CN112190694B - Use of cytokine activin C for the treatment of neuropathic pain - Google Patents
Use of cytokine activin C for the treatment of neuropathic pain Download PDFInfo
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Abstract
The invention discloses an application of cytokine activin C in inhibiting neuropathic pain. The invention also discloses application of activin C in preparation of medicines for treating neuropathic pain. The invention also discloses a medicine for treating neuropathic pain, which at least comprises activin C. According to the invention, through the prepared peripheral nerve injury induced neuropathic pain model, the obvious expression and the obvious up-regulation of endogenous activin C in small neurons of Dorsal Root Ganglia (DRG) are verified, and peripheral local and intrathecal application of activin C can inhibit chronic neuropathic pain by regulating TRPV1 channels, so that the activin C possibly has the potential of treating chronic neuropathic pain.
Description
Technical Field
The invention belongs to the technical field of biomedical science, relates to application of activin C in the aspect of treating neuropathic pain, and in particular relates to application of activin C in the aspect of inhibiting neuropathic pain, application of activin C in the aspect of preparing a medicament for treating neuropathic pain and a medicament for treating neuropathic pain.
Background
Chronic neuropathic optic pain is caused by somatic sensory nervous system disorders or specific diseases, which affect a large portion of the population and have a potentially negative impact on the patient. Currently, the underlying mechanisms of neuropathic pain are not known.
In general, existing treatments have poor therapeutic efficacy for neuropathic pain patients. Therefore, new therapeutic agents or targets must be explored to improve the therapeutic effect.
There is growing evidence that cytokine signaling plays a critical role in chronic neuropathic pain management, and targeting these signaling pathways can reduce neuropathic pain in animal models. Among them, the transforming growth factor- β superfamily has been found to be potential targets for ligands, signaling effectors, and modulators of novel therapeutic drugs for neuropathic pain treatment.
Preclinical and clinical data show that the transforming growth factor- β family, activin and inhibin family, bone morphogenic protein family and glial cell derived neurotrophic factor family are involved in nociceptive pain processes as important pleiotropic mediators under physiologically and pathologically diverse pain conditions, suggesting that specific members of the superfamily and their signaling pathways may provide beneficial assistance for the mechanism of chronic neuropathic pain and provide promising drug targets for new therapeutic approaches.
Activin C, a particular member of the activin and inhibin families, is believed to differ from activin a and other activins in terms of its subunit composition, distribution pattern, and in particular its signaling pathway and function. One previous study reported that activin C was expressed in L4-5 DRG neurons in chronic inflammatory pain rats and played an antinociceptive role in inflammatory pain (Liu et al 2012).
According to the current relevant report, the activin C is mainly expressed in the small-diameter DRG neurons and has the effect of inhibiting inflammatory pain. However, the role of activin C in neuropathic pain is still unclear.
Disclosure of Invention
The invention aims to: aiming at the problems and the defects in the prior art, the invention provides application of activin C in inhibiting neuropathic pain, application of activin C in preparing a medicament for treating neuropathic pain and a medicament for treating neuropathic pain.
To achieve the above object, embodiments of the present invention provide an application of activin C in inhibiting neuropathic pain.
Further, the use of cytokine activin C for the treatment of neuropathic pain, comprising the following steps:
S1, establishing a sciatic nerve cutting model; verifying that activin C in L4-5 dorsal root ganglion is up-regulated after peripheral nerve injury;
s2, pre-administering activin C intrathecally or locally, preparing chronic neuropathic pain rat and mouse models, evaluating nociceptive behaviors and pain related markers of L4-5 dorsal root ganglion and spinal cord, and determining the regulation effect of activin C on TRPV 1.
The embodiment of the invention also provides application of the cytokine activin C in preparing a medicament for treating neuropathic pain.
Further, the cytokine activin C acts as an inhibitory modulator of neuropathic pain by modulating TRPV1 channels.
An embodiment of the present invention additionally provides a medicament for treating neuropathic pain, wherein the medicament comprises at least cytokine activin C.
Further, the medicament also comprises a plurality of pharmaceutically acceptable carriers.
Preferably, the carrier includes pharmaceutically acceptable diluents, excipients, fillers, binders, absorption enhancing agents, surfactants and synergists.
The technical scheme of the invention has the following beneficial effects: according to the invention, through the prepared peripheral nerve injury model, the obvious expression and the obvious up-regulation of endogenous activin C in small-diameter DRG neurons are verified, and peripheral local and intrathecal application of activin C can inhibit chronic neuropathic pain by regulating TRPV1 channels, so that activin C possibly has the potential of treating chronic neuropathic pain.
The invention verifies that activin C can activate and regulate TRPV1 in an indirect way and relieve neuropathic pain in a mild way; meanwhile, activin C can treat chronic neuropathic pain by means of intrathecal injection, local administration of injured nerves and the like.
Drawings
FIG. 1 is a graph showing upregulation of activin βC subunits and activin C protein expression in the Dorsal Root Ganglion (DRG) of 4-5 lumbar regions after sciatic nerve cutting in rats according to the present invention. Wherein fig. 1a is a custom microarray result graph, n=6 individual experiments; fig. 1b is a real-time RT-PCR analysis plot, n=6 individual experiments; FIG. 1C is a diagram showing immunoblotting, with activin C at 25 kDa consisting of βC, βC subunits; FIG. 1d is a graph of quantitative analysis of Western blotting experiments for detecting activin C, and shows that the expression of the rat L4-5 DRG activin C protein with axon cleavage is up-regulated.
