CN108703963B - Application of anethole in preparing medicine for treating neuropathic pain - Google Patents

Abstract

The invention discloses an application of Anethole (Anethole) in preparing a medicine for treating neuropathic pain. The experimental result of the invention shows that when the anethole with the dose of 500mg/kg is used in the safe dose range, the anethole with the dose of 500mg/kg can obviously relieve the allodynia and the hyperalgesia induced by the chronic compressive injury model of the sciatic nerve, improve the histopathological change of the sciatic nerve and regulate and control the protein expression of inflammatory cytokines, and the anethole is proved to have the functions of relieving the neuropathic pain caused by the chronic compressive injury and promoting the functional recovery of the nerve.

Description

Application of anethole in preparing medicine for treating neuropathic pain

Technical Field

The invention relates to an application of anethole, in particular to an application of anethole in preparing a medicine for treating neuropathic pain.

Background

Neuropathic pain (neuropathic pain) is a chronic pain syndrome with a complex pathogenesis, usually pain caused by abnormal or damaged nervous system function, and is often seen in autoimmune diseases (such as multiple sclerosis), metabolic diseases (such as diabetic neuropathy), infections (postherpetic neuralgia), vascular diseases (such as stroke), nerve compression, nerve trauma, cancer, etc. Neuropathic pain afflicts 1/6 of people in the world, causes not only physical pain and dysfunction, but also mental and psychological disorders such as depression and anxiety, has serious influence on life and quality of life of patients, and brings huge burden to families and society. In recent years, with the rapid development of pathology, physiology, molecular biology and clinical treatment technology of pain, the research of neuropathic pain has been greatly advanced. At present, most of the main treatment medicines for neuropathic pain are antidepressants, non-steroidal anti-inflammatory drugs, opioid drugs and the like, but the medicines seriously affect the life quality of patients due to various adverse reactions, thereby limiting the clinical use of the medicines. Therefore, the generation mechanism of the neuropathic pain is further researched, and the medicine for treating the neuropathic pain, which has high safety and good clinical curative effect, is found on the basis, so that the medicine has important academic value and practical significance.

The fructus Foeniculi is dried mature fruit of Foeniculum vulgare L.of Foeniculum of Umbelliferae. All plants can be used for medicine. Is one of the important traditional Chinese medicines commonly used, and has the effects of dispelling cold, relieving pain, regulating qi and harmonizing stomach according to the Chinese pharmacopoeia (one part). According to the literature report, the medicine has the functions of resisting inflammation, resisting oxidation, relieving pain and resisting hepatic fibrosis.

The current research on chemical components of fennel is mainly focused on the research on the components of volatile oil. The extract of fennel oil and the main components of anethole and anisaldehyde. Wherein Anethole (AN) is also called 1-methoxy-4-propenyl benzene, anisole, and its molecular formula is C ₁₀ H ₁₂ And (O). In recent years, domestic and foreign researches have proved that anethole has pharmacological effects of anti-inflammation, analgesia, antioxidation, anti-diabetic metabolic abnormality, immunoregulation, neuroprotection and the like, but the protective effect on neuropathic pain is not reported in documents at present. The compound has extremely high potential value and social significance when being developed into a medicament for treating neuropathic pain.

Disclosure of Invention

The invention aims to provide the application of anethole in preparing a medicament for treating neuropathic pain through researching the pharmacological action of the anethole.

The invention achieves the purpose through the following technical scheme:

the invention provides an application of anethole in preparing a medicine for treating neuropathic pain, wherein the structural formula of the anethole is shown as a formula (1):

in particular, the neuropathic pain is neuropathic pain of peripheral nerve injury.

Specifically, the single application dose of anethole is limited to a dose that does not cause central inhibition.

Specifically, the single application dosage of the anethole is 125-500mg/kg.

Preferably, the dosage of the anethole for single application is 250-500mg/kg.

Preferably, the single application dose of the anethole is 500mg/kg.

Specifically, the dosage form of the medicine is a pharmaceutically allowable oral dosage form or injection dosage form.

In particular to the application of the anethole as the only active ingredient in preparing the medicine for treating neuropathic pain.

The application of the anethole in preparing the medicine for treating neuropathic pain provided by the invention has the following beneficial effects:

(1) The anethole can obviously relieve mechanical allodynia, cold allodynia and thermal allodynia;

(2) The anethole can improve injured sciatic nerves, increase nerve conduction velocity and sensory nerve action potential amplitude, obviously inhibit the expression of proinflammatory cytokines TNF-alpha, IL-6 and IL-1 beta protein in spinal cord tissues and promote the expression of IL-10 protein.

