CN109620819B

CN109620819B - Application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing medicine for improving senile macular degeneration

Info

Publication number: CN109620819B
Application number: CN201811563225.2A
Authority: CN
Inventors: 刘健康; 李媛; 冯智辉; 曹瑞军; 黄娜
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2021-06-22
Anticipated expiration: 2038-12-20
Also published as: CN109620819A

Abstract

The application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing the medicine for improving the senile macular degeneration of retina, wherein the (1,2, 3-trimethoxy benzene) -acrylketone is used as an antioxidant for activating biphase and enhancing mitochondrial function, has an inhibiting effect on mitochondrial function damage caused by acrolein, has an inhibiting effect on cell activity reduction caused by the acrolein and has an inhibiting effect on cell death caused by the acrolein. The action of the (1,2, 3-trimethoxy benzene) -propenone is completed by activating biphase, has wide pharmacological actions of resisting cancer, inflammation, bacteria and virus, treating diabetes, protecting nerves and the like, and belongs to a high-efficiency, safe and simplified medicament.

Description

Application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing medicine for improving senile macular degeneration

Technical Field

The invention belongs to the fields of biology and medicine, and particularly relates to application of (1,2, 3-trimethoxy benzene) -propenone in preparation of a medicine for improving senile macular degeneration.

Background

Age-related macular degeneration (AMD), one of the blinding eye diseases occurring in the elderly, has an unknown etiology and may be associated with a variety of factors including chronic macular light injury, inheritance, metabolism, ethnicity, diet, and the like. The results of the studies indicate that the onset of AMD is strongly associated with atrophy and degeneration of the Retinal Pigment Epithelium (RPE) cells. RPE cells are located between the neural retina and the choroid, and under normal circumstances, play critical roles in many important ocular functions, such as phagocytosis of the photoreceptor outer segment, physiological adsorption of the neural retina, maintenance of the blood retina, and transport and secretion of numerous ions, growth factors, and cytokines. Its degeneration can induce a number of retinal diseases, including AMD.

When RPE cells are damaged, mature RPE cells stop proliferating, resulting in an increase in morphology. In this pathological situation, the loss or deterioration of function of RPE cells eventually leads to the impairment of photoreceptors associated therewith, resulting in the deterioration of vision. In AMD, this disease, RPE cells lose their ability to effectively degrade the light-sensitive waste material, resulting in the accumulation of the waste material on the retina. Dysfunctional RPE cells also cause a deterioration in photoreceptor function, which can cause a great deal of damage to vision.

Besides, there are other hypotheses: (1) theory of oxidative damage: the retinal oxygen consumption is much higher than other tissues, and the photoreceptor cell outer segments are rich in polyunsaturated fatty acids and are therefore more susceptible to oxidative stimuli when exposed to light for long periods of time, and the oxidative damage mechanism of AMD has been proposed. (2) Vascular pattern mechanisms: vascular model AMD is considered a vascular disease, primarily characterized by primary choroidal vascular perfusion abnormalities, with secondary retinal pigment epithelial cell damage leading to AMD. Vascular models emphasize the importance of arteriosclerosis and hypertension in the pathogenesis of AMD, and similar risk factors and pathogenesis are believed to exist between AMD and arteriosclerosis. (3) The genetics and genetics say that: the study shows that AMD has certain family tendency, about 1/4AMD occurrence is determined by genetic factors, and in recent 10 years, several genes are found to be related to the AMD, such as CFH, C2, ABC1, ABCA4, EFEMP1, VMD2, TIMP3 and the like. Clinical studies on family aggregates, monozygotic twins patients, etc. have shown that genetic factors dominate the development of AMD.

Current treatments for age-related macular degeneration are as follows: the type of AMD that clinically causes severe irreversible damage to vision is usually wet AMD, characterized primarily by the formation of Choroidal Neovascularization (CNV). The treatment of wet AMD aims to destroy and inhibit new blood vessels, and various treatment methods such as laser photocoagulation, transpupillary thermotherapy, photodynamic therapy, surgery and radiotherapy have limited effects, so that the purpose of delaying further vision decline is often achieved, and the requirement of effectively controlling the disease process is difficult to achieve.