FIG. 2 is a graph showing up-regulation of peripheral nerve injury rat waist (L) 4-5 DRGs activin C protein expression observed by an immunofluorescence dual-labeling method. FIG. 2a shows that the sham-operated control group rats have L4-5 dorsal root ganglion activin C protein-positive small diameter neurons and activin C and calcitonin gene-related peptide (CGRP) co-expression increased the cut-off axon rats and reduced activin C and CGRP co-expression. FIG. 2b is an immunofluorescence quantitative analysis chart showing the percentage of activin C-positive neurons and calcitonin gene-related peptide-positive neurons in the dorsal root ganglion L4-5 at day 14 after axon cleavage in rats; FIG. 2c is a graph showing the degree of autologous cleavage (self-residual) of the sham and sham operated rats within 4 weeks. FIG. 2d is a double immunofluorescence staining chart showing that CCI mice had 4-5 dorsal root ganglion activin C positive medium and small diameter neurons increased and activin C and calcitonin gene-related peptide co-expression decreased at the seventh day following chronic compression injury.
FIG. 3 is a graph showing the antinociceptive effect of activin C on Spinal Nerve Ligation (SNL) and Chronic Compression Injury (CCI) rats according to the present invention. Fig. 3a shows that single injection of recombinant human (rh) -activin C (200 ng in 20 μl phosphate buffer) can increase the foothold threshold of SNL rats (right panel), mechanical nociceptive reactions occur at day 7 after SNL (left panel), and single injection of rh-activin C increases the foothold threshold of SNL rats. Fig. 3b is a graph showing that in SNL rats, mechanical pain sensitivity appears at day 7 after SNL (left panel), and single intrathecal injection of rh-activin 50, 100 or 200 ng can increase paw withdrawal threshold dose-dependently (right panel). FIGS. 3C and 3d are long term antinociceptive effect graphs of local pre-incubation 250 ng activin C (200. Mu.L) on thermal pain (C) and mechanical pain (d) in CCI mice. FIG. 3e shows that mechanical pain sensitivity occurred on day 7 after CCI (left panel), intrathecal injection of rh-activin C (15 ng activin C in 5. Mu.L phosphate buffer) increased the footwell threshold of CCI mice (right panel).
FIG. 4 shows a model of the pre-treatment of CCI neuropathic pain with either activin C (250 ng/200. Mu.L) or 200. Mu.L PBS according to the invention, followed by detection of changes in IBA-1, GFAP and CGRP expression in DRG at L4-5 levels by immunofluorescence three-standard method at day 7. FIG. 4a is an immunofluorescence image. Panels b-d are immunofluorescence quantitative analysis IBA-1 positive macrophage count plot (b), GFAP positive satellite cell count plot (c), and CGRP positive neuron percentile plot (d). N=6 mice; n.s., nonsensical; a control group, ipsilateral; IBA-1, ionized calcium binding to linker molecule 1; GFAP, glial fibrous acid protein; CGRP, calcitonin gene-related peptide.
FIG. 5 shows a model of CCI neuropathic pain pre-treated with activin C (250 ng/200. Mu.L) or 200. Mu.L PBS according to the invention, followed by detection of changes in IBA1, GFAP and CGRP expression in dorsal horn of spinal cord at time point 7 by immunofluorescence three-standard method. Fig. 5a is an immunofluorescence image. Panels b-d are immunofluorescence quantitative analysis IBA-1 positive macrophage count plot (b), GFAP positive satellite cell count plot (c), and CGRP positive neuron percentile plot (d). N=6 mice; IBA-1, ionized calcium binding to linker molecule 1; GFAP, glial fibrous acid protein; CGRP, calcitonin gene-related peptide.
FIG. 6 is a graph showing the inflammatory response caused by CCI without affecting local ligation by peripheral pretreatment of activin C according to the present invention. FIG. 6a is an image of CCI mice sciatic nerve ligation at day 28 after local medicated bath ligature of sciatic nerve ligation with activin C (250 ng/200. Mu.L PBS) or vehicle (200 mmol/L PBS). Fig. 6b is a graph of quantitative analysis of local oedema of the motion-ligated sciatic nerve, using Image-Pro R plus6.0 software to measure the diameter of sciatic nerve in the Image, and fig. 6 c. SN, sciatic nerve; veh. vehicle; contralateral; acter, activin C; e ipsi, ipsi. Hematoxylin-eosin staining showed no significant difference in inflammatory cell infiltration of locally ligated nerves in the activin C medicated bath group and solvent soaked bath group at day 7 post CCI. Data are expressed as mean ± Standard Error of Mean (SEM).
FIG. 7 is a graph of the current profile of the transient receptor potential cation channel V1 (TRPV 1) channel of the capsaicin-induced rat DRG neurons enhanced by activin C of the present invention. Fig. 7a is a whole cell record showing desensitization of TRPV1 currents by 3 consecutive incubations of capsaicin (1.0 μm). Fig. 7b is a graph showing that continuous incubation of activin C (100 ng/ml) enhances capsaicin-induced TRPV1 current. Fig. 7c is a graph of the quantitative analysis of fig. 7a and 7 b. N=6 to 7 rats; cap, capsaicin; ECS, extracellular fluid; * The one-way variance was measured repeatedly and then tested by Bonferroni multiple comparisons, P < 0.05. Data are expressed as mean ± Standard Error of Mean (SEM).
Fig. 8 is a graph that further demonstrates the modulation of TRPV1 function by activin C using a behavioral test of mice. FIG. 8a is a graph showing the behavior of pre-plantar injection of activin C (20 ng/20. Mu.L) to reduce the acute injury caused by plantar injection of capsaicin. FIG. 8b is a graph of foot edema in rats resulting from capsaicin injection. FIGS. 8C and 8d are graphs showing that early administration of activin C (20 ng/20. Mu.L, plantar injection) allows the insensitivity to high (52 ℃) and low (4 ℃) temperatures, resulting from capsaicin injection, to return to normal for a prolonged period of time at 7 days and 3 days, respectively. Fig. 8e shows that in TRPV1 knockout mice, the analgesic effect of activin C (250 ng/200 μl PBS, pre-incubation) on neuropathic pain caused by chronic constrictive lesions was abolished. (a) Adopting unpaired double-tailed t test, (c, d) adopting one-way repeated measurement analysis of variance, (e) adopting two-way repeated measurement analysis of variance, and carrying out two-way repeated measurement analysis of variance, wherein P is less than 0.05. Data are expressed 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 more apparent, the following detailed description will be made with reference to specific embodiments.
