Calcitonin Gene-related Peptide Protein Research Articles

Unlocking the potential of luteolin: A natural migraine management approach through network pharmacology

BackgroundLuteolin, a natural flavonoid, exhibits antioxidant and anti-inflammatory properties and has been investigated for potential health benefits. Its focus on migraine management arises from its ability to mitigate neuroinflammation, a key factor in migraine attacks. MethodspkCSM and Swiss ADME were employed to assess luteolin's pharmacokinetic properties, revealing challenges such as low water solubility and limited skin permeability. OSIRIS Property Explorer is used to check the toxicity. Ligand binding simulations indicated luteolin's potential to interact with calcitonin gene related peptide proteins, crucial in migraine pathophysiology. DisGeNet identified common targets related to migraine, with subsequent network analysis emphasizing promising targets. Results and DiscussionLuteolin demonstrated good intestinal absorption but faced BBB limitations, suggesting a potential for oral administration but questioning direct brain impact. Nanoformulation was proposed to address solubility challenges, emphasizing the need for in vivo validation. The highest binding affinity with CGRP proteins PDBID: 6PFO (−7.63 kcal/mol) suggested a potential for migraine treatment, requiring empirical confirmation. Enrichment network analysis illustrated luteolin's potential in migraine treatment, emphasizing key targets such as PTGS2, AKT1, ESR1, MMP2, and MMP9. Luteolin shows promise for migraine management, evident in its pharmacokinetic, toxicological profiles, and interactions with CGRP proteins. Challenges like low solubility suggest the need for nanoformulations and empirical validation. Target identification and network analysis offer insights, highlighting potential therapeutic avenues in migraine treatment. ConclusionLuteolin holds promise in migraine management, necessitating further research for translation into effective interventions, considering its neuroprotective potential in broader neurological conditions.

Cryo-EM Structure of the Human Amylin 1 Receptor in Complex with CGRP and Gs Protein.

Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.

PLGA Containing Human Adipose-Derived Stem Cell-Derived Extracellular Vesicles Accelerates the Repair of Alveolar Bone Defects via Transfer of CGRP.

Calcitonin gene-related peptide (CGRP) is an important neuropeptide expressed in the nerve fibers during bone repair. Here, we aimed to pinpoint the role of CGRP in the osteogenic differentiation property of human periodontal ligament stem cells (hPDLSCs) and the resultant repair of alveolar bone defect. The key factor related to the osteogenic differentiation of hPDLSCs was retrieved from the GEO database. After extraction from hADSCs (hADSC-EVs) and identification, EVs were subjected to coculture with hPDLSCs, in which the expression patterns of CGRP and osteogenic differentiation marker proteins (ALP, RUNX2, and OCN), as well as ALP activity, were detected. A novel cell-free tissue-engineered bone (TEB) comprised of PLGA/pDA and hADSC-EVs was implanted into the rats with alveolar bone defects to evaluate the repair of alveolar bone defects. CGRP was enriched in hADSC-EVs. hADSCs delivered CGRP to hPDLSCs through EVs, thereby promoting the osteogenic differentiation potential of hPDLSCs. The PLGA/pDA-EV scaffold released EVs slowly, and its implantation into the rat alveolar bone defect area significantly induced bone defect repair, which was reversed by further knockdown of CGRP. In conclusion, our newly discovered cell-free system consisted of hADSC-EVs, and PLGA/pDA scaffold shows promising function in repairing alveolar bone defects.

Comparison of stimulation-induced local changes of expression of local Meckel cell-related proteins and neuropeptides and number of mast cells among acupuncture and moxibustion alone or in combination in chronic atrophic gastritis rats

