect of advanced glycation end products (AGEs) on AMPK include, but are not limited to, preservatives, antioxidants, flavoring agents, coloring agents, surfactants, and polymeric compounds.

In certain embodiments, illustrative examples of the polymer compound that can be used in the present disclosure include, but are not limited to, natural polymer compounds and synthetic polymer compounds.

in certain embodiments, illustrative examples of pharmaceutically acceptable excipients that can be used in the pharmaceutical compositions of the present disclosure for reducing the inhibitory effect of advanced glycation end products (AGEs) on SIRT1 include, but are not limited to, preservatives, antioxidants, flavorants, colorants, surfactants, and polymeric compounds.

in certain embodiments, illustrative examples of pharmaceutically acceptable excipients that can be used in the pharmaceutical compositions of the present disclosure for reducing the AMPK inhibitory effect of high concentrations of glucose include, but are not limited to, preservatives, antioxidants, flavoring agents, coloring agents, surfactants, and polymeric compounds.

in certain embodiments, illustrative examples of pharmaceutically acceptable excipients that can be used in the disclosed pharmaceutical compositions for inhibiting advanced glycation end products (AGEs) induced increase in Reactive Oxygen Species (ROS) include, but are not limited to, preservatives, antioxidants, flavoring agents, coloring agents, surfactants, and polymeric compounds.

in certain embodiments, illustrative examples of pharmaceutically acceptable excipients that can be used in the pharmaceutical compositions of the present disclosure for treating or preventing diabetic kidney disease include, but are not limited to, preservatives, antioxidants, flavors, colorants, surfactants, and polymeric compounds.

Hereinafter, the present disclosure will be explained in detail by the following examples in order to better understand various aspects of the present application and advantages thereof. It should be understood, however, that the following examples are not limiting and are merely illustrative of certain embodiments of the present disclosure.

Examples

Example 1

Mulberroside A (MBA) reduces the inhibition of Advanced glycation end products (AGEs) on AMPK and SIRT1 in HUVEC cells and the induced generation of Reactive Oxygen Species (ROS)

Experimental materials:

cell: human Umbilical Vein Endothelial Cells (HUVEC) (ATCC)

Mulberroside a (Mulberroside a, MBA): from Shanghai Dingrui chemical industry (Shanghai) Co., Ltd

Advanced glycation end products (AGEs): prepared according to the literature method (Xu et al, Journal of Ethnopharmacology137(2011)359-

Antibody: AMPK antibody ((Cell Signaling Technology) CST, cat No. 5832 s); SIRT1 antibody (Santa Cruz, cat # SC74504)

Reactive Oxygen Species (ROS) detection kit (chemiluminescence method): purchased from Shanghai Biyuntian Biotechnology Co., Ltd, cat #: s0033

The experimental method comprises the following steps:

(1) spreading HUVEC cells in a porous cell culture plate with a glass slide spread at the bottom, and treating the cells according to experimental groups after the cells are attached stably;

(2) grouping experiments: each test was divided into three groups, namely a normal group, a model group (AGEs: 300. mu.g/mL), and an MBA treatment group (AGEs 300. mu.g/mL + MBA 40. mu.M), and the test was administered and incubated for 24 hours;

(3) detecting the total amount of AMPK and SIRT1 protein in cells: after 24 hours of incubation, the following procedure was performed for the indicated cells:

1) cell fixation and perforation: removing cell well culture solution, washing cells with PBS, fixing with 4% paraformaldehyde, washing with PBS, perforating with 0.25% triton X-100, and washing with PBS;

2) blocking incubation primary antibody: blocking with 2% BSA for 30 min, washing twice with PBS, adding primary antibody (AMPK or SIRT-1 antibody) diluted at a ratio of 1:200, and incubating overnight at 4 deg.C;

3) and (3) secondary antibody incubation: removing the primary antibody on the third day of the experiment, washing with PBS, adding a fluorescent secondary antibody with the same species as the primary antibody, incubating at 37 ℃ in a dark place, and washing with PBS;

4) dyeing and sealing the core: adding 0.5 mu g/mL DAPI (PBS) for dyeing (keeping out of the light), washing with PBS, taking out the cover glass attached with cells from the PBS, placing the cover glass in the place where the cover glass is kept out of the light for air drying, and dropwise adding an anti-fluorescence quenching sealing tablet;

5) and (5) observing under a laser confocal microscope, and taking a picture.