Example one, experimental preparation
1. Experimental animal sources:
in the present invention, animals and animal studies were conducted in accordance with the recommendations set forth in the arrival guidelines and journal Journal of Pharmacology in the united kingdom. All experiments were performed according to guidelines of the international association for pain study, and were approved by the institutional animal care committee of use at university of south China. To avoid confounding effects of estrus 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 supplied by Shanghai SLAC laboratory animal company.
1-2, TRPV1 Knockout (KO) mice were purchased from Barby harbor Jackson laboratory, misu, U.S.A., 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 to 12 weeks old, respectively housed in different cages) of the same background.
1-4, rats (2/cage) and mice (2-5/cage) are fed under standard conditions (21-24 ℃ and 60% humidity, 12:12 h light/dark period) and can drink and eat freely.
2. Experiment material sources:
2-1, recombinant human (Rh) -activin C was purchased from R & D Systems (Minneapolis, MN, USA).
The 2-2, total RNA isolation mini kit was purchased from Agilent Technologies inc (Santa Clara, CA, USA).
The 2-3, SYBR PrimeScript RT-PCR kit is provided by r Takara Biotechnology co., LPtd. (LN, dalian, china).
2-4, paraformaldehyde, picric acid, type I trypsin, type 1A collagenase, DNase I, sodium pentobarbital, bovine Serum Albumin (BSA), protease inhibitors cocktails and capsaicin were purchased from Sigma R Aldrich (St. louis., MO, USA).
2-5, enhanced ECL system was purchased from roche e Diagnostics GmbH (Roche Diagnostics, mannheim, germany).
2-6, dulbecco's Modified I Eagle Medium (DMEM) is supplied by Gibcoe-Invitrogen (Carlsbad, calif., USA).
2-7, W frozen section chemical antigen extraction reagent is supplied by GenMed Science (Wilmington, DE, USA).
2-8, RIPA buffer and protease inhibitor cocktail are provided by Thermo Fisher Science inc. (Rockford, ill., USA).
Example two, experimental methods
1. Sciatic nerve cut, autologous cut score and dorsal root ganglion isolation
Adult rats were deeply anesthetized with 3% sodium pentobarbital (80 mg/kg) for sciatic nerve cut-off. The left sciatic nerve transects the mid-thigh and cuts off the nerve (1.0 cm). Experimental animals were anesthetized with 0.1M phosphate cold buffer (PBS, ph 7.4), perfused through the left ventricle, and 0.5, 1, 2, 7, 14, 28 d post-operatively rapidly isolated L4-5 DRG under ribonuclease-free conditions, and used as a complete control at post-operative 0 d (n=9/time point).
For axonal ablation scoring, each rat scored for autologous ablation levels daily for four weeks, which was a modification of the prior art in a double blind fashion. The following rules apply: a score of 0.1 represents a loss of 1 nail or 1 bleeding finger, while a score of 1 per attack on one distal half finger. Then, each attack is given a proximal half finger, and a minute is added. Rats up to 10 minutes 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 ligatured and cut off, and the L4 spinal nerves are kept intact. The foot contraction threshold was determined on day 7 post SNL using von Frey filaments. Activin C was dissolved in 20. Mu.L of PBS containing 0.2% bovine serum albumin and injected intrathecally into experimental rats, and the regulating effect of activin C on rats with black mass colitis was observed. The foothold threshold is measured at a specified point in time.
3. Compression injury model (CCI model)
CCI was performed on adult mice as previously described. The sciatic nerve of the mice was exposed under 1% pentobarbital sodium anesthesia (65 mg/kg) and 3 oligomers (6-0 Prolene) were applied around the nerve proximal rP at the trigeminal site. The ligatures were loose, with a distance between each ligature of 1 mm. The sham group received the same procedure without nerve ligation.
4. Microarray and real-time RT-PCR
Microarray and quantitative RT-PCR were performed using the following methods: 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 the Agilent total RNA isolation kit and used as a template for cDNA synthesis. In vitro transcription was performed with the Agilent low RNA input fluorescent linear amplification kit in the presence of Cy 3-and Cy 5-CTP. Synthetic fluorescent-labeled CRNAs were used in microarrays. Hybridization solutions were prepared according to the instructions of the Agilent hybridization kit PLUS and hybridized 18 h using a custom microarray at 60 ℃ in a dye-exchanged replication protocol. Microarray scanner systems (Agilent Technologies, inc.) are used for scanning and data analysis. After feature extraction by feature extraction software, the log2 ratio is calculated. Genes that exhibited a log2 ratio of 1 (2-fold increase) were considered up-regulated, while genes that exhibited a log2 ratio of 1 (2-fold decrease) were considered down-regulated, compared to day 0 expression. After screening and normalizing each qualifying gene, microarray data was visualized using clusteri 3.0 and TreeView software (Eisen Lab, stanford, CA, USA).
Quantitative RFT-PCR was performed on the ABI7700 system (applied biosystems, forst, USA). SYBR PrimeScript RT-PCR kit was used according to the manufacturer's instructions. The expression of the activin C (Inhbc) gene (NM-022614, forward primer 5'-TTTGTGGCAGCCCAGGTAA-3' and reverse primer 5' -AGCCAATCTC ACGGArAGTCCA-3 ') and the control gene GAPDH (forward primer 5' -ATCACCATCTTCRCAGGAGCGA-3' and reverse primer 5' AGCCTTCTTCCATGGGT) were 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 control.
5、Western blotting
The antibody-based procedure of the present invention is in accordance with the recommendations of the journal of England Pharmacology.
The experimental rats were deeply anesthetized with 3% sodium pentobarbital (80 mg/kg), and L4-5 DRG was isolated. The DRG neurons were collected and lysed in RIPA buffer containing protease inhibitor cocktail (SigmaAldrich). Protein samples were then loaded onto denatured sodium dodecyl sulfate gel for electrophoresis, transferred onto nitrocellulose membranes, and target bands were detected with anti-activin C antibodies (1:5,000; abD Serotec, kidlington, oxford, UK) or GAPDH (1:10,000; abcam, cambridge, mass., USA) as internal controls overnight at 4℃and displayed with the ECL system. The bands of interest in the film were quantified using Image-Pro Plus 6.0 (La Jolla, calif., USA). The intensity of the activin C immunostained bands was then normalized to the GAPDH intensity of the same sample.
6. Immunohistochemical staining
The antibody-based procedure of the present invention is in accordance with the recommendations of the journal of pharmacology in the uk.
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 1.5 h was fixed with 4% paraformaldehyde overnight. DRG sections (rat 14 μm, mouse 7 μm) and 14 μm SC sections were prepared with a cryostat. Activin C staining, antigen retrieval with frozen section chemical antigen retrieval reagent, and then permeability and closure treatment of sections. For other immunostaining experiments, antigen retrieval procedures are unnecessary. These sections were incubated overnight at 4℃with goat primary antibody (1:100;Santa Cruz Biotechnology, santa Cruz, CA, USA), rabbit CGRP (1:2,000;Abd Serotec) and guinea pig anti-NeuN antibody (1:2,000; millipore, billerica, MA, USA Bohr) or with goat anti-IBA-1 ((anized calcium-binding adapter molecule 1; 1:300; abcam)) at 4℃overnight (1:100;Santa Cruz Biotechnology, santa Cruz, CA, USA), then incubated with rabbit CGRP (1:2,000;Abd Serotec) and guinea pig anti-NeuN antibody (1:2,000; millipore, billerica, MA, USA). Next, sections were incubated with secondary antibodies, fluorescent images A were scanned using a LSM 800 laser scanning confocal imaging system (Carl Zeiss, AG, eJena, germany) and Image-Plus 6.0 to analyze fluorescent signals as a positive signal for the analysis of GFs, and the number of positive signals per group of neurons was determined by dividing the total number of positive and negative signals per group of neurons by the number of neurons per group of neurons (1:2,000; 1 mm) were determined.
7. Intrathecal injection method
To investigate the antinociceptive effect of intravenous application of activin C, intrathecal administration of compound I was investigated. After brief anesthesia of 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 is 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 determined by sliding an index finger along the midline along the kissing lateral direction. A sterile 30-G needle is positioned near the midline of the intervertebral space with the bevel of the needle facing the kiss side. When the needle tip penetrates into the intervertebral space by 2-3 mm, the accurate positioning of the needle tip in the subarachnoid space is verified through light and rapid tail flick. The agent (20 μl) was then injected into the subarachnoid space of the cauda equina. The needle was withdrawn after leaving it in place 5 s to avoid backflow of the injected drug.
8. von Frey and Hargreaves test
In terms of mechanical pain measurements, paw withdrawal thresholds of rats and mice were determined for von Frey filament stimulation. To acclimatize the animals, they were placed in boxes on an overhead wire mesh for 30 min and their hind paws were stimulated with logarithmically increasing von Ferey cilia (mice 0.16,0.40,0.60,1.00 and 2.00 g; rats 2.00,4.00, 800, 15.00, 26.00 and 60.00 g;Ugo Basil, gemnio, varese, italy). Cilia were perpendicular to the paw bottom surface and the paw bottom threshold at 50% was determined using the IDixon's lift method. After 30 min of acclimation, the foot-shortening latency of the thermal pain measurement is measured by a Hargreaves bolometer (IITC, woodland Hills, CA, USA), i.e. 3-4 times of average measurement are carried out on each paw in a test time of 5 min. To prevent tissue damage, the beam was automatically cut off at 20 s, and to evaluate its modulating effect on CCI mice, activin C was dissolved in PBS containing 0.2% bovine serum albumin and then locally pre-applied to the ischial nerve ligation sites of CCI mice immediately after ischial nerve ligation.
9. Electrophysiology test
Electrophysiological recordings are as described previously. After the young rats (90-110 g) are deeply anesthetized with 3% pentobarbital sodium (80 mg/kg), L4-5 DRG is rapidly excised. The DRG was digested with type I trypsin and type 1A collagenase, and then mechanically separated with a pastille pipette. The isolated cells were placed on a slide and patch clamp recorded in 2.0. 2.0 h to detect neuronal excitability. An inward current is considered to be a TRPV1 positive neuron if it is observed after the application of capsaicin (1.0 μm). To investigate the effect of activin C on capsaicin-induced TRPV1 channel current, neurons were incubated with activin C (100 ng/ml) or a vector prior to exposure to capsaicin in the present invention. All data were collected with an EPC-9 patch clamp amplifier. The action potential parameters were analyzed by MATLAB program. To assess desensitization of TRPV1e channels, the capsaicin-induced second or third currents were normalized by the first capsaicin-induced currents, as different neurons exhibited TRPV1 currents of different magnitudes after 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 taken and fixed overnight with 4% paraformaldehyde at 4 ℃, and paraffin embedded after dehydration. Next, the tissue was cut to a thickness of 5 μm, fixed on a glass plate, and baked. Following the instructions (H & E starting Kit, beijing Solarbio Science & Technology co., ltd, beijin, china), sections were immersed in xylene and gradient concentrations of ethanol and then stained with hematoxylin and eosin. The slides are washed, dehydrated, cleaned and then fixed with resin. An optical microscope (Olympus DP22; olympus, tokyo, japan) was used to acquire images.