The invention proves that the anethole has the function of treating neuropathic pain caused by chronic compressive injury of sciatic nerve for the first time, and can be used for preparing the medicine for treating neuropathic pain.

Drawings

FIG. 1 is a graph of the mechanistic paw withdrawal reflex threshold for neuropathic pain in mice with anethole.

FIG. 2 is a graph of cold foot-lifting times of anethole on neuropathic pain in mice.

FIG. 3 is a graph of the heat-shrinkable paw reflex latency of anethole to neuropathic pain in mice.

FIG. 4 is a graph of the mechanical tenderness threshold of anethole on neuropathic pain in mice.

FIG. 5 is a composite action potential diagram of anethole on sciatic nerve of neuropathic pain in mice (FIG. 5A is a sham operation group, FIG. 5B is a model group, FIG. 5C is a pregabalin group, FIG. 5D is an anethole (125 mg/kg) group, FIG. 5E is an anethole (250 mg/kg) group, and FIG. 5F is an anethole (500 mg/kg) group).

FIG. 6 is a statistical plot of sensory nerve conduction velocity of anethole on neuropathic pain in mice.

FIG. 7 is a statistical plot of the amplitude of the complex action potential of anethole on sciatic nerve of neuropathic pain in mice.

FIG. 8 is a graph showing the change in the sciatic nerve structure of ANNEX in HE-stained neuropathic pain mice (FIG. 8A is a sham-operated group, FIG. 8B is a model group, FIG. 8C is a pregabalin group, FIG. 8D is an anethole (125 mg/kg) group, FIG. 8E is an anethole (250 mg/kg) group, and FIG. 8F is an anethole (500 mg/kg) group).

Fig. 9 shows the effect of anethole on TNF- α protein expression levels in neuropathic painful spinal cord tissue in mice (compare to sham: ^## p<0.01, comparison with model group: ^* p<0.05， ^** p<0.01

)。

FIG. 10 is a graph showing the effect of anethole on the level of IL-6 protein expression in neuropathic pain spinal cord tissue in mice (compared to sham: ^## p<0.01, comparison with model group: ^* p<0.05， ^** p<0.01

)。/>

FIG. 11 shows the effect of anethole on the level of IL-1. Beta. Protein expression in neuropathic painful spinal cord tissue in mice (compare to sham: ^## p<0.01, comparison with model set: ^* p<0.05， ^** p<0.01

)。

FIG. 12 is a graph showing the effect of anethole on the level of IL-10 protein expression in neuropathic pain spinal cord tissue in mice (compared to sham: ^## p<0.01, comparison with model group: ^* p<0.05， ^** p<0.01

)。

Detailed Description

The present invention will be described in further detail with reference to examples, in which anethole used is a compound represented by the above formula (1) and is commercially available.

Example 1

The application of the anethole in preparing the medicine for treating the neuropathic pain is disclosed, wherein the neuropathic pain is the neuropathic pain caused by peripheral injury, the single application dose of the anethole is 125mg/kg of a mouse, and the dosage form of the medicine is an oral dosage form.

Example 2

The application of the anethole in preparing the medicine for treating the neuropathic pain is disclosed, wherein the neuropathic pain is the neuropathic pain caused by peripheral injury, the single-application dose of the anethole is 250mg/kg for a mouse, and the dosage form of the medicine is an oral dosage form.

Example 3

The application of the anethole in preparing the medicine for treating the neuropathic pain is disclosed, wherein the neuropathic pain is the neuropathic pain caused by peripheral injury, the single application dose of the anethole is 500mg/kg of a mouse, and the dosage form of the medicine is an oral dosage form.

The following animal experiments further illustrate the effects of the above examples 1 to 3:

1. experimental Material

1.1 animal treatment

Male ICR mice, 18-22g, purchased from the experimental animals center of ningxia medical university, animal production license number: NCXK (Ning) 2015-0001. The feeding conditions include standard feed, tap water, room temperature at 24 + -2 deg.C, humidity of 50-60%, and daily illumination and dark time of 12 hr respectively. Before the experiment, the animals were subjected to experiment environment adaptation 3.

