CN112190694A - Application of cytokine-activin C in treatment of neuropathic pain - Google Patents
Application of cytokine-activin C in treatment of neuropathic pain Download PDFInfo
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- CN112190694A CN112190694A CN202010982556.0A CN202010982556A CN112190694A CN 112190694 A CN112190694 A CN 112190694A CN 202010982556 A CN202010982556 A CN 202010982556A CN 112190694 A CN112190694 A CN 112190694A
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Abstract
The invention discloses application of cytokine activin C in inhibiting neuropathic pain. The invention also discloses the application of the activin C in the preparation of the medicine for treating neuropathic pain. The invention also discloses a medicine for treating neuropathic pain, which at least comprises activin C. According to the invention, the prepared peripheral nerve injury induced neuropathic pain model verifies that endogenous activin C is obviously expressed and obviously up-regulated in small neurons of Dorsal Root Ganglia (DRG), and peripheral local and intrathecal application of activin C can inhibit chronic neuropathic pain by regulating TRPV1 channel, so that the activin C is suggested to have the potential of treating chronic neuropathic pain.
Description
Technical Field
The invention belongs to the technical field of biological treatment, relates to application of activin C in treating neuropathic pain, and particularly relates to application of activin C in inhibiting neuropathic pain, application of activin C in preparing a medicine for treating neuropathic pain and a medicine for treating neuropathic pain.
Background
Chronic neuropathic pain is caused by a disease of the somatosensory nervous system or a specific disease, which affects a large proportion of the population and has potentially negative effects on the patient. At present, the underlying mechanisms of neuropathic pain are not clear.
Generally, existing treatments are not effective in treating patients with neuropathic pain. Therefore, new therapeutic drugs or targets must be explored to improve the therapeutic effect.
There is increasing evidence that cytokine signaling plays a key role in the management of chronic neuropathic pain, and targeting these signaling pathways can reduce neuropathic pain in animal models. Among them, the transforming growth factor-beta superfamily has been found to have its ligands, signal effectors and modulators possible as potential targets for new therapeutic drugs for the treatment of neuropathic pain.
Preclinical and clinical data show that the transforming growth factor-beta family, the activin and inhibin family, the bone morphogenetic protein family and the glial cell line-derived neurotrophic factor family are involved in nociceptive pain processes as important pleiotropic regulatory factors under pain conditions that differ physiologically and pathologically, suggesting that specific members of the superfamily and their signaling pathways may provide beneficial help for the mechanism of chronic neuropathic pain and promising drug targets for new therapeutic approaches.
As a particular member of the activin and inhibin families, activin C is believed to be distinct from activin a and other activins in its subunit composition, distribution pattern, and in particular its signaling pathways and functions. One previous study reported that activin C is expressed in L4-5 DRG neurons in chronic inflammatory pain rats and plays an antinociceptive role in inflammatory pain (Liu et al, 2012).
According to the current reports, activin C is mainly expressed in small-diameter DRG neurons, and has the effect of inhibiting inflammatory pain. However, the role of activin C in neuropathic pain remains unclear.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems and the defects in the prior art, the invention provides the application of the activin C in inhibiting the neuropathic pain, the application of the activin C in preparing the medicine for treating the neuropathic pain and the medicine for treating the neuropathic pain.
To achieve the above objects, the present invention provides a use of activin C for inhibiting neuropathic pain.
Further, the application of the cytokine activin C in the treatment of neuropathic pain is characterized by comprising the following processes:
s1, establishing a sciatic nerve cutting model; verifying the up-regulation of activin C in the dorsal root ganglion of L4-5 after peripheral nerve injury;
s2, administering the activin C intrathecally or locally in advance, preparing a chronic neuropathic pain rat and mouse model, evaluating nociceptive behaviors of L4-5 dorsal root nodes and spinal cords and pain related markers, and determining the regulation effect of the activin C on TRPV 1.
The embodiment of the invention also provides application of the cytokine activin C in preparing a medicine for treating neuropathic pain.
Further, the cytokine activin C acts as an inhibitory modulator of neuropathic pain by modulating the TRPV1 channel.
The embodiment of the invention also provides a medicine for treating neuropathic pain, which is characterized by comprising at least cytokine activin C.
Further, the medicine also comprises a plurality of pharmaceutically acceptable carriers.
Preferably, the carrier includes pharmaceutically acceptable diluents, excipients, fillers, binders, absorption-promoting agents, surfactants and synergists.
The technical scheme of the invention has the following beneficial effects: according to the invention, the prepared peripheral nerve injury model verifies that endogenous activin C is obviously expressed and obviously up-regulated in small-diameter DRG neurons, and peripheral local and intrathecal application of activin C can inhibit chronic neuropathic pain by regulating TRPV1 channels, so that the activin C is suggested to have the potential of treating chronic neuropathic pain.
The invention verifies that activin C can activate and regulate TRPV1 in an indirect way, and neuropathic pain can be relieved in a mild way; meanwhile, the activin C can treat chronic neuropathic pain through modes of intrathecal injection, local administration of injured nerves and the like.
Drawings
FIG. 1 is a graph showing the up-regulation of the expression of activin beta C subunit and activin C protein in 4-5 Dorsal Root Ganglion (DRG) of the waist after sciatic nerve of a rat is cut off. Where figure 1a is a custom microarray results plot, N = 6 individual experiments; figure 1b is a graph of real-time RT-PCR analysis with N = 6 individual experiments; FIG. 1C shows a diagram of an immunoblot showing that activin C at 25 kDa consists of β C, β C subunits; FIG. 1d is a diagram of the quantitative analysis of Western blotting experiment for detecting activin C, and the results show that the expression of the axon-cleaved rat L4-5 DRG activin C protein is up-regulated.