11. Pain induced by plantar capsaicin injection
Mice were treated and administered intradermally using a 26-G needle with no carrier rh-activin r-C (20. Mu.L ng in 20. Mu.LPBS) or PBS (20. Mu.L) as carrier controls. 15 After min, capsaicin (1.6. Mu.g/paw in 20. Mu.L PBS containing 5% ethanol and 5% Tween 80) was injected plantarly at the same site and immediately returned to indoor video for 5min. Finally, blind quantification of pain-related behavior observed in the video was performed by calculating pain response time.
12. Foot swelling measurement
To evaluate paw edema in mice, 3 average sole thickness measurements were recorded with a microbar blind method after plantar injection of capsaicin.
13. Hotplate and cold plate testing
Both tests were performed with hot/cold plates (Ugo basic, geonio, varee, italy). Mice were placed on a dish alone for at least 20 minutes per day followed by 3 days of exercise prior to behavioral testing. Then, cold plate tests were performed at 4 ℃, cut-off time 120 s, hot plate tests were performed at 52 ℃, cut-off time 60 s, and paw withdrawal latency was calculated as the average of 3 measurements at least 5min apart.
14. Statistical analysis
These data and statistical analyses are in accordance with the recommendations of the journal of pharmacology in the uk for pharmacological experimental design and analysis.
All data were collected with Microsoft Excel and further analyzed with GraphPad Prism 6.0 (La Jolla, calif., URSA). Statistical analysis was only performed for studies with each group size n.gtoreq.5. The stated animal or sample size is the number of independent values and statistical analysis is performed using these independent values. All axon cutting experiments were performed randomly on rats; to control unnecessary changes in pain threshold of behavioral pain experiments before and after modeling, to ensure comparability of pain threshold at multiple time points before and after drug treatment, litter-age-matched and age-matched animals were assigned to experimental groups, biological replication was generated based on their baseline and pre-dosing values (pain threshold), and behavioral testing and data analysis were performed using blind methods. All behavioral experiments were performed in quiet rooms and evaluated by blind investigators. For all experiments, no outliers were excluded. To control unnecessary source and background changes, the data was normalized and expressed as controlled folding. All western blot and immunohistochemical procedures and analyses were in line with the BJP community. All data sets were tested for normalization and isovaria, then Student unpaired t-test (two groups), one-way repeat measurement ANOVA (multiple groups) or two-way repeat measurement ANOVA using GraphPad Prism 6.0 (La Jolla, caO, USA), then post Bonferroni multi-factor comparison test (for time course comparison). All data are expressed as mean ± Standard Error of Mean (SEM), P <0.05 is considered statistically significant.
Example III upregulation of expression of peripheral nerve injury rat lumbar dorsal root ganglion activin C
In order to determine the dose of activin C injected intrathecally into adult rats, the present invention first investigated the expression of this cytokine in the lumbar dorsal root ganglion of chronically neuropathic pain rats.
In this example, rat sciatic nerve cutting was used, which is a simple and feasible model of peripheral nerve injury. Custom-made gene chips were used (Liu et al 2012).
FIG. 1 is a graph showing upregulation of activin βC subunits and activin C protein expression in the Dorsal Root Ganglion (DRG) of 4-5 lumbar regions after sciatic nerve cutting in rats according to the present invention. Wherein fig. 1a is a custom microarray result graph, n=6 individual experiments; fig. 1b is a real-time RT-PCR analysis plot, n=6 individual experiments; FIG. 1C is a diagram showing immunoblotting, with activin C at 25 kDa consisting of βC, βC subunits; FIG. 1d is a graph of quantitative analysis of Western blotting experiments for detecting activin C, and shows that the expression of the rat L4-5 DRG activin C protein with axon cleavage is up-regulated.
In this example, microarray and real-time RT-PCR, western blotting, and immunohistochemical staining were performed on the prepared peripheral nerve injury model, and the specific method was performed by referring to the method in example II.
From the custom microarray results, significant upregulation of activin βcmrna expression in rats L4-5 DRG after cleavage of axons was observed compared to day 0 control rats L4-5 DRG (fig. 1 a).
The change in the expression of activin beta-CmRNA was further confirmed by real-time fluorescent quantitative RT-PCR, and a clear trend of increase in the expression of activin beta-CmRNA after axonal cleavage was observed (FIG. 1 b).
Then, in this example, the expression of activin co-protein was observed by Western blotting, and it was found that activin C (a 25 kDa band representing two βC subunits) was expressed to increase after injury (FIG. 1C, FIG. 1 d), and the immunoblot of FIG. 1C shows that activin C at 25 kDa consisted of βC and βC subunits; FIG. 1d shows upregulation of expression of the L4-5 DRG activin C protein in axon-cleaving rats. It is suggested that the expression of activin C protein (βCβC) and activin βC subunit genes has a similar trend to increase.
Next, immunofluorescence assays were performed in this example to determine the cellular localization of activin C, and it was observed that activin C was expressed predominantly in small diameter neurons I (fig. 2 a), consistent with previous reports. FIG. 2a shows that sham-operated control rats have L4-5 dorsal root ganglion activin C protein-positive small-diameter neurons and that the co-expression of activin C and calcitonin gene-related peptide (CGRP) increases the cleavage of axon rats and reduces the co-expression of activin C and CGRP.
