CN111494379A

CN111494379A - Application of piperine in improving early olfactory disorder of Parkinson's disease

Info

Publication number: CN111494379A
Application number: CN202010538454.XA
Authority: CN
Inventors: 杨慧; 高歌; 李若林
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2020-06-13
Filing date: 2020-06-13
Publication date: 2020-08-07

Abstract

The invention relates to a new application of piperine in medicines, in particular to an application of piperine in improving early-stage dysosmia of Parkinson's disease, which comprises the steps of 1) treating central nervous system diseases, 2) treating dysosmia accompanied by the Parkinson's disease, 3) inhibiting cell death, 4) degrading α -syn and p- α -syn, and experiments prove that the piperine can degrade α -syn and p- α -syn, so that the toxic effect of the piperine is reduced, the early-stage dysosmia of a PD model is improved, and further the occurrence of late-stage dyskinesia is prevented.

Description

Application of piperine in improving early olfactory disorder of Parkinson's disease

Technical Field

The invention relates to the field of medicines, in particular to application of piperine in improvement of early olfactory disorder of Parkinson's disease, and particularly relates to mechanism research of piperine in improvement of pathological change of Parkinson's disease.

Background

Parkinson's Disease (PD) is one of the most common neurodegenerative diseases in clinic, the main clinical manifestations of PD are resting tremor, muscular rigidity, bradykinesia and unstable posture with high disability rate, PD is slow in onset, occult, progressive exacerbation, the preclinical period of which can reach 10-20 years and is accompanied by a series of non-motor symptoms, wherein olfaction is one of non-motor symptoms at the early stage of PD, more than 90% of PD patients are accompanied by different degrees of dysosmia, and the hypofunction of olfaction can occur 20 years before the typical motor symptoms of PD and is accompanied by the whole course of the disease, α -synuclein (α -synuclein, α -syn) is the main component of the characteristic physical marker pathway predispositions of PD (L ew bodices, L Bs), which is also the first discovered PD causative gene, the pathological α -cadaver first reaches the early stage of the Olfactory Bulb (OB) and the olfactory bulb, the central cortex of PD, which is the primary pathological brain and then the pathological cortical region of the early stage of PD which is the clinical manifestation of the pathological disorder.

When dyskinesia of PD patients occurs, the death number of dopaminergic neurons reaches 40% -60%, and striatal dopamine is reduced by 80%, and the disease is in the difficult-to-rescue stage. Therefore, if intervention can be performed early when non-motor symptoms appear in early stage of disease, the intervention is an effective means for treating the disease or preventing the disease course from developing, and the search and development of a novel medicine with high curative effect and low side effect for intervention in early stage of PD provides a new direction for PD treatment. At present, no therapeutic medicine for preventing the progress of the disease exists in PD, and the commonly used means mainly comprise: drug therapy, surgical therapy, cell transplantation, transgenic technology, etc., which are mainly aimed at the motor symptoms, and evidence of evidence-based medicine (EBM) indicates that drug therapy is still the most commonly used and effective treatment at present. The first choice of medicine for treating PD is MEDUOBA with levodopa as the main component, the medicine can only be used for symptomatic treatment to improve symptoms, and the prognosis of patients is not obviously improved, so that the medicine is still a treatment means for treating symptoms but not root causes, and the treatment effect is smaller and smaller along with the prolonging of the application time and the increasing of the dosage of the medicine, and the toxic and side effects are larger and larger.

In recent years, the open utilization of traditional Chinese medicines has a research prospect, a large number of researches prove that the traditional Chinese medicines can achieve the aim of treating the Parkinson's disease through the effects of protecting substantia nigra cells, improving the content of neurotransmitters, inhibiting oxidative stress reaction, reducing excitatory toxicity and the like, and compared with western medicines, the traditional Chinese medicines have lower toxic and side effects.

Piper longum is a plant of Piper genus of Piperaceae family, and its dried mature fruit ear is used as traditional Chinese, Mongolian and Tibetan medicines, is pungent in taste and hot in nature, and is clinically used for treating symptoms such as stomach and abdomen psychroalgia, anorexia, dyspepsia, kidney cold, cold diarrhea, emesis, etc. Piper longum is rich in alkaloids, amides, lignans, terpenoids, sterols and other compounds, wherein the alkaloids and amides are about 35 kinds, and Piperine (PIPERIne) is one of the main monomer components in Piper longum. The pharmacological action of the piperine is wide, so that the piperine has the effects of resisting oxidation, immunoregulation, tumors and the like, and has different degrees of antagonistic action on convulsion, prostatic cancer and epilepsy, but related researches on the piperine monomer in the treatment of olfaction disorder of a Parkinson disease model are not reported in the literature.