To compare the effects of acupuncture and moxibustion alone or in combination on the number of mast cells and expression levels of cytoketatin 18 (CK18) and CK19 (marker of Meckel cells), and calcitonin gene-related peptide (CGRP), neuropeptide-Y (NPY) and bradykinin (BK) in the local acupoint area of rats with chronic atrophic gastritis (CAG). Fifty male SD rats were randomly divided into normal, CAG model, moxibustion, acupuncture and acupuncture+moxi-bustion groups (10 rats in each group). The CAG model was established by gavage of 1-methyl-3-nitro-1-nitrosoguanidine (170 μg/mL,1 mL/100 g, once a week) and 40% ethanol solution (twice a week) for 12 consecutive weeks. After successful establishment of CAG model, moxibustion, manual acupuncture or acupuncture+moxibustion was applied to bilateral "Zusanli" (ST36) and "Zhongwan"(CV12) for 15 min, once daily for 14 consecutive days. At the end of the experiment, the gastric mucosal tissues were collected for observing histopathological changes of gastric mucosa after H.E. staining, and the tissues of the stimulated ST36 region collected for detecting the expression levels of CK18, CK19, CGRP, NPY and BK and the number of mast cells in the local ST36 region by immunohistochemistry. Compared with the normal group, the number of mast cells, the expression levels of CK19, NPY and BK in the ST36 area were significantly increased (P<0.05), and the expression level of CGRP was apparently decreased (P<0.05) in the model group. In comparison with the model group, the number of mast cells and the expression levels of CGRP and NPY in the moxibustion group, the expression of CGRP in the acupuncture group, and the number of mast cells, as well as the expression levels of CK18, CK19 and CGRP in the acupuncture+moxibustion group were significantly up-re-gulated (P<0.05). The effect of acupuncture combined with moxibustion was obviously superior to that of moxibustion or acupuncture in up-regulating the expression of CK18 and CK19 (P<0.05) and superior to that of moxibustion in down-regulating BK expressio level (P<0.05). No significant changes were found in the expression of CK18 after modeling (vs the normal group), in the expression levels of CK18, CK19 and BK after moxibustion and acupuncture (vs the model group), in the number of mast cells and expression of NPY after acupuncture (vs the model group), and in the expression levels of NPY and BK after acupuncture+moxibustion (vs the model group) (P>0.05). H.E. staining showed infiltration of many lymphocytes in the gastric mucosa and submucosal layers, atrophy and necrosis of lots of main cells with vacuole-like changes, and disordered arrangement of the atrophic glands in the model group, which was milder particularly in the acupuncture + moxibustion group. Acupuncture combined with moxibustion of ST36 can up-regulate the levels of local CK18, CK19 and CGRP proteins and number of mast cells, moxibustion may up-regulate the levels of CGRP and NPY and number of mast cells, while acupuncture may up-regulate the expression of CGRP in the local stimulated area in CAG rats.

Calcitonin gene related peptide modified mesenchymal stem cells reduce restenosis after carotid balloon injury in rats

This work aimed to investigate the effects of calcitonin gene-related peptide (CGRP)-modified mesenchymal stem cells (MSCs) on vascular stenosis in carotid balloon-injured rats. The CGRP gene labeled with EGFP was transfected into bone marrow MSCs, and CGRP protein expression in MSCs was confirmed by immunofluorescence assays. A rat carotid balloon injury model was established using a surgical method. CGRP-modified MSCs were orthotopically transplanted into the injured area of the rats. At 28 days after cell transplantation, EGFP and CD31 expression was detected by immunofluorescence staining. Hematoxylin-eosin (H&E) staining was used to detect the intima/media area of the injured carotid artery stenosis site, and the expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. MSCs from rat bone marrow positively expressed CD29 and negatively expressed CD45. In vivo immunofluorescence staining showed that EGFP expression was significantly increased at the vascular injury site of rats transplanted with MSC_CGRP compared with the control group on the 28th day after cell transplantation and that the damaged vessels exhibited continuous CD31 expression. H&E staining showed that the vascular intimal proliferation area of rats transplanted with MSC_CGRP was significantly reduced compared with that of other groups. Furthermore, the immunohistochemistry results showed that PCNA expression in the endothelium of the MSC_CGRP group was lower than that of the other groups. Therefore, MSCs transfected with the CGRP gene can express the CGRP protein, and the implantation of CGRP-modified MSCs at the damaged site after carotid balloon-induced injury can reduce the neointimal area.

Transient Receptor Potential Channel 4 Small-Molecule Inhibition Alleviates Migraine-Like Behavior in Mice.

Migraine is a common neurological disorder with few available treatment options. Recently, we have demonstrated the role of transient receptor potential cation channel subfamily C member 4 (TRPC4) in itch and the modulation of the calcitonin gene-related peptide (CGRP), a biomarker and emerging therapeutic target for migraine. In this study, we characterized the role of TRPC4 in pain and evaluated its inhibition as anti-migraine pain therapy in preclinical mouse models. First, we found that TRPC4 is highly expressed in trigeminal ganglia and its activation not only mediates itch but also pain. Second, we demonstrated that the small-molecule inhibitor ML204, a specific TRPC4 antagonist, significantly reduced episodic and chronic migraine-like behaviors in male and female mice after injection of nitroglycerin (NTG), a well-known migraine inducer in rodents and humans. Third, we found a significant decrease in CGRP protein levels in the plasma of both male and female mice treated with ML-204, which largely prevented the development of chronic migraine-like behavior. Using sensory neuron cultures, we confirmed that activation of TRPC4 elicited release of CGRP, which was significantly diminished by ML-204. Collectively, our findings identify TRPC4 in peripheral sensory neurons as a mediator of CGRP release and NTG-evoked migraine. Since a TRPC4 antagonist is already in clinical trials, we expect that this study will rapidly lead to novel and effective clinical treatments for migraineurs.