(4) Intracellular Reactive Oxygen Species (ROS) detection: according to the instructions of a Reactive Oxygen Species (ROS) detection kit. After the administration and incubation for 24 hours, removing the cell well culture solution, adding a DCFH-DA solution, incubating, then washing with PBS, fixing the cells with 4% paraformaldehyde, washing with PBS, fixing the glass slide attached with the cells on a cover glass, and observing and taking a picture under a laser confocal microscope;

(5) and (4) analyzing results: the collected cell images are converted into corresponding signal data by software analysis. Analysis of differences between experimental groups was performed using ANOVA or t-test.

The experimental results are as follows:

(1) inhibitory effect of mulberroside A on reduction of advanced glycation end products (AGEs) on HUVEC cells AMPK and SIRT1

AGEs (300. mu.g/mL) treated HUVEC cells for 24 hours inhibited the intracellular levels of AMPK and SIRT1 proteins, and the addition of MBA (40. mu.M) significantly inhibited AGEs-induced reduction of intracellular AMPK (FIG. 1) and SIRT1 (FIG. 2) proteins.

(2) Mulberroside A (MBA) inhibits the induced generation of advanced glycosylation end products (AGEs) on ROS in HUVEC cells

AGEs (300. mu.g/mL) treated HUVEC cells for 24 hours significantly increased the oxidative stress of the cells, as evidenced by an increase in the oxidative stress product Reactive Oxygen Species (ROS), while the addition of MBA (40. mu.M) significantly inhibited AGEs-induced increases in intracellular ROS (FIG. 3).

Example 2

Activation effect of mulberroside A, oxyresveratrol and resveratrol on HEK cell AMPK under normal and high-sugar conditions

Experimental materials:

cell: human embryonic kidney cell 293(Human embryo kidney cells, HEK293) (ATCC)

Total AMPK cell kit (Total AMPK cellular kit-500T) (Cisbio)

Phospho-AMPK (Thr172) cell kit (Phospho-AMPK (Thr172) cellular kit-500T) (Cisbio)

Glucose: purchased from Sigma, cat # G7021

Mulberroside A: from Shanghai Dingrui chemical industry (Shanghai) Co., Ltd

Oxidizing resveratrol: from Shanghai Dingrui chemical industry (Shanghai) Co., Ltd

Resveratrol: from Shanghai Dingrui chemical industry (Shanghai) Co., Ltd

The experimental method comprises the following steps:

(1) spreading HEK293 cells in a porous cell culture plate, and processing the cells according to experimental groups after the cells are attached stably;

(2) grouping experiments: the experiments were divided into 3 groups, experimental group 1: resveratrol (RES); experimental group 2: oxyresveratrol (OXY) group, experimental group 3: mulberroside A (MBA) group. In each large group, two subsets of treatment conditions were divided, with the cell culture medium containing 5.5mM glucose subset and 30mM glucose subset. Each compound was tested for its effect at different final concentrations.

(3) And (3) cell administration treatment: cells were incubated for 24 hours at different concentrations of each compound and glucose, grouped according to the above experiments.

(4) Intracellular total AMPK (T-AMPK) and activated state phosphorylated AMPK (P-AMPK) assays: according to the kit operation instructions. Cell well supernatants were removed, cells were washed with PBS, lysed with lysis buffer, the appropriate amount was removed, incubated overnight with the corresponding HTRF premixed P-AMPK or T-AMPK antibodies, respectively, and the fluorescence emission values in the 650 and 620nM wavelength bands were read using a plate reader.

(5) And (4) analyzing results: the 650/620nM fluorescence ratio was calculated, and the ratio of phosphorylated AMPK (P-AMPK) to total AMPK was calculated.

The experimental results are as follows:

as shown in fig. 4, human embryonic kidney cells 293(HEK293) were cultured in a medium containing normal glucose (5.5mM) for 24 hours, and added with Resveratrol (RES) and Oxyresveratrol (OXY) at different concentrations to increase intracellular AMPK activity in a dose-dependent manner, as indicated by an increase in phosphorylated AMPK (P-AMPK) concentration. When HEK293 is cultured in a culture medium with high glucose concentration (30mM) for 24 hours, compared with the result of normal cell culture, the P-AMPK of phosphorylation AMPK (P-AMPK) of cells cultured by high sugar is obviously reduced, and the reduction amplitude is inhibited by adding Resveratrol (RES) and Oxyresveratrol (OXY). Mulberroside A (MBA) has no effect on AMPK activity in HEK293 cells under normal and high-concentration glucose culture conditions. The results show that Resveratrol (RES) and Oxyresveratrol (OXY) have the effects of activating the AMPK activity of HEK293 cells and reducing the inhibition of high-concentration glucose on the AMPK activity of the cells.

