CN113616794A

CN113616794A - Function of Panx1 protein in preparation of drugs for preventing and treating neuropathic pain

Info

Publication number: CN113616794A
Application number: CN202111003210.2A
Authority: CN
Inventors: 韦中亚; 陈罡; 王茜; 凌卓敏
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2021-08-30
Filing date: 2021-08-30
Publication date: 2021-11-09

Abstract

The invention discloses an effect of gap junction protein Panx1 in preparation of drugs for preventing and treating neuropathic pain, belonging to the technical field of biological medicines. The invention determines that Panx1 is mainly expressed in Schwann cells, and the expression level of the Panx1 can be obviously increased due to chronic neurological pathological damage; the inhibitor and the mimic peptide of Panx1 can obviously inhibit mechanical pain and thermal pain caused by chronic neuropathic injury of sciatic nerve, and the inhibitor probenecid of Panx1 can obviously inhibit the expression of Panx1 caused by injury. This provides strong basic research evidence for the clinical use of Panx1 inhibitors in the treatment of chronic neuropathic pain patients in the future.

Description

Function of Panx1 protein in preparation of drugs for preventing and treating neuropathic pain

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an effect of gap junction protein Panx1 in preparation of a medicine for preventing and treating neuropathic pain.

Background

Neuropathic pain is a chronic pain caused by primary damage and dysfunction of the nervous system. The clinical characteristics of the medicine are strong stubborn, high incidence rate and long course of disease, and hyperalgesia still persists for weeks, months or even years. The key problem of the current treatment of chronic neuropathic pain is that the generation and development mechanisms are not completely clear. Over the past years of research, which has focused on neurons, it was discovered that peripheral sensitization, which results from increased excitability of primary sensory neurons, and central sensitization, which results from inflammatory mediators acting on central neurons, are important pathological bases for the onset and maintenance of neuropathic pain. But increasing evidence in recent years suggests that: glial cells play an important role in the development and progression of neuropathic pain. Glial cells mainly include glial cells of the central nervous system: astrocytes, oligodendrocytes and microglia; and glial cells of the peripheral nervous system: schwann cells and satellite cells. The research shows that: in the brain and spinal cord, after nerve injury, activated microglia and activated astrocytes can release various pain-related cytokines, chemotactic molecules and other substances to regulate the excitability of neurons and promote the occurrence, development and maintenance of chronic neuropathic pain; and inhibiting the activation of microglia and astrocytes can block or relieve various chronic neuralgia. In the peripheral nervous system, satellite cells surround dorsal root neurons, which activate glial cells earlier than in the central nervous system, playing an important role in the development of neuropathic pain. However, schwann cells secrete a variety of pain-associated pro-inflammatory/anti-inflammatory factors after peripheral nerve injury, but their role and possible mechanism in the development, progression and maintenance of neuropathic pain is still poorly understood.

Pannexins (panxs) is another family of gap junction proteins that was discovered following Connexins. It is a large pore membrane channel with some permeability to pain-related ATP and other signaling molecules. Pannexins family members include mainly pannexin1, pannexin2 and pannexin3, of which pannexin1(Panx1) is of great interest and is widely expressed in glial cells and neurons of the nervous system. The research shows that: activation of Panx1 in astrocytes, microglia, and dorsal root ganglia plays a regulatory role in the development, progression, and treatment of various types of pain.

Disclosure of Invention

The invention aims to provide the function of the gap junction protein Panx1 in preparing medicines for preventing and treating neuropathic pain, provide more powerful evidence for using Panx1 as a treatment target, and simultaneously aim to further enrich the physiological and pathological functions of Schwann cells and participate in the molecular regulation mechanism of chronic neuropathic pain.

The invention determines that Panx1 is mainly expressed in Schwann cells, and the expression level of the Panx1 can be obviously increased due to chronic neurological pathological damage; the inhibitor and the mimic peptide of Panx1 can obviously inhibit mechanical pain and thermal pain caused by chronic neuropathic injury of sciatic nerve, and the inhibitor probenecid of Panx1 can obviously inhibit the expression of Panx1 caused by injury. This provides strong basic research evidence for the clinical use of Panx1 inhibitors in the treatment of chronic neuropathic pain patients in the future.

