Application of small molecule 18: 1 LPE in preparation of medicine for preventing and / or treating Parkinson's disease

Abstract

The invention belongs to the technical field of medicines for resisting Parkinson's disease, and particularly relates to application of micromolecule 18: 1 LPE in preparation of medicines for preventing and / or treating Parkinson's disease. The invention particularly provides application of small molecule 18: 1 LPE in preparation of a medicine for preventing and / or treating Parkinson's disease. The micromolecule 18: 1 LPE can increase the phosphorylation level of Bcl-2 and activate an autophagy pathway, so that pathological aggregation of alpha-syn is relieved, the neuroprotective effect is achieved, and PD motion symptoms are relieved. Therefore, the small molecule 18: 1 LPE has the prospect of being developed into the medicine for preventing and / or treating the Parkinson's disease. The invention provides a new thought and scientific basis for preventing and treating neurodegenerative diseases such as Parkinson's disease by activating an autolysosome approach.

Description

Technical Field

[0001] This invention belongs to the field of anti-Parkinson's disease drug technology, and in particular relates to the application of a small molecule 18:1 LPE in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Its main clinical manifestations are motor symptoms, such as resting tremor, muscle rigidity, dyskinesia, and postural instability, as well as non-motor symptoms such as constipation and olfactory dysfunction. This is likely due to the abnormal accumulation of alpha-synuclein (α-syn) in dopaminergic neurons in the substantia nigra pars compacta, leading to the progressive loss of dopaminergic neurons. Although α-syn also exists in normal brain tissue, its abnormal accumulation disrupts cell function, causing neuronal death and ultimately resulting in the gradual loss of motor and cognitive functions. Currently, PD treatment primarily involves temporarily suppressing motor symptoms, which cannot effectively slow the progression of neurodegeneration, and long-term medication use has side effects.

[0003] In many neurodegenerative diseases, the autophagy-lysosome pathway (ALP) plays a crucial role in degrading misfolded and abnormally aggregated proteins. Therefore, the use of drugs with autophagy activity may slow down aggregate formation and neurodegeneration, potentially benefiting patients with Parkinson's disease (PD). Beclin 1 is a key regulator of autophagy; however, its expression gradually declines with age. Furthermore, during periods of high-intensity activity in the central nervous system, the autophagy-lysosome pathway (ALP) is prone to dysfunction. Impaired ALP may promote the accumulation of toxic α-synuclein, leading to the release of inflammatory factors and increased oxidative stress, further exacerbating damage and death of dopaminergic neurons, creating a vicious cycle in the progression of PD.

[0004] Studies have found that the apoptosis inhibitor Bcl-2 can bind to Beclin 1 in the BH3 domain, thereby preventing Beclin 1-dependent autophagy. Phosphorylation of either Bcl-2 or Beclin 1 can block the interaction between Beclin 1 and Bcl-2, subsequently dissociating Bcl-2 from the key autophagy protein Beclin 1. This forms the Beclin 1-Vps34-PI3K multiprotein complex, activating autophagy and restoring ALP's ability to clear α-synuclein. Therefore, restoring ALP function by regulating the autophagy pathway has become one of the core research directions in PD treatment. Summary of the Invention

[0005] The purpose of this invention is to provide the application of a small molecule 18:1 LPE in the preparation of drugs for the prevention and / or treatment of Parkinson's disease. The small molecule 18:1 LPE can increase the phosphorylation level of Bcl-2, activate the autophagy pathway, thereby reducing the pathological accumulation of α-synuclein, exerting a neuroprotective effect, and alleviating the motor symptoms of PD.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the use of a small molecule 18:1 LPE in the preparation of drugs for the prevention and / or treatment of Parkinson's disease, wherein the structural formula of the small molecule 18:1 LPE is shown in Formula I:

[0007] Formula I.

[0008] Furthermore, the Parkinson's disease drug is used to treat α-synuclein-induced Parkinson's disease.