Disclosure of Invention

The invention provides a new application of piperine as a medicament for treating olfactory disorder of Parkinson disease. Piperine (Piperine) is an alkaloid with molecular formula of C₁₇H₁₉NO₃The piperine has the molecular weight of 285.34, exists in dried nearly ripe or ripe fruits of piper longum (Piperlongu m L.) of Piperaceae, the piperine is white crystal powder, has the melting point of 130-133 ℃, is dissolved in acetic acid, benzene, ethanol and chloroform, is slightly soluble in ether, and is almost insoluble in water and petroleum ether.

The new application of the piperine medicine provided by the invention comprises the following steps:

1) treating central nervous system disorders;

2) treating olfactory disorders associated with Parkinson's disease;

3) inhibiting neuronal cell death;

4) degradation α -synuclein (α -synuclein, α -syn) and phosphorylation α -synuclein (p- α -syn).

In the above application, the disease of the central nervous system may be Parkinson's disease.

The present invention further provides a pharmaceutical composition comprising piperine. The composition can comprise 1-1000mg of piperine, and any one of pharmaceutical excipients according to requirements, and the composition can be orally taken, injected and externally used, and the preferable composition form is tablets, capsules, granules, oral liquid and the like.

The invention further provides a medicament for treating α -syn-induced parkinsonism accompanied by olfactory disorders, which comprises piperine.

The present invention further provides a medicament for inhibiting neuronal cell death, comprising piperine.

The invention further provides a drug for degrading α -syn and p- α -syn, which comprises piperine.

The beneficial effects of the present invention are further illustrated by experimental data below.

The invention adopts Thy 1-SNCA mice (α -syn transgenic mice, TG mice, purchased from Jackson L ab) as disease models of PD, the mice present olfactory disorder at 6 months of age and dyskinesia at 12 months of age, which indicates that the mice meet the pathogenesis of PD.

Experiments prove that the olfaction disorder of TG mice of 6 months of age can be obviously improved and the motor symptoms can be prevented by the piperine through the behavioral detection of each group of mice, the α -syn and p- α -syn are subjected to immunohistochemical staining and Western immunoblotting detection in the olfactory bulb, the expression of α -syn and p- α -syn can be reduced by the piperine, and the PIP intervention does not influence the transcription level of h- α -syn through further RT-PCR detection, so that the PIP is prompted to promote the degradation of α -syn and p- α -syn.

In order to further determine the mechanism that piperine reduces the expression of α -syn, relieves the toxic effect of the piperine, improves early olfactory disorder of a PD model and further prevents late dyskinesia from occurring, the invention adopts a α -syn overexpression injury cell model, namely a transfection α -syn plasmid to treat an SK-N-SH cell line.

Experiments prove that α -syn plasmid is transfected for 24 hours, piperine (with the final concentration of 25 mu M) is added to act for 24 hours, cell viability is detected by an MTT method, cytotoxicity is detected by a L DH method, the results show that piperine can remarkably relieve cell viability reduction and cytotoxicity caused by α -syn overexpression, α -syn and p- α -syn are detected, the results show that piperine can reduce the expression of α -syn and p- α -syn, further RT-PCR detection shows that PIP intervention does not influence the transcription level of h- α -syn, and PIP is prompted to promote the degradation of α -syn and p- α -syn.

The experiments show that piperine can degrade α -syn and p- α -syn, reduce the toxic effect of piperine, improve early olfactory disorder of a PD model and further prevent late dyskinesia.

Drawings

FIG. 1 is a graph of PIP improving early olfactory impairment in Thy-1 α -syn transgenic mice, which is graphically depicted as the oral administration of

PIP

25, 50, 100mg/kg for 6 weeks in Thy1 α -syn transgenic mice.

The diagram shows that the Thy1 α -syn transgenic mice are treated by

PIP

25, 50 and 100mg/kg orally for 6 weeks, after the olfactory sense of the mice is obviously improved, 100mg/kg of PIP is continuously given for continuous gavage for treatment, the olfactory sense and the motor behavior of each group of mice are detected once every 3 months, and the dopamine content of the striatum of each group of mice of 12 months of age is detected by a high performance liquid chromatography method.

