CN117695374A - Biological medicine for treating parkinsonism - Google Patents
Biological medicine for treating parkinsonism Download PDFInfo
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- CN117695374A CN117695374A CN202410103602.3A CN202410103602A CN117695374A CN 117695374 A CN117695374 A CN 117695374A CN 202410103602 A CN202410103602 A CN 202410103602A CN 117695374 A CN117695374 A CN 117695374A
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Abstract
The invention provides a biological medicine for treating parkinsonism, belonging to the technical field of neurobiology. According to the invention, in vitro experiments show that rakicidin A can significantly improve the survival rate of a cell model of the 6-OHDA induced parkinsonism and inhibit oxidative stress. In vivo experiments show that compared with a parkinsonism model group, rakicidin A can increase the movement distance of mice, improve the expression of tyrosine hydroxylase in brain tissues and improve dyskinesia. Therefore, rakicidin A can protect dopaminergic neurons through an antioxidant stress effect and exert the treatment effect of the parkinsonism, so that the rakicidin A can be developed into a novel therapeutic medicament for the parkinsonism.
Description
Technical Field
The invention belongs to the technical field of neurobiology, and particularly relates to a biological medicine for treating parkinsonism.
Background
Parkinson's disease, commonly known as paralysis agitans, is a common neurodegenerative disease that occurs mainly in the middle-aged and elderly people. Symptoms of patients fall into two main categories: first, clinical manifestations in sports, including resting tremor, bradykinesia, muscle stiffness, balance disturbances, and postural instability; second, clinical manifestations of non-motor aspects such as constipation, nocturia, hyposmia, sleep disorders, depression anxiety, illusion, and cognitive disorders.
Currently, there is no consistent conclusion regarding the causative agent and mechanism of parkinson's disease. Parkinson's disease has been known for many years, and during this period, the knowledge of parkinson's disease is also gradually in depth, and factors such as oxidative stress, abnormal mitochondrial respiratory chain, aggregation of specific proteins and the like can induce parkinson's disease, and meanwhile, the parkinson's disease is possibly guided by the synergistic effect of various mechanisms. Among them, oxidative stress caused by mitochondrial damage is considered to be a major cause of parkinson's disease.
In recent years, the prevalence rate of parkinson's disease gradually rises, however, the existing means for treating PD and medicines only can achieve a slowing effect but cannot be cured, and some relatively common western medicine treatment medicines can maintain the dopamine content of the brain of a patient, but have quite large side effects and are easy to generate drug resistance. Therefore, there is an urgent need to intensively study and search for new drugs to effectively slow down the occurrence of oxidative stress, providing a more powerful means for treating parkinson's disease.
Disclosure of Invention
rakicidinA is a depsipeptide compound produced by marine micromonospora, and the chemical structural formula of the depsipeptide compound is shown as the following chemical structural formula (I):
the existing research shows that rakicidin A has excellent anti-tumor effect, but other researches on the rakicidin A do not appear, and the invention aims to provide a novel effect of rakicidin A in parkinsonism treatment so as to prepare a biological medicament for treating parkinsonism.
In order to achieve the above purpose, the present invention provides the following technical solutions:
firstly, the invention provides a biological medicine for treating parkinsonism, which is a liquid biological medicine, and the liquid biological medicine consists of a depsipeptide compound rakicidinA and physiological saline, wherein the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
Preferably, the concentration of the depsipeptide compound rakicidinA in the liquid biological medicine is more than or equal to 50 mug/ml.
Preferably, the concentration of the depsipeptide compound rakicidinA in the liquid biopharmaceutical is 50-200 μg/ml.
Preferably, the liquid biopharmaceutical is administered orally.
Secondly, the invention provides application of a depsipeptide compound rakicidinA in preparing a biological medicament for treating parkinsonism, which is characterized in that the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
Preferably, the depsipeptide compound rakicidinA achieves a parkinson's disease therapeutic effect by inhibiting oxidative stress injury.
Secondly, the invention provides application of a depsipeptide compound rakicidinA in preparing a biological medicament for inhibiting parkinsonism caused by oxidative stress injury induced by 6-OHDA, wherein the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
Preferably, the biological medicine is a liquid biological medicine, the liquid biological medicine consists of a depsipeptide compound rakicidinA and physiological saline, and the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
Preferably, the concentration of the depsipeptide compound rakicidinA in the liquid biopharmaceutical is 50-200 μg/ml.
Preferably, the liquid biopharmaceutical is administered orally.
Secondly, the invention provides an application of the depsipeptide compound rakicidinA in inhibiting nerve cell oxidative damage induced by 6-OHDA, wherein the application is to pre-treat nerve cells for 24 hours by using a culture medium containing 50-200 mug/ml of the depsipeptide compound rakicidinA to reduce the nerve cell oxidative damage induced by 6-OHDA; the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
The invention has the beneficial effects that:
in vitro experiments show that rakicidinA has no toxicity to PC12 cells, and the medium and high concentration of rakicidinA can remarkably improve the survival rate of a cell model of the 6-OHDA induced parkinsonism and inhibit the oxidative stress of nerve cells.
Compared with the parkinsonism model group, the rakicidinA can increase the spontaneous movement distance of mice, improve the expression of Tyrosine Hydroxylase (TH) in brain tissues and improve the movement disorder symptoms.
Therefore, the invention discovers that rakicidinA can protect dopaminergic neurons through the antioxidation stress effect, thereby playing a new function of the treatment effect of the parkinsonism, and compared with the existing chemical medicines, the rakicidinA is a marine natural product and has small toxic and side effects.
Drawings
FIG. 1 shows the results of the detection of the cytotoxic effects of varying concentrations of rakicidin A on nerve cell PC 12;
FIG. 2 shows the results of the detection of the protective effect of different concentrations of rakicidin A on 6-OHDA-induced parkinsonism cell model;
FIG. 3 shows the results of the detection of oxidative stress injury inhibition of 6-OHDA-induced parkinsonism cell model by different concentrations of rakicidin A;
FIG. 4 shows the results of detection of the motor behavior effects of rakicidin A on a 6-OHDA induced Parkinson's disease mouse model;
FIG. 5 shows the results of detection of the effect of rakicidin A on TH protein expression in brain tissue of a 6-OHDA-induced Parkinson's disease mouse model.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
EXAMPLE 1 cytotoxicity assay of rakicidin A on PC12 cells
PC12 cells in logarithmic growth phase were grown at 5×10 per ml 5 Density of individual single cell suspensions were prepared, inoculated into 96 well plates, and 100 μl of suspension was added per well; the well plate was placed at 37℃with 5% CO 2 Culturing for 24 hours in a cell culture box, and removing the culture medium by using a liquid transfer device after cells are attached to the bottom of a culture dish;
then, adding rakicidin A solution dissolved in DMEM complete medium respectively, wherein the final concentration is 1, 10, 50, 100, 150 and 200 mug/ml, culturing for 24 hours after drug treatment, setting 5 repeated holes for each concentration treatment, and setting a blank group only added with the medium and a control group only added with the DMEM complete medium;
subsequently, 20. Mu.L of MTT solution was added to each well and incubated for 4 hours. After the incubation, the incubation was gently and slowly extracted, 150 μl DMSO (dimethyl sulfoxide) was added to each well, and gently shaken for 15 minutes;
finally, absorbance values at 490nm were measured for each well using a microplate reader and the effect of rakicidin a on PC12 cells was assessed by calculating cell viability.
EXAMPLE 2 protection of cell models of 6-hydroxydopamine (6-OHDA) -induced Parkinson's disease by varying concentrations of rakicidin A
The experiment is divided into a control group, a 6-OHDA group, a low-concentration rakicidin A (1 mug/ml) +6-OHDA group, a medium-concentration rakicidin A (50 mug/ml) +6-OHDA group, a high-concentration rakicidin A (200 mug/ml) +6-OHDA group and a blank group;
first, PC12 cells in logarithmic growth phase were grown at 5X 10 cells per ml 5 Density of individual single cell suspensions were prepared, inoculated into 96 well plates, and 100 μl of suspension was added per well; the well plate was placed at 37℃with 5% CO 2 Culturing for 24 hours in a cell culture box, and removing the culture medium by using a liquid transfer device after cells are attached to the bottom of a culture dish;
then, according to the experimental group, adding the complete culture medium of DMEM with the final concentration of 1, 50 and 200 mug/ml, treating the drugs for 24 hours, adding the complete culture medium of DMEM into the control group and adding the complete culture medium of DMEM into the 6-OHDA group;
then, according to the experimental group, adding a DMEM complete medium containing 100 mu mol/L6-OHDA into each well, and treating the cells for 24 hours;
subsequently, 20. Mu.L of MTT solution was added to each well and incubated for 4 hours; after the incubation, the incubation was gently and slowly extracted, 150 μl DMSO (dimethyl sulfoxide) was added to each well, and gently shaken for 15 minutes;
finally, absorbance values at 490nm were measured for each well using a microplate reader and the effect of rakicidin a on cell viability in the parkinsonism cell model PC12 was measured by calculating cell viability.