Disclosure of Invention

The invention solves the problem of providing the application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing the medicine for improving the senile macular degeneration, wherein the (1,2, 3-trimethoxy benzene) -acrylketone can effectively improve the function of mitochondria, eliminates the active oxygen of the mitochondria and exerts the antioxidant activity by up-regulating the expression of the diphase gene; the (1,2, 3-trimethoxy benzene) -acrylketone as an antioxidant targeting mitochondria can restore mitochondrial damage and oxidative stress in RPE cells caused by acrolein, obviously improve cell activity, protect retina function, and prevent and intervene the occurrence and development of age-related macular degeneration of retina by resisting oxidation and protecting mitochondrial function.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the application of (1,2, 3-trimethoxy benzene) -propenone in preparing the medicine for improving the senile macular degeneration of retina, wherein the structural formula of the (1,2, 3-trimethoxy benzene) -propenone is as follows:

the application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing the medicine for improving the senile macular degeneration is characterized in that the (1,2, 3-trimethoxy benzene) -acrylketone enhances the functions of mitochondria by improving the mitochondrial membrane potential and the aerobic metabolism capability of the mitochondria.

The application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing the medicine for improving the senile macular degeneration is characterized in that the (1,2, 3-trimethoxy benzene) -acrylketone activates a biphasic enzyme system, removes mitochondrial active oxygen, enhances the antioxidant activity of cells and has the antioxidant function.

The application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing the medicine for improving the senile macular degeneration is characterized in that the (1,2, 3-trimethoxy benzene) -acrylketone can inhibit the oxidative damage of RPE cells and the death of the RPE cells caused by acrolein, has the function of protecting the retinal cells and improves the senile macular degeneration caused by the oxidative damage.

Compared with the prior art, the invention has the following beneficial technical effects:

the invention discloses a new synthesized antioxidant targeting mitochondria, discloses that (1,2, 3-trimethoxy benzene) -propenone can obviously inhibit RPE death caused by oxidative damage, has obvious retina cell protection function, explains that (1,2, 3-trimethoxy benzene) -propenone can prevent the occurrence and development of age-related macular degeneration caused by oxidative damage through antioxidation and improvement of mitochondrial function, and has good application prospect.

The chalcone compound has great flexibility and can combine with different receptors, so that the chalcone compound has wide biological activity, such as anticancer, anti-inflammatory, antibacterial, antiviral, diabetes treatment, neuroprotection and the like.

The chalcone analogue has the strongest biological activity and lower toxic and side effects compared with other synthetic compounds. The compound is (1,2, 3-trimethoxybenzene) -propenone (Tak, (1,2,3-trimethylbenzene) -acrylone), and a charged group, a methoxy group or a hydroxyl group on a benzene ring of a chalcone compound are added, so that the capacity of synthesizing glutathione in cells can be greatly improved. The biosynthesis of constitutive and inducible glutathione is closely related to the transcriptional activity of Nrf2, which shows that the charged groups on the benzene ring can also increase the transcriptional activity of Nrf 2. Three methoxy groups are modified on each benzene ring of the (1,2, 3-trimethoxybenzene) -propenone, so that the (1,2, 3-trimethoxybenzene) -propenone is supposed to have strong antioxidant activity for promoting transcription of Nrf2 and synthesis of glutathione.

Drawings

FIG. 1 is a bar graph showing the non-toxic side effects of (1,2, 3-trimethoxybenzene) -propenone on human RPE cells, wherein FIG. 1A is a bar graph showing the effect of (1,2, 3-trimethoxybenzene) -propenone on the number of human RPE cells; FIG. 1B is a bar graph of the effect of (1,2, 3-trimethoxy-phenyl) -propenone on human RPE activity.

FIG. 2 shows that (1,2, 3-trimethoxy-benzene) -propenone can improve mitochondrial function. Wherein 2A is a bar graph of the effect of (1,2, 3-trimethoxy-phenyl) -propenone on human RPE cell mitochondrial membrane potential; FIGS. 2B and 2C are graphs of the effect of (1,2, 3-trimethoxy-phenyl) -propenone on mitochondrial number.