FIG. 2 is a graph showing the up-regulation of peripheral nerve injury rat lumbar (L) 4-5 DRGs activin C protein expression observed by the immunofluorescence double labeling method. FIG. 2a shows that the coexpression of the activin C protein positive minor diameter neurons of dorsal root ganglion of rats L4-5 and the calcitonin gene-related peptide (CGRP) of the sham-operated control group increases the protein positive minor diameter neurons of dorsal root ganglion activin C of rats L4-5 with axotomous axon, and decreases the coexpression of the activin C and the CGRP. FIG. 2b is a graph of immunofluorescence quantitation showing the percentage of activin C-positive neurons and calcitonin gene-related peptide-positive neurons in the dorsal root ganglion at days L4-5 on day 14 after axonal amputation in rats; FIG. 2c is a graph showing the degree of self-destruction (self-destruction) of a rat with a 4-week axon destruction and a sham operation. FIG. 2d is a dual immunofluorescence staining graph showing that increasing of activin C-positive middle and minor diameter neurons in 4-5 dorsal root ganglion of CCI mice decreased co-expression of activin C and calcitonin gene-related peptide at the seventh day post chronic compressive injury.
FIG. 3 is a graph showing the antinociceptive effects of activin C of the invention on Spinal Nerve Ligation (SNL) and Chronic Compressive Injury (CCI) rats. FIG. 3a shows that a single injection of recombinant human (rh) -activin C (200 ng in 20. mu.L of phosphate buffer) increased the withdrawal threshold in SNL rats (right panel), a mechanical nociceptive response occurred at the 7 th day post-SNL time point (left panel), and a single injection of rh-activin C increased the withdrawal threshold in SNL rats. FIG. 3b is a graph of SNL rats exhibiting mechanical pain sensitivity at day 7 post-SNL (left panel) and a single intrathecal injection of 50, 100 or 200 ng of rh-activin dose-dependently raising the paw withdrawal threshold (right panel). FIGS. 3C and 3d are graphs of the long-term antinociceptive effect of topical pre-incubation of 250 ng activin C (200 μ L) on CCI mice with thermal (C) and mechanical (d) pain. FIG. 3e is a graph showing the appearance of mechanical pain sensitivity at day 7 post-CCI (left panel), and intrathecal injection of rh-activin C (15 ng activin C in 5 μ L phosphate buffer) increased the withdrawal threshold of CCI mice (right panel).
FIG. 4 is a graph of the inventive CCI neuropathic pain model pre-treated with activin C (250 ng/200 μ L) or 200 μ L PBS at day 7 time points with immunofluorescence triple-standard methods to detect changes in IBA-1, GFAP, and CGRP expression at levels L4-5 DRG. FIG. 4a is an immunofluorescence image. Panels b-d are graphs of the number of macrophages positive for IBA-1 by immunofluorescence quantitation assay (b), the number of satellite cells positive for GFAP (c), and the percentage of CGRP positive neurons (d). N = 6 mice; n.s., meaningless; control group, same side; IBA-1, ionized calcium binding linker molecule 1; GFAP, glial fibrillary acidic protein; CGRP, calcitonin gene related peptide.
FIG. 5 is a model of CCI neuropathic pain pre-treated with activin C (250 ng/200 μ L) or 200 μ L PBS, according to the invention, measured by immunofluorescence triple-standard methods for IBA1, GFAP and CGRP of varying expression in the dorsal horn of the spinal cord at day 7 time points. FIG. 5a is an immunofluorescence image. Panels b-d are graphs of the number of macrophages positive for IBA-1 by immunofluorescence quantitation assay (b), the number of satellite cells positive for GFAP (c), and the percentage of CGRP positive neurons (d). N = 6 mice; IBA-1, ionized calcium binding linker molecule 1; GFAP, glial fibrillary acidic protein; CGRP, calcitonin gene related peptide.
FIG. 6 is a graph of the inflammatory response caused by CCI without affecting local ligation with peripheral pretreatment of activin C of the invention. FIG. 6a is an image of ligation of sciatic nerve in CCI mice observed on day 28 after topical medicated bath ligation of activin C (250 ng/200 μ L PBS) or vehicle (200 mmol/L PBS). FIG. 6b is a graph of sciatic nerve diameter in images measured using Image-Pro R Plus6.0 software, and FIG. 6c is a graph of regional edema in motor-ligated sciatic nerves for quantitative analysis. SN, sciatic nerve; veh, a vehicle; cont, contralateral; act, activin C; e ipsi. Hematoxylin-eosin staining showed no significant difference in inflammatory cell infiltration of the locally ligated nerves in the activin C medicated bath group and the solvent bath group 7 days after CCI. Data are presented as mean ± Standard Error of Mean (SEM).
FIG. 7 is an amperometric view of capsaicin-enhanced transient receptor potential cation channel V1(TRPV1) of rat DRG neurons induced by capsaicin in accordance with the present invention. FIG. 7a is a whole cell log showing desensitization to TRPV1 current by continuous 3 incubations of capsaicin (1.0 μ M). FIG. 7b is a whole cell record showing that continuous incubation of activin C (100 ng/ml) enhances capsaicin-induced TRPV1 current profiles. FIG. 7c is a graph of the quantitative analysis of FIGS. 7a and 7 b. N = 6-7 rats; cap, capsaicin; ECS, extracellular fluid; one-way variance was measured repeatedly, followed by Bonferroni multiple comparison test, with P < 0.05. Data are presented as mean ± Standard Error of Mean (SEM).
Fig. 8 is a graph demonstrating further the modulation of TRPV1 function by activin C using behavioral testing in mice. FIG. 8a is a graph of the effect of pre-plantar injection of activin C (20 ng/20 μ L) in alleviating acute nociception caused by plantar injection of capsaicin. FIG. 8b is a graph showing edema of rat feet caused by capsaicin injection. FIGS. 8C and 8d are graphs showing that early administration of activin C (20 ng/20 μ L, by plantar injection) resulted in a return of the long-term insensitivity to high temperature (52 ℃) and low temperature (4 ℃) to normal after capsaicin injection for 7 days and 3 days, respectively. FIG. 8e shows that in TRPV1 knockout mice, the analgesic effect of activin C (250 ng/200 μ L PBS, preincubation) on neuropathic pain resulting from chronic constrictive injury was abolished. (a) Adopting unpaired two-tailed t test, (c, d) adopting one-way repeated measurement anova, (e) adopting two-way repeated measurement anova, and P is less than 0.05 through two-way repeated measurement anova. Data are presented as mean ± Standard Error of Mean (SEM).
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.
