Application of 2-[4-hydroxy-3-(4-hydroxyphenyl) benzyl]-4-(4-hydroxyphenyl) phenol in intervention of early lesion of Parkinson's disease

Abstract

The invention belongs to the technical field of medicines, and discloses application of 2-[4-hydroxy-3-(4-hydroxyphenyl) benzyl]-4-(4-hydroxyphenyl) phenol as shown in a formula (I) in intervention of early lesion of Parkinson's disease. Specifically, the invention relates to 2-[4-hydroxy-3-(4-hydroxyphenyl) benzyl]-4-(4-hydroxyphenyl) phenol and a pharmaceutically acceptable salt thereof, and application of a pharmaceutical composition of the compound in preparation of drugs for intervening early lesion of Parkinson's disease. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medical technology and mainly relates to the application of 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol, a compound separated from Gastrodia elata, in intervening in the early lesions of Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain and a significant decrease in dopamine content in the striatum. The pathological hallmarks are the presence of Lewy bodies and Lewy neurites in the remaining neurons, the main component of which is aggregated α-synuclein. The pathogenesis of PD is still unclear.

[0003] Clinically, drug treatment for PD is mainly aimed at patients in the middle and late stages, and is mainly symptomatic treatment, which cannot delay or reverse pathological progression. Therefore, it is urgent to find effective drugs for early control and / or delaying the neurodegenerative process of PD.

[0004] The rhizome of Gastrodia elata is a traditional precious Chinese medicine with multiple biological effects. Tianma Gouteng decoction can be used as an additional treatment for PD patients. Compound 20C, i.e., compound of formula (I), is a tetramerized benzyl compound extracted from Gastrodia elata, called 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol (structure shown in formula (I)). Previous studies have shown that 20C exhibits neuroprotective effects in a cell model damaged by rotenone, alleviates behavioral disorders in an MPTP mouse model and a 6-OHDA rat model, and reduces the loss of midbrain dopaminergic neurons. However, there has been no report on whether the compound of formula (I) can intervene in the early lesions of Parkinson's disease.

[0005] The applicant discovered that the compound of formula (I) can inhibit the aggregation of Parkinson's disease pathological protein α-synuclein in vitro; in the mouse PD early lesion model constructed based on the "gut-brain axis" theory, the compound of formula (I) effectively inhibited the aggregation and modification of pathological α-synuclein in gastric tissue and brain tissue, showing a protective effect on neurons.

[0006] Summary of the invention

[0007] The technical problem solved by the present invention is to provide a 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol as shown in formula (I) and a pharmaceutically acceptable salt thereof for use in the preparation of a drug for intervening in the early stages of Parkinson's disease. The 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol as shown in formula (I) has the characteristics of inhibiting α-synuclein aggregation in vivo and in vitro, exerting a neuroprotective effect, and the like.

[0008] In order to solve the technical problem of the present invention, the present invention adopts the following technical solution:

[0009] The first aspect of the technical solution of the present invention is to provide the use of 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol and its pharmaceutically acceptable salts as shown in the general formula (I) in the preparation of drugs for intervening in the early lesions of Parkinson's disease.

[0010]

[0011] The second aspect of the technical solution of the present invention is to provide a use of a pharmaceutical composition in the preparation of a drug for intervening in the early stages of Parkinson's disease, characterized in that the pharmaceutical composition contains 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol as shown in formula (I) and a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier or excipient as an effective ingredient,

[0012]

[0013] The compound represented by the above formula (I) can be extracted by conventional phytochemical methods or synthesized by conventional medicinal chemistry methods.