FIG. 3 is a graph showing that (1,2, 3-trimethoxy-phenyl) -propenone activates diphase and decreases intracellular Reactive Oxygen Species (ROS) levels. Wherein FIG. 3A is a bar graph of (1,2, 3-trimethoxy benzene) -propenone inducing biphase gene mRNA expression; FIG. 3B is a gray scale graph of (1,2, 3-trimethoxy benzene) -propenone induced expression of biphase gene protein; FIG. 3C is a bar graph of (1,2, 3-trimethoxy-phenyl) -propenone decreasing intracellular ROS levels.

FIG. 4 is a graph showing that (1,2, 3-trimethoxy-phenyl) -propenone protects against RPE cell death caused by oxidative damage. Wherein FIG. 4A is a bar graph of (1,2, 3-trimethoxy-phenyl) -propenone for cell viability protection; FIG. 4B is a bar graph of (1,2, 3-trimethoxy-phenyl) -propenone for mitochondrial membrane potential protection; FIG. 4C is a fluorescent plot of (1,2, 3-trimethoxy-phenyl) -propenone for apoptosis protection.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

1. Experimental Material

(1,2, 3-trimethoxy benzene) -propenone ((1,2,3-trimethylbenzene) -acrylone) is; glutamic acid, MTT, JC-1, DCFH-DA purchased from sigma corporation; TRIzol reagent was purchased from Invitrogen corporation; RNA reverse transcription kits and SYBR fluorescent dyes were purchased from gangbao bio-inc. The RNA primer sequence is ordered and synthesized from Xian Ongkexi biological limited company; the Hoechst kit and the IP lysate are purchased in Biyun days; apoptosis kits were purchased from BD bio; primary antibodies for NQO-1, HO-1, Nrf2, Keap1 were purchased from Santa Cruz. Horseradish peroxidase-conjugated secondary antibodies against mice, rabbits, goats were purchased from Jackson ImmunoResearch Laboratories.

2. Experimental cell culture and model establishment

Human Retinal Pigment epithelial Cells (RPE) were derived from the American ATCC cell bank. Cells were cultured in a medium containing 95% air and 5% CO₂Constant temperature at 37 deg.C, wet, sterile incubator. The cells were cultured in high-glucose DMEM/F12 medium containing 10% bovine serum. All experiments used cells from fourth to eighth passage.

3. Experimental methods

(1) MTT assay

After the cells treated by the drug are cultured in a cell culture box for a corresponding time, the original culture medium is sucked out, a serum-free culture medium containing 0.5mg/ml MTT is added, after the cells are incubated in the culture box for 4 hours, the cells are washed once by PBS, DMSO is added for dissolution, and the light absorption value of the cells is detected at the wavelength of 490 nm.

(2) Mitochondrial membrane potential detection

After the cells treated with the drug are cultured in a cell culture box for a corresponding time, the original culture medium is sucked out, a serum-free culture medium containing 5 mu g/ml JC-1 is added, the cells are incubated in the culture medium for 1h, washed once by PBS, and the fluorescence intensity of the cells is detected at the wavelengths of 485/535nm and 485/590 nm.

(3) Detection of active oxygen

After the cells treated by the medicine are cultured in a cell culture box for a corresponding time, sucking out the original culture medium, adding a serum-free culture medium containing a 5DCFH-DA probe, incubating in the culture medium for 30min, washing twice by PBS, adding 200 mu L of cell lysate, scraping the cells by cell scraping, oscillating for 15s, carrying out ice bath for 10min, repeating the steps for three times, centrifuging for 10min at 4 ℃, 13000g, taking the supernatant, and detecting the fluorescence intensity of the supernatant at the wavelength of 485/590 nm.