FIG. 3 shows that PIP intervention leads Thy-1 α -syn transgenic mice to pathologically distributed α -syn sparsely at olfactory bulb parts, and as shown in the figure, after Thy1- α -syn transgenic mice are treated by oral administration of

PIP

25, 50 and 100mg/kg for 6 weeks, the olfactory bulbs of the mice are taken and the expression of h- α -syn and p- α -syn is detected by an immunohistochemical staining method.

FIG. 4 shows PIP degradation Thy-1 α -syn transgenic mouse olfactory locus pathologic α -syn, which is shown in the specification that Thy1 α -syn transgenic mouse is treated by oral administration of PIP 25, PIP 50 and PIP 100mg/kg for 6 weeks, mice olfactory locus is taken, h- α -syn and p- α -syn levels are detected by a western blot method, and RNA level of h- α -syn in olfactory locus of each group of animals is detected by RT-PCR.

Figure 5 is PIP mitigation α -syn induced cytotoxicity a graphical representation of drug concentration windows and time windows for PIP screened on SK-N-SH cell lines using the method of MTT to detect cell viability and L DH to detect cytotoxicity.

Figure 6 shows PIP mitigation α -syn induced increase in cell death rate graphically, cells were PI/Hoechst stained with PI-stained cells in red, Hoechst stained total cells in blue, and statistical red/blue values were used to assess cell death rate on SK-N-SH cell lines after injury modeling using α -syn overexpression followed by treatment with 25 μ M PIP for 24 h.

FIG. 7 shows that PIP treatment sparsely distributes pathological α -syn in SK-N-SH cells, and α -syn treatment is performed for 24h on SK-N-SH cell lines, 25 μ M PIP is used for treatment, and after 24h of co-culture, expression of α -syn and p- α -syn are detected by an immunohistochemical staining method.

FIG. 8 is a graph showing that PIP treatment degrades the level of pathological α -syn in SK-N-SH cells after α -syn treatment for 24h, treatment with 25 μ M PIP, and 24h co-culture on SK-N-SH cell lines, and the levels of α -syn and p- α -syn were measured by western blot and the level of h- α -syn RNA in each group of cells was measured by RT-PCR.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.

Example 1 piperine PIP alleviates α -syn transgenic Parkinson's disease model mice for early olfactory impairment and prevents late motor symptoms from occurring.

Diluting PIP to 3.75, 7.5 and 15mg/ml with 1.2% DMSO, using 2% DMSO as a control, performing intragastric administration at a dosage of 25, 50 and 100mg/kg, detecting olfactory behaviors after 6 weeks of administration, continuing to give 100mg/kgPIP after PIP treatment effectively relieves olfactory disorder, and detecting olfactory behaviors and motor behaviors of each group of mice at 9 and 12 months of age.

2. Evaluating the olfactory behaviors of the mice of each group through a food burial experiment, an open field experiment and a sugar water preference experiment; the rod rotating experiment and the pole climbing experiment detect the motion behavior of each group of mice.

Food burial experiments are commonly used for olfactory detection of mice. The method comprises the steps that a mouse is fasted for 12-14h before olfactory detection, during detection, a piece of sweeled cereal is buried at a position 0.5cm below a padding of a mouse cage to be detected, the mouse is placed into the mouse cage to be detected, and the time from the time when the mouse is placed into the mouse cage to the time when the mouse finds food is recorded. The shorter the food-seeking time, the better the olfactory ability of the mouse.

Open field experiments were performed for 3 days, each corner of a test box (72 × 72 × 30cm) was used as the test area (3 × 3 × 3cm), each mouse was placed in the box for 15min before the experiment to accommodate the environment, three experimental corners were placed with control (water), another corner was placed with test substance (sweeled cereal, or urine from female mice or Paprika), the mice were placed in the center of the box, the experiment was performed for 5min, and the time of each mouse at each corner was recorded.

The experiment is carried out for 3 days, each mouse is raised in a single cage, two bottles of 1% sucrose solution are given to each mouse at the 1 st 24h, 1 bottle of clear water, 1 bottle of 1% sucrose solution and 3 rd 24h, clear water and sucrose solution are exchanged, and the drinking amount of each bottle of water of the mouse in the period is calculated.