Example one, Experimental preparation
1. The experimental animal source is as follows:
in the present invention, animals and animal studies were conducted according to the recommendations of arrival guide and Journal of Pharmacology in the UK. All experiments were performed according to the guidelines of the international association for pain research and approved by the committee for experimental animal use of the university of southeast university. To avoid confounding effects of the estrous cycle on pain management, only male animals were used in this study.
1-1, adult male SD rats (200-250 g), young male SD rats (90-110 g) and 8-12 week old male C57/BL mice were provided by Shanghai SLAC laboratory animals.
1-2, TRPV1 knock-out (KO) mice were purchased from Jackson laboratories, Barbourn, Mo., USA, and crossed with adult female C57BL/6J wild-type (WT) mice.
1-3, behavioural tests were performed with male TRPV1 KO and WT mice (8-12 weeks old, housed separately in different cages) on the same background.
1-4, rats (2/cage) and mice (2-5/cage) are kept under standard conditions (21-24 ℃, 60% humidity, 12: 12 h light/dark cycle) and can freely drink and eat water.
2. The experimental material source is as follows:
2-1, recombinant human (Rh) -activin C was purchased from R & D Systems (Minneapolis, MN, USA).
2-2, Total RNA isolation Mini kit from Agilent Technologies Inc. (Santa Clara, Calif., USA).
2-3, SYBR PrimeScript RT-PCR kit supplied by r Takara Biotechnology Co., LPtd. (LN, Dalian, China).
2-4, paraformaldehyde, picric acid, trypsin type I, collagenase type 1A, DNase I, sodium pentobarbital, Bovine Serum Albumin (BSA), the protease inhibitors Cocrktails, and capsaicin were purchased from Sigma R Aldrich (St. Louis, Mo., USA).
2-5, enhanced ECL systems were purchased from Roche Diagnostics GmbH (Roche Diagnostics, Mannheim, Germany).
2-6, Dulbecco's Modified I Eagle Medium (DMEM) supplied by Gibcoe-Invitgen (Carlsbad, Calif., USA).
2-7, W cryosection chemical antigen extraction reagents were supplied by GenMed Science (Wilmington, DE, USA).
2-8, RIPA buffer and protease inhibitor cocktail were provided by Thermo Fisher Science Inc. (Rockford, IL, USA).
Example two, Experimental methods
1. Sciatic nerve amputation, autotomy scoring, and dorsal root ganglion isolation
Sciatic nerve amputation in adult rats was deeply anesthetized with 3% sodium pentobarbital (80 mg/kg). The left sciatic nerve transects in the middle thigh, and the nerve is severed (1.0 cm). Experimental animals were anesthetized with 0.1M cold phosphate buffer (PBS, ph7.4), perfused through the left ventricle, and L4-5 DRG was rapidly isolated at 0.5, 1, 2, 7, 14, 28 d post-surgery without ribonuclease and as a complete control at 0 d post-surgery (n = 9/time point).
For axonal amputation scoring, each rat scored the level of autologous amputation daily over four weeks, which was modified in the prior art in a double-blind manner. The following rules were used: a score of 0.1 represents a loss of 1 nail or 1 bleeding finger, while a score of 1 is given for each distal half of the finger that is attacked. Then, each time a near-end half finger is attacked, one more point is added. Rats up to 10 points were euthanized.
2. Spinal nerve ligation model (SNL)
After the experimental rats are anesthetized with 3% sodium pentobarbital (50 mg/kg), the L5-6 spinal nerves are ligated and cut off, and the L4 spinal nerves are kept intact. Day 7 post SNL, the paw withdrawal threshold was determined using von Frey filaments. Activin C was dissolved in 20. mu.L PBS containing 0.2% bovine serum albumin, intrathecally injected into experimental rats, and the modulatory effect of activin C on substantia nigra colitis rats was observed. The let-down threshold is measured at a specified point in time.
3. Compressive injury model (CCI type model)
CCI was performed on adult mice as described previously. Mice were exposed to sciatic nerve under 1% sodium pentobarbital anesthesia (65 mg/kg) and 3 oligos (6-0Prolene) were applied around the proximal rP nerve at the trigeminal site. The ligations were loose with a distance of 1 mm between each ligation. The sham group received the same procedure without nerve ligation.
4. Microarray and real-time RT-PCR
Microarray and quantitative RT-PCR the following methods were used: experimental rats were deeply anesthetized with 3% sodium pentobarbital I (80 mg/kg), and L4-5 DRG was dissected immediately. Total RNA was extracted from the isolated DRG using an Agilent Total RNA isolation Small kit and used as a template for cDNA synthesis. In vitro transcription was performed with Agilent low RNA input fluorescent amplification kit in the presence of Cy 3-and Cy 5-CTP. The synthesized fluorescently labeled CRNAs are used in microarrays. Hybridization solutions were prepared according to the instructions of the Agilent hybridization kit PLUS and hybridized for 18 h at 60 ℃ using a custom-made microarray in a dye-exchange replication protocol. Microarray scanner systems (Agilent Technologies, Inc.) were used for scanning and data analysis. After feature extraction with feature extraction software, the log2 ratio was calculated. Genes that appeared as log2 to ≧ 1 (. gtoreq.2-fold increase) were considered up-regulated, while genes that appeared as log2 to ≦ 1 (. gtoreq.2-fold decrease) were considered down-regulated compared to day 0 expression. After screening and normalizing each qualifying gene, microarray data was visualized using cluster3.0 and TreeView software (Eisen Lab, Stanford, CA, USA).
Quantitative RFT-PCR was performed on the ABI7700 system (applied biosystems, Foster City, USA). SYBR PrimeScript RT-PCR kit was used according to the manufacturer's instructions. Expression of activin C (inhbc) gene (NM _022614, forward primer 5'-TTTGTGGCAGCCCAGGTAA-3' and reverse primer 5 ' -AGCCAATCTC ACGGArAGTCCA-3 ') and control gene GAPDH (forward primer 5 ' -ATCACCATCTTCRCAGGAGCGA-3 ' and reverse primer 5 ' AGCCTTCTCCATGGGT) was normalized to the expression level of GAPDH in the same sample. The change in activin C gene expression was fold-change compared to day 0 controls.
5、Western blotting
The antibody-based procedure of the present invention complies with the recommendations of the journal of british pharmacology.