(4) Detection of apoptosis

Apoptosis was detected with a Heochst dye: culturing cells with a 12-well plate, culturing the cells treated by the drug in a cell culture box for a corresponding time, sucking out the original culture medium, washing the cells twice by PBS, adding 500 mu L of fixing solution, fixing for 30min at room temperature, washing the cells three times by PBS, adding 500 mu L of Heochst dye solution for 5min each time, dyeing for 10min by a shaking table, washing the cells three times by PBS, observing and photographing under a fluorescence normal microscope.

(5) mRNA content detection

The detection is carried out by adopting a reverse transcription RNA-real-time fluorescence quantitative PCR method. The specific method comprises the following steps:

1) RNA extraction

12-well cell culture plate, adding 500 μ L TRIzol reagent into each well, shaking at room temperature for 5min, adding 100 μ L chloroform (1/5 total volume) to extract protein, mixing vigorously for 15s, standing at room temperature for 15min, centrifuging at 13,000g and 4 deg.C for 20 min. Transferring the upper aqueous phase to another RNase-Free EP tube, adding equal volume of isopropanol, mixing, standing at room temperature for 15min, centrifuging at 13,000g and 4 deg.C for 20min, and removing the supernatant. Add 500. mu.L of pre-cooled 75% ethanol, mix by inversion, centrifuge for 10min at 13,000g,4 ℃. The supernatant was discarded, ethanol was evaporated and completely evaporated, and dissolved in 10-20. mu.l of DEPC water. The concentration of the reverse transcription reaction solution is detected and determined by an ultraviolet spectrophotometer and used for reverse transcription.

2) Reverse transcription of RNA

The transcription volume was 20. mu.l, 500ng of RNA was removed, 4. mu.l of 5 XTMaster Mix was added, the volume was made up to 20. mu.l with DEPC water, incubated for 60min at 37 ℃ and allowed to act for 15s at 80 ℃. Placing at-20 ℃ for later use.

3) Real-time fluorescent quantitative PCR (Real-time PCR)

The SYBR Green method is used for carrying out the method, and the reaction system comprises 1 mu l of cDNA and 5 mu l of 2

Premix Ex Taq^TMII, 0.5. mu.l of the upstream and downstream primer mixture (10. mu.M), and 10. mu.l of sterile water was added. Reaction conditions were as described, 95 ℃ melting for 10min, 40 cycles of PCR (each cycle consisting of 95 ℃ 30s, 55 ℃ 30s, 72 ℃ 20s), and finally melting curves were observed (95 ℃ 15s, 60 ℃ 15s, 95 ℃ 15 s).

(6) Protein detection

1) Protein extraction

Adding 100 mu L of IP lysate into each well of a 6-well cell culture plate, scraping cells by using a cell scraper, oscillating for 15s, carrying out ice bath for 10min, repeating the operation for three times in such a way that the ice bath time is at least more than 30min, centrifuging for 10min at 4 ℃ at 13000g, taking supernatant, carrying out protein quantification by using a BCA method, leveling, adding 5X loading buffer and mercaptoethanol, boiling for 10min, and preserving the protein at-80 ℃ for later use.

2)Western blot

Performing gel running by using 10% acrylamide gel, wherein the loading amount of protein is 20 mu g, performing transfer printing by using a PVDF membrane, sealing, incubating a primary antibody, standing overnight at 4 ℃, cleaning the primary antibody, incubating a secondary antibody, cleaning the secondary antibody at room temperature for 1h, and performing chemiluminescence.

(7) Statistical analysis

Results are expressed as Mean ± s.e.m, and data analysis using One Way-ANOVA analysis method, significant statistical significance was p <0.05 and p < 0.01.

Example one

The (1,2, 3-trimethoxy benzene) -acrylketone has no toxic or side effect on human RPE cells

Human RPE cells were treated with (1,2, 3-trimethoxy-phenyl) -propenone for 24 hours at concentrations of 0.1, 1, 5, 10. mu.M. Crystal violet staining measures the change in cell number, and the fuel specifically attaches to the cell surface, and the relative change in cell number can be analyzed by comparing the absorbance. As shown in fig. 1A, crystal violet staining analysis showed that various concentrations of (1,2, 3-trimethoxy-phenyl) -propenone produced no change in the number of RPE cells. Meanwhile, the relative viability of the cells was measured by the MTT method, as shown in FIG. 1B, the viability of the cells was not significantly changed after the cells were pretreated with (1,2, 3-trimethoxy phenyl) -propenone. These results show that the compound has no toxic side effects on RPE cells. Results are expressed as Mean ± s.e.m, and statistical results represent three independent experiments.