Chemotherapy-induced peripheral neuropathy is promoted by enhanced spinal insulin-like growth factor-1 levels via astrocyte-dependent mechanisms

BackgroundChemotherapy-induced peripheral neuropathy (CIPN) is a common and intractable complication in chemotherapy-receiving patients. Insulin-like growth factor-1 (IGF-1) is a popular neurotrophin with various functions, such as maintaining neuronal survival and synaptic functioning in the central nervous system. Therefore, we hypothesized that the IGF-1 signaling pathway could be a candidate target for treating CIPN. MethodsWe established the CIPN model by injecting mice intraperitoneally with oxaliplatin and assessed IGF-1 protein expression, its receptor IGF1R, phospho-IGF1R (p-IGF1R), interleukin-17A (IL-17A), tumor necrosis factor-α (TNF-α), and calcitonin gene-related peptide (CGRP) in the lumbar spinal cord with Western blot and immunofluorescence. To examine the effect of IGF-1 signaling on CIPN, we injected mice intrathecally or intraperitoneally with mouse recombinant IGF-1 (rIGF-1). ResultsIGF-1 protein expression decreased significantly in the spinal cord on D3 and D10 (the 3rd and 10th days after beginning oxaliplatin chemotherapy) and was co-localized with astrocytes primarily in the lumbar spinal cord, whereas IGF1R was predominantly expressed on neurons. Both intrathecally- and intraperitoneally-administered rIGF-1 relieved the chemotherapy-induced pain-like behavior and reduced IL-17A, TNF-α, and CGRP protein expressions in the spinal cord. ConclusionOur results indicate a vital role for IGF-1 signaling in CIPN. Targeting IGF-1 signaling could be a potent therapeutic strategy for treating CIPN in clinical settings.

Effect of electroacupuncture pretreatment on transient receptor potential vanilloid 1(TRPV1)/calcitonin gene-related peptide(CGRP) signal and NF-κB p65 protein expression in rats with acute myocardial ischemia

To observe the effect of electroacupuncture(EA)pretreatment on transient receptor potential vanilloid 1(TRPV1)/calcitonin gene-related peptide(CGRP)signal and nuclear factor-κB p65 (NF-κB p65) protein expression in myocardial tissue of acute myocardial ischemic injury (AMI) rats, and to investigate the possible mechanism of electroacupuncture pretreatment against AMI. A total of 60 adult male SD rats were randomly divided into blank control, sham operation, model and EA pretreatment groups, 15 rats in each group. The acute myocardial ischemia model was established by ligating the left anterior descending (LAD)branch of the coronary artery in the model group and EA pretreatment group, while threading but no ligating at left anterior descending branch of the coronary artery was applied in the sham operation group. In the EA pretreatment group, bilateral "Neiguan"(PC6) acupoints were selected, with intensity of 2 mA and frequency of 2 Hz/100 Hz, for 20 min, once daily for 7 days before modeling. Electrocardiogram (ECG) was recorded by physiological signal acquisition system, and the ST segment potential offset values of standard Ⅱ lead were analyzed before surgery，30 min and 24 h after operation. The TTC staining was used to observe the percentage of myocardial infarction area. The HE staining was used to observe the pathological changes of myocardial tissue and the degree of inflammatory cell infiltration. And Western blot was used to detect TRPV1/CGRP signal and NF-κB p65 protein expression levels in myocardial tissue. Compared with the sham operation group, the ECG-J point potential in the model group was significantly increased at 30 min and decreased at 24 h after operation (P<0.05), myocardial infarction area increased significantly (P<0.05), the myocardial fibers were obviously disordered, inflammatory cell infiltration was obvious, and the expressions of TRPV1，CGRP and NF-κB p65 proteins were all increased (P<0.05). Compared with the model group, the EA pretreatment group was decreased in the ECG-J point potential at 30 min after operation(P<0.05), significantly reduced in myocardial infarction area (P<0.05), improved in the morphology of myocardial fibers, reduced ininflammatory cell infiltration, and increased in the protein expressions of TRPV1 and CGRP in myocardium (P<0.05), significantly decreased in the protein expression of NF-κB p65 (P<0.05). EA pretreatment may enhance TRPV1/CGRP signaling, down-regulate NF-κB p65 protein expression, reduce myocardial inflammatory response status, improve AMI injury, and reduce myocardial infarction area.

Effects of electroacupuncture on miR-34a-5p/SIRT1 signaling in the trigeminal ganglion of rats with migraine

To investigate the effect of electroacupuncture(EA) on miR-34a-5p, silent mating type information regulation 2 homolog-1 (SIRT1) and nuclear factor-κB subunit p65 (NF-κB p65) in the trigeminal ganglion of rats with migraine, so as to explore the mechanisms of EA underlying prevention of migraine. Male SD rats were randomly divi-ded into normal, sham operation, model, EA, and EA plus EX527(a SIRT1 inhibitor) groups, with 10 rats in each group. The rat model of migraine was established by electrical stimulation of the trigeminal ganglion. Before modeling, EA was applied at "Waiguan"(TE5) and "Fengchi"(GB20) for 20 min each time, once a day for 5 consecutive days, and intraperitoneal injection of EX527 (5 mg/kg) every day simultaneously. Serum prostaglandin E2 (PGE2) and calcitonin gene-related peptide (CGRP) concentrations were measured by enzyme-linked immunosorbent assay. The levels of miR-34a-5p, SIRT1 and interleukin-1β (IL-1β) mRNA，and protein expression of SIRT1, IL-1β, NF-κB p65, NF-κB Ac-p65 and cyclooxygenase-2 （COX2） in trigeminal ganglia were detected by real-time quantitative PCR and Western blot, separately. The serum concentrations of PGE2 and CGRP， the expression of miR-34a-5p, IL-1β mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression in the trigeminal ganglion were remarkably increased (P<0.05), while the SIRT1 mRNA and protein decreased (P<0.05) in the model group in contrast to the normal group. Following EA intervention， the serum PGE2 and CGRP concentrations, miR-34a-5p expression, IL-1β mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression were significantly down-regulated (P<0.05), and SIRT1 mRNA and protein significantly up-regulated (P<0.05). Compared with the EA group, the serum concentrations of PGE2 and CGRP, IL-1β mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expressions increased (P<0.05), and SIRT1 protein decreased (P<0.05) in the EA plus EX527 group. In migraine rats, EA can inhibit miR-34a-5p expression in the trigeminal ganglion, increase SIRT1 expression, down-regulate IL-1β/COX2 inflammation signals, reduce PGE2 synthesis, and thus redue CGRP released from the peripheral terminals, which may be one of the mechanisms of EA in preventing migraine.

Hejie Zhitong prescription promotes sleep and inhibits nociceptive transmission-associated neurotransmitter activity in a rodent migraine model

BackgroundMigraine is painful disease in which neurotransmitters related to pain transmission play an important role. Hejie Zhitong prescription (HJZT) has been used in the clinic as an effective prescription for the treatment of migraine for many years. Our team aimed to further explore its antimigraine mechanism based on previous research results and to explore the inhibitory effect of HJZT on the transmission of pain related to nitroglycerine (NTG)-induced migraine as well as the synergistic effect of HJZT with pentobarbital sodium on promoting sleep.MethodsSixty mice were randomly assigned to groups and received the corresponding interventions. Sleep latency and sleep time were recorded to calculate the incidence of sleep. Forty-eight Wistar rats were randomly assigned and administered an intervention corresponding to their group. Calcitonin gene-related peptide (CGRP), serotonin (5-HT), substance P (SP), and cholecystokinin (CCK) levels were measured using ELISAs. Levels of the cannabinoid receptor type 1 (CB1R) and cyclooxygenase-2 (COX-2) protein were assessed using immunohistochemistry. The expression of the CGRP and CCK mRNAs in the midbrain and trigeminal ganglion (TG) were measured using real-time quantitative PCR.ResultsHJZT promoted the occurrence of sleep in mice. HJZT downregulated COX-2 expression in the midbrain and TG of rats but upregulated the expression of the CB1R, and decreased the plasma level of the CGRP protein and expression of its mRNA in the midbrain and TG. It also downregulated the expression of the CCK mRNA in the midbrain and TG. The high-dose HJZT treatment increased plasma 5-HT levels, but did not induce changes in the plasma levels of the SP or CCK protein.ConclusionsHJZT exerts a synergistic effect with pentobarbital sodium on promoting sleep. As for anti-migraine, HJZT can inhibits the expression of nociceptive transmission-associated neurotransmitters, including 5-HT, CGRP and CCK, which may be related to its upregulation of CB1R and downregulation of COX-2.

Construction of calcitonin gene-related peptide-modified mesenchymal stem cells and analysis of their effects on the migration and proliferation of vascular smooth muscle cells.