[0009] The present invention also provides a drug for treating Parkinson's disease, wherein the drug uses a small molecule 18:1 LPE as the active ingredient and also contains a pharmaceutically acceptable carrier, and is formulated into a pharmaceutically acceptable dosage form.

[0010] Furthermore, the carrier includes solid, liquid, and semi-solid carriers.

[0011] Furthermore, the dosage forms include tablets, capsules, injections, and drops.

[0012] The beneficial effects of this invention are as follows: This invention reveals that the small molecule 18:1 LPE can increase the expression level of Bcl-2 phosphorylated protein, causing Bcl-2 to dissociate from Beclin 1. The dissociated Beclin 1 participates in the formation of the Beclin 1-Vps34-PI3K multiprotein complex, activating autophagy and degrading abnormally aggregated α-synuclein, thereby rescuing dopaminergic neurons and ultimately exerting a neuroprotective effect, alleviating motor symptoms of Parkinson's disease. Therefore, the small molecule 18:1 LPE shows promise as a drug for the prevention and / or treatment of Parkinson's disease. This invention will provide new ideas and scientific evidence for the prevention and treatment of neurodegenerative diseases such as Parkinson's disease through the activation of the autophagy-lysosomal pathway. Attached Figure Description

[0013] Figure 1 The effect of 18:1 LPE on motor coordination ability (rotarod fatigue test) in MPTP-induced PD mouse model was studied. Figure 1 (a) is the experimental flowchart; Figure 1(b) Changes in mouse body weight during the modeling period; Figure 1 (c) Fatigue rotator test - number of drops; Figure 1 (d) represents the fatigue rotator test - drop latency; This means P < 0.05.

[0014] Figure 2 The effect of 18:1 LPE on motor coordination in MPTP-induced PD mouse models (balance beam and pole climbing experiments), among which... Figure 2 (a) is the time taken to climb to the bottom of the pole in the pole climbing experiment; Figure 2 (b) is the time for turning around during the pole climbing experiment; Figure 2 (c) is the balance beam experiment; This represents P < 0.05; This means P < 0.01.

[0015] Figure 3 The effect of 18:1 LPE on damage in A53T α-syn cells, among which, Figure 3 (a) Effect of 10 μM, 20 μM, and 30 μM 18:1 LPE on the survival of A53T-transfected SH-SY5Y cells as detected by CCK-8 assay; Figure 3 (b) Effect of LDH on the survival of SH-SY5Y cells transfected with A53T; This represents P < 0.05; This represents P < 0.01; This represents P < 0.001; This means P < 0.0001.

[0016] Figure 4 The effect of 18:1 LPE on P-α-Syn and apoptosis in A53T α-syn cells, among which, Figure 4 (a) To detect the effect of 18:1 LPE on the expression level of P-α-Syn protein in SH-SY5Y cells transfected with A53T; Figure 4 (b) To detect the effect of 18:1 LPE on the expression level of cleaved Caspase-3 protein in SH-SY5Y cells transfected with A53T; This represents P < 0.01; This represents P < 0.001; This means P < 0.0001.

[0017] Figure 5 The effect of 18:1 LPE on oxidative stress levels in A53T α-syn cells, among which, Figure 5 (a) Effect of 18:1 LPE on GSH levels in A53T SH-SY5Y transfected cells; Figure 5 (b) Effect of 18:1 LPE on MDA levels in A53T SH-SY5Y transfected cells; This represents P < 0.05; This represents P < 0.01; This means P < 0.0001.

[0018] Figure 6 The effect of 18:1 LPE on autophagic flux in A53T α-syn cells, where, Figure 6 (a) Changes in p62 and LC3Ⅱ / Ⅰ protein expression levels in SH-SY5Y cells transfected with A53T by 18:1 LPE; Figure 6 (b) Effects of 18:1 LPE on LC3Ⅱ / Ⅰ protein expression levels in A53T-transfected SH-SY5Y cells after the addition of a late autophagy inhibitor; This represents P < 0.001; This means P < 0.0001.

