Calcitonin Gene-related Peptide mRNA Research Articles

Subarachnoid Hemorrhage Depletes Calcitonin Gene-Related Peptide Levels of Trigeminal Neurons in Rat Dura Mater.

Subarachnoid hemorrhage (SAH) remains a major cause of cerebrovascular morbidity, eliciting severe headaches and vasospasms that have been shown to inversely correlate with vasodilator calcitonin gene-related peptide (CGRP) levels. Although dura mater trigeminal afferents are an important source of intracranial CGRP, little is known about the effects of SAH on these neurons in preclinical models. The present study evaluated changes in CGRP levels and expression in trigeminal primary afferents innervating the dura mater 72 h after experimentally induced SAH in adult rats. SAH, eliciting marked damage revealed by neurological examination, significantly reduced the density of CGRP-immunoreactive nerve fibers both in the dura mater and the trigeminal caudal nucleus in the medulla but did not affect the total dural nerve fiber density. SAH attenuated ex vivo dural CGRP release by ~40% and in the trigeminal ganglion, reduced both CGRP mRNA levels and the number of highly CGRP-immunoreactive cell bodies. In summary, we provide novel complementary evidence that SAH negatively affects the integrity of the CGRP-expressing rat trigeminal neurons. Reduced CGRP levels suggest likely impaired meningeal neurovascular functions contributing to SAH complications. Further studies are to be performed to reveal the importance of impaired CGRP synthesis and its consequences in central sensory processing.

Analgesic and anti-inflammatory effects of galangin: a potential pathway to inhibit transient receptor potential vanilloid 1 receptor activation.

Galangin, commonly employed in traditional Chinese medicine for its diverse medicinal properties, exhibits potential in treating inflammatory pain. Nevertheless, its mechanism of action remains unclear. Mice were randomly divided into 4 groups for 7 days: a normal control group, a galangin-treated (25 and 50 mg/kg), and a positive control celecoxib (20 mg/kg). Analgesic and anti-inflammatory effects were evaluated using a hot plate test, acetic acid-induced writhing test, acetic acid-induced vascular permeability test, formalininduced paw licking test, and carrageenan-induced paw swelling test. The interplay between galangin, transient receptor potential vanilloid 1 (TRPV1), NF-κB, COX-2, and TNF-α proteins was evaluated via molecular docking. COX- 2, PGE2, IL-1β, IL-6, and TNF-α levels in serum were measured using ELISA after capsaicin administration (200 nmol/L). TRPV1 expression in the dorsal root ganglion was analyzed by Western blot. The quantities of substance P (SP) and calcitonin gene-related peptide (CGRP) were assessed using qPCR. Galangin reduced hot plate-induced licking latency, acetic acid-induced contortions, carrageenantriggered foot inflammation, and capillary permeability in mice. It exhibited favorable affinity towards TRPV1, NF- κB, COX-2, and TNF-α, resulting in decreased levels of COX-2, PGE2, IL-1β, IL-6, and TNF-α in serum following capsaicin stimulation. Galangin effectively suppressed the upregulation of TRPV1 protein and associated receptor neuropeptides CGRP and SP mRNA, while concurrently inhibiting the expression of NF-κB, TNF-α, COX-2, and PGE2 mRNA. Galangin exerts its anti-inflammatory pain effects by inhibiting TRPV1 activation and regulating COX-2, NF-κB/TNF-α expression, providing evidence for the use of galangin in the management of inflammatory pain.

Effect of acupuncture on the expression of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome.

To observe the effect of acupuncture on the expressions of neuropeptides and related inflammatory factors in rats with diarrhea-predominant irritable bowel syndrome(IBS-D), so as to explore the mechanism of acupuncture in the treatment of IBS-D. Male Wistar rats were randomly divided into blank group, model group, medication group, and acupuncture group, with 6 rats in each group. Except for the blank group, the other groups were subjected to 14-day "acetic acid enema + restraint stress" to establish the IBS-D rat model. After successful modeling, the medication group received gavage of pinaverium bromide(15 mg/kg) once a day, and the acupuncture group received acupuncture at "Baihui"(GV20) and bilateral "Tianshu"(ST25), "Shangjuxu"(ST37), "Zusanli"(ST36), and "Taichong"(LR3) for 20 min every day, both groups were treated continuously for 14 days. The general state of the rats in each group was observed, and the body weight of the rats was measured. The open-field experiment was conducted to measure the horizontal and vertical movements, and the number of fecal pellets of rats. The histopathological morphology of hypothalamus and colon of rats was observed by HE staining. Toluidine blue staining was used to observe and count the mast cells(MCs) in the colon tissue of rats. ELISA was used to detect the serum contents of tumor necrosis factor-α(TNF-α) and interleukin(IL)-10. Real-time fluorescence quantitative PCR was performed to detect the mRNA expressions of calcitonin gene-related peptide(CGRP) in the hypothalamus and colon tissue. Western blot was used to detect the expressions of corticotropin-releasing factor(CRF) in the hypothalamus and colon tissue. HE staining showed that there was inflammatory cell infiltration in the lamina propria of colon in the model group, and it was reduced in the other groups. Compared with the blank group, the model group showed significantly decreased body weight, decreased walking distance and upright times in open field experiment, decreased serum IL-10 contents(P<0.05, P<0.01), increased fecal pellet number (P<0.01), increased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions and CRF expressions in the hypothalamus and colon tissue(P<0.01). Compared with the model group, both medication and acupuncture groups showed significantly increased body weight, walking distance and upright times in the open-field experiment, and serum IL-10 contents(P<0.01, P<0.05), significantly decreased fecal pellet number (P<0.05), significantly decreased MC number in the colon tissue, serum TNF-α contents, and CGRP mRNA expressions in the hypothalamus and colon tissue(P<0.01)；at the same time, the acupuncture group showed significantly decreased CRF expressions in the hypothalamus and colon tissue(P<0.01, P<0.05). There was no significant difference in the above indicators between the medication group and the acupuncture group. Acupuncture can improve the general and emotional state, inflammatory response, and neuropeptide expression in rats with IBS-D, and alleviate the symptoms of IBS-D, which may be related to the regulation of neuropeptides and inflammatory factors levels.

Calcitonin Gene-Related Peptide mRNA Synthesis in Trigeminal Ganglion Neurons after Cortical Spreading Depolarization.

Migraine is a debilitating neurovascular disorder characterized by recurrent headache attacks of moderate to severe intensity. Calcitonin gene-related peptide (GGRP), which is abundantly expressed in trigeminal ganglion (TG) neurons, plays a crucial role in migraine pathogenesis. Cortical spreading depolarization (CSD), the biological correlate of migraine aura, activates the trigeminovascular system. In the present study, we investigated CGRP mRNA expression in TG neurons in a CSD-based mouse migraine model. Our in situ hybridization analysis showed that CGRP mRNA expression was observed in smaller-sized neuronal populations. CSD did not significantly change the density of CGRP mRNA-synthesizing neurons in the ipsilateral TG. However, the cell sizes of CGRP mRNA-synthesizing TG neurons were significantly larger in the 48 h and 72 h post-CSD groups than in the control group. The proportions of CGRP mRNA-synthesizing TG neurons bearing cell diameters less than 14 μm became significantly less at several time points after CSD. In contrast, we found significantly greater proportions of CGRP mRNA-synthesizing TG neurons bearing cell diameters of 14-18 μm at 24 h, 48, and 72 h post-CSD. We deduce that the CSD-induced upward cell size shift in CGRP mRNA-synthesizing TG neurons might be causative of greater disease activity and/or less responsiveness to CGRP-based therapy.

Shaoyao Gancao decoction alleviates the central hyperalgesia of recurrent NTG-induced migraine in rats by regulating the NGF/TRPV1/COX-2 signal pathway

Ethnopharmacological relevanceShaoyao Gancao Decoction (SGD) is well known as an effective prescription for analgesia composed of two herbs, and is noted as traditional Chinese medicine morphine. It is widely used in various conditions causing pain, including migraine. However, there is currently no research exploring the mechanism of action in the treatment of migraines. Aim of the studyThe current research was devised to determine the underlying regulatory mechanism of SGD, by verifying its role in the NGF/TRPV1/COX-2 signal pathway. Materials and methodsThe active components in SGD were identified by UHPLC-MS. A migraine model was prepared by subcutaneous (s.c.) injection of nitroglycerin (NTG) into the neck to detect migraine-like behavior, orbital hyperalgesia threshold changes, and the therapeutic effect of SGD. The mechanism of SGD in remedying migraine was studied through transcriptome sequencing (RNA-seq), which was further validated utilizing Elisa, Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting (WB) experiments. ResultsIn the SGD chemical composition analysis, 45 components were identified including gallic acid, paeoniflorin and albiforin. In the behavioral experiments, SGD treatment significantly decreased the score of migraine-like head scratching in the NTG-induced migraine model (Mod) rats, while the hyperalgesia threshold increased outstandingly on days 10, 12, and 14 (P < 0.01, P < 0.001 or P < 0.0001). In migraine biomarkers experiment, compared with the Mod group, the 5-hydroxytryptamine (5-HT) contents were outstandingly enhanced by SGD treatment, while nitric oxide (NO) contents were markedly declined (P < 0.01). In the RNA-seq test, the down-regulated genes of SGD inhibiting hyperalgesia migraine included the neurotrophic factor (NGF) and transient receptor potential vanillic acid subfamily protein 1 receptor (TRPV1). The down-regulation pathway is the inflammatory mediator regulation of TRP channels. In gene set enrichment analysis (GSEA), SGD decreased the over-expression of protooncogene tyrosine-protein kinase Src (SRC) and TRPV1 in this pathway, and the two genes clustered at its lower end, with similar functions. PPI network results show that NGF interacts with TRPV1. Further verification shows that when compared with Mod group, the plasma cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) protein expression levels and the dura mater calcitonin gene-related peptide (CGRP), extracellular signal-regulated kinase (ERK), p-ERK, SRC and NGF protein expression levels in the SGD group were remarkably decreased (P < 0.01, P < 0.001 or P < 0.0001), and the expression level of TRPV1 protein showed a downward trend (P = 0.06). The expression levels of COX-2, NO, CGRP, TRPV1, SRC and NGF mRNA in the dura mater was overtly down-regulated (P < 0.05, P < 0.01 or P < 0.001). ConclusionsSGD has a significant inhibitory effect on the NGF/TRPV1/COX-2 signaling pathway that mediates central hyperalgesia migraine, thus suggesting the molecular mechanism of SGD in improving the symptoms of migraine may be related to the central hyperalgesia neurotransmitter that regulates the pathogenesis of migraine.

Expression of neurotransmitters, vasculogenesis markers and myosin heavy chain isoforms in the masseter muscle of senescence-accelerated mouse prone 8 mice.

Learning and memory deficits and pathologic changes in the hippocampus caused by toothlessness and soft diet feeding are related to reduced masseter muscle (MM) function. Myosin heavy chain (MyHC) isoform expression in the MM also changes under different chewing conditions. The neurotransmitter calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor A (VEGF-A) are involved in MM formation. However, the relationship between CGRP, VEGF-A and MyHC isoforms in the MM in the senescence-accelerated mouse prone 8 (SAMP8) strain, a model of learning and memory deficits, remains unclear. Changes in CGRP, VEGF-A, vasculogenesis marker, and MyHC isoform mRNA expression in the MMs of aging SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice was investigated through quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. qRT-PCR revealed obviously high CGRP levels in the SAMP8 mouse MM (p<0.001). MyHC-IId/x mRNA expression in the MM was higher in 24-week-old SAMP8 mice than 24-week-old SAMR1 mice (p<0.001) but lower in slow-MyHC SAMP8 mice than SAMR1 mice (p<0.001). CGRP mRNA was observed on the muscle fibers of the SAMP8 mouse MM but not the SAMR1 mouse MM through in situ hybridization. Principal component analysis (PCA) revealed strong positive contributions of SAMP8-MyHC-IId/x, SAMP8-CGRP, SAMR1-MyHC-emb, SAMR1-CGRP, SAMR1-VEGF-A, SAMR1-CD31, SAMP8-VEGF-A, and SAMP8-CD31 in the MM at 12 and 24 weeks. CGRP is also key for the MyHC-IId/x and slow-MyHC patterns in the MMs of SAMP8 mice.