Example 3

Therapeutic effect of mulberroside A on diabetic kidney disease of mice

Experimental materials:

test compounds: mulberroside a (Mulberroside a, MBA): from Shanghai Dingrui chemical industry (Shanghai) Co., Ltd

Experimental animals: male C57BL/6 Diabetic Kidney Disease (DKD) mice

The experimental method comprises the following steps:

model establishment and administration mode: streptozotocin (STZ) was injected subcutaneously 48 hours after neonatal mice and female mice were nursed for four weeks. A total of 12 hyperglycemic male mice were picked four weeks later, randomly divided into 2 groups, and High Fat Diet (HFD) feeding was started. After 1 week, experiment group 2 orally drenches 5 mg/kg/day detection compound for 49 days, and experiment group 1 drenches blank solvent group

The experimental detection indexes are as follows: during animal model establishment and administration, and after animal sacrifice, kidney and liver are collected to determine the following indexes:

animal weight and feed intake

Animal serology biochemistry and kidney function detection: serum blood glucose, total Triglycerides (TG), Blood Urea Nitrogen (BUN) and Creatinine (CREA) concentrations were measured.

Total volume of urine, urinary Creatinine (CREA), urine albumin content (mALB) was determined by the Elisa method for 24-hour urine.

Histopathological analysis:

kidney: PAS staining, pathological analysis of glomerular injury, calculation of glomerular sclerosis index.

Results of the experiment

The mice injected with STZ in the experiment have the insulin secretion reduced because part of islet beta-cells are damaged, so that the blood sugar of the newborn mice is increased and exceeds the normal value, and the mice become diabetic mice in 1 month after the mice are injected with STZ. Under the condition of continuous high-fat feeding, the weight, the blood sugar and the serum triglyceride of the mouse are gradually increased, the kidney function and the tissues are damaged, and the mouse becomes a Diabetic Kidney Disease (DKD) mouse.

(1) Mulberroside A remarkably improves lipid metabolism balance of high-fat-fed mice

Mice in the administered group had significantly reduced Triglyceride (TG) compared to the blank control group (fig. 5).

(2) Mulberry bark glucoside A can significantly improve renal function of Diabetic Kidney Disease (DKD) mice and inhibit renal tissue injury

In the experiment, blood and urine of the diabetic kidney disease mouse are collected and subjected to biochemical detection on the 49 th day of the last administration of the mulberroside A. The results show that the mulberroside A significantly reduces the serum Creatinine (CREA) level of the mice (figure 6) and has the trend of reducing the blood urea nitrogen level (figure 7) compared with the blank control group; mice were significantly reduced in total urine volume at 24 hours (figure 8), urinary microalbumin (mALB) (figure 9) and total urinary Creatinine (CREA) (figure 10).

After the experiment was completed, the mice were sacrificed, kidneys were harvested, formalin fixed, and kidney histopathological analysis was performed. Mulberroside a significantly inhibited glomerulosclerosis lesions compared to the blank control results (fig. 11 and 12).

In the present disclosure, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.

From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the disclosure. Such variations and modifications are intended to fall within the scope of the appended claims of this disclosure.

Claims

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing the inhibitory effect of advanced glycation end products (AGEs) on AMPK:

2. use of a compound represented by formula (II) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for reducing the AMPK inhibitory effect of high concentration glucose:

3. use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing the inhibitory effect of advanced glycation end products (AGEs) on SIRT 1:

4. use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting the increase of Reactive Oxygen Species (ROS) induced by advanced glycation end products (AGEs):

5. use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of diabetic kidney disease:

6. use of a compound of formula (II) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of diabetic kidney disease:

7. a pharmaceutical composition for reducing the AMPK inhibitory effect of advanced glycation end products (AGEs) comprising an inhibitory effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof or a combination thereof and a pharmaceutically acceptable excipient:

8. a pharmaceutical composition for reducing the inhibitory effect of advanced glycation end products (AGEs) on SIRT1, comprising an inhibitory effective amount of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof or a combination thereof, and a pharmaceutically acceptable excipient:

9. a pharmaceutical composition for reducing AMPK inhibition by high concentration glucose comprising an inhibitory effective amount of a compound represented by formula (II) or a pharmaceutically acceptable salt thereof or a combination thereof and a pharmaceutically acceptable adjuvant:

10. a pharmaceutical composition for inhibiting advanced glycation end products (AGEs) induced Reactive Oxygen Species (ROS) increase, comprising an inhibitory effective amount of a compound represented by formula (I):

11. a pharmaceutical composition for treating or preventing diabetic kidney disease, comprising a therapeutically or prophylactically effective amount of a compound represented by formula (I), formula (II), or a pharmaceutically acceptable salt thereof, or a combination thereof, and a pharmaceutically acceptable adjuvant: