Application of clenbuterol hydrochloride in preparation of medicine for treating Parkinson's disease

Abstract

According to the application of the clenbuterol hydrochloride in preparation of the medicine for treating the Parkinson's disease, the concentration of the clenbuterol hydrochloride in the medicine for treating the Parkinson's disease is 1-20 [mu] M. It is proved for the first time that clenbuterol hydrochloride can play a neuroprotective role by improving an electrophysiological mechanism: (1) MPP + induced voltage gating K + current I-V curve abnormity is significantly improved, and a cell repolarization function is improved; and (2) the K + channel current is specifically increased. The invention provides an important experimental basis for developing novel anti-Parkinson's disease drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of clenbuterol hydrochloride in preparing a drug for treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD), a neurodegenerative disorder, significantly impacts patients' health and well-being. The disease presents primarily with movement disorders, with core symptoms including bradykinesia, resting tremor, muscle rigidity, and postural balance impairment. Notably, PD patients also commonly experience a variety of non-motor symptoms, such as mood disorders (depression, anxiety, apathy), cognitive decline (dementia), autonomic dysfunction (constipation), and sleep disturbances, which significantly reduce patients' quality of life.

[0003] The pathological manifestations of PD patients are the abnormal aggregation of α-synuclein (α-syn) in neurons and the formation of Lewy bodies, which leads to the loss of dopamine neurons in the substantia nigra and striatum of the brain and the reduction of dopamine nerve signals. Current clinical drug treatments for Parkinson's disease are mainly based on strategies to regulate the imbalance of neurotransmitters in Parkinson's disease: (1) Dopaminergic replacement therapy: for example, levodopa, as a dopamine precursor, crosses the blood-brain barrier and is converted into dopamine in the striatum, compensating for dopamine deficiency in the substantia nigra-striatal pathway, improving movement disorders, and partially inhibiting pathological low-frequency oscillation activity in the cortical-basal ganglia loop; (2) Dopamine receptor stimulation: Pramipexole, ropinirole, etc. regulate the excitability balance of the basal ganglia pathway by activating D2 / D3 receptors, reducing abnormal synchronized discharges in the subthalamic nucleus; (3) Cholinergic antagonist therapy: for example, benzhexol selectively blocks the M1 muscarinic receptors of striatal cholinergic interneurons, correcting the acetylcholine hyperactivity caused by dopamine deficiency, thereby improving abnormal neuronal electrical activity.

[0004] Cellular ion channels are fundamental components of neural electrical activity, and abnormal regulation of ion channel currents plays a key role in the pathogenesis of Parkinson's disease. Potassium channel dysfunction may lead to abnormal aggregation of α-synuclein, enhanced oxidative stress and mitochondrial damage, and damage and death of dopaminergic neurons. Furthermore, the interaction between α-synuclein and ion channels further exacerbates the neurodegenerative process. Therefore, regulating ion channel function may be a potential target for Parkinson's disease treatment. However, the effects of the commonly used Parkinson's disease drugs mentioned above on neuronal electrophysiology are mostly indirect or broad-spectrum, lacking specificity.

[0005] Clenbuterol hydrochloride is a selective β2-adrenergic receptor agonist, clinically used to treat bronchospasm caused by respiratory diseases such as bronchial asthma and chronic obstructive pulmonary disease (COPD). Recent studies have shown that this drug may have a potential protective effect on dopaminergic neurons through mechanisms such as regulating mitochondrial function and alleviating oxidative stress. The study of clenbuterol hydrochloride in this study modulating the electrophysiological characteristics of Parkinson's disease model cells provides a new research direction for exploring its application in Parkinson's disease. Summary of the Invention

[0006] To solve the above problems, the present invention provides the use of clenbuterol hydrochloride in the treatment of Parkinson's disease by improving the voltage-gated K + The IV curve characteristics of the current improve the cell repolarization function and specifically enhance the potassium ion channel (K + ) current, thereby exerting a neuroprotective effect. The specific plan is as follows:

[0007] The use of clenbuterol hydrochloride in the treatment of Parkinson's disease, wherein the clenbuterol hydrochloride is purchased from Solarbio, and the molecular structure is as follows:

[0008]

[0009] The present invention selects MPP + (1-methyl-4-phenylpyridinium ion)-induced human neuroblastoma SH-SY5Y cell line is used to construct an in vitro model of Parkinson's disease. This model has the following significant features: based on the advantages of human origin of the SH-SY5Y cell line, catecholaminergic neuronal characteristics and good experimental operability, it has become the preferred in vitro model for studying the pathogenesis of Parkinson's disease and drug screening; and MPP + Under induction, the typical pathological changes of Parkinson's disease can be stably reproduced, including abnormal aggregation of α-synuclein and a significantly increased rate of neuronal apoptosis.

[0010] The application of clenbuterol hydrochloride in the preparation of drugs for treating Parkinson's disease was studied in the above-mentioned SH-SY5Y cell model.