[0014] Beneficial technical effects

[0015] The present invention proves through pharmacological experiments that the compound shown in formula (I) can inhibit α-synuclein aggregation in vitro; in a mouse PD early lesion model constructed based on the "gut-brain axis" theory, the total amount of α-synuclein in mouse gastric tissue, α-synuclein aggregates, α-synuclein nitration modification and ubiquitin content are reduced; the content of brainstem α-synuclein and nitrated α-synuclein is reduced, and the protective effect on 5-hydroxytryptamine neurons in the raphe nucleus is exerted. The above experiments show that the compound has a pharmacological effect of intervening in the early lesions of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The compound of formula (I) inhibits α-synuclein aggregation in vitro

[0017] Figure 2The compound of formula (I) reduces the content of α-synuclein in the stomach of mice

[0018] Figure 3 The compound of formula (I) reduces the content of nitrated α-synuclein in the stomach of mice

[0019] Figure 4 The compound of formula (I) reduces the content of proteinase K-insoluble α-synuclein aggregates in mouse stomach

[0020] Figure 5 The compound of formula (I) reduces the content of ubiquitin in the stomach of mice

[0021] Figure 6 The compound of formula (I) reduces the content of α-synuclein and nitrated α-synuclein in the brain stem of mice

[0022] Figure 7 The compound of formula (I) reduces the damage of serotonergic neurons in the raphe nucleus of mice Specific implementation method:

[0023] The present invention uses in vitro experiments to determine the effect of the compound of formula (I) on the β-folding content formed by the aggregation of α-synuclein, and detects the effect of the compound on the aggregation of α-synuclein; at the same time, based on the "gut-brain axis" theory, a mouse PD early lesion model is constructed to detect the effect of the compound on the pathological modification and aggregation of α-synuclein in the stomach and brain of the animal model, and the effect on the number of brainstem neurons, and determine the neuroprotective effect of the compound, so as to provide an experimental basis for the development of the compound as a drug that can intervene in the early lesions of Parkinson's disease.

[0024] The following experimental examples can further explain the present invention, but do not limit the present invention in any way.

[0025] Experimental Example 1. Effect of the compound of formula (I) on α-synuclein aggregation in vitro.

[0026] The experimental groups are as follows:

[0027] Monomer group (α-Syn monomer): 2 mg / ml recombinant human α-synuclein monomer;

[0028] Preformed fibril group (α-Syn PFF): DMSO was added at 1% v / v to the 2 mg / ml recombinant human α-synuclein monomer solution, and the mixture was shaken at 1000 rpm at 37°C for 7 days before testing.

[0029] Positive drug Anle138b group: 100 μmol / L Anle138b was added to 2 mg / ml recombinant human α-synuclein monomer solution, shaken at 37°C and 1000 rpm for 7 days before testing;

[0030] Compound of formula (I) group: 1 μmol / L, 10 μmol / L and 100 μmol / L of compound of formula (I) were added to 2 mg / ml recombinant human α-synuclein monomer solution, shaken at 37°C and 1000 rpm for 7 days and then tested;

[0031] After shaking, 10 μl of solution was taken from each group, 30 mM ThT solution was added, and fluorescence was detected after incubation at 37°C in the dark for 1 hour. The detection conditions were: excitation wavelength 440 nm, emission wavelength 480 nm. There were three replicate wells in each group, and the fluorescence intensity of each group was normalized to the absolute value of the fluorescence of the prefabricated fibril group as 100%.

[0032] result( Figure 1 ) showed that after 7 days of shaking at 37°C and 1000rpm, the ThT fluorescence of the preformed fibril group was significantly enhanced, indicating that a large number of β-folds were formed in the protein system and α-synuclein aggregation occurred. As the concentration of formula (I) increased, the fluorescence intensity of the formula (I) compound group decreased significantly and showed concentration dependence. The inhibitory effect of formula (I) at a concentration of 100μmol / L on α-synuclein aggregation was similar to that of the positive drug Anle138b.

[0033] Experimental Example 2. Protective effect of the compound of formula (I) in the early lesion model of PD in mice.