Example 3
ROS content detection
The experiment was divided into a control group, a 6-OHDA group, a low concentration rakicidin A (1. Mu.g/ml) +6-OHDA group, a medium concentration rakicidin A (50. Mu.g/ml) +6-OHDA group, and a high concentration rakicidin A (200. Mu.g/ml) +6-OHDA group;
first, PC12 cells in logarithmic growth phase were grown at 5X 10 cells per ml 5 Density of individual single cell suspensions were prepared, inoculated into 96 well plates, and 100. Mu.L of suspension was added to each well. The well plate was placed at 37℃with 5% CO 2 Culturing for 24 hours in a cell culture box, and removing the culture medium by using a liquid transfer device after cells are attached to the bottom of a culture dish;
then, according to the experimental group, adding the complete culture medium of DMEM with the final concentration of 1, 50 and 200 mug/ml, treating the drugs for 24 hours, adding the complete culture medium of DMEM into the control group and adding the complete culture medium of DMEM into the 6-OHDA group;
then, according to the experimental group, adding a DMEM complete medium containing 100 mu mol/L6-OHDA into each well, and treating the cells for 24 hours;
subsequently, the ROS content was detected using a DCFH-DA (2 ',7' -dichlorobenzene dihydrofluorescein diacetate method) kit, and the DCFH-DA was diluted to a final concentration of 10. Mu. Mol/L in a DMEM medium at a ratio of 1:1000; carefully blotting the cell culture broth, adding 50 μl of DCFH-DA working solution into each well, and incubating at 37deg.C for 20 min; after the incubation, carefully washing the cells for 3 times by using a serum-free DEME medium, and washing off DCFH-DA which does not enter the cells;
finally, the absorbance value (emission wavelength 525 nm) at 488nm of each well was measured using a microplate reader, and the percentage of each group to the control group content was calculated.
EXAMPLE 4 construction of a 6-OHDA parkinsonism mouse model
Healthy male C57BL/6 mice of 10 weeks of age and body weight ranging from 20 to 25g were randomly divided into three groups, respectively: control, model and experimental groups; preparing 0.15% sodium pentobarbital solution, and injecting the anesthetized mice intraperitoneally according to the dosage of 0.2mL/10 g;
then preparing 2 mug/mug of 6-OHDA solution, fixing a deeply anesthetized mouse on a stereotactic instrument device, cutting off the scalp of the mouse, finding a bregma origin, determining a drilling position on the left side by using 2.2mm behind the bregma and 1.4mm beside a midline and 4.7mm below a dura, slowly pushing a microsyringe needle to the coordinate position, and injecting 1ml of 6-OHDA (normal saline containing 2% ascorbic acid in equal amount is injected into a control group) at a constant speed within 2 min; after the injection is finished, a proper amount of penicillin is smeared at the wound, the operation line is sutured, and the operation line is put back into a clean cage for continuous feeding.
The next day after injection, the experimental group was given rakicidin A (50. Mu.g/g) by gavage, and the control group and the model group were given an equivalent amount of physiological saline by gavage once daily for 10 times.
Example 5 open field experiment to examine the Effect of rakicidin A on motor behavior in the Parkinson mouse model
After 14 days of modeling, each mouse was placed in a separate open area, the behavioral parameters of the mice were recorded for 5min, the movement distances of the mice at the center and the periphery were recorded using a computer, and the total distance was calculated.