1.2 Experimental drugs and instruments

Anethole (Shanghai leaf Biotech Co., ltd.) was prepared into a suspension with CMC-Na, and the concentration of the mother liquor was 500mg/mL, and it was used as it was. Pentobarbital sodium (Sigma-Aldrich Co.), pregabalin capsules (Peucel pharmaceuticals Co., ltd.), von Frey filiments (Danmic Global, USA), cold plate apparatus (BIOSEB scientific apparatus, france), PL-200 thermal stinger (Doudotai scientific Co., ltd.), transmission electron microscope (H-7650 Hitachi, tokyo, japan), rabbit anti-TNF-alpha, IL-6 (from Proteitech Co., ltd.), rabbit anti-IL-1 beta and IL-10 polyclonal antibody (from Abcam Co., ltd.), enzyme reader (1510, thermo Fisher Co., ltd.), electrophoresis apparatus, electrotransfer (Powerpac basic, bio-Rad, USA) and gel imaging analyzer (JS-860B, shanghai Peqing Co., ltd.).

1.3 grouping and administration of Experimental animals

Mice were randomly divided into sham surgery groups, chronic stress injury (CCI) model groups, different doses of anethole (125 mg/kg, 250mg/kg, 500 mg/kg) and pregabalin positive drug groups. After surgery modeling, the mice were dosed with different doses of anethole and pregabalin positive drug (dose 0.1ml/10g body weight, gavage, once every 12 hours). The sham operation group and the model group were administered the same amount of CMC-Na. Pharmacodynamic evaluations such as behavioral, electrophysiological, histopathology and molecular biology were performed on days 0, 7,8, 10, 12 and 14 after CCI molding.

1.4 establishment of mouse Chronic Compressive Injury (CCI) model

An animal model of neuropathic pain caused by chronic constrictive injury is established by ligating sciatic nerve of a mouse. After weighing the mice, the mice were anesthetized by intraperitoneal injection using 0.8% sodium pentobarbital injection at a dose of 0.1ml/10 g. After anesthesia, the prone position of a mouse is placed on a sterilized operating table, hair and skin are cut at the junction of the right hip and the thigh, skin surface sterilization is carried out by iodophor disinfectant, an incision of about 1.5cm is cut at the junction along the walking direction of the sciatic nerve by a surgical scissors, biceps femoris and gluteus are separated bluntly along muscle lines by a glass pricking needle, the sciatic nerve trunk is exposed, 3 lax ligatures with the interval of 1mm are carried out on the exposed sciatic nerve trunk by 4-0 medical chromium goat intestine which is sterilized and soaked by normal saline, and the muscle layer and the skin are sutured layer by layer. In order to avoid tissue necrosis caused by excessive ligation force, the ligation of the lateral limb should be subject to slight tremor to avoid disturbance of the blood flow of the epineurium.

2. Procedure of experiment

(one) determination of behavioural parameters

1.1 Experimental methods:

measuring mechanical paw reflex threshold (PWT): placing an organic glass box (22 × 12 × 22 cm) on a metal screen, after the mouse adapts for 15min in the box, vertically stimulating the middle part of the sole of the hind limb of the mouse by using a von Frey cilium mechanical stimulator for a duration of less than or equal to 4 seconds, and regarding the foot lifting or licking behavior of the mouse as a positive reaction, otherwise, as a negative reaction. The stimulation is repeated 10 times (with a time-time interval of 3-5 s) at each intensity from small to large, and the intensity of about 5 times of the occurrence of the foot contraction reflex is taken as PWT.

Measuring the cold foot lifting times: in a quiet environment, the mouse is placed on a metal cold plate of a cold plate instrument at the temperature of 4 ℃, the movement of the mouse is limited by a glass cover, and after the mouse adapts to the quiet state for about 5min, the foot lifting times of the operative side limb of the mouse within 5min are observed and recorded, namely the cold contraction foot reflection times.

Determination of heat-shrinkable paw reflex latency (PWL): the organic glass box is placed on a glass plate with the thickness of 3mm, and the mouse is placed in the organic glass box to move freely for 30min so as to adapt to the testing environment and temperature. The soles of the mice were irradiated with a PL-200 model thermal pain stimulator. The time taken from the start of irradiation to the appearance of leg lift in the mice was PWL. The stimulation site is the surgical side hind paw portion that is in close proximity to the glass plate. When the rear claw moves, the irradiation is stopped. The irradiation auto-off time was 20sec to prevent tissue damage. The intensity of the thermal stimulus remained the same throughout the experiment. Stimulation was repeated three times and the PWL was averaged.