Lentiviral expression vectors for calcitonin gene-related peptide (CGRP) were used to transfect rat bone marrow mesenchymal stem cells (MSCs). After assessing the biological characteristics of proliferation and aging in MSCs transfected with CGRP, we observed the effects of the CGRP-modified rat MSCs on the migration and proliferation of rat vascular smooth muscle cells (VSMCs) in vitro. Rat MSCs were isolated, cultured in vitro, and identified by flow cytometry. A CGRP recombinant lentivirus was transfected into MSCs. The transfection efficiency was determined by fluorescence microscopy and flow cytometry, and CGRP in MSCs was detected by real-time quantitative PCR, ELISA, and immunofluorescence. The proliferation and senescence of CGRP-modified MSCs were evaluated by MTT assay and beta-galactosidase staining. VSMCs were isolated, cultured in vitro, and identified by immunofluorescence. CGRP-modified MSCs and VSMCs were cocultured in a Transwell system. The proliferation and migration of VSMCs were evaluated by scratch testing and the MTT method. Rat bone marrow MSCs showed a spindle-shaped morphology, adherent growth in vitro, positive CD29 and CD90 expression, and negative CD45 expression. CGRP was stably expressed in MSCs after 48h of recombinant lentivirus transfection. CGRP mRNA and protein secretion in CGRP recombinant lentivirus-transfected MSCs were higher than that in control MSCs. Immunofluorescence showed that CGRP protein could be expressed in CGRP-modified MSCs. The proliferation ability and senescence rates did not differ between lentivirus-transfected MSCs and untransfected MSCs. Rat VSMCs expressed α-SMA protein and exhibited a spindle-shaped morphology and adherent growth in vitro. In Transwell coculture experiments, scratch testing of VSMCs showed that CGRP-modified MSCs could reduce VSMC proliferation and migration. The CGRP gene can be stably expressed in MSCs after CGRP recombinant lentivirus transfection. CGRP recombinant lentivirus transfection has little effect on the proliferation or senescence of MSCs, and CGRP-modified MSCs can inhibit the proliferation and migration of VSMCs. These results lay a foundation for research on the use of CGRP gene-engineered MSCs in restenosis therapy.

NF-κB-Associated Pain-Related Neuropeptide Expression in Patients with Degenerative Disc Disease.

The role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) has been highlighted in mechanisms underlying inflammatory and neuropathic pain processes. The present study was designed to investigate whether NF-κB signaling is associated with pain-related neuropeptide expression in patients with chronic back pain related to degenerative disc disease (DDD). Intervertebral disc (IVD) tissues were collected from forty DDD patients undergoing disc replacement or fusion surgery, and from eighteen postmortem (PM) control subjects. RELA, NFKB1, CGRP, TAC1, TRPV1, and MMP-3 gene expression were analyzed by RT-qPCR, while NF-κB subunit RelA and NF-κB1–DNA binding in nuclear extracts and calcitonin gene related peptide (CGRP), substance P (SP), and transient receptor potential, subfamily V, member 1 (TRPV1) protein levels in cytosolic extracts of tissues were assessed by enzyme-linked immunosorbent assay (ELISA). An upregulated NF-κB1–DNA binding, and higher CGRP and TRPV1 protein levels were observed in DDD patients compared to PM controls. In DDD patients, NF-κB1–DNA binding was positively correlated with nuclear RelA levels. Moreover, NF-κB1–DNA binding was positively associated with TRPV1 and MMP-3 gene and SP and TRPV1 protein expression in DDD patients. Our results indicate that the expression of SP and TRPV1 in IVD tissues was associated with NF-κB activation. Moreover, NF-κB may be involved in the generation or maintenance of peripheral pain mechanisms by the regulation of pain-related neuropeptide expression in DDD patients.

Antagonism of Transient Receptor Potential Ankyrin Type-1 Channels as a Potential Target for the Treatment of Trigeminal Neuropathic Pain: Study in an Animal Model.

Transient receptor potential ankyrin type-1 (TRPA1) channels are known to actively participate in different pain conditions, including trigeminal neuropathic pain, whose clinical treatment is still unsatisfactory. The aim of this study was to evaluate the involvement of TRPA1 channels by means of the antagonist ADM_12 in trigeminal neuropathic pain, in order to identify possible therapeutic targets. A single treatment of ADM_12 in rats 4 weeks after the chronic constriction injury of the infraorbital nerve (IoN-CCI) significantly reduced the mechanical allodynia induced in the IoN-CCI rats. Additionally, ADM_12 was able to abolish the increased levels of TRPA1, calcitonin gene-related peptide (CGRP), substance P (SP), and cytokines gene expression in trigeminal ganglia, cervical spinal cord, and medulla induced in the IoN-CCI rats. By contrast, no significant differences between groups were seen as regards CGRP and SP protein expression in the pars caudalis of the spinal nucleus of the trigeminal nerve. ADM_12 also reduced TRP vanilloid type-1 (TRPV1) gene expression in the same areas after IoN-CCI. Our findings show the involvement of both TRPA1 and TRPV1 channels in trigeminal neuropathic pain, and in particular, in trigeminal mechanical allodynia. Furthermore, they provide grounds for the use of ADM_12 in the treatment of trigeminal neuropathic pain.