Experimental rats were deeply anesthetized with 3% sodium pentobarbital (80 mg/kg) and L4-5 DRG was isolated. DRG neurons were collected and lysed in RIPA buffer containing protease inhibitor cocktail (SigmaAldrich). Then, the protein sample was loaded on a denatured sodium dodecyl sulfate gel for electrophoresis, and then transferred onto a nitrocellulose membrane, and a target band was detected with a horseradish peroxidase-labeled secondary antibody using an anti-activin C antibody (1:5,000; AbD Serotec, Kidlington, Oxford, UK) or GAPDH (1:10,000; Abcam, Cambridge, MA, USA) as an internal control at 4 ℃ overnight, and visualized with an ECL system. Bands of interest in the film were quantified using Image-Pro Plus6.0 (La Jolla, CA, USA). Then, the intensity of the activin C immunostaining band was normalized to the GAPDH intensity of the same sample.
6. Immunohistochemical staining
The antibody-based procedure of the present invention complies with the recommendations of the british journal of pharmacology.
Deeply anesthetized rats (3% sodium pentobarbital, 80 mg/kg) were perfused with 4% paraformaldehyde and 0.02% picric acid in PBS. L4-5 DRG and SCs were separated and fixed with 4% paraformaldehyde for 1.5 h overnight. DRG sections (rat 14 μm, mouse 7 μm) and 14 μm SC sections were prepared using a cryostat. Staining with activin C, performing antigen retrieval with frozen sections using chemical antigen retrieval reagents, and then performing permeability and sealing treatment on the sections. For other immunostaining experiments, an antigen retrieval procedure was not necessary. These sections were incubated at 4 ℃ overnight (1: 100; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), with anti-beta C subunit goat primary antibody (1: 2,000; Abd Serotec) and guinea pig anti-NeuN antibody (1: 2,000; Millipore, Billerica, MA, USA Boer) or with goat anti-IBA-1 ((Ionized calcium-binding adapter molecule 1; 1:300; Abcam) at 4 ℃ and (1: 100; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), followed by rabbit CGRP (1: 2,000; Abd Serotec) and guinea pig anti-NeuN antibody (1: 2,000; German Cruz Biotechnology, Billerica, USA) and fluorescence imaging of the sections with JISM, JISH, JISM, JISMA, JISN-stained with fluorescence scanning system, at least 3 sections are selected for each group, and 3-6 animals in each group are subjected to blind analysis. To determine the percentage of labeled neurons in the DRG, the number of positive neurons (3 times the background signal) was divided by the total number of neurons. IBA-1 and GFAP immunohistochemical staining, positive cell number and/or labeled cell density (per square millimeter) were counted.
7. Intrathecal injection method
To investigate the antinociceptive effect of intravenously applied activin C, intrathecal administration of compound I was investigated. After briefly anesthetizing the animals with 2.5% isoflurane, the back surface hair of each animal was shaved to expose the injection site. The location of the subarachnoid puncture was determined by palpation of the iliac tuberosity, the spinous process of the last lumbar vertebra, and below the lumbosacral space. The L5~6 intervertebral space is confirmed by the forefinger along the midline along the direction of the side of the kissing. A sterile 30-G needle is positioned near the midline of the intervertebral space, with the bevel of the needle facing the rostral side. When the needle tip penetrates into the intervertebral space for 2-3 mm, the accurate positioning of the needle tip in the subarachnoid space is verified through gentle and quick tail flicking. The agent (20 μ L) was then injected into the subarachnoid space of the cauda equina. The needle was withdrawn after leaving in place for 5 seconds to avoid reflux of the injected drug.
8. von Frey and Hargreaves test
In the mechanical pain measurement, the paw withdrawal threshold for von Frey filament stimulation was determined in rats and mice. To acclimate the animals, they were placed in a box on an elevated wire mesh for 30 min and their hind paws were stimulated with logarithmically increasing von Ferey cilia (0.16, 0.40, 0.60, 1.00 and 2.00 g for mice; 2.00, 4.00, 800, 15.00, 26.00 and 60.00 g for rats; Ugo base, Gemonio, Varesee, Italy). Cilia were perpendicular to the paw bottom surface and the 50% paw bottom threshold was determined using the IDixon's ascent and descent method. After 30 min acclimation, the paw withdrawal latency for thermal pain measurements was determined using a Hargreaves bolometer (IITC, Woodland Hills, Calif., USA), i.e., 3-4 measurements per paw on average over a 5min test period. To prevent tissue damage, the beam was automatically switched off at 20 s, and to evaluate its modulatory effect in CCI mice, activin C was dissolved in PBS containing 0.2% bovine serum albumin, and then locally pre-applied to sciatic nerve ligations of CCI mice immediately after sciatic nerve ligation.
9. Electrophysiological testing
The electrophysiological recording was as described previously. After deep anesthesia of young rats (90-110 g) with 3% sodium pentobarbital (80 mg/kg), L4-5 DRG was rapidly excised. DRG was digested with trypsin type I and collagenase type 1A and then mechanically separated with a Pasteur pipette. The isolated cells were placed on a glass slide and patch clamp recordings were performed over 2.0 h to detect neuronal excitability. If an inward current was observed after capsaicin (1.0 μ M) application, it was considered to be a TRPV1 positive neuron. To investigate the effect of activin C on capsaicin-induced TRPV1 channel current, neurons were incubated with activin C (100 ng/ml) or vehicle prior to exposure to capsaicin in the present invention. All data were collected using an EPC-9 type patch clamp amplifier. The action potential parameters were analyzed using the MATLAB program. To assess desensitization of the TRPV1e channel, capsaicin-induced secondary or tertiary currents were normalized to primary capsaicin-induced currents, since different neurons exhibited different magnitudes of TRPV1 current upon exposure to capsaicin.
10. Hematoxylin eosin staining
Deeply anesthetized mice (2% sodium pentobarbital, 100 mg/kg) were perfused with 4% paraformaldehyde and 0.02% picric acid in PBS. The sciatic nerve of the mice was fixed with 4% paraformaldehyde overnight at 4 ℃, dehydrated and embedded in paraffin. Next, the tissue was cut to a thickness of 5- μm, fixed on a glass plate, and dried. Sections were soaked in xylene and graded ethanol and then stained with hematoxylin and eosin according to the instructions (H & E Staining Kit, Beijing Solarbio Science & Technology co., Ltd, Beijing, China). Slides were washed, dehydrated, cleaned and then fixed with resin. Images were acquired using an optical microscope (Olympus DP22; Olympus, Tokyo, Japan).