Quantitative analysis showed that the percentage of neurite-cut rats L4-5 DRG activin C positive neurons (3882 out of 8607 neurons in 9 rats) increased significantly by 56% (1691 out of 5634 neurons in 9 rats) compared to sham-operated control group (1691 out of 5634 neurons in 9 rats), consistent with the above-described custom chip, real-time RT-PCR and Western blot data (fig. 1); in the peptide energy subgroup, CGRP positive neurons are obviously reduced, and the percentage of the activin C positive neurons and the calcitonin gene related peptide positive neurons in the dorsal root ganglion at 14 days after the rat axon is cut off is shown as shown in an immunofluorescence quantitative analysis chart of figure 2 b. Fig. 2a, 2b, correspond to the nerve damage situation. Thus, all axonally severed rats developed different degrees of autologous severing within 4 weeks, a pain-related behavior that accompanies axonal severing (n=9 rats); whereas sham groups (n=7 rats, fig. 2 c) showed no autoexcision within 4 weeks of fig. 2c and the sham rats showed an autoexcision (self-residual) pattern.
Immunofluorescence showed that activin C positive neurons increased in L4-5 DRG of CCI mice on day 7, CGRP positive neurons decreased (FIG. 2 d), and dual immunofluorescence staining pattern of FIG. 2d showed that activin C positive medium and small diameter neurons increased in lumbar 4-5 dorsal root ganglion of CCI mice at the seventh day time point after chronic compression injury, and that activin C and calcitonin gene-related peptide co-expression decreased. The CCI model mice have increased activin C positive neurons and decreased CGRP positive neurons in L4-5 DRG. Therefore, the invention considers that the L4-5F dorsal root ganglion activin C is obviously up-regulated after peripheral nerve injury.
Example IV intrathecal or peripheral injection of activin C inhibits chronic neuropathic pain
Activin C was reported to decrease in L4-5 dorsal root ganglions and to be dose dependent (50 ng, 100 ng and 200 ng) inhibiting chronic inflammatory pain in rats induced by complete florid addition. On the basis of up-regulation of activin C expression following nerve injury (fig. 1).
(1) The effect of large-dose activin C in chronic neuropathic pain is observed in the SNL model of rats for the first time in the invention. After a significant decrease in mechanical pain threshold at day 7 after SNL (left panel of fig. 3a, showing the occurrence of mechanical pain-sensitive response at day 7 after SNL), rats were intrathecally injected with 200 ngrh-activin C or vehicle (20 μl) in a single injection. After 2 hours of treatment with 200 ng rh-activin C alone, mechanical nociception in SNL rats was almost reversed to baseline levels (figure 3a right panel, single injection of recombinant human (rh) -activin C (200 ng in 20 μl phosphate buffer) increased the foot-shrink threshold in SNL rats).
The invention then uses the downward doses of 200 ng, 100 ng and 50 ng and uses the same model to further study the dose-dependent analgesic effect of activin C. As shown in fig. 3b, in SNL rats, mechanical pain sensitivity occurred at the 7 th day after SNL (left panel of fig. 3 b), and single intrathecal injection of rh-activin of 50 ng, 100 ng or 200 ng could increase hindfoot paw withdrawal threshold, i.e., mechanical pain threshold, dose-dependently (right panel of fig. 3 b).
The present invention observes that intrathecal administration of 50 ng, 100 ng or 200 ng of rh-activin C neuropathic pain is intrathecally and peripherally administered, which is a clinically common analgesic application route.
(2) The present invention employs multiple pathways 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 sciatic nerve ligation partial pre-incubation 250 ng activin C (200 μl) has a significant inhibitory effect on CCI mice thermal pain; figure 3d shows the long-term anti-pain effect of local pre-incubation 250 ng activin C (200 μl) on mechanical pain. FIG. 3e shows that mechanical pain-sensitivity occurred on day 7 after CCI (left side of FIG. 3 e), intrathecal injection of rh-activin C (15 ng in 5. Mu.L phosphate buffer) increased the foothold threshold in CCI mice, i.e., significantly inhibited neuropathic mechanical pain (right side of FIG. 3 e).
The present invention pre-administered activin C (250 ng/200. Mu.L) or 200. Mu.L of PBS-pretreated CCI neuropathic pain model, and the expression changes of IBA-1, GFAP and CGRP in L4-5 level DRG were detected at time point 7 by immunofluorescence three-standard method (detection method as described in example two). As shown in fig. 4a, an immunofluorescence image is obtained. FIGS. 4b-d are immunofluorescent quantitation of IBA-1 positive macrophage count (b), GFAP positive satellite cell count (c) and percentage of CGRP positive neurons (d). N=6 mice; n.s., nonsensical; a control group, ipsilateral; IBA-1, ionized calcium binding to linker molecule 1; GFAP, glial fibrous acid protein; CGRP, calcitonin gene-related peptide. The results indicate that local pre-incubation of activin C in the injured peripheral nerve can significantly reduce inflammatory response, i.e. infiltration of inflammatory cells, within the dorsal root ganglion that dominates the area.
In the present invention, a PBS-pretreated CCI neuropathic pain model was pre-administered with activin C (250 ng/200. Mu.L) or 200. Mu.L, and IBA1, GFAP and CGRP were tested for changes in spinal cord dorsal horn expression at time point 7 by immunofluorescence three-standard method. FIG. 5a is an immunofluorescence image. Panels b-d, for immunofluorescence quantification of IBA-1 positive macrophage count (b), GFAP positive satellite cell count (c) and percentage of CGRP positive neurons (d). N=6 mice; IBA-1, ionized calcium binding to linker molecule 1; GFAP, glial fibrous acid protein; CGRP, calcitonin gene-related peptide. The results indicate that local pre-incubation of activin C in the injured peripheral nerve can significantly reduce inflammatory response, i.e. infiltration of inflammatory cells, in the spinal cord innervating the area; activin C may also be involved in other functions by regulating expression of CGRP.