Measuring a mechanical tenderness threshold value: mice were placed in a special plastic fixation cylinder and a mechanical tenderness tester was used to apply a continuously increasing pressure at a constant rate to the plantar aspect of the hind paw of the mouse. When the hind paw is retracted or hoarsed, the pressurization is stopped and the pressure value at that time is read, and the pressure value is used as the nociceptive threshold.

1.2 Experimental results:

there were no significant differences in the pre-operative PWT (fig. 1), cold foot lift times (fig. 2), PWL (fig. 3), and mechanical tenderness threshold (fig. 4) for each group of mice. On day 7 after surgery, compared with the sham group, the PWT, PWL and mechanical tenderness thresholds of the remaining mice were all significantly decreased (p < 0.01), and the cold foot raising times were significantly increased (p < 0.01). At 8, 10, 12 and 14 days after the model building, compared with the model group, PWT, PWL and mechanical tenderness threshold values of anethole (500 mg/kg) group and pregabalin (40 mg/kg) group are obviously increased, and cold foot raising times are obviously reduced (p is less than 0.05); in the anethole (250 mg/kg) group, 12 days and 14 days after the model building, the PWT, PWL and the mechanical tenderness threshold value are all obviously increased, and the cold foot raising frequency is obviously reduced (p <0.05, p < 0.01) compared with the model group; the anisole (125 mg/kg) group had no significant difference in the individual behavioural parameters.

(II) electrophysiological measurement

2.1 Experimental methods:

observation of electrophysiological activity of sciatic-peroneal nerve: after anesthesia, the same incision was opened at the time of molding, muscle and fascia were separated bluntly to expose the sciatic nerve trunk to the sciatic incisal track and distal catgut ligation site, and the sciatic nerve was protected with warm paraffin oil during the separation process. The acupuncture needle is made into a hook shape and is connected to an alligator clip of a stimulation electrode, the hook-shaped acupuncture needle connected to the alligator clip is hooked on an sciatic nerve trunk close to an sciatic notch, recording needle electrodes are respectively inserted into ankle parts and muscles between the second and third toes of a foot of a mouse, reference electrodes are placed in gastrocnemius muscles innervated by sciatic nerves, and the distance between the two reference electrodes is about 5mm. In the test process, physiological saline is used as a conductive medium and is supplemented in time according to specific conditions. The electrodes are connected with a BL-420F biological function experiment system, the experiment item selects the measurement of the nerve trunk excitation conduction velocity in the sub-option of the muscle nerve experiment, and the parameter values are set. The following data were measured and recorded: (1) the action potential latency recorded by the two electrodes and the difference (the time difference between the two peaks) thereof, namely the difference Deltat between the latencies; (2) the distance s between the two recording electrodes; (3) the compound muscle action potentials amplitude (CMAP amplitudes) is the distance between the action potential peaks and troughs. And (1) and (3) in the data are respectively selected from three action potential oscillograms for data statistics, and the average value is taken as the final statistical data. The Nerve Conduction Velocity (NCV) is calculated as the difference between the distance between the recording electrodes and the upper latency, i.e., NCV = s/Δ t. The sensory nerve conductance gradient (SNCV) is calculated according to this formula.

Observation of sciatic-peroneal nerve electrophysiological activity: the action potential of the sciatic-peroneal nerve, i.e., sensory nerve action potentials (SNAs), was induced by inserting a reference electrode into the anterior fibular muscle of the lateral limb of the mouse in the same manner as above. The difference Δ t in latency, the amplitude of the compound muscle action potential (CMAP amplitudes), and the distance between the electrodes, s, were recorded. The motor nerve conduction velocity (SNCV) is calculated according to the NCV calculation formula.

2.2 Experimental results:

the amplitude of the sciatic-peroneal action potential of the mice in the model group was significantly decreased and the latency was significantly increased compared to the sham group 14 days after the model creation (fig. 5), and a-f represent the sham group, the model group, the pregabalin group, and the anethole group (125, 250, 500 mg/kg), respectively. The compound muscle action potential amplitude and sensory nerve conduction velocity were significantly reduced in the model group mice compared to the sham-operated group (p < 0.01). The compound muscle action potential amplitude and sensory nerve conduction velocity were significantly increased in the anethole (125, 250, 500 mg/kg) group and pregabalin (40 mg/kg) group mice on 14 days post-molding compared to the model group (. P <0.05,. P < 0.01); the mice in the anethole (125 mg/kg) group showed no significant change in the combined muscle action potential amplitude and sensory nerve conduction velocity 7 days after administration (fig. 6 and 7).