11. Pain induced by plantar injection of capsaicin
Mice were treated and habituated and vehicle-free rh-activin r-C (20 ng in 20. mu.LPBS) or PBS (20. mu.L) was given intradermally using a 26-G needle as a vehicle control. After 15 min, the same site was intraplanted with capsaicin (1.6. mu.g/paw in 20. mu.L PBS containing 5% ethanol and 5% Tween 80) and immediately returned to videotaping for 5 min. Finally, pain-related behavior observed in the video was blindly quantified by calculating pain response time.
12. Foot swelling measurement
To evaluate the mouse paw edema, 3 mean paw thickness measurements were recorded blindly with a microcard after plantar injection of capsaicin.
13. Hot and cold plate testing
Both tests were performed with hot/cold plates (Ugo Basile, Gemonio, Varese, Italy). Mice were placed on trays individually for at least 20 minutes per day, followed by habituation for 3 days prior to behavioral testing. The cold plate test was then performed at 4 ℃ with a cut-off time of 120 s, the hot plate test at 52 ℃ with a cut-off time of 60 s, and the paw withdrawal latency was calculated as the average of 3 measurements at intervals of at least 5 min.
14. Statistical analysis
These data and statistical analyses are in accordance with the "journal of pharmacological tests" recommendations for the design and analysis of pharmacological experiments.
All data were collected with Microsoft Excel and further analyzed with GraphPad Prism 6.0(La Jolla, CA, URSA). Statistical analysis was performed only for studies with each group size n ≧ 5. The declared animal or sample size is the number of independent values and statistical analysis is performed using these independent values. Rats were used randomly for all axon-cutting experiments; to control the unnecessary changes in pain threshold of behavioral pain tests before and after modeling, to ensure comparability of pain threshold at multiple time points before and after drug treatment, nest-age matched and age matched animals were assigned to experimental groups, bioreplicates were generated based on their baseline and pre-dose values (pain threshold), and behavioral testing and data analysis were performed blindly. All behavioral experiments were performed in a quiet room and evaluated by blind investigators. For all experiments, no outliers were excluded. To control unwanted sources and background variations, the data were normalized and represented as controlled folds. All western blot and immunohistochemical procedures and analyses were in accordance with the BJP society. All data sets were tested for normality and equal variance, followed by Student unpaired t-tests (two panels), one-way repeated measures ANOVA (multiple panels), or two-way repeated measures ANOVA using GraphPad Prism 6.0(La Jolla, CaO, USA), followed by a post-group Bonferroni multifactor comparative test (for time-course comparisons). All data are expressed as mean ± Standard Error of Mean (SEM), P <0.05 is considered statistically significant.
EXAMPLE III Up-regulation of peripheral nerve injury rat lumbar dorsal root ganglion activin C expression
In order to determine the dose of activin C injected intrathecally in adult rats, the present inventors first investigated the expression of this cytokine in the lumbar dorsal root ganglion of chronically neuropathic pain rats.
Rat sciatic nerve amputation is used in this example, and is a simple and feasible peripheral nerve injury model. A custom gene chip was used (Liu et al, 2012).
FIG. 1 is a graph showing the up-regulation of the expression of activin beta C subunit and activin C protein in 4-5 Dorsal Root Ganglion (DRG) of the waist after sciatic nerve of a rat is cut off. Where figure 1a is a custom microarray results plot, N = 6 individual experiments; figure 1b is a graph of real-time RT-PCR analysis with N = 6 individual experiments; FIG. 1C shows a diagram of an immunoblot showing that activin C at 25 kDa consists of β C, β C subunits; FIG. 1d is a diagram of the quantitative analysis of Western blotting experiment for detecting activin C, and the results show that the expression of the axon-cleaved rat L4-5 DRG activin C protein is up-regulated.
In this example, microarray, real-time RT-PCR, Western blotting, and immunohistochemical staining were performed on the prepared peripheral nerve injury model, and the specific method was performed according to the method in example two.
From the custom microarray results, a significant up-regulation of activin β CmRNA expression in rat L4-5 DRG after axonal dissection was observed compared to day 0 control rats L4-5 DRG (FIG. 1 a).
The changes in activin β CmRNA expression were further confirmed by real-time fluorescent quantitative RT-PCR and a trend was observed for a significant increase in activin β CmRNA expression following axonal excision (fig. 1 b).
Then, in this example, Western blotting was used to observe the expression of activin co-protein, and it was found that activin C (a 25 kDa band representing two β C subunits) was increased in expression after injury (fig. 1C, fig. 1d), and the immunoblot in fig. 1C shows that activin C at 25 kDa consisted of β C, β C subunits; FIG. 1d shows upregulation of neurite-cleaved rat L4-5 DRG activin C protein expression. Suggesting that the expression of activin C protein (beta C) and activin beta C subunit gene have similar increasing trend.
Next, this example was subjected to immunofluorescence assay to determine the cellular localization of activin C and observed to be expressed predominantly in small diameter neurons I (fig. 2a), consistent with previous reports. FIG. 2a shows that the coexpression of activin C and calcitonin gene-related peptide (CGRP) in rats L4-5 in the sham-operated control group increases protein-positive small-diameter neurons of dorsal root ganglion activin C in rats L4-5 with axotomous axons, and decreases the coexpression of activin C and CGRP.
Quantitative analysis showed that the percentage of neurite-severed rats L4-5 DRG activin C-positive neurons (3882 out of 8607 neurons in 9 rats) increased significantly-56% (1691 out of 5634 neurons in 9 rats) on day 14 compared to sham controls (1691 out of 5634 neurons in 9 rats), consistent with the custom chip, real-time RT-PCR and Western blot data described above (figure 1); in the peptidergic subgroup, CGRP positive neurons were significantly reduced, as shown in the immunofluorescence quantitative analysis chart of fig. 2b, showing the percentage of activin C-positive neurons and calcitonin gene-related peptide-positive neurons in the dorsal root ganglion at L4-5 days 14 after rat axonal amputation. Fig. 2a, fig. 2b, consistent with a nerve injury condition. Therefore, all axon-severed rats developed different degrees of autodissection within 4 weeks, a pain-related behavior with axon dissection (n = 9 rats); in the sham-operated group (n = 7 rats, fig. 2c), no autodissection was observed, as shown by the 4-week-old excised axons in fig. 2c and the degree of autodissection (self-remaining) in the sham-operated rats.