Example 6
Western blot examined the effect of rakicidin A on TH expression in brain tissue of Parkinson mouse model
Oxidative damage results in a decrease in Tyrosine Hydroxylase (TH) protein, which is closely related to loss of dopamine system function in parkinson's disease, and loss of dopamine is one of the main causes of symptoms of parkinson's disease, and thus the present invention examined the effect of rakicidin a on TH expression in a parkinsonism mouse model.
First, protein extraction is performed:
after the mice are sacrificed, 50mg of brain tissue of the mice is taken, 500 μl of RIPA added with PMSF is added, after full lysis, the mixture is centrifuged at 12000g for 10 minutes at 4 ℃, and the supernatant is rapidly sucked into a precooled 1.5mL centrifuge tube; protein quantification is carried out by BCA method, and finally, protein samples are added into a 5×loading buffer and heated in a boiling water bath for 10 minutes, so as to complete protein sample preparation;
gel electrophoresis and electrotransformation:
12% separation gel and 5% compression gel were prepared and cast into SDS-PAGE gels. Subsequently, adding an appropriate amount of pre-cooled 1 Xelectrophoresis buffer, and adding the total intracellular protein extract and cytoplasmic proteins into lanes (including pre-stained protein Maker and samples), respectively; performing electrophoresis under 80V for about 30 min until the sample enters the separation gel layer, and then adjusting the voltage to 120V to continue electrophoresis; when the target protein band reaches the appropriate position (reference to the position of the pre-stained protein Maker), electrophoresis is stopped.
The PVDF membrane is cut according to the size of the gel, soaked in methanol for 1 min, then soaked in a transfer buffer solution, and meanwhile, the filter paper is soaked in the transfer buffer solution for 15 min. The gel, PVDF membrane and filter paper are arranged in layers to ensure that constant pressure membrane transfer is started after removing bubbles.
Antibody incubation and development:
after completion of the transfer, the membrane was put into 5% nonfat dry milk for 1 hour, and then residual liquid on the surface of the membrane was sufficiently washed with TBST buffer. Placing the film into a self-sealing bag by using a sealing machine, sealing three edges, adding TH and beta-actin primary antibodies into the bag, removing bubbles as much as possible, sealing the bag mouth, and placing the bag at 4 ℃ overnight; subsequently, the self-sealing bags were cut open and the membranes were washed 3 times for 10 minutes each with TBST buffer.
Next, the membrane was replaced in a closed bag, a proper amount of secondary antibody of appropriate concentration was added, the mouth of the bag was closed, and the membrane was incubated at room temperature for 1 hour. The self-sealing bag was again cut off and the membrane was washed 3 times with TBST buffer for 10 minutes each. During this period, equal volumes of chemiluminescent reagents, liquid A and liquid B, were mixed.
After the mixed solution is prepared, the membrane protein surface is fully contacted with the mixed solution downwards; development was performed after waiting for 5 minutes.
Experimental results:
example 1 the cytotoxic effects of varying concentrations of rakicidin A on nerve cell PC12 were examined and the results obtained are shown in FIG. 1. The results show that in the concentration range of 1-200 mug/ml, rakicidin A has no obvious effect on PC12 cell survival. This suggests that rakicidin a does not have significant cytotoxicity to PC12 cells in this concentration range, which lays a foundation for further pharmacodynamic studies of rakicidin a.
Example 2 the protective effect of varying concentrations of rakicidin A on 6-hydroxydopamine (6-OHDA) -induced cell models of Parkinson's disease was examined and the results obtained are shown in FIG. 2. The results showed that the cell viability of the parkinsonism cell model supplemented with 6-OHDA was significantly reduced compared to the normal cultured cells of the control group, confirming the successful establishment of the cell model. When treated with low, medium and high concentrations of rakicidin a, it was found that at low concentrations, rakicidin a did not effectively increase the cell viability of model cells, whereas medium and high concentrations of rakicidin a significantly increased the cell viability of model cells, showing a clear cytoprotective effect.
Example 3 the inhibition of oxidative stress in a cell model of parkinson's disease induced by 6-hydroxydopamine (6-OHDA) by rakicidin a at different concentrations was examined and the results obtained are shown in figure 3. Similar to the results of fig. 2, the 6-OHDA-added parkinsonism model showed a significant increase in ROS content compared to the control group, and the low concentration of rakicidin a also had no significant inhibitory effect on ROS. Meanwhile, it can be seen that when the rakicidin A with high concentration is treated in use, the ROS content can be effectively reduced, so that a certain inhibition effect is generated on oxidative stress caused by 6-OHDA, and a certain protection effect is exerted on nerve cells.