Electroacupuncture Relieves Visceral Hypersensitivity by Down-regulating Mast Cell Number，PAR-2/TRPV 1 Signaling, etc. in Colonic Tissue of Rats with Irritable Bowel Syndrome

To observe the effect of electroacupuncture (EA) stimulation of "Shangjuxu" (ST 37, Lower Confluent point) and "Tianshu" (ST 25, Front-Mu point) on visceral pain and expression of colonic tryptase(Try), proteinase-activated receptor 2(PAR-2)，transient receptor potential channel vanilloid subfamily member 1 (TPRV 1)，substance P (SP) and calcitonin gene-related peptide (CGRP) in rats with irritable bowel syndrome (IBS), so as to explore its mechanisms underlying improvement of IBS. Forty male Sprague Dawley rats were equally randomized into normal control (control), model, medication and EA groups (n＝10 in each). The IBS model was established by chronic acute combining stress (CACS, water deprivation, fasting, tail clamping, forced swimming in ice water, restraint, etc.) for 21 days. Rats of the medication group were treated by gavage of Pinaverium Bromide (1 mg/mL, 15 mg/kg), once daily for 14 d. EA (10 Hz/50 Hz, 0.2－0.3 mA) was applied to bilateral ST 37 and ST 25 for 30 min, once daily for 14 d. The muscular withdrawal reflex (AWR) of both abdomen and buttock was detected by colorectal distension (CRD) with a water-filled balloon for examining the visceral hypersensitivity. The number of mast cells in the colonic tissue was counted after toluidine blue stain. The immunoactivity of colonic Try was determined by immunochemistry and the expression of colonic PAR-2, TRVP 1, SP and CGRP proteins detected by Western blot. After modeling, the body weight was significantly decreased in IBS rats of the model, medication and EA groups compared with their own individual pre-treatment and with the control group (P<0.01), and markedly higher in both medication and EA groups than in the model group (P<0.05, P<0.01). The intra-colonic volume thresholds for inducing abdominal and hip AWR were significantly lower in the model group than in the normal control group (P<0.01), and obviously higher in both medication and EA groups than in the model group (P<0.05，P<0.01). The AWR scores of intra-colorectal balloon at volumes of 0.5, 1.0 and 1.5 mL of water were significantly higher in the model group than in the control group (P<0.01), and considerably lower in the EA and medication groups than in the model group (P<0.01). The number of colonic MC and the expression levels of colonic Try, PAR-2, TRPV 1, SP and CGRP proteins were significantly higher in the model group than in the control group (P<0.01), and obviously decreased in both medication and EA groups relevant to the model group (P<0.01). Comparison between the medication and EA groups showed that the decreased expression levels of colonic PAR-2, TRPV 1, SP and CGRP proteins were significantly lower in the EA group than in the medication group (P<0.05), but no significant differences were found between the two groups in intra-colonic volumes for inducing AWR, AWR scores, body weight, and colonic MC number and Try immunoactivity levels (P>0.05)．. EA of ST 37 and ST 25 can relieve visceral hypersensitivity in IBS rats, which may be associated with its effects in down-regulating the number of MC and the expression of PAR-2, TRVP 1, SP, CGRP and Try proteins in the colonic tissue.

Association of cough hypersensitivity with tracheal TRPV1 activation and neurogenic inflammation in a novel guinea pig model of citric acid-induced chronic cough.

ObjectiveThis study was performed to establish a novel model of citric acid-induced chronic cough in guinea pigs and to investigate the pathogenesis of cough hypersensitivity.MethodsHealthy conscious guinea pigs inhaled citric acid (0.4 M) for 3 minutes twice daily for 25 days. Cough reactivity was evaluated, substance P (SP) and calcitonin gene-related peptide (CGRP) in bronchoalveolar lavage fluid were detected, and transient receptor potential cation channel subfamily V member 1 (TRPV1) protein expression in the trachea and bronchus was determined. Tracheal and bronchial tissues were examined for TRPV1.ResultsInhalation of 0.4 M citric acid increased coughing in a time-dependent manner: coughing peaked at 15 days and reached the lowest level at 25 days. This was accompanied by similar changes in SP, CGRP, and TRPV1 protein expression. TRPV1 was mainly observed in the mucosal and submucosal layer of the trachea and bronchi. The areas of TRPV1 positivity in the trachea and bronchi of citric acid-treated animals were significantly larger than in the control group.ConclusionsRepeated inhalation of citric acid can be employed to establish a chronic cough model in guinea pigs. Cough hypersensitivity in this model is related to tracheal TRPV1 activation and neurogenic inflammation.