A Single Episode of Cortical Spreading Depolarization Increases mRNA Levels of Proinflammatory Cytokines, Calcitonin Gene-Related Peptide and Pannexin-1 Channels in the Cerebral Cortex

Cortical spreading depolarization (CSD) is the neuronal correlate of migraine aura and the reliable consequence of acute brain injury. The role of CSD in triggering headaches that follow migraine aura and brain injury remains to be uncertain. We examined whether a single CSD occurring in awake animals modified the expression of proinflammatory cytokines (Il1b, TNF, and Il6) and endogenous mediators of nociception/neuroinflammation-pannexin 1 (Panx1) channel and calcitonin gene-related peptide (CGRP), transforming growth factor beta (TGFb) in the cortex. Unilateral microinjury of the somatosensory cortex triggering a single CSD was produced in awake Wistar rats. Three hours later, tissue samples from the lesioned cortex, intact ipsilesional cortex invaded by CSD, and homologous areas of the contralateral sham-treated cortex were harvested and analyzed using qPCR. Three hours post-injury, intact CSD-exposed cortexes increased TNF, Il1b, Panx1, and CGRP mRNA levels. The strongest upregulation of proinflammatory cytokines was observed at the injury site, while CGRP and Panx1 were upregulated more strongly in the intact cortexes invaded by CSD. A single CSD is sufficient to produce low-grade parenchymal neuroinflammation with simultaneous overexpression of Panx1 and CGRP. The CSD-induced molecular changes may contribute to pathogenic mechanisms of migraine pain and post-injury headache.

Calcitonin Gene-Related Peptide and Calcitonin mRNA Expression in Periaqueductal Gray Area in the Formalin Test in Rats

Background: Calcitonin gene-related peptide (CGRP) and rat calcitonin (rCT) play critical roles in descending pain control systems. Objectives: The present research aimed to evaluate the effect of the intracerebroventricular (ICV) administration of CGRP and rCT on the mRNA expression of CGRP and rCT peptides in the periaqueductal gray (PAG) of healthy rats in the formalin test. Methods: A total of 24 male Sprague Dawley rats were categorized into four groups (n = 6). One week after stereotaxic surgery, 1.5 nmol CGRP or rCT peptides were injected (ICV) once daily for 7 days. After 20 min from the last injection, the right foot of the animal was injected subcutaneously with 2.5% formalin. Pain-related behaviors were recorded immediately for 60 min. The PAG nucleus was then removed to assess the changes made in the mRNA expression of the CGRP and rCT. Results: ICV injection of CGRP or rCT reduced pain in the different phases of the trial. ICV injection of rCT induced the expression of rCT mRNA in the PAG area (P &lt; 0.05). However, ICV injection of rCT had no significant effect on the CGRP mRNA expression in the PAG area. Moreover, following the ICV injection of CGRP, the expression of rCT mRNA increased in the PAG area (P &lt; 0.05). It is noteworthy that the ICV injection of CGRP caused a significant effect on the CGRP mRNA expression in the PAG area (P &lt; 0.05). Conclusions: The ICV injections of CGRP and rCT peptides decreased pain in the formalin test. Higher mRNA expression of these peptides in the PAG might be a possible mechanism for this observation.

Sex Differences in CGRP Regulation and Function in the Amygdala in a Rat Model of Neuropathic Pain