[0019] Figure 7 The effect of 18:1 LPE on the expression levels of Beclin1 and P-Bcl-2 proteins in A53T-transfected SH-SY5Y cells; This represents P < 0.05; This represents P < 0.001; This means P < 0.0001. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the various embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0021] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0022] Example 1: Small molecule 18:1 LPE improves motor coordination in MPTP-induced PD mouse model (fatigue rotarod test). The clinical manifestations of Parkinson's disease (PD) include four core motor symptoms: bradykinesia, resting tremor, rigidity, and postural instability, as well as non-motor symptoms such as gastrointestinal disturbances and decreased sense of smell. To elucidate the effect of 18:1 LPE on MPTP-induced PD motor coordination in mice, this study established a subacute Parkinson's disease model in 8-week-old C57BL / 6J mice by intraperitoneal injection of MPTP hydrochloride for seven consecutive days. During the modeling period, daily changes in mouse body weight and the mice's condition were recorded. On day eight, a 14-day treatment regimen of 10 mg / kg 18:1 LPE via gavage was initiated. Figure 1 a). First, weight changes during the modeling period were recorded. The results showed that the body weight of the model group (MC) and the treatment group (LPE, both receiving intraperitoneal injection of MPTP for seven consecutive days) was significantly lower than that of the NC control group receiving intraperitoneal injection of normal saline. This suggests that the model was successful. Figure 1 b).

[0023] Subsequently, to assess changes in the fatigue tolerance of mice, this embodiment further conducted a fatigue rotarod test on the mice. Figure 1 The results showed that, compared with the NC control group, the MC model group mice had a shorter duration of endurance on the fatigue rotundus and a higher number of falls within five minutes. LPE administration significantly increased the duration of endurance on the fatigue rotundus and reduced the number of falls, indicating that LPE administration improved the MPTP-induced endurance decline in mice.

[0024] Example 2: Small molecule 18:1 LPE restores motor coordination in MPTP-induced PD mouse model (balance beam test and pole climbing test). The balance beam test and pole climbing test are classic methods for evaluating motor coordination in mice and are also important indicators for assessing motor behavior in PD model mice. This example uses the pole climbing test and balance beam test to study the motor coordination ability of MPTP-induced PD mice induced by 18:1 LPE. The pole climbing test (… Figure 2 (ab) Compared with the NC control group, the time taken for PD mice in the MC model to climb from the bottom to the top of the pole remained unchanged, while the time to turn around at the top of the pole was slightly prolonged, but this was not statistically significant. This was determined through the balance beam test ( Figure 2c) The results showed that, compared with the NC control group, the MC model group mice took longer to walk across the 50cm balance beam, and after treatment with 18:1 LPE, they could walk across it more quickly. These results indicate that 18:1 LPE can improve the motor coordination ability of MPTP-induced PD mice.

[0025] Example 3: Small molecule 18:1 LPE alleviates cell damage induced by A53T α-syn overexpression. SH-SY5Y cells are widely used as a cell model for studying pathological disorders (PD). A PD cell model was established by transiently transfecting SH-SY5Y cells with a plasmid encoding the A53T mutant α-syn and culturing them for 24 hours to simulate the pathological aggregation-induced cell damage of α-syn. Adding 18:1 LPE and culturing for another 24 hours showed that 30 μM of 18:1 LPE significantly reduced cell damage. This was confirmed by the CCK8 assay. Figure 3 a) After SH-SY5Y cells expressed A53T a-syn, cell viability decreased significantly, but after treatment with 18:1 LPE, cell viability increased in a dose-dependent manner. Simultaneously, lactate dehydrogenase (LDH) also demonstrated ( Figure 3 (b) 18:1 LPE alleviated damage in SH-SY5Y cells transfected with A53T a-syn in a dose-dependent manner.