Examples 5 and 6 examined the effect of rakicidin A on motor behavior and TH protein expression in a 6-hydroxydopamine (6-OHDA) -induced mouse model of Parkinson's disease. As can be seen from fig. 4 and 5, the spontaneous movement distance of the model mice is significantly reduced compared with that of the normal control group, and TH protein expression in brain tissues is significantly reduced, confirming the successful establishment of the 6-OHDA injury model. Compared with the model group, the spontaneous movement distance of the mice in the experimental group with the rakicidin A administered by the stomach is improved, and the expression of TH protein in brain tissues is obviously improved. The above results show that rakicidin a can ameliorate motor impairment symptoms in parkinsonian mice, which may be associated with rakicidin a inhibiting oxidative stress and thereby protecting dopaminergic neurons.
The protection effect of rakicidin A on the parkinsonism model is examined through an in vitro and in vivo experimental system. Example 1 shows that rakicidin A was not significantly toxic to PC12 cells at concentrations ranging from 1-200 μg/ml. Example 2 shows that medium and high concentration of rakicidin A significantly improved survival of 6-OHDA induced parkinsonism cell model. Example 3 it was further found that medium and high concentrations of rakicidin A inhibited the oxidative stress induced by 6-OHDA. In vivo experiments, rakicidin A increased spontaneous locomotor distance in 6-OHDA mice and increased TH expression levels in substantia nigra tissues compared to model groups (examples 5 and 6). This suggests that rakicidin a may protect dopaminergic neurons by antioxidant action, thereby ameliorating motor dysfunction in parkinsonian mice. In conclusion, the present study initially validated the protective potential of rakicidin a against parkinson's disease, so that rakicidin a could be prepared as a novel therapeutic drug for parkinson's disease.
Claims (10)
1. The biological medicine for treating the parkinsonism is characterized in that the biological medicine is a liquid biological medicine, the liquid biological medicine consists of a depsipeptide compound rakicidinA and physiological saline, and the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
2. The biopharmaceutical for treating parkinson's disease according to claim 1, wherein the concentration of the depsipeptide compound rakicidinA in the liquid biopharmaceutical is 50 μg/ml or more.
3. A biopharmaceutical for use in the treatment of parkinson's disease according to claim 2, wherein the concentration of the depsipeptide compound rakicidinA in said liquid biopharmaceutical is 50-200 μg/ml.
4. A biopharmaceutical for use in the treatment of parkinson's disease according to claim 3, wherein said liquid biopharmaceutical is administered orally.
5. The application of the depsipeptide compound rakicidinA in preparing the biological medicine for treating the parkinsonism is characterized in that the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
6. The use of a depsipeptide compound rakicidinA according to claim 5 for the preparation of a biopharmaceutical for the treatment of parkinson's disease, said depsipeptide compound rakicidinA achieving a parkinson's disease treatment effect by inhibiting oxidative stress damage.
7. The application of a depsipeptide compound rakicidinA in preparing a biological medicament for inhibiting parkinsonism caused by oxidative stress injury induced by 6-OHDA is characterized in that the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
8. The biological medicine for treating the parkinsonism caused by the oxidative stress injury induced by the 6-OHDA is characterized by being a liquid biological medicine, wherein the liquid biological medicine consists of a depsipeptide compound rakicidinA and physiological saline, and the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
9. A biopharmaceutical for use in the treatment of parkinson's disease caused by 6-OHDA-induced oxidative stress injury according to claim 8, wherein the concentration of rakicidinA in the liquid biopharmaceutical is 50-200 μg/ml.
10. Use of a depsipeptide compound rakicidinA for inhibiting 6-OHDA-induced oxidative damage of nerve cells, characterized in that the use is to pre-treat nerve cells with a medium containing 50-200 μg/ml of the depsipeptide compound rakicidinA for 24h to reduce 6-OHDA-induced oxidative damage of nerve cells; the structure of the depsipeptide compound rakicidinA is shown as a chemical structural formula (I).
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