The amygdala has emerged as a key player in the emotional response to pain and pain modulation. The lateral and capsular regions of the central nucleus of the amygdala (CeA) represent the “nociceptive amygdala” due to their high content of neurons that process pain-related information. These CeA divisions are the targets of the spino-parabrachio-amygdaloid pain pathway, which is the predominant source of calcitonin gene-related peptide (CGRP) within the amygdala. Changes in lateral and capsular CeA neurons have previously been observed in pain models, and synaptic plasticity in these areas has been linked to pain-related behavior. CGRP has been demonstrated to play an important role in peripheral and spinal mechanisms, and in pain-related amygdala plasticity in male rats in an acute arthritis pain model. However, the role of CGRP in chronic neuropathic pain-related amygdala function and behaviors remains to be determined for both male and female rats. Here we tested the hypothesis that the CGRP1 receptor is involved in neuropathic pain-related amygdala activity, and that blockade of this receptor can inhibit neuropathic pain behaviors in both sexes. CGRP mRNA expression levels in the CeA of male rats were upregulated at the acute stage of the spinal nerve ligation (SNL) model of neuropathic pain, whereas female rats had significantly higher CGRP and CGRP receptor component expression at the chronic stage. A CGRP1 receptor antagonist (CGRP 8-37) administered into the CeA in chronic neuropathic rats reduced mechanical hypersensitivity (von Frey and paw compression tests) in both sexes but showed female-predominant effects on emotional-affective responses (ultrasonic vocalizations) and anxiety-like behaviors (open field test). CGRP 8-37 inhibited the activity of CeA output neurons assessed with calcium imaging in brain slices from chronic neuropathic pain rats. Together, these findings may suggest that CGRP1 receptors in the CeA are involved in neuropathic pain-related amygdala activity and contribute to sensory aspects in both sexes but to emotional-affective pain responses predominantly in females. The sexually dimorphic function of CGRP in the amygdala would make CGRP1 receptors a potential therapeutic target for neuropathic pain relief, particularly in females in chronic pain conditions.

Promotion of corneal angiogenesis by sensory neuron-derived calcitonin gene-related peptide

The normal cornea has no blood vessels but has abundant innervation. There is emerging evidence that sensory nerves, originated from the trigeminal ganglion (TG) neurons, play a key role in corneal angiogenesis. In the current study, we examined the role of TG sensory neuron-derived calcitonin gene-related peptide (CGRP) in promoting corneal neovascularization (CNV). We found that CGRP was expressed in the TG and cultured TG neurons. In the cornea, minimal CGRP mRNA was detected and CGRP immunohistochemical staining was exclusively co-localized with corneal nerves, suggesting corneal nerves are likely the source of CGRP in the cornea. In response to intrastromal suture placement and neovascularization in the cornea, CGRP expression was increased in the TG. In addition, we showed that CGRP was potently pro-angiogenic, leading to vascular endothelial cell (VEC) proliferation, migration, and tube formation in vitro and corneal hemangiogenesis and lymphangiogenesis in vivo. In a co-culture system of TG neurons and VEC, blocking CGRP signaling in the conditioned media of TG neurons led to decreased VEC migration and tube formation. More importantly, subconjunctival injection of a CGRP antagonist CGRP8-37 reduced suture-induced corneal hemangiogenesis and lymphangiogenesis in vivo. Taken together, our data suggest that TG sensory neuron and corneal nerve-derived CGRP promotes corneal angiogenesis.

Effect of Fish Stock Density on Hormone Genes Expression from Brain and Gastrointestinal Tract of Salmo salar.