Example two

The (1,2, 3-trimethoxy benzene) -propenone can improve mitochondrial function

Mitochondrial membrane potential is one of important indicators of mitochondrial function, and higher mitochondrial membrane potential indicates better mitochondrial function, while mitochondrial membrane potential tends to decrease much at the time of cell death. We used the classical JC-1 lipophilic cationic dye to detect mitochondrial membrane potential. JC-1 exists in a polymer form in a mitochondrial membrane and generates red fluorescence, and JC-1 cannot be gathered in mitochondria and exists in a monomer form and generates green fluorescence. The red light/green light ratio can reflect the intensity of the membrane potential of the mitochondrial membrane, and the higher the ratio, the stronger the membrane potential. As shown in FIG. 2A, mitochondrial membrane potential was significantly increased at both 5 and 10 μ M treatment concentrations 24 hours after treatment of RPE cells with (1,2, 3-trimethoxy-phenyl) -propenone. The number of mitochondria is used as another index of mitochondrial function, and the detection is completed by a method combining a classical mito-tracker dye with a flow cytometer, as shown in fig. 2B, a red curve shows the number of mitochondria in an untreated cell of a drug, a green curve shows the number of mitochondria treated with 1 μ M of the drug, a purple curve shows the number of mitochondria treated with 10 μ M of the drug, and the purple curve is obviously shifted relative to the untreated cell, so that the number of mitochondria remarkably increased is confirmed, and the drug can improve the number of mitochondria in an RPE cell. FIG. 2B shows that the drug increases the number of mitochondria within the cell in a time-dependent manner. These data suggest that (1,2, 3-trimethoxy-phenyl) -propenone can improve mitochondrial function. Results are expressed in Mean ± s.e.m, statistical results represent three independent experiments; p <0.05, p < 0.01.

EXAMPLE III

The (1,2, 3-trimethoxy benzene) -propenone can effectively enhance the antioxidant activity of cells

Reactive Oxygen Species (ROS) refers to a group of chemically active compounds having oxygen-containing groups, with free radicals being the most predominant ROS. ROS exist in many forms, such as superoxide anion (O2)^-· OH), hydroxyl radical (· OH), hydrogen peroxide (H2O2), and the like.

ROS are produced in many parts of the cell, but the vast majority (approximately 90%) of ROS are from mitochondria. Mitochondrial ROS production is the result of oxidative phosphorylation. Excessive ROS can cause oxidative stress and cause damage to RPE cells, ultimately leading to the development of AMD. An antioxidant system is also present in a human body, and can be activated under specific conditions to increase the antioxidant capacity of the body and eliminate excessive ROS, so that the system is a two-phase enzyme system. The system is composed of a series of enzymes, such as glutathione-S-transferase (GST for short), cysteine glutamate ligase (GCL for short), Epoxide Hydrolase (EH), heme oxygenase (HO-1 for short), quinone oxidoreductase (NQO 1 for short) and the like. The expression of the enzymes is induced, and the related diseases caused by various foreign substances can be effectively caused.

As shown in FIG. 3A, (1,2, 3-trimethoxy benzene) -propenone treated cells rapidly induced mRNA expression of diphase genes such as HO-1, GCLm for 6 hours. While FIG. 3B shows that the protein content of the biphase gene, such as HO-1, increases dramatically with 24 hours of treatment. Finally, as shown in fig. 3C, intracellular ROS levels were significantly reduced. These data show that (1,2, 3-trimethoxy-phenyl) -propenone is effective in enhancing cell antioxidant activity. Results are expressed in Mean ± s.e.m, statistical results represent three independent experiments; p <0.05, p < 0.01.