[0034] Main materials and methods

[0035] (1) Experimental reagents and instruments: compound of formula (1), sodium carboxymethyl cellulose (Sigma), isoflurane (Shenzhen Reward Life Science Co., Ltd.), RIPA lysis buffer (Beijing Prilai Gene Technology Co., Ltd.), protease inhibitor (Beijing Prilai Gene Technology Co., Ltd.), phosphatase inhibitor (APE), protein quantification kit (Beijing Prilai Gene Technology Co., Ltd.), protein marker (Thermo), 5× loading buffer (Beijing Pulilai Gene Technology Co., Ltd.), bovine serum albumin (Roche), TH antibody (Millipore), TPH2 antibody (Abcam), α-synuclein antibody (Abcam), n-syn antibody (Thermo), GAPDH antibody (proteintech), proteinase K (Abcam), HRP-goat anti-rabbit IgG (KPL), HRP-goat anti-mouse IgG (KPL), ECL ultrasensitive luminescent liquid (Beijing Pulilai Gene Technology Co., Ltd.), 3% hydrogen peroxide (Sigma); DAB colorimetric solution (Wuhan Saiweier Biotechnology Co., Ltd.), hematoxylin stain (Shanghai Aibixin Biotechnology Co., Ltd.); small animal anesthesia machine (Midmark), high-speed tissue grinder (Wuhan Kangtao Technology Co., Ltd.), refrigerated centrifuge (Beckman Coulter), frozen section machine (Leica), paraffin section machine (Leica), protein electrophoresis instrument (Beijing Liuyi Instrument Factory), chemiluminescence imager (Shanghai Tianneng Life Science Co., Ltd.).

[0036] (2) Preparation of the compound of formula (I) and blank solvent: Weigh 250 mg of the powder of the compound of formula (I), 250 mg of sodium carboxymethylcellulose (CMC-Na) powder, and 50 mL of ultrapure water. Grind the compound of formula (I) into fine powder in a mortar, add CMC-Na powder in small amounts and grind continuously until the powder of the compound of formula (I) and the CMC-Na powder are uniformly mixed, add ultrapure water in small amounts and grind continuously until a uniform suspension is ground. The blank solvent is a 0.5% CMC-Na solution, prepared by the same grinding method. The prepared compound of formula (I) and blank solvent are stored at 4°C. The entire preparation process of the compound of formula (I) needs to be operated in the dark.

[0037] (3) Experimental animals: SPF grade C57BL / 6N mice (Beijing Weitonglihua Experimental Animal Technology Co., Ltd.), weighing 19-21 g, male, were housed in the animal room of the Institute of Materia Medica, Chinese Academy of Medical Sciences, with 12 h light and dark alternation, temperature 22±1°C, relative humidity 55±5%, and free access to food and water. The mice were housed in separate cages on the day of arrival, and all experiments followed the regulations of the Experimental Ethics Committee of the Chinese Academy of Medical Sciences.

[0038] Preparation, grouping and drug administration of early PD lesion model in mice

[0039] Normal mice were randomly divided into three groups, namely AAV-NC group, AAV-syn group, and AAV-syn+20C group. The anesthesia state of the mice was maintained with isoflurane, and the mice were fixed on the operating table in a supine position. After disinfection with 75% alcohol, an incision of about 1 cm was made in the left upper abdomen of the mice to free the stomach of the mice. Three injection sites were selected in the glandular stomach of the mice, near the forestomach, near the pylorus, and in the middle. 1 μL of viral vector was injected at each site (negative control viral vector was injected in the AAV-NC group, and AAV9-SNCA viral vector was injected in the AAV-syn group and AAV-syn+20C group). The injection lasted for 2 minutes, and the needle was retained for 1 minute. After the injection, the stomach was returned to the abdominal cavity, penicillin solution was added for antibacterial, and the incision was sutured. On the third day after the model was constructed, oral administration was started according to the weight of the mice. The AAV-syn+20C group was given a solution of the compound of formula (I) at a dose of 50 mg / kg, and the AAV-NC group and AAV-syn group were given an equal volume of blank solvent. Behavioral testing, sampling, and subsequent pathological and biochemical tests were performed 4 weeks after administration.