(III) HE staining for observing histopathological changes of sciatic nerve

3.1 Experimental methods:

after baking sciatic nerve tissue sections overnight in an incubator at 80 ℃, hematoxylin-eosin staining (HE staining) was performed:

(1) dewaxing: sequentially soaking in xylene I (10 min) and xylene II (10 min);

(2) rehydration: rehydrating gradient ethanol for 2-4 min respectively (anhydrous I, anhydrous II, 95%,85%,80% and 70%), and cleaning the surface ethanol with distilled water;

(3) dyeing: soaking in a dye vat containing hematoxylin dye for 4min, taking out the slide, washing with running water, differentiating in 1% hydrochloric acid alcohol solution for 5s, washing with running water, placing in eosin dye vat for 2min, and washing with water for 3 times;

(4) and (3) dehydrating: sequentially soaking in 70% ethanol (30 s), 80% ethanol (30 s), 95% ethanol (30 s), anhydrous ethanol I (2 min), and anhydrous ethanol II (2 min);

(5) and (3) transparency: xylene I (2 min) and xylene II (2 min);

(6) sealing: the sections were dropped with neutral gum, mounted, and histopathological changes were observed by light microscopy.

3.2 Experimental results:

as shown in FIG. 8: A-F respectively represent sham operation group, model group, pregabalin group, and anethole group (125, 250, 500 mg/kg). The neuron cell bodies in the sham operation group are full, closely arranged and uniformly dyed; the neuron gaps of the model group are increased, the cells are arranged loosely, a large number of vacuoles appear, the cell bodies are reduced, and the staining is not uniform; the neuron changes of anethole (250, 500 mg/kg) group and pregabalin (40 mg/kg) group are improved, the arrangement is compact, and the vacuole number is obviously reduced. While the anethole (125 mg/kg) group was not significantly changed compared to the model group.

(IV) detecting the expression of TNF-alpha, IL-6, IL-1 beta and IL-10 protein in the spinal cord of a mouse with neuropathic pain by Western blot

4.1 Experimental methods:

extracting total protein by using a Katy total protein extraction kit, measuring the total protein concentration of a sample by using a BCA protein content detection kit, and calibrating the unified protein concentration. SDS-polyacrylamide gel electrophoresis was carried out by wet-transferring a nitrocellulose membrane (NC membrane). And (4) taking out the nitrocellulose membrane after the membrane conversion is finished, and sealing the nitrocellulose membrane in 5% skimmed milk powder sealing liquid for 1h. And after the sealing, incubating the primary antibody diluted by 5% skim milk powder, standing overnight at 4 ℃, rewarming for 1h at room temperature, washing the membrane, and incubating the secondary antibody. NC membranes were washed 3 times with PBST for 10min each. And (3) dropwise adding a protein chemiluminescence agent (ECL), fixing the NC membrane in the film box, and pressing the NC membrane into the film for exposure. Taking out the film, placing into developing solution and fixing solution for 1min respectively, and cleaning with clear water. Gel image analysis imaging system (culture, JS-860B) scans and image analyzes each band of interest on the film.

7.2 Experimental results:

as shown in FIGS. 9 to 12, compared with the sham surgery group, the TNF-alpha, IL-6 and IL-1 beta protein expression was significantly increased in the model group, and the IL-10 protein expression was significantly decreased (# # P < 0.01); as shown in fig. 7-10, the spinal cord tissue TNF- α, IL-6, IL-1 β protein expression was significantly reduced in mice of the anethole (500 mg/kg) group compared to the model group (plep <0.05, < 0.01); as shown in fig. 10, IL-10 protein expression was significantly reduced in spinal cord tissue in mice in the anethole (500 mg/kg) group compared to the model group (. P <0.05,. P < 0.01). The protective effect of the anethole is suggested to reduce the expression and activation of TNF-alpha, IL-6 and IL-1 beta through up-regulating the expression of IL-10 protein, and play an anti-inflammatory role, thereby having protective effect on neuropathic pain caused by chronic compressive injury of mice.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.

Claims (1)

1. The application of the anethole as the only effective component in preparing the medicine for treating neuropathic pain caused by chronic compressive injury of sciatic nerve is characterized in that: the structural formula of the anethole is shown as the formula (1):

the compound of the formula (1),

the single application dosage of the anethole is 500mg/kg, and the dosage form of the medicine is a pharmaceutically allowable oral dosage form, an injection dosage form or a powder injection.

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