Research of the heart protective effect of exercise precondition mediated by calcitonin gene related peptide on acute exhaustion rats

To observe the heart protective effect of exercise preconditioning (EP) in the acute exhaustion exercise (EE) rats, and explore its action mechanism further. Eighty healthy male Sprague-Dawley (SD) rats were divided into control group (C group), EP group, EE group, and EP+EE group randomly, with 20 rats in each group. The rats in EP and EP+EE groups were trained for 3 weeks according to the daily swimming for 60 minutes (swimming 15 minutes, resting 5 minutes, repeating 3 times) with 6 days each week. The rats in EE and EP+EE groups on the last 1 day after 3 weeks, 3% weight heavy weight was carried once for swimming EE. Two hours after the last EE, abdominal aortic blood and heart was harvested, the levels of serum MB isoenzyme of creatine kinase (CK-MB) and calcitonin gene related peptide (CGRP) were determined by enzyme linked immunosorbent assay (ELISA); the ultrastructure of myocardium was observed by optical microscopy; the levels of myocardial malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by ELISA, the mRNA expression of myocardial CGRP was assayed by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression of myocardial CGRP was assayed by Western Blot. Compared with C group, the levels of serum CK-MB and myocardial MDA were significantly increased, serum CGRP content, myocardial SOD activity, and mRNA and protein expressions of myocardial CGRP were significantly decreased in EE group and EP+EE group. Compared with EE group, the levels of serum CK-MB and myocardial MDA in EP+EE group were decreased [CK-MB (U/L): 13.11±0.77 vs. 15.55±0.90, MDA (μmol/L): 389.57±49.60 vs. 709.08±160.49], the level of serum CGRP, and mRNA and protein expressions of myocardium CGRP were increased [serum CGRP (ng/L): 120.41±9.07 vs. 97.97±9.05, CGRP mRNA (2 -ΔΔCT): 0.45±0.09 vs. 0.14±0.02, CGRP protein (gray value): 0.78±0.08 vs. 0.41±0.04, all P < 0.05], the degree of myocardial injury was obviously alleviated. There was no significant difference in the indexes between the EP group and C group. EP has the heart protective effect for the acute EE rats, and the mechanism is closely related to the endogenous protective substance CGRP.

The role of the transient receptor potential ankyrin type-1 (TRPA1) channel in migraine pain: evaluation in an animal model

BackgroundClinical and experimental studies have pointed to the possible involvement of the transient receptor potential ankyrin type-1 (TRPA1) channels in migraine pain. In this study, we aimed to further investigate the role of these channels in an animal model of migraine using a novel TRPA1 antagonist, ADM_12, as a probe.MethodsThe effects of ADM_12 on nitroglycerin-induced hyperalgesia at the trigeminal level were investigated in male rats using the quantification of nocifensive behavior in the orofacial formalin test. The expression levels of the genes coding for c-Fos, TRPA1, calcitonin gene-related peptide (CGRP) and substance P (SP) in peripheral and central areas relevant for migraine pain were analyzed. CGRP and SP protein immunoreactivity was also evaluated in trigeminal nucleus caudalis (TNC).ResultsIn rats bearing nitroglycerin-induced hyperalgesia, ADM_12 showed an anti-hyperalgesic effect in the second phase of the orofacial formalin test. This effect was associated to a significant inhibition of nitroglycerin-induced increase in c-Fos, TRPA1 and neuropeptides mRNA levels in medulla-pons area, in the cervical spinal cord and in the trigeminal ganglion. No differences between groups were seen as regards CGRP and SP protein expression in the TNC.ConclusionsThese findings support a critical involvement of TRPA1 channels in the pathophysiology of migraine, and show their active role in counteracting hyperalgesia at the trigeminal level.

The effect of melatonin on gene expression of calcitonin gene-related peptide and some proinflammatory mediators in patients with pure menstrual migraine.

The neuropeptide calcitonin gene-related peptide (CGRP), a potent vasoactive and a marker of trigeminal inflammation, has been considered as an important mediator in various types of migraine such as pure menstrual migraine. Earlier studies have shown that CGRP can modulate the synthesis and release of other inflammatory factor including nitric oxide (NO) and interleukin-1beta (IL-1β) from trigeminal ganglion glial cells. Exogenous melatonin protects the tissues from inflammatory damages. The goal of this study was to determine the anti-inflammatory effects of melatonin on the CGRP expression, inducible nitric oxide synthase (iNOS) activity, NO, and IL-1β release in cultured peripheral blood mononuclear cells (PBMCs) from pure menstrual migraine patients and healthy subjects. This study was performed on 12 pure menstrual migraine patients and 12 age-and sex-matched healthy subjects. PBMCs were isolated and treated with melatonin for 12h at pharmacological dose. Gene expression was evaluated by real-time PCR. CGRP and IL-1β proteins in culture supernatant were determined by ELISA method. iNOS activity in PBMCs was determined by colorimetric assays. Total nitrite as an indicator of NO concentrations in the culture supernatants was measured using Griess method. We found that melatonin treatment significantly decreases mRNA expression of CGRP release, NO production, and iNOS activity in the patient groups. Taken together, it appears that melatonin reduces inflammation through decreasing CGRP level and iNOS activity in the patients with migraine; however, further studies are needed in this regard.