Drawings

FIG. 1 shows the result of the expression of Panx1 in sciatic nerve caused by chronic neuropathic injury. Wherein: a is the expression condition of Panx1, Panx2 and Panx3 of normal sciatic nerve detected by real-time quantitative PCR experiment; B-D are changes in the expression of Panx1, Panx2, and

Panx3 mRNA

4 and 14 days after nerve injury; e is the change in expression of

Panx

14 and 14 days after immunoblot detection of nerve injury; f is the result of statistical analysis of the E picture.

FIG. 2 shows that chronic neuropathic injury causes increased expression of Schwann cell Panx 1. Wherein: a is the immunofluorescence co-labeling result of the neurofilament markers NF200 and Panx1 in the sciatic nerve; b is the immunofluorescence co-labeling experiment result of the dorsal root ganglion markers Nissl and Panx 1; c is an immunofluorescence co-labeling experiment result of Schwann cell markers S100 beta and Panx1 in sciatic nerve after CCI injury; d is the result of statistical analysis of the C picture.

FIG. 3 shows the Panx1 inhibitors CBX, probenecid and mimetic peptides¹⁰Panx1 can significantly inhibit mechanical pain and thermal pain caused by nerve injury.

Fig. 4 shows that the Panx1 inhibitor probenecid significantly inhibited the expression of Panx1 caused after injury. Wherein: a is PBS after Sham group and CCI, and the expression of the sciatic nerve Panx1 changes after 3 days and 7 days of probenecid; b is the result of statistical analysis of the A picture.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

Previous studies by the inventors have found that schwann cells cultured in vitro predominantly express Panx1, which plays an important regulatory role in the process of hypotonic-induced ATP release. Thus, the inventors speculate that schwann cell Panx1 may be a targeting molecule for neuropathic pain modulation.

The method firstly utilizes a sciatic nerve Chronic Compression Injury (CCI) mouse pain model, utilizes qPCR to detect the expression change of Pannexins after injury, and utilizes immunoblotting and immunofluorescence to further detect the expression change of Panx1 after injury; the Panx1 inhibitors, Carbexolone (CBX), probenecid and pannexin1, were administered subperioneally 10 days after CCI injury¹⁰Panx1, which measures changes in mechanical and thermal pain in mice, and changes in Panx1 expression at various time points after administration of probenecid. The effects of Panx1 of Schwann cells in neuropathic pain can be known, more basic research bases can be provided for Panx1 as a clinical treatment target, and the functions of Schwann cells and cells of neuropathic pain can be further enrichedThe molecular mechanism.

The specific experimental method is as follows:

1. constructing a chronic compressive injury model (CCI) of sciatic nerve: the mouse is continuously anesthetized by isoflurane gas on an operation platform, after shaving and disinfection, the skin is longitudinally cut above the outer side of the femur, muscle is bluntly separated along muscle lines, sciatic nerve is exposed, peripheral tissues are dissociated, three ligatures are performed at the position of about 5 mm near the trigeminal branch of the sciatic nerve by 6.0 surgical suture lines, the distance is about 1 mm, the blood circulation degree of the adventitia is not influenced, the skin is sutured after the incision is closed, and the mouse is disinfected. Both surgical implementation and post-operative animal care were in accordance with the national institutes of health (USA) laboratory animal care and use guidelines (NIH Publication number 85-23, revisised 1996). The experimental animals used were approved by the animal ethics committee of university of southeast university (No. S20180806-002).

2. Real-time quantitative PCR: extracting total RNA at sciatic nerve injury positions of each group of experimental mice by adopting a Trizol method, carrying out total RNA reverse transcription by using a TaKaRa kit after quantifying 1 mu g respectively, synthesizing a first cDNA chain, and quantitatively detecting the expression of each subtype of Panxs by using an SYBR Green method.