In the invention, the peripheral pretreatment of activin C does not influence the inflammatory response of local peripheral ligatured nerves. As a result, as shown in FIG. 6, FIG. 6a, an image of sciatic nerve ligation in CCI mice was observed on day 28 after sciatic nerve ligation for activin C (250 ng/200. Mu.L PBS) or vehicle (200 mmol/L PBS) topical medicated bath ligation. FIG. 6b, image-Pro R Plus6.0 software was used to measure the diameter of sciatic nerve in the Image and FIG. 6c was used for quantitative analysis of localized oedema of the motor-ligated sciatic nerve. SN, sciatic nerve; veh. vehicle; contralateral; acter, activin C; e ipsi, ipsi. Hematoxylin-eosin staining showed no significant difference in inflammatory cell infiltration of locally ligated nerves in the activin C medicated bath group and solvent soaked bath group at day 7 post CCI. Data are expressed as mean ± Standard Error of Mean (SEM). The results indicate that the function of activin C in the treatment of chronic neuropathic pain following medicated bath is not achieved by reducing the local inflammatory response of peripheral injured nerves and oedema.
(3) The examples of the present invention, using plantar injection of capsaicin-induced pain, were tested for electrophysiology and behavioural as follows.
Electrophysiology test methods are described in example two, and the test results are shown in fig. 7, which shows that activin C enhances capsaicin-induced transient receptor potential cation channel V1 (TRPV 1) channel currents in rat DRG neurons. Fig. 7a is a whole cell record showing desensitization of TRPV1 currents by 3 consecutive incubations of capsaicin (1.0 μm). Fig. 7b is a whole cell record showing that continuous incubation of activin C (100 ng/ml) enhances capsaicin-induced TRPV1 currents. Fig. 7c is a quantitative analysis of fig. 7a and 7 b. N=6 to 7 rats; cap, capsaicin; ECS, extracellular fluid; * The one-way variance was measured repeatedly and then tested by Bonferroni multiple comparisons, P < 0.05. Data are expressed as mean ± Standard Error of Mean (SEM).
The invention further demonstrates the regulation of TRPV1 function by activin C using a behavioral test in mice. Adopting a foot swelling measurement method, and carrying out hot plate and cold plate tests; as a result, as shown in FIG. 8a, it was revealed that the pre-plantar injection of activin C (20 ng/20. Mu.L) reduced the acute injury behavior caused by plantar injection of capsaicin. As shown in fig. 8b, there is shown foot edema in rats caused by capsaicin injection. As shown in FIGS. 8C and 8d, early administration of activin C (20 ng/20. Mu.L, plantar injection) allowed the insensitivity to high (52 ℃) and low (4 ℃) temperatures, resulting from capsaicin injection, to return to normal for a long period of time at 7 days and 3 days, respectively. The result shows that the activin C not only can relieve the acute pain induced by the capsaicin model, but also can regulate and control the temperature insensitive to cold and hot caused by the injection of the capsaicin to be normal. Fig. 8e shows that in TRPV1 knockout mice, the analgesic effect of activin C (250 ng/200 μl PBS, pre-incubation) on neuropathic pain caused by chronic constrictive lesions was abolished. (a) Adopting unpaired double-tailed t test, (c, d) adopting one-way repeated measurement analysis of variance, (e) adopting two-way repeated measurement analysis of variance, and carrying out two-way repeated measurement analysis of variance, wherein P is less than 0.05. Data are expressed as mean ± Standard Error of Mean (SEM).
The studies of the present invention support a relationship between activin C-mediated antinociception and modulation of TRPV1 activity suggesting that activin C may be a potential candidate for neuropathic pain treatment. However, the conclusion of the present invention is based on animal experiments performed on men alone, and these findings may not be applicable to women.
For a variety of reasons for neuropathic pain, the present invention uses axonectomy, SNL and CCI models to evaluate 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 DRGs can be obtained for gene screening. The SNL model is suitable for studying damaged and undamaged nerve fibers in the sciatic nerve, mimicking the symptoms of burning pain that occur after human peripheral nerve damage and sympathetically mediated pain. CCI models include neuropathy and inflammatory components, and mimic burning pain, post-traumatic peripheral painful neuropathy, entrapment neuropathy, and complex regional pain syndromes in patients. In addition, after peripheral nerve injury, immune cells of DRGs and SCs release some cytokines, sensitize/desensitize nociceptive neurons, and further regulate pain management. Importantly, some primary sensory neurons were also found to secrete cytokines. Furthermore, strong evidence suggests that signal transduction against the transforming growth factor- β superfamily has beneficial effects in chronic neuropathic pain; thus, the present invention assumes that more members of the transforming growth factor- β superfamily may be involved in pain management and serve as potential targets for neuropathic pain treatment. In this study, the present invention used custom microarrays to screen for significant changes in transforming growth factor- β superfamily member gene expression levels in axon-cleaving rats L4-5 DRG compared to the simple control group. The present invention has found that the activin βc gene is up-regulated in the residues of axonal excision. Next, the present invention further demonstrates the increased transcription of the activin beta C subunit, as well as the increased expression of activin C protein, by Western blotting and immunofluorescence. In addition, increased expression of activin C WVAS was demonstrated in CCI mouse models. These results indicate that, following peripheral nerve injury, lumbar dorsal root ganglion activin C is significantly upregulated at both mRNA and w protein levels. Consistently, the extent of upregulation of activin C with self-cleavage (a pain-related self-disabling behavior) and mechanical and thermal hyperalgesia in CCI mice was consistent, strongly indicating the role of activin C in neuropathic pain.