Electro-acupuncture promotes the proliferation of neural stem cells and the survival of neurons by downregulating miR-449a in rat with spinal cord injury.

The aim of this study is to investigate the mechanism of electro-acupuncture (EA) on the recovery of injured spinal cord. Rats were randomly divided into normal control, sham-operated, SCI, SCI+EA group and T10 segment spinal cord injury (SCI) rat model was established by the modified Allen's method. After 7 days, the mRNA and protein expression of Nestin, neuron specific nuclear protein (NeuN) and calcitonin gene related peptide (CGRP) were detected by real time RT-PCR, Western blot and immunohistochemistry respectively. The protein expression of cleaved caspase 3, Bax, Bcl-2, TNF-α and IL-1β were also detected by Western blot. MicroRNA 449a (miR-449a) expression was also compared. Further, 12 SCI rats were randomly divided into EA and miR subgroups (EA + miR-449a agomir injection). The expression of Nestin, NeuN, CGRP, cleaved caspase 3, Bax, Bcl-2, TNF-α, IL-1β and miR-449a was compared. The direct interaction of miR-449a and CGRP mRNA was assessed by dual luciferase reporter assay. At day 7, compared with sham-operated group, miR-449a expression in SCI group was significantly increased (P < 0.05), and NeuN and CGRP mRNA and protein expression was markedly decreased (P < 0.05), but protein levels of Nestin, cleaved caspase 3, TNF-α, IL-1β and the ratio of Bax/Bcl-2 in SCI group were significantly increased (P < 0.05). The EA treatment significantly reduced miR-449a level and cleaved caspase 3, TNF-α, IL-1β level and the ratio of Bax/Bcl-2 (P < 0.01), but substantially increased Nestin, NeuN and CGRP expression (P < 0.05 or 0.01). High level of miR-449a in miR subgroup was accompanied by decreased expression of Nestin, NeuN and CGRP and increased expression of cleaved caspase 3, TNF-α, IL-1β and elevation of the ratio of Bax/Bcl-2 (P < 0.05), suggesting miR-449a inhibits the effects of EA on NSCs and neurons. Luciferase reporter assay showed that miR-449a bound to the 3' UTR of CGRP, and thereby regulated CGRP expression. In conclusion, EA promotes proliferation of neural stem cells and the survival of neurons by downregulation of miR-449a expression.

Elevated expression of transient receptor potential vanilloid type1 in dorsal root ganglia of rats with endometriosis.

Pain is the most pronounced complaint of women with endometriosis, however the underlying mechanism is still poorly understood. In the present study, the authors evaluate the effect of transient receptor potential vanilloid type 1 (TRPV1) of dorsal root ganglia (DRG) on endometriosis-associated pain. A total of 36 SD rats were randomly divided into a sham group (n=9) and a Model group (n=27), accepted auto-transplanted pieces of fat or uterus to the pelvic cavity. At 4 weeks, the Model group was randomly subdivided into the following groups: ENDO group (no treatment, n=9), BCTC group (Model + BCTC, an antagonist of TRPV1, n=9), Vehicle group (Model + cyclodextrin, the vehicle of BCTC, n=9). Tail-flick test was performed prior to surgery, 1 h prior to and following treatment of BCTC or cyclodextrin. The expression of TRPV1, substance P (SP), calcitonin gene-related peptide (CGRP) in L1-L6 DRG was measured via immunohistochemistry, western blotting and RT-qPCR. The results indicated that the Model group exhibited a significant decrease in tail flick latency compared to pre-surgical baseline, and the expression of TRPV1, SP, CGRP protein and mRNA in L1-L6 DRG significantly increased compared to the sham group. BCTC significantly improved tail flick latency, and downregulated the expression of TRPV1, SP and CGRP protein and mRNA levels in L1-L6 DRG compared to ENDO group. However, there were no significant differences of those in Vehicle group compared with the ENDO group. Taken together, the current study provides evidence that TRPV1 expressed in DRG may serve an important role in endometriosis-associated pain.