The Panxs primers were as follows:

Panx1 sense sequence: CCTCATTAACCTCATTGTGTAT (SEQ ID NO.1)

anti-sense sequence： CATTGTAGCCTTCAGACTTG (SEQ ID NO.2)；

Panx2 sense sequence: CATTGTAGCCTTCAGACTTG (SEQ ID NO.3)

anti-sense sequence: CTCCTGCTGGATGTCTAG (SEQ ID NO.4)；

Panx3 sense sequence: CTCAGATTATGGACTATGAACAC (SEQ ID NO.5)

anti-sense：TCAGAAGGTAACTTGGAGAAT (SEQ ID NO.6)；

18S sense sequence：GACAGGATTGACAGATTGATAG (SEQ ID NO.7)

anti-sense：CGTTATCGGAATTAACCAGAC (SEQ ID NO.8)；

GAPDH sense sequence：TCCATGACAACTTTGGCATTG (SEQ ID NO.9)

anti-sense：CAGTCTTCTGGGTGGCAGTGA (SEQ ID NO.10)。

3. immunoblotting: after the animals are treated, sciatic nerves at the ligation site of CCI are extracted, cell lysate containing protease inhibitor is added, an ultrasonic cell disruptor performs ultrasonic treatment on ice for 5 s, then the sciatic nerves are disrupted on ice for 30 min, the sciatic nerves are centrifuged at 14000 rpm at 4 ℃ for 30 min, supernatant is taken, and after quantification is performed by using a BCA protein quantification kit, 5 x loading buffer with the volume of 1/4 is added, boiling water bath is performed for 10 min, and the sciatic nerves are subpackaged at-20 ℃ for storage. After electrophoresis on 10% separation gel, membrane transfer is carried out, the membrane is sealed in 5% skimmed milk for 2 h at room temperature, primary anti-Panx1 (1:200, rabbit, Abcam) and anti-GAPDH (1: 10000, mouse, Sigma) are added for incubation overnight at 4 ℃, the membrane is washed for 3 times by TBST, 10 min each time, anti-rabbit HRP-labeled secondary antibody (1: 1000) is incubated for 2 h at room temperature, ECL color development and exposure detection by a chemiluminescence imager.

4. And (3) performing fluorescent staining on immune tissues: after the mice are deeply anesthetized by 1% pentobarbital sodium, limbs are fixed, the heart is exposed, the limbs and the liver of the mice are perfused to be white by using normal saline, then the perfusate is changed into 4% PFA, the mice are perfused to be hardened, sciatic nerves are carefully taken out, the 4% PFA is placed, the fixation is carried out for 8 to 12 hours at the temperature of 4 ℃, and then the mice are changed into 30% sucrose which is freshly prepared to be dehydrated to be sunk to the bottom (about 2 to 4 days). And longitudinally cutting the sciatic nerve into 12 mu m/piece by using a freezing slicer, sticking the sciatic nerve to a glass slide coated with positive charges, and waiting for subsequent fluorescent staining of the immune tissue.

The specific steps of the fluorescent staining of the immune tissue are as follows: washing with 0.01M PBS for 3 times, each for 10 min, and breaking membrane with 0.3% Triton X-100 at room temperature for 10 min; washing with 0.01M PBS for 3 times, each for 10 min, and blocking with 10% BSA at room temperature for 2 hr; the following antibodies were diluted with 0.1M PBS containing 3% BSA and 0.1% Triton X-100: anti-NF200 (1: 500, mouse, Sigma), anti-S100 beta (1: 400, mouse, Sigma), anti-Panx1 (1: 300, rabbitt, Abcam), incubation at 4 ℃ for 1-2 days, washing with 0.01M PBS for 3 times, 10 min each time, then selecting appropriate fluorescently labeled secondary antibody according to the primary antibody, labeling 2 h at room temperature in the absence of light, washing with 0.01M PBS for 3 times, 10 min each time. Nissl staining of dorsal root ganglia (1:200, Thermo Fish) was performed at room temperature for 20 min, the washing process was the same as that after incubation with secondary antibody, and finally 90% glycerol was used for mounting, and the mounting was performed at room temperature, and image acquisition was performed using a confocal laser microscope.