Simple SummaryVarious long-term stress conditions may exist in fish cultivation, damaging the physiological responses that regulate the fish growth and feed. Different signalers connect the brain with the gastrointestinal tract, including the perception of stress factors for the regulation of physiological responses. Here, we evaluated the effect of varying culture densities of Salmo salar post-smolt on the gene expression of some brain and gastrointestinal hormone signalers. We found that high stock densities could promote the levels of molecules associated with feed inhibition, which could be related to the stress pathway regulated by corticoids. Thus, the expression of these peptide hormones could be used as biological markers to improve production practices in fish aquaculture.A variety of long-term stress conditions may exist in fish cultivation, some of which are so severe that fish can no longer reestablish homeostasis. In teleost fish, the brain and gastrointestinal tract integrate signals that include the perception of stress factors regulating physiological responses, such as social stress by fish population density, where peripheral and central signals, such as peptide hormones, are the main regulators. Therefore, we proposed in this study to analyze the effect of different stock densities (SD) in the gene expression of brain neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP), together with the gastrointestinal peptide hormones leptin (Lep), vasointestinal peptide (VIP), and protachykinin-1 (Prk-1) in Salmo salar post-smolt. The coding sequence of S. salar VIP and Prk-1 precursors were firstly cloned and characterized. Then, the mRNA expression of these genes, together with the NPY, Lep, and CGRP genes, were evaluated in post-smolts kept at 11 Kg/m3, 20 Kg/m3, and 40 Kg/m3. At 14 days of culture, the brain CGRP and liver leptin mRNA levels increased three and tenfold in the post-smolt salmons kept at the highest SD, respectively. The high levels of leptin were kept during all the fish culture experiments. In addition, the highest expression of intestine VIP mRNA was obtained on Day 21 in the group of 40 Kg/m3 returning to baseline on Day 40. In terms of stress biochemical parameters, cortisol levels were increased in the 20 Kg/m3 and 40 Kg/m3 groups on Day 40 and were the highest in the 20 Kg/m3 group on Day 14. This study provides new insight into the gastrointestinal signals that could be affected by chronic stress induced by high stock density in fish farming. Thus, the expression of these peptide hormones could be used as molecular markers to improve production practices in fish aquaculture.

Characteristic expression of CGRP and osteogenic and vasculogenic markers in the proximal and distal regions of the rib during male mouse development

BackgroundNeuropeptide calcitonin gene-related peptide (CGRP) is a neurotransmitter related to vasculogenesis and osteogenesis during bone formation and organ development. From the foetal period to the postnatal period, the thorax, which is necessary for lung respiration, forms. The thorax exhibits the same cartilage ossification as the bones of the extremities, but a specific system within the thorax exists as costal cartilage after birth. The relationship among CGRP, osteogenesis and vasculogenic markers in the two rib locations during thorax formation is not fully understood. Materials and methodsIn our study, male mice were used to provide ribs under different development conditions on various embryonic days (E12. 5, E14.5, and E17.5) and postnatal days (P1 and P5). The mRNA expression levels of CGRP, vascular endothelial growth factor (VEGF-A), type 1 collagen (Col1a-1), type 2 collagen (Col2a1), neuropeptide Y (NPY), osteocalcin (OCN) and osteopontin (OPN) were analysed by qRT-PCR. We also analysed the mRNA expression of CGRP, VEGF-A and OPN by in situ hybridization. Multivariate modelling with principal component analysis (PCA) was performed to estimate the interactions among the quantitative real-time RT-PCR data. ResultsThe mRNA expression levels of CGRP, VEGF-A, Col2a, Col1a-1, OCN, and NPY in the male mouse rib gradually increased during development. An antisense probe for CGRP mRNA was strongly detected in the central region of the mouse rib at E12.5 and the hypertrophic and ossification zones at E17.5 by in situ hybridization. VEGF-A was also located in the same region as CGRP at E12.5 and E17.5. OPN was strongly detected at the rib formation stage from E14.5 to E17.5. The expression of CGRP also differed between the proximal and distal regions of the rib at E17.5. As demonstrated by in situ hybridization, CGRP continuously participates in cartilage formation in the distal regions of the rib after birth. The PCA revealed that the mRNA expression of CGRP was related to that of Col1a-1 and VEGF-A during rib formation. ConclusionThis study shows that CGRP is involved in vascular and bone formation during rib development and may also be involved in cartilage formation after birth. The findings suggest that CGRP may temporarily participate in bone formation and continuously participate in cartilage formation in the rib, which may also be related to the formation of the anterior thoracic wall after birth

Effect of Chinese Medicine Xinmaitong on Blood Pressure in Spontaneously Hypertensive Rats.