[0040] (3) Measurement methods and results

[0041] Western blot

[0042] Weigh the tissue with a balance, wash with ice-cold PBS, dry the surface water with filter paper, weigh it, add lysis buffer at a ratio of 1:10 (i.e. 1 mg tissue plus 10 μL lysis buffer, lysis buffer formula: RIPA lysis buffer, phosphatase inhibitor, protease inhibitor prepared according to their respective instructions), put grinding beads in the EP tube, and use a tissue grinder to break the tissue (frequency 60 Hz, running time 60 s). The homogenate was placed on ice for 30 minutes, vortexed once every 10 minutes, and centrifuged at 4°C, 12000×g for 30 minutes. The supernatant was collected and BCA protein quantification was performed, and the remaining samples were mixed at a ratio of protein supernatant: 5× loading buffer = 4:1, and boiled at 100°C for 10 minutes in a metal bath to fully denature the protein. The denatured protein was added to the pre-prepared 15% polyacrylamide gel loading well and separated by SDS-PAGE electrophoresis, with a concentrated gel voltage of 80V and a separation gel voltage of 120V. Stop electrophoresis until the front of bromophenol blue dye reaches the end of the gel. Transfer the protein from the gel to the PVDF membrane using the wet method, with a transfer current of 300 mA and a time of 60 min. Take out the PVDF membrane, put it in 5% skim milk powder, and block it on a shaker at room temperature for 2 h, then add the primary antibody and incubate it overnight at 4°C. The next day, wash the membrane with TBS-T 3 times, 10 min / time, add the secondary antibody labeled with horseradish peroxidase, and incubate it at room temperature for 2 h. Then wash the membrane with TBS-T 3 times, 10 min / time. Add ECL super-sensitive luminescent liquid and expose it in a luminometer. The results show that the compound of formula (1) can reduce the expression of α-synuclein in mouse gastric tissue ( Figure 2 ) and nitrated α-synuclein ( Figure 3 ); reduce the content of brainstem α-synuclein and nitrated α-synuclein ( Figure 6 ).

[0043] Immunohistochemistry After the mice were perfused by heart, the brain and stomach tissues were carefully removed and continued to be fixed by immersion in 4% paraformaldehyde. After dehydration with gradient sucrose polyformaldehyde solution, they were used for subsequent section staining analysis. Paraffin sections were dewaxed in two xylenes for 15 minutes, then hydrated in gradient alcohol (100%, 95%, 80%, 75%, 60% ethanol) for 5 minutes, washed with pure water for 5 minutes, heated in citric acid antigen retrieval solution for 10 minutes for antigen retrieval, and frozen sections can be directly subjected to antigen retrieval. After the sections were cooled naturally, they were washed 3 times with 0.1M PBS, 5 minutes / time, permeabilized in 0.5% TritonX-100 permeabilization solution for 15 minutes, washed 3 times with 0.1M PBS, 5 minutes / time, incubated with 3% hydrogen peroxide for 30 minutes, washed 3 times with 0.1M PBS, 5 minutes / time, blocked with 8% BSA for 30 minutes, and incubated with primary antibody at 4°C overnight. The next day, after the slices were rewarmed, they were washed with PBS-T for 3 times, 10 min / time, incubated with the corresponding secondary antibody at room temperature for 2 h, washed with PBS-T for 3 times, 10 min / time, and developed with DAB colorimetric solution. The nuclei were stained with hematoxylin according to the experimental needs. The slices were then immersed in 60%, 75%, 80%, 95%, and two 100% ethanol for 2 min, immersed in two xylenes for 3 min to dehydrate, and sealed with neutral gum. The results showed that the compound of formula (1) can reduce the expression of α-synuclein in mouse gastric tissue ( Figure 2 ), nitrated α-synuclein ( Figure 3 ) and PK-insoluble α-synuclein aggregates ( Figure 4 ) content, and reduced the ubiquitin content in the stomach of mice ( Figure 5 ) and protects 5-HT neurons in the raphe nucleus ( Figure 7 ).

Claims

1. Use of 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol as shown in formula (I) and its scientifically acceptable salt in the preparation of drugs for intervening in the early stage of Parkinson's disease, 2. Use of a pharmaceutical composition in the preparation of a drug for intervening in the early stages of Parkinson's disease, characterized in that: The pharmaceutical composition contains 2-[4-hydroxy-3-(4-hydroxyphenyl)benzyl]-4-(4-hydroxyphenyl)phenol as shown in formula (I) and a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient as an effective ingredient.

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