5. Sub-cranial injection: mice model CCI were anesthetized by longitudinal incision of the skin over the lateral aspect of the femur, muscle blunt dissection, exposure of the sciatic nerve, and slow injection of 6 μ l PBS, a Panx 1-related inhibitor and a mimetic peptide (including the inhibitor carbexolone (CBX, 100 μ M), probenecid, 500 μ M and pannexin1 mimetic peptide) under the sciatic nerve by a micro-syringe pump at the first ligation of CCI¹⁰Panx1, 100 μ M), stop the needle for about 5-10 min after injection is complete, close the incision, suture the skin and sterilize.

6. And (3) pain ethological detection: all animal behavioral experiments were performed using a "double-blind" experimental design. Screening mechanical pain threshold mean value of a mouse before a CCI model to be about 1.0, and screening thermal pain threshold mean value of the mouse to be 10 s; changes in mechanical and thermal pain were measured on the tenth day post injury, following the subpial administration of Panx1 inhibitor and mimetic peptide.

(1) Mechanical allodynia test: the 50% withdrawal threshold was estimated by the up-down method using von Frey filaments: and (3) placing an organic transparent glass box on a metal screen, after the mouse adapts to the organic glass box for 30 min, vertically stimulating the middle part of the hind limb sole of the mouse by von Frey fiber filaments for 1-2 s, and regarding the mouse as a positive reaction when the mouse raises or licks the foot, or regarding the mouse as a negative reaction.

(2) Thermal hyperalgesia experiments: the organic glass box is placed on a glass plate with the thickness of 3 mm, and the thenar of the mouse is irradiated to the part tightly attached to the glass plate by thermal radiation stimulation according to the Hargreaves method. The time from the start of irradiation until the appearance of the mice raised the feet was recorded with a cut-off time of 20 s.

The results are as follows:

1. chronic neuropathic injury of sciatic nerve causes increased expression of Schwann cell Panx1

Real-time quantitative PCR results showed that the expression of Panx1 was significantly higher in normal mouse sciatic nerve than in Panx2 and Panx3 (fig. 1A), while chronic neuropathic pathological injury (CCI) caused significantly increased expression of Panx1 (at least for 14 days), while the expression of Panx2 and Panx3 was decreased (fig. 1B-D). Immunoblot results further confirmed from protein levels that nerve injury significantly increased the expression of Panx1 (fig. 1E-F). To further demonstrate that the increased expression of Panx1 following injury was primarily increased expression of Panx1 in schwann cells, the inventors examined the expression of Panx1 in the sciatic nerve and DRG, and the results of immunofluorescence double-label experiments (fig. 2A-B) showed that Panx1 was primarily expressed in the soma of the dorsal root ganglion (Nissl marker) and less expressed in the processes (NF 200 marker); good co-localization with schwann cell marker S100 β, while expression of schwann cell Panx1 was significantly increased at both 4 and 14 days post-CCI.

2. The Panx1 inhibitor can remarkably relieve mechanical pain and thermal pain caused by chronic neuropathic pathological injury

Behavioral experiments demonstrated that the Panx1 inhibitor carbenoxolone (cbx), probenecid and pannexin1 mimetic was injected subcapsular to sciatic nerve 10 days after chronic neuropathic injury¹⁰Panx1 can remarkably inhibit mechanical pain and thermal pain caused by nerve injury, and the effect can be maintained for 3-4 days (figure 3).

3. Inhibition of probenecid by Panx1 significantly reduces the up-regulation of Panx1 expression caused by nerve injury

The results of immunofluorescence experiments show that: after nerve injury, the expression of Panx1 was significantly increased compared to the sham group (sham group) when the inhibitors probenecid and PBS were administered, but the expression level of Panx1 was significantly lower than that of the PBS group 3 days after administration of probenecid (fig. 4).

Sequence listing

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Claims

The Panx1 protein can be used for preparing or screening drugs for preventing and treating neuropathic pain.
The function of the Panx1 protein inhibitor in preparing medicines for preventing and treating neuropathic pain.
3. Use according to claim 2, characterized in that: the Panx1 protein inhibitor is carbenoxolone, probenecid or mimic peptide¹⁰Panx 1。