Immunofluorescence shows that activin C positive neurons and CGRP positive neurons are increased in L4-5 DRG on day 7 of CCI mice, and a double immunofluorescence staining chart of figure 2d shows that activin C positive medium and small diameter neurons of CCI mice dorsal root ganglion 4-5 are increased at the seventh day after chronic compressive injury, and coexpression of activin C and calcitonin gene-related peptide is reduced. Increase of activin C positive neurons and decrease of CGRP positive neurons in the CCI model mice L4-5 DRG. Therefore, the invention considers that the L4-5F dorsal root node activin C is obviously up-regulated after the peripheral nerve injury.
Example four intrathecal or peripheral injection of activin C inhibits chronic neuropathic pain
Activin C was reported to be reduced in the dorsal root ganglia at L4-5 and to be dose-dependent (50 ng, 100 ng and 200 ng) in inhibiting chronic inflammatory pain in rats induced by the complete Floieder addition. Based on upregulation of activin C expression following nerve injury (figure 1).
(1) In the invention, the effect of activin C in chronic neuropathic pain is observed in a rat SNL model by adopting a large dose of activin C for the first time. After a significant decrease in the mechanical pain threshold at day 7 post-SNL (fig. 3a left panel, showing the mechanical hyperalgesia at the 7 th day time point post-SNL), rats were injected intrathecally with 200 ngrh-activin C or vehicle (20 μ L) in a single injection. After 2 hours of treatment with 200 ng rh-activin C alone, the mechanical hyperalgesia in SNL rats was almost reversed to baseline levels (FIG. 3a, right panel, a single injection of recombinant human (rh) -activin C (200 ng in 20. mu.L phosphate buffer) increased the withdrawal threshold in SNL rats).
The present invention then further investigated the dose-dependent analgesic effect of activin C using 200 ng, 100 ng and 50 ng downward doses, and using the same model. As shown in FIG. 3b, in SNL rats, mechanical hyperalgesia appeared at the 7 th day post-SNL (left panel of FIG. 3 b), and a single intrathecal injection of 50 ng, 100 ng or 200 ng of rh-activin dose-dependently increased the hind paw withdrawal threshold, i.e., the mechanical pain threshold (right panel of FIG. 3 b).
The present invention observes that 50 ng, 100 ng or 200 ng of rhactivin C neuropathic pain administered intrathecally and peripherally is a clinically common route of analgesic application.
(2) The present invention employs multiple approaches and three models of neuropathic pain to determine the effect of activin C on neuropathic pain. As shown in fig. 3C and 3d, fig. 3C shows that 250 ng of activin C (200 μ L) was significantly inhibitory to CCI mouse hot pain by local pre-incubation with sciatic nerve ligation; figure 3d shows the long-term anti-pain effect of topical pre-incubation of 250 ng activin C (200 μ L) on mechanical pain. FIG. 3e shows that mechanical hyperalgesia occurred 7 days after CCI (left side of FIG. 3 e), and intrathecal injection of rh-activin C (15 ng in 5. mu.L phosphate buffer) increased the withdrawal threshold of CCI mice, i.e. significantly inhibited neuropathic mechanical pain (right side of FIG. 3 e).
The present invention pre-administers activin C (250 ng/200 μ L) or 200 μ L PBS pre-treated CCI neuropathic pain models and measures IBA-1, GFAP and CGRP for changes in expression at L4-5 levels in DRG using immunofluorescence triple-standard protocol (assay as described in example II) at day 7 time points. FIG. 4a shows an immunofluorescence image. FIGS. 4b-d, immunofluorescence quantitation of IBA-1 positive macrophage numbers (b), GFAP positive satellite cell numbers (c), and percentage of CGRP-positive neurons (d). N = 6 mice; n.s., meaningless; control group, same side; IBA-1, ionized calcium binding linker molecule 1; GFAP, glial fibrillary acidic protein; CGRP, calcitonin gene related peptide. This result indicates that local pre-incubation of activin C in damaged peripheral nerves can significantly reduce the inflammatory response, i.e. infiltration of inflammatory cells, within the dorsal root ganglia that innervate this area.
In the present invention, the changes in expression of IBA1, GFAP and CGRP in dorsal horn of spinal cord were measured by immunofluorescence triple-standard method at day 7 in CCI neuropathic pain model pre-treated with activin C (250 ng/200 μ L) or 200 μ L of PBS. FIG. 5a is an immunofluorescence image. Panels b-d, immunofluorescence quantitation of IBA-1 positive macrophage numbers (b), GFAP positive satellite cell numbers (c), and percentage of CGRP positive neurons (d). N = 6 mice; IBA-1, ionized calcium binding linker molecule 1; GFAP, glial fibrillary acidic protein; CGRP, calcitonin gene related peptide. This result indicates that local pre-incubation of activin C in damaged peripheral nerves can significantly reduce the inflammatory response, i.e. infiltration of inflammatory cells, within the spinal cord that innervates this area; activin C may also be involved in other functions by regulating the expression of CGRP.
The activin C peripheral pretreatment of the present invention does not affect the inflammatory response of the local peripheral ligated nerve. Results are shown in FIG. 6, FIG. 6a, which is an image of ligation of sciatic nerve of CCI mice observed on day 28 after local medicated bath ligation of activin C (250 ng/200 μ L PBS) or vehicle (200 mmol/L PBS). FIG. 6b, which is a measurement of diameter of sciatic nerve in an Image using Image-Pro R Plus6.0 software, and FIG. 6c, which is a quantitative analysis of regional edema of motor-ligated sciatic nerve. SN, sciatic nerve; veh, a vehicle; cont, contralateral; act, activin C; e ipsi. Hematoxylin-eosin staining showed no significant difference in inflammatory cell infiltration of the locally ligated nerves in the activin C medicated bath group and the solvent bath group 7 days after CCI. Data are presented as mean ± Standard Error of Mean (SEM). This result indicates that the function of activin C in the treatment of chronic neuropathic pain after medicated bath is not achieved by reducing local inflammatory responses and edema in peripheral damaged nerves.