[0011] The present invention uses CCK-8 cytotoxicity experiments to confirm that 1μM, 20μM, 40μM, and 60μM clenbuterol hydrochloride treatment has no significant effect on cell viability and has low cytotoxicity. 1μM, 20μM, 40μM, and 60μM clenbuterol hydrochloride treatment for 48 hours can reverse MPP. + The SH-SY5Y cell viability was decreased by induced treatment. Among them, 1μM and 20μM clenbuterol hydrochloride treatment had a better effect on rescuing cell viability, showing good biosafety.

[0012] Furthermore, the present invention uses the whole-cell patch clamp technique to detect the effect of clenbuterol on cell voltage-gated K + The results showed that 1μM and 20μM clenbuterol hydrochloride could significantly improve the voltage-gated K + Current IV curve characteristics, improve cell repolarization function, and specifically increase K + Channel current.

[0013] The present invention has the following beneficial effects compared to the prior art:

[0014] This invention demonstrates for the first time that clenbuterol hydrochloride can exert neuroprotective effects by improving electrophysiological mechanisms in the concentration range of 1μM to 20μM: (1) Significantly improve MPP + Induced voltage-gated K + Abnormal current IV curve, improving cell repolarization function; (2) specifically increasing K + Channel current. There are currently no reports on clenbuterol hydrochloride regulating the electrophysiological characteristics of Parkinson's disease model cells. This study provides important experimental basis for the development of new anti-Parkinson's disease drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Clenbuterol hydrochloride increases voltage-gated K in an in vitro cell model of Parkinson's disease + Current IV curve diagram.

[0016] Figure 2 Increase K of Clenbuterol Hydrochloride in an in vitro cell model of Parkinson's disease + Current statistics graph. DETAILED DESCRIPTION

[0017] In order to make the technical means, inventive features, and objectives of the present invention easier to understand, the technical solution of the invention is further described below in conjunction with one embodiment and specific implementation method of the use of clenbuterol hydrochloride in the treatment of Parkinson's disease given in the present invention.

[0018] Example 1: Cytotoxicity assay of clenbuterol hydrochloride using the CCK-8 method, including:

[0019] (1) Collect cells in the logarithmic phase and adjust the cell suspension concentration. Add 100 μL of cell suspension to wells 2-6 of columns 2-11 of a 96-well plate (column 2 is the untreated group, columns 3-11 are the drug-treated groups). Add 100 μL of cell culture medium to row 7 (as the zero well), and fill the edge columns with 100 μL of sterile PBS. Pre-incubate the culture plate in a 37°C, 5% CO2 incubator for 24 hours.

[0020] (2) Columns 3-6 of the 96-well plate were treated with 1 μM, 20 μM, 40 μM, and 60 μM clenbuterol hydrochloride, respectively, and column 7 was treated with 1 mM MPP. + Treatment, add 1μM clenbuterol hydrochloride + 1mM MPP to columns 8-11 respectively + , 20μM clenbuterol hydrochloride + 1mM MPP + , 40μM clenbuterol hydrochloride + 1mM MPP + , 60μM clenbuterol hydrochloride + 1mM MPP + After treatment, the culture plate was placed in a 37°C, 5% CO2 incubator for incubation.

[0021] (3) 48 hours after drug addition, add 10 μL of CCK-8 solution to each well in columns 2 to 11. Be careful not to form bubbles in the wells when adding the solution.

[0022] (5) Incubate the culture plate in an incubator for 2 hours.

[0023] (6) After measuring the absorbance at 450 nm using an enzyme-labeled instrument, calculate the cell viability (%) = (A(drug-added well) - A(zero-adjusted well) / A(undrug-added well) - A(zero-adjusted well)) × 100%.

[0024] Table 1 shows the toxicity test of clenbuterol hydrochloride at different concentrations on the in vitro cell model of Parkinson's disease. The CCK-8 test results in Table 1 show that 1μM and 20μM clenbuterol hydrochloride treatments have a better effect on rescuing cell viability [MPP + Group: (47.65±0.51)% vs 1μM clenbuterol hydrochloride + MPP + Group: (64.03±0.71)%; MPP + Group: (47.65±0.51)% vs 20μM clenbuterol hydrochloride group + MPP + group: (72.75±0.76)%, p<0.01]. Moreover, under normal culture conditions, treatment with 1μM and 20μM clenbuterol hydrochloride for 48 h had no significant effect on cell viability [1μM clenbuterol hydrochloride group (99.06±3.04)% vs control group (100.20±1.23)%; 20μM clenbuterol hydrochloride group (100.10±0.95)% vs control group (100.20±1.23)%, p>0.05], indicating that the drug has low cytotoxicity within the effective concentration range.

[0025]

[0026] Example 2: SH-SY5Y / MPP after treatment with clenbuterol hydrochloride +Detection of voltage-gated potassium current in cells. Includes:

[0027] (1) Inoculate SH-SY5Y cells into lysine-coated 12-well plate cell slides and allow the cells to proliferate to 50%-60% of the slide.