Rod-rotating experiments and pole-climbing experiments are commonly used for detecting the movement coordination ability of mice. The rod rotating experiment is to place the mouse on a roller of a rod rotating instrument and avoid the mouse from falling, the roller is set to be in an acceleration mode, the speed is gradually increased from 4rpm to 40rpm within 5min, the lower sensing platform is correspondingly stopped when the animal falls down, and the latency period of falling from the roller is recorded. Longer time indicates stronger mouse movement ability. The climbing experiment was carried out by fixing an iron ball of 2.5cm diameter to an iron rod of 1cm diameter and 50cm length and winding gauze around the iron rod to increase friction. The mice were placed in a head-down position with their hind paws grasping the top of the iron rod and the time it took for each mouse to climb down from the beginning to the bottom of the rod. The shorter the time, the stronger the motor coordination ability of the mouse.

3. Immunofluorescence measures the levels of olfactory bulb h- α -syn and p- α -syn.

Immunofluorescence is the principle of applying immunology basic principle-antigen-antibody reaction, namely the principle of antigen and antibody specific binding, the fluorescent pigment which does not affect the activity of antigen-antibody is marked on the antibody (or antigen), and after the fluorescent pigment is combined with the corresponding antigen (or antibody), a specific fluorescent reaction is presented under a fluorescence microscope, and the antibody is researched for positioning, qualification and relative quantification, in the experiment, the contents of h- α -syn and p- α -syn in the mouse olfactory bulb are evaluated by immunofluorescence, and the steps are as follows:

(1) the animals are fixed by perfusion, brains are taken immediately, and then are put into 4 percent paraformaldehyde solution containing 20 percent and 30 percent of sucrose in sequence and then are fixed at 4 ℃ overnight;

(2) freezing the slices to 30 μ M, and putting the slices into 0.01M PBST solution (pH7.2-7.5) for staining;

(3) soaking and washing the slices in 0.01M PBST solution for three times for 5 min;

(4) slicing into 0.01M citrate antigen repairing solution, and repairing at 95 deg.C for 10 min;

(5) the slices are soaked and washed for three times for 10min in PBST;

(6) sealing the section in 5% normal sheep serum at room temperature for 1h (inhibiting non-specific staining), discarding the serum, and keeping the section straight

Adding appropriately diluted primary antibody h- α -syn, p- α -syn (0.01M PBST dilution), and incubating at 4 ℃ overnight;

(7) soaking and washing the slices in PBST for three times, 5min each time;

(8) slicing into 1:500 fluorescent secondary antibody (diluted by 0.01M PBST) and keeping out of the sun for 1h at room temperature;

(9) soaking and washing the slices in PBST for three times, 5min each time;

(10) slicing into 10000Hoechst at 1:1, staining nuclei in dark for 5-10min, and keeping the temperature at room temperature;

(11) soaking and washing the slices in PBST for three times, 5min each time;

(12) mounting the slices, sealing the slices with 70% glycerol, and storing at 4 ℃ in a dark place.

4. Results of the experiment

The experimental animal is a 6-month-old Thy1 α -syn transgenic mouse, piperine PIP is orally taken for treatment, the dosage is 25, 50 and 100mg/kg, olfactory behaviors are detected after 6 weeks of administration, the results of olfactory behaviors show that olfactory disorders of the 6-month-old Thy1 α -syn transgenic mouse occur, and the olfactory disorders of the mouse are relieved to different degrees after the PIP is administered for 6 weeks.

After detecting that the PIP treatment effectively relieves the olfactory disorder, the PIP treatment is continuously given to 100mg/kg, and the olfactory behavior and the motor behavior of each group of mice are detected when the mice are 9 and 12 months old.

The levels of h- α -syn and p- α -syn of olfactory bulb parts are detected by immunohistochemical and western blot methods, the results show that the levels of h- α -syn and p- α -syn can be reduced by PIP treatment with different doses, and RT-PCR detection finds that PIP intervention does not influence the transcription level of h- α -syn, and the PIP is prompted to promote the degradation of α -syn and p- α -syn.

Example 2 piperine PIP inhibits α -syn-induced cell death.

The cell lines used in the following examples are as follows:

SK-N-SH cell lines: human neuroblastoma cells (ATCC, cryopreserved in this chamber) (American Type cultureselection);

the experimental procedure in the following examples is as follows:

1. cell viability was measured by the method of MTT.