[0026] Example 4: Small molecule 18:1 LPE reduces α-synuclein accumulation and weakens apoptosis. Phosphorylation at serine 129 is crucial in the development of α-synuclein pathology. It promotes the formation of fibrils, which subsequently induce the formation of α-synuclein-rich insoluble inclusion bodies in the cytoplasm of brain cells, becoming a marker of pathological α-synuclein in PD. Therefore, this study examined the expression level of P-α-Syn by 18:1 LPE. The results showed that SH-SY5Y cells overexpressing A53T α-synuclein had significantly increased phosphorylation at S129, and the expression level of Cleaved Caspase-3 protein was also significantly increased. After 24 hours of treatment with 18:1 LPE, phosphorylation at S129 and Cleaved Caspase-3 were reduced in a dose-dependent manner, without affecting caspase-3 levels. Figure 4 ab).

[0027] Example 5: Small molecule 18:1 LPE reduces oxidative stress levels in cells overexpressing A53T α-syn. High levels of oxidative stress can accelerate the accumulation of α-synuclein, leading to neuronal death. Reduced glutathione (GSH) is one of the most important intracellular antioxidants, maintaining intracellular redox balance; therefore, changes in GSH levels can reflect the oxidative stress state of cells. Figure 5 a. 18:1 LPE treatment significantly increased intracellular GSH levels, enhanced cellular antioxidant activity, and improved oxidative stress levels. Similarly, MDA, a commonly used indicator of oxidative stress, was examined, and the results showed that 18:1 LPE treatment could dose-restrain cellular MDA levels (…). Figure 5 b) Example 6: Small molecule 18:1 LPE induces autophagy and degradation of α-syn Aberrant deposition of synuclein is a significant pathological change in PD, and ALP plays a crucial role in its degradation. Detection of autophagy-related proteins LC3b and p62 showed that… Figure 6 a) In SH-SY5Y cells overexpressing A53T α-syn, the conversion of LC3Ⅰ to LC3Ⅱ was reduced, while p62 accumulation was observed, indicating inhibition of the autophagy pathway. However, after treatment with 18:1 LPE, the autophagic flux typically increased, with LC3Ⅰ to LC3Ⅱ conversion, and more p62 was consumed in the autophagic flux. To further confirm whether the degradation of A53T α-syn by 18:1 LPE is mediated through autophagy, we investigated the effect of the autolysosomal inhibitor bafilomycin A1 (Baf A1). When cells were stimulated with the late autophagy inhibitor Baf A1, intracellular autolysosomal degradation was inhibited; the observed changes in LC3B-II at this time only represented changes in the number of autophagosomes. Figure 6 b. When using Baf A1, compared with the absence of 18:1 LPE, the protein expression level of A53T α-syn cells overexpressing 18:1 LPE increased during the LC3Ⅰ to LC3Ⅱ conversion, indicating that this small molecule can increase the level of autophagosomes.

[0028] Example 7: Small molecule 18:1 LPE increases autophagy by activating Bcl-2. Beclin1 can interact with Bcl-2, and phosphorylation of Bcl-2 can block their interaction. Dissociated Beclin1 activates autophagy by forming a Beclin 1-Vps34-PI3K multiprotein complex. Therefore, this example examined the protein expression levels of P-Bcl-2 and Beclin1. The results showed that, compared with SH-SY5Y cells overexpressing A53T α-syn, the protein expression levels of P-Bcl-2 and Beclin1 increased in a dose-dependent manner after 18:1 LPE treatment. Figure 7 ).

[0029] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. The use of small molecule 18:1 LPE in the preparation of drugs for the prevention and / or treatment of Parkinson's disease, wherein the structural formula of the small molecule 18:1 LPE is shown in Formula I: Formula I.

2. The application according to claim 1, characterized in that, The aforementioned Parkinson's disease drug is used to treat α-synuclein-induced Parkinson's disease.

3. A drug for treating Parkinson's disease, characterized in that, The drug uses 18:1 LPE as its active ingredient and also contains a pharmaceutically acceptable carrier, and is formulated into a pharmaceutically acceptable dosage form.

4. The application according to claim 3, characterized in that, The carrier includes solid, liquid, and semi-solid carriers.

5. The application according to claim 3, characterized in that, The dosage forms include tablets, capsules, injections, and drops.

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