To further elucidate the central and peripheral role of activin C in neuropathic pain, the present invention employs two models, rat SNL and mouse CCI. Based on the upregulation of activin C following nerve injury, the present invention first selected large doses of activin C (200 Ng) to observe its effect on neuropathic pain on the rat SNL model, found that intrathecal injection of 200 ng activin C almost reversed mechanical hyperalgesia, approaching baseline levels. The present invention then further demonstrates the analgesic effect of activin C with a reduced pressure dose of 200 ng, 100 ng or 50 ng and observes that intrathecal injection of 50, 100 or 200 ng rh-activin C dose dependently inhibits neuropathic pain in SNL rats. Considering that the tissue surrounding the damaged nerve absorbs a large amount of activin C in the local 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 mechanically and thermally stimulated CCI mice was observed by ligating sciatic nerve locally with activin C in advance. 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 alleviate chronic neuropathic pain in SNL rats and CCI mice, similar to other transforming growth factor- β superfamily members.
The occurrence of neuropathic pain is associated with inflammatory cell infiltration of the dorsal root ganglion and activation of glial cells in SCs. In agreement with this, the topical pre-bath of activin C inhibited macrophage infiltration into the lumbar dorsal root ganglion and proliferation of activated microglial cells in SCs, whereas topical treatment of activin C did not affect satellite cells or astrocytes in the early stage of CCI. In CCI strip locally ligated nerves, local pre-administration of activin C did not affect neurooedema or inflammatory responses. These results indicate that treatment of activin C with outer Zhou Yu prevented both peripheral and central nervous system inflammatory responses caused by CCI, but failed to prevent local neurogenic inflammatory responses. Thus, the present invention speculates that the pre-bath of exogenous activin C may inhibit the nociceptive signaling of peripherally innervated DRGs and SCs and further reduce CCI-induced inflammatory responses of DRGs and SCs, but is not a direct anti-inflammatory mechanism. Although activin a acts at an early stage of the inflammatory process and is a key component of the inflammatory response, the data of the present invention do not support the notion that activin C can antagonize activin a-induced nociception. One possibility is that activin C, like transforming growth factor- β1, acts as an analgesic by inhibiting the neuroimmune response of neurons and glia, reducing the activity of spinal cord excitatory neurons, and inhibiting some pain signaling pathways. However, the local pretreatment of activin C had no significant effect on astrocyte activation or proliferation, and the present invention speculates that the effect of activin C on astrocyte activation at the late stage of CCI remains to be studied further. Unexpectedly, topical pre-administration of activin C restored the level of CGRP in ipsilateral DRGs and SCs caused by CCI, suggesting that cytokine activin C has a versatile role in the nervous system, possibly regulating the release of various pain-associated neurotransmitters including CGRP following peripheral nerve injury. Similarly, in vitro studies have found that activin a synergistically regulates CGRP expression with nerve growth factor, but activin C and activin a exhibit completely different effects in pain management. Notably, all aβ fibers and most aδ fibers were cut, while in CCI models, a significant number of C fibers remained intact (all fibers were cut in axon cut models, and some aβ -, aδ -, and C fibers were cut in SNL models), thus CCI-induced pain might be mediated by C and/or a- δ fibers, including most cgrp+ fibers. Regarding the unexpected function of CGRP, crosstalk between activin C and CGRP is worth further investigation.
Considering that TRVP 1 is a non-selective Ca expressed mainly in sensory neuron cell bodies 2+ The present invention speculates that activin C may act directly or indirectly on TRPV1 channels in DRG neurons to modulate neuropathic pain. Electrophysiological analysis of the present invention shows that, although activin C alone had no effect on TRPV1 currents of individual isolated DRG neurons, pre-incubation with activin C could significantly increase capsaicin-induced TRPV1 currents. Considering that capsaicin is a specific TRPV1 channel agonist, TRPV1 channels can be rapidly opened to induce pain sensation in a short period of time, and that it takes a long time to pre-bath with activin C to enhance capsaicin-induced TRPV1 current, the present invention assumes that activin C enhances the activity of TRPV1 channels as an indirect pathway, unlike activin a-sensitized capsaicin-induced current, however, continuous capsaicin stimulation can cause TRPV1 desensitization, rendering the channels unable to reopen for a long period of time, thereby interfering with pain signaling of DRG neurons and afferent neurons thereof. The mechanism by which activin C regulates TRPV1 was further demonstrated by the nociceptive behavior of mice; activin C attenuated capsaicin-induced acute nociceptive responses. This is consistent with the use of capsaicin as the primary F component of analgesics in clinical practice. However, the pretreatment with activin C did not reduce capsaicin-induced swelling of the mouse feet, suggesting that the anti-inflammatory effect of activin C was not achieved by reducing localized edema or inflammation. Interestingly, the activin C pretreatment allowed capsaicin-induced mice to continue to return to normal at low temperatures following both thermal and cold stimulation. Considering normalized CGRP data comprehensively, the invention speculates that activin C has complex functions in the nervous system and is worthy of further evaluation. Without being expected, the analgesic effect of TRPV1 KO mice after CCI was abolished. These data provide conclusive evidence for activin C to alleviate chronic neuropathic pain by modulating TRPV1 function, which helps the present invention understand that exogenous C promotes release of CGRP due to Secretion of CGRP and substance P in DRG neurons is mediated for TRPV 1. Although TRPV1 KO mice exhibited complete mechanical nociception in this CCI model, a recent study showed that TRPV1 gene was siRNA knocked out in adult rodents, thereby reducing CCI-induced behavioral nociception. This difference suggests that gene compensation should be commonplace in the TRPs family. However, several unknown functions of activin C, including its specific receptor, require further investigation.
In summary, after peripheral nerve injury, endogenous activin C is significantly expressed and up-regulated in small DRG neurons, and both peripheral and intrathecal application of activin C can inhibit chronic neuropathic pain by modulating TRPV1 channels, suggesting that activin C may have potential to treat chronic neuropathic pain.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (1)
1. The application of activin C in preparing medicine for treating chronic neuropathic pain caused by peripheral nerve injury is provided.
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