(3) The following electrophysiological and behavioral tests were performed using the pain induced by plantar injection of capsaicin in the examples of the invention.
Electrophysiological test method as described in example two, the test results are shown in fig. 7, showing that activin C enhances capsaicin-induced transient receptor potential cation channel V1(TRPV1) channel currents in rat DRG neurons. Figure 7a is a whole cell record showing desensitization to TRPV1 current by continuous 3 incubations of capsaicin (1.0 μ M). FIG. 7b is a whole cell record showing that continuous incubation of activin C (100 ng/ml) enhances the capsaicin-induced TRPV1 current. FIG. 7c is a quantitative analysis of FIGS. 7a and 7 b. N = 6-7 rats; cap, capsaicin; ECS, extracellular fluid; one-way variance was measured repeatedly, followed by Bonferroni multiple comparison test, with P < 0.05. Data are presented as mean ± Standard Error of Mean (SEM).
The present invention further demonstrates the modulation of TRPV1 function by activin C using behavioral testing of mice. Adopting a foot swelling measuring method, and carrying out hot plate and cold plate tests; the results are shown below, as shown in FIG. 8a, that pre-plantar injection of activin C (20 ng/20 μ L) reduced acute nociceptive behavior caused by plantar injection of capsaicin. As shown in FIG. 8b, edema of the rat foot caused by capsaicin injection was revealed. As shown in FIGS. 8C and 8d, the prolonged insensitivity to high temperature (52 ℃) and low temperature (4 ℃) caused by capsaicin injection was normalized by administering activin C (20 ng/20 μ L, by plantar injection) in advance for 7 days and 3 days, respectively. The result shows that the activin C not only can relieve acute pain induced by a capsaicin model, but also can control the insensitivity to cold and hot temperatures caused by capsaicin injection to be normal. FIG. 8e shows that in TRPV1 knockout mice, the analgesic effect of activin C (250 ng/200 μ L PBS, preincubation) on neuropathic pain resulting from chronic constrictive injury was abolished. (a) Adopting unpaired two-tailed t test, (c, d) adopting one-way repeated measurement anova, (e) adopting two-way repeated measurement anova, and P is less than 0.05 through two-way repeated measurement anova. Data are presented as mean ± Standard Error of Mean (SEM).
The present study supports a relationship between activin C-mediated antinociception and modulation of TRPV1 activity, suggesting that activin C may be a potential candidate for the treatment of neuropathic pain. However, the present invention concluded that these findings may not be applicable to women, based on animal experiments performed only on men.
Since neuropathic pain has a variety of causes, the present invention uses the axotomy, SNL and CCI models to assess the effect of activin C on neuropathic pain. The nerve cutting model is reliable and has good repeatability, and can be used for clinically simulating phantom limb pain. In addition, more DRG can be obtained for gene screening. The SNL model is useful for studying damaged and undamaged nerve fibers in the sciatic nerve, mimicking the symptoms of burning pain that occurs after damage to the peripheral nerve and sympathetically mediated pain in humans. The CCI model includes neuropathy and inflammatory components and mimics the patient's burning pain, post-traumatic peripheral painful neuropathy, entrapment neuropathy, and complex regional pain syndrome. In addition, following peripheral nerve injury, immune cells of DRG and SCs release some cytokines, sensitizing/desensitizing the nociceptive neurons, and further modulating pain management. Importantly, some primary sensory neurons were also found to secrete cytokines. Furthermore, there is strong evidence that signal transduction directed to the transforming growth factor- β superfamily has a beneficial effect in chronic neuropathic pain; thus, the present invention hypothesizes that more members of the transforming growth factor- β superfamily may be involved in pain management and serve as potential targets for the treatment of neuropathic pain. In this study, the present invention used a custom microarray to screen for significant changes in the expression levels of transforming growth factor- β superfamily member genes in axon-severed rats L4-5 DRG compared to the control group alone. The present inventors have found that the activin β C gene is up-regulated in expression in the residue of axonal ablation. Next, the present invention further confirmed the increased transcription of activin β C subunit and the increased expression of activin C protein by Western blotting and immunofluorescence. In addition, increased expression of activin C WVAS was demonstrated in a CCI mouse model. These results indicate that, following peripheral nerve injury, lumbar dorsal root ganglion activin C is significantly upregulated in mRNA and w-protein levels. Consistently, upregulation of activin C was consistent with the extent of autotomy (a pain-associated self-disabling behavior) and mechanical and thermal hyperalgesia in CCI mice, strongly suggesting a role for activin C in neuropathic pain.
To further elucidate the central and peripheral role of activin C in neuropathic pain, the present invention employed two models, rat SNL and mouse CCI. Based on the up-regulation of activin C after nerve injury, the invention firstly selects a large dose of activin C (200 Ng) to observe the influence of activin C on neuropathic pain on a rat SNL model, and discovers that 200 Ng of activin C injected in a sheath cavity almost reverses mechanical hyperalgesia and approaches to a baseline level. Then, the present invention further confirmed the analgesic effect of activin C using a hypotensive dose of 200 ng, 100 ng or 50 ng, and it was observed that intrathecal injection of 50, 100 or 200 ng of rh-activin C inhibited neuropathic pain in SNL rats dose-dependently. Given that the tissue surrounding the damaged nerve absorbs a significant amount of activin C in the topical bath, the present invention selects a large dose (250 ng) to ensure that the damaged nerve can absorb a sufficient dose. The antinociceptive effect of activin C on both mechanical and heat stimulated CCI mice was observed by local pre-administration of activin C through the ligature of the sciatic nerve. In addition, a single intrathecal injection of activin C inhibited established mechanical pain in CCI mice. These data indicate that central or peripheral administration of activin C can relieve chronic neuropathic pain in SNL rats and CCI mice, similar to other TGF- β superfamily members.