[0028] (2) According to the drug screening concentration in Example 1, 1 mM MPP was added to the cultured cells. + , 1 mM MPP + and 20 μM clenbuterol hydrochloride for 24 h.

[0029] (3) After 24 hours of drug treatment, the current-voltage (IV) relationship of each group of cells was detected using the whole-cell patch clamp technique. The experimental system configuration is as follows:

[0030] Extracellular fluid:

[0031] 140 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 10 mM HEPES (pH adjusted to 7.4 with Tris-base, osmolarity 290-300 mOsm).

[0032] Electrode liquid:

[0033] 140 mM potassium gluconate, 5 mM potassium chloride, 10 mM hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.2 mM ethylene glycol diethyl ether diaminetetraacetic acid (EGTA), 2 mM magnesium chloride, 4 mM MgATP, 0.3 mM Na2GTP, and 10 mM disodium creatine phosphate (pH adjusted to 7.3 with KOH, osmolarity 280-300 mOsm).

[0034] Electrode preparation:

[0035] Borosilicate glass capillaries (Sutter Instruments, outer diameter 1.5 mm, inner diameter 0.86 mm) were pulled using a P97 needle puller.

[0036] After the electrode is filled with liquid, it is positioned on the cell membrane surface by a micromanipulator. Negative pressure is applied to form a high-resistance seal. After capacitance compensation, the membrane is broken and the whole-cell mode is entered.

[0037] (4) Use MultiClamp700B to detect the current value of SH-SY5Y cells with a voltage of -100mV to +40mV.

[0038] (5) Count the current values ​​of SH-SY5Y cells at different voltages and plot the IV curve using GraphPadPrism8. Figure 1 .

[0039] like Figure 1 As shown, under voltage clamp recording (-100 mV to +40 mV), SH-SY5Y cells exhibited typical voltage-dependent K + Current characteristics. Compared with the blank control group, MPP + The IV curve of the treatment group shifted significantly downward, K + The current amplitude was significantly reduced (p < 0.01), indicating that MPP + By inhibiting K + Channel opening or changes in its voltage-dependent activation properties interfere with K + Channel function, resulting in impaired cell repolarization ability. + In the injury model, intervention with 1μM and 20μM clenbuterol hydrochloride partially restored MPP + Induced K + The channel current was abnormal, while the treatment of SH-SY5Y cells with 1μM and 20μM clenbuterol hydrochloride alone had no significant effect on the basal K⁺ current IV curve, suggesting that the drug specifically corrects the MPP in the concentration range of 1μM to 20μM. + Induced K + Channel activation disorder, improves cell repolarization function, and thus plays a potential neuroprotective role.

[0040] Clenbuterol hydrochloride intervention partially restored MPP + Induced K + The channel current decreases. Figure 2 As shown, under voltage clamp +40mV recording, MPP + K of the treatment group + The current is significantly reduced, [MPP + group: (215.4±40.37) pA vs control group: (469.3±25.65) pA, p<0.01], in MPP + In the injury model, intervention with 1μM and 20μM clenbuterol hydrochloride partially restored MPP + Induced K + The channel current decreases, [MPP + group: (215.4±40.37) pA vs 1μM clenbuterol hydrochloride group: (347±9.02) pA; MPP +group: (215.4±40.37) pA vs 20μM clenbuterol hydrochloride group: (402.8±18.03) pA, p<0.01]. However, the treatment of SH-SY5Y cells with 1μM and 20μM clenbuterol hydrochloride alone had no significant effect on the basal K + The above results indicate that clenbuterol hydrochloride selectively reverses MPP in the range of 1 to 20 μM. + Induced K + Channel dysfunction without affecting normal cells' K + This property makes it potentially useful in the treatment of neurodegenerative diseases such as Parkinson's disease, avoiding the side effects of extensive regulation of ion channels.

[0041] It should be understood that the above-described specific embodiments of the present invention are merely illustrative or illustrative of the principles of the present invention and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents thereof.

Claims

1. Use of clenbuterol hydrochloride in the preparation of a drug for treating Parkinson's disease, wherein the molecular structure of clenbuterol hydrochloride is as follows: ， It is characterized in that The concentration of the clenbuterol hydrochloride in the drug for treating Parkinson's disease is 1-20 μM.

2. The use of clenbuterol hydrochloride as claimed in claim 1 in a drug for treating Parkinson's disease, characterized in that: Clenbuterol hydrochloride significantly improves SH-SY5Y cell MPP + Induced voltage-gated K + Abnormal current IV curve, improving cell repolarization function and specifically increasing K + Channel current.

3. The use of clenbuterol hydrochloride as claimed in claim 1 or 2 in a drug for treating Parkinson's disease, characterized in that: The drug clenbuterol hydrochloride was used at 20 μM.

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