The MTT method is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit the blue-purple crystalline Formazan in the cells, and dead cells do not have the function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at 490nm wavelength by enzyme linked immunosorbent detector, which can indirectly reflect living cell number. Within a certain range of cell number, MTT crystal formation is proportional to the cell number, i.e., the greater the absorbance, the more active it is. The method comprises the following steps:

(1) logarithmic growth phase cells were seeded in 96-well plates at about 100. mu.l/well (about 1 × 10)⁴One), 5% CO at 37 ℃₂Continuously culturing in a cell culture box;

(2) treating 96-well plate cells according to experimental requirements, and discarding the culture medium after the treatment is finished;

(3) mu.l MTT solution was added to each well, and the volume was adjusted to 100. mu.l using medium, and the mixture was left at 37 ℃ with 5% CO₂Culturing for 4 hours in the incubator;

(4) carefully absorbing and removing the supernatant, adding 100 mu l DMSO into each well, and shaking the mixture on a shaking table for 10min in a dark place to completely dissolve the generated particles;

(5) and reading the absorbance value of the pore plate at 490nm wavelength by using an enzyme-labeling instrument, and performing statistical mapping.

2. The cytotoxicity was measured by lactate dehydrogenase release rate measurement (L DH method).

L DH (lactate dehydrogenase) is a stable cytosolic enzyme, is present in all cells and is rapidly released into the cell culture medium when the cell membrane is damaged L DH activity is catalyzed by two enzymes L DH oxidizing lactate to pyruvate and reacting pyruvate with tetrazolium INT to formazan crystals, the increase in formazan crystals in the culture medium is directly related to the increase in the number of cells lysed the formazan crystals dye is water soluble and can be detected with a spectrophotometer at 490nm wavelength and by measuring the activity of L DH in the cell culture supernatant, the degree of cell damage can be determined and the assay is sensitive, convenient and accurate and is suitable for a wide variety of cytotoxic assays as follows:

(1) transfer each group of cell supernatants to a new 96-well plate, 100 μ Ι per well;

(2) adding 100 mu l of reaction mixture into each hole, and incubating for 30min at room temperature in a dark place;

(3) after incubation, adding 50 μ l of stop solution into each well, and shaking on a shaking table in a dark place for 10 min;

(4) the microplate reader reads the absorbance value of the well plate at 490nm wavelength, analyzes the data and makes statistics and maps.

3. Cell death rate was measured by PI/Hochest staining.

Hochest33342 is a blue specific dye that binds to cellular DNA, penetrates live cell membranes, and labels both live and dead cells. The PI dye can not enter cells with intact cell membranes, namely, living cells resist the PI (propidium iodide) dye, and necrotic cells are damaged at an early stage due to the integrity of the membrane and can be stained by the PI dye to emit red fluorescence. Based on these properties, double staining with PI/Hoechst is often used to detect the proportion of cell death. The larger the ratio, the higher the cell death rate.

4. Results of the experiment

After α -syn is used for transfecting SK-N-SH cells for 24h, piperine PIP is used for treatment, and then the cells are incubated for 24h together, and MTT cell viability detection and L DH cytotoxicity detection are carried out.

The result shows that the cell death rate is obviously increased after the α -syn is used for treating the cells, and the cell death rate is improved after the PIP is treated.

The levels of α -syn and p- α -syn in each group of cells are detected by an immunohistochemical and western blot method, the result shows that PIP treatment can reduce the levels of α -syn and p- α -syn, and further RT-PCR detection finds that PIP intervention does not influence the transcription level of h- α -syn, and the PIP is prompted to promote the degradation of α -syn and p- α -syn.

Claims

1. Use of piperine in preparing medicine for treating central nervous system diseases is provided.

2. The use according to claim 1, wherein the central nervous system disorder is parkinson's disease.

3. Application of piperine in preparing medicine for treating dysosmia accompanied with Parkinson disease is provided.

4. Use of piperine in the preparation of a medicament for inhibiting cell death.

5. Application of piperine in preparing medicines for degrading α -syn and p- α -syn is provided.

6. A medicament for the treatment of central nervous system disorders comprising piperine.

7. A medicament for treating olfactory disorders caused by parkinson's disease, comprising piperine.

8. A medicament for inhibiting cell death comprising piperine.

9. A drug that degrades α -syn and p- α -syn, comprising piperine.