Neuropathic pain occurs in association with inflammatory cell infiltration of the dorsal root ganglion and activation of glial cells in the SCs. In line with this, topical pre-bath of activin C inhibits macrophage infiltration into the lower back root ganglion and proliferation of activated microglia in SCs, whereas topical treatment with activin C does not affect satellite cells or astrocytes in the early stages of CCI. In nerves with local ligation of CCI strips, local predosing of activin C did not affect the neuroedema or inflammatory response. These results indicate that peripheral preconditioning activin C blocks CCI-induced inflammatory responses in the peripheral and central nervous systems, but not local neurogenic responses. Thus, the present invention speculates that pre-bathing with exogenous activin C may inhibit nociceptive signaling of peripheral nerves to innervating DRG and SCs, and further reduce the inflammatory response of DRG and SCs by CCI, but not a direct anti-inflammatory mechanism. Although activin a plays a role in the early stages of the inflammatory process and is a key component of the inflammatory response, the data of the present invention do not support the idea that activin C can antagonize activin a-induced hyperalgesia. One possibility is that activin C, like tgf- β 1, acts as an analgesic by suppressing neuroimmune responses in neurons and glial cells, reducing the activity of spinal excitatory neurons, and inhibiting some pain signaling pathways. However, local pretreatment of activin C had no significant effect on astrocyte activation or proliferation, and the present inventors speculated that the effect of activin C on astrocyte activation in the late stage of CCI remains to be studied further. Unexpectedly, topical predosing of activin C restored normal CGRP levels in ipsilateral DRG and SCs as a result of CCI, suggesting a multifaceted role for cytokine activin C in the nervous system, potentially modulating the release of various pain-associated neurotransmitters, including CGRP, following peripheral nerve injury. Similarly, in vitro studies found that activin a and nerve growth factor synergistically regulate the expression of CGRP, but activin C and activin a exhibit completely different roles in pain management. Notably, all a β fibers and most a δ fibers were severed, while in the CCI model, a large number of C fibers remained intact (all fibers were severed in the axon-severed model, and some a β -, a δ -, and C fibers were severed in the SNL model), and therefore, CCI-induced pain may be mediated by C and/or a- δ fibers, including most CGRP + fibers. With respect to the unexpected function of CGRP, the crosstalk between activin C and CGRP deserves further investigation.
It is considered that TRVP 1 is a non-selective Ca expressed mainly in sensory neuron cells2+The channel, which transmits pain signals in a sensitized state, the present invention speculates that activin C may act directly or indirectly with the TRPV1 channel in DRG neuron to modulate neuropathic pain. Electrophysiological analysis of the present invention showed that while activin C alone had no effect on TRPV1 current in individual isolated DRG neurons, pre-incubation with activin C significantly increased capsaicin-induced TRPV1 current. Considering that capsaicin is a specific TRPV1 channel agonist that rapidly opens the TRPV1 channel to induce pain in a short period of time and that a long time is required for pre-bathing with activin C to enhance capsaicin-induced TRPV1 current, the present invention assumes that activin C enhances the activity of the TRPV1 channel as an indirect pathway, unlike activin a-sensitive capsaicin-induced current, however, continued capsaicin stimulation causes desensitization of TRPV1, rendering the channel non-reopening for a long period of time, thereby interfering with pain signaling from DRG neurons and their afferent neurons. The mechanism by which activin C modulates TRPV1 was further demonstrated by the nociceptive behaviour of mice; activin C attenuates capsaicin-induced acute nociceptive responses. This is consistent with the clinical practice of using capsaicin as the major F component of analgesics. However, activin C pretreatment did not reduce capsaicin-induced foot swelling in mice, suggesting that the anti-inflammatory effects of activin C were not achieved by reducing local edema or inflammation. Interestingly, activin C pretreatment resulted in thermal and cold stimulationThe mice induced by the later capsaicin continued to return to normal at low temperature. By comprehensively considering normalized CGRP data, the invention conjectures that activin C has complex functions in the nervous system and is worthy of further evaluation. As expected, the analgesic effect of TRPV1 KO mice was abolished after CCI. These data provide conclusive evidence that activin C relieves chronic neuropathic pain by modulating TRPV1 function, and help the present invention understand that exogenous C promotes the release of CGRP, as TRPV1 mediates the secretion of CGRP and substance P in DRG neurons. Although TRPV1 KO mice exhibited complete mechanical hyperalgesia in this CCI model, a recent study showed that TRPV1 gene in adult rodents was siRNA knock-out, thereby reducing CCI-induced behavioral hyperalgesia. This difference suggests that gene complementation should be common among TRPs families. However, several unknown functions of activin C, including its specific receptor, require further investigation.
In summary, following peripheral nerve injury, endogenous activin C is significantly expressed and significantly upregulated in small DRG neurons, and both peripheral and intrathecal applications of activin C inhibit chronic neuropathic pain by modulating the TRPV1 channel, suggesting that activin C may have potential for the treatment of chronic neuropathic pain.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. Use of cytokine activin C for inhibiting neuropathic pain.
2. Activin C for use according to claim 1 in the treatment of neuropathic pain, characterised in that it comprises the following processes:
s1, establishing a sciatic nerve cutting model; verifying the up-regulation of dorsal root ganglion activin C of 4-5 (L4-5) waists after peripheral nerve injury;
s2, administering the activin C intrathecally or locally in advance, preparing a chronic neuropathic pain rat and mouse model, evaluating nociceptive behaviors of L4-5 dorsal root nodes and spinal cords and pain related markers, and determining the regulation effect of the activin C on TRPV 1.
3. Application of activin C in preparation of medicines for treating neuropathic pain is provided.
4. The use of activin C according to claim 3 for the preparation of a medicament for the treatment of neuropathic pain, wherein activin C acts as an inhibitory modulator of neuropathic pain by modulating the TRPV1 channel.
5. A medicament for the treatment of neuropathic pain, said medicament comprising at least activin C.
6. The agent for the treatment of neuropathic pain according to claim 5, further comprising a plurality of pharmaceutically acceptable carriers.
7. The medicament for treating diseases related to angiogenesis excessive according to claim 6, wherein the carrier comprises pharmaceutically acceptable diluents, excipients, fillers, binders, absorption promoters, surfactants and synergists.
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