Example four

(1,2, 3-trimethoxy benzene) -propenone can inhibit acrolein-induced RPE cell death

Acrolein is one of the main harmful substances in smoke generated during smoking and is also a key substance for inducing oxidative damage of cells, and epidemiology shows that smoking is one of the key pathogenic factors for the onset of AMD. As shown in fig. 4A, treatment with acrolein can drastically reduce the mitochondrial membrane potential in RPE cells, while advanced treatment with (1,2, 3-trimethoxy-phenyl) -propenone can effectively protect the mitochondrial membrane potential of RPE cells. Also as shown in FIG. 4B, (1,2, 3-trimethoxy-phenyl) -propenone was effective in inhibiting the decrease in RPE cell activity caused by acrolein. Fig. 4C shows that acrolein treatment significantly induced cell death, apoptotic bodies appeared in the cells, and (1,2, 3-trimethoxy-phenyl) -propenone effectively restored the normal state of the cells, preventing the occurrence of cell death. These data heads show that (1,2, 3-trimethoxy-phenyl) -propenone is effective in inhibiting acrolein-induced cell death. Results are expressed in Mean ± s.e.m, statistical results represent three independent experiments; p <0.05, p < 0.01.

The experimental results prove that the (1,2, 3-trimethoxy benzene) -acrylketone can inhibit cell damage caused by oxidative damage by improving the antioxidant and mitochondrial aerobic metabolic capacity in RPE cells, thereby playing a role in protecting retina. The following applications may be made:

application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing medicine for improving retinal macular degeneration caused by aging and oxidative damage.

Furthermore, the medicine is a medicine for improving the respiratory function of mitochondria.

The medicine can improve the membrane potential of mitochondria and the aerobic metabolism capability of mitochondria.

Furthermore, the medicine is an antioxidant medicine which can not only remove active oxygen in cells, but also can remove active oxygen generated by electron leakage of a mitochondrial respiratory chain in a targeted mode.

Furthermore, the medicine is a medicine for activating a diphase enzyme system and improving the expression of the diphase enzyme gene so as to play the role of antioxidation.

Furthermore, the drug is a drug having a protective effect on the cytotoxic reaction caused by oxidative stress.

The medicine can inhibit cell activity reduction and cell apoptosis increase caused by oxidative damage.

In the aging process, the injury of a large number of toxic substances in the environment is borne, the ROS is increased, the oxidative damage is aggravated to be a main manifestation form, and the activation of a two-phase enzyme system in vivo, the enhancement of an antioxidant function and the promotion of mitochondrial activity are effective ways for resisting diseases. And (1,2, 3-trimethoxy benzene) -acrylketone shows good characteristics of inhibiting oxidative stress and mitochondrial injury in the RPE cell injury experiment. Therefore, (1,2, 3-trimethoxy benzene) -acrylketone has good application prospect in preventing eye diseases such as senile macular degeneration and the like.

The following applications may be made:

application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing medicine for preventing and treating retinal cell injury. The medicament is used for treating cell damage caused by oxidative stress.

Application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing medicine or health product for preventing senile macular degeneration.

The embodiments given above are preferable examples for implementing the present invention, and the present invention is not limited to the above-described embodiments. Any non-essential addition and replacement made by the technical characteristics of the technical scheme of the invention by a person skilled in the art belong to the protection scope of the invention.

Claims

The application of (1,2, 3-trimethoxy benzene) -acrylketone in preparing the medicine for improving the senile macular degeneration is characterized in that the structural formula of the (1,2, 3-trimethoxy benzene) -acrylketone is as follows:

the (1,2, 3-trimethoxy benzene) -acrylketone enhances the functions of mitochondria by improving the membrane potential of the mitochondria and the aerobic metabolism capability of the mitochondria;

the (1,2, 3-trimethoxy benzene) -acrylketone activates a diphasic enzyme system, removes active oxygen of mitochondria, enhances the antioxidant activity of cells and has the antioxidant function;

the (1,2, 3-trimethoxy benzene) -acrylketone can inhibit RPE cell oxidative damage, inhibit RPE cell death caused by acrolein, protect retinal cells, and improve senile macular degeneration caused by oxidative damage.