Objective To investigate the effect of traditional Chinese antihypertensive compound Xinmaitong on blood pressure and vasoactive factors of vasoconstrictor endothelin-1 (ET-1) and vasodilator calcitonin gene related peptide (CGRP) in spontaneously hypertensive rats (SHRs) with early stage hypertension. Methods Twenty male SHRs were randomly divided into two groups: 10 for hypertensive control group and 10 for hypertensive treatment group. In addition, 10 Wistar rats were used as the normal control group without any intervention. SHRs of hypertensive treatment group were orally treated with Xinmaitong, while the hypertensive control group was treated with the normal saline (NS) for a total of eight weeks. The blood pressure in SHRs was examined before and after the end of the eight-week study. After treatment, the rats were killed and the blood samples were collected to measure plasma levels of ET-1 and CGRP by ELISA method, respectively. Meanwhile, the aorta rings were isolated for measuring the mRNA expression of ET-1 and CGRP by PCR. Moreover, the protein levels of ET-1 and CGRP were studied by immunohistochemical. Results Daily oral administration of Xinmaitong resulted in significant fall in the SHRs' blood pressure, including systolic and diastolic blood pressures (SBP and DBP), mean blood pressure (MBP), and pulse pressure (PP). The plasma ET-1 levels were reduced and CGRP increased. In parallel, the mRNA and protein expression of ET-1 were decreased, whereas the mRNA and protein expression of CGRP were enhanced in SHRs treated with Xinmaitong. Conclusion The present study demonstrated for the first time that Xinmaitong leads to the fall in blood pressure of SHRs and that this antihypertensive effect is, at least in part, due to improvement of arterial tone.

Inhibition of NR2A reduces calcitonin gene-related peptide gene expression induced by cortical spreading depression in rat amygdala

Despite robust evidence on the role of calcitonin gene-related peptide (CGRP) in migraine via both central and peripheral actions, relatively less is known about how CGRP in the limbic system is involved in migraine progression. This study investigated whether CGRP production machinery exists in the two key limbic regions including hippocampus and amygdala using cortical spreading depression (CSD) as a model of migraine and whether such alteration by CSD is sensitive to N-methyl-d-aspartate (NMDA) receptor regulation in rats. A single or repetitive CSD was induced by topical application of KCl and monitored using electrophysiological methods. The NR2A-containing NMDA receptor antagonist, NVP-AAM077, or its vehicle, was perfused into the contralateral cerebroventricular ventricle of rat. Quantitative PCR was used to measure CGRP mRNA levels in the ipsilateral and contralateral hippocampus and amygdala after CSD events and compared to respective sham treatments. The results showed that neither a single CSD nor repetitive CSD affected CGRP mRNA levels in both the contralateral and ipsilateral hippocampus at 24 h post CSD induction. Differently, significant elevation of CGRP gene expression was observed in the ipsilateral amygdala at 24 h post multiple CSD, but not contralateral side, and not post-single CSD. Further results showed that the CSD-induced CGRP gene expression in the amygdala was markedly reduced by NVP-AAM077 and this reduction corresponded to a reduced cortical susceptibility to CSD in rats. We conclude that repetitive CSD events induce CGRP gene expression in amygdala, which is sensitive to NR2A regulation.

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.

Calcitonin gene-related peptide facilitates sensitization of the vestibular nucleus in a rat model of chronic migraine

BackgroundVestibular migraine has recently been recognized as a novel subtype of migraine. However, the mechanism that relate vestibular symptoms to migraine had not been well elucidated. Thus, the present study investigated vestibular dysfunction in a rat model of chronic migraine (CM), and to dissect potential mechanisms between migraine and vertigo.MethodsRats subjected to recurrent intermittent administration of nitroglycerin (NTG) were used as the CM model. Migraine- and vestibular-related behaviors were analyzed. Immunofluorescent analyses and quantitative real-time polymerase chain reaction were employed to detect expressions of c-fos and calcitonin gene-related peptide (CGRP) in the trigeminal nucleus caudalis (TNC) and vestibular nucleus (VN). Morphological changes of vestibular afferent terminals was determined under transmission electron microscopy. FluoroGold (FG) and CTB-555 were selected as retrograde tracers and injected into the VN and TNC, respectively. Lentiviral vectors comprising CGRP short hairpin RNA (LV-CGRP) was injected into the trigeminal ganglion.ResultsCM led to persistent thermal hyperalgesia, spontaneous facial pain, and prominent vestibular dysfunction, accompanied by the upregulation of c-fos labeling neurons and CGRP immunoreactivity in the TNC (c-fos: vehicle vs. CM = 2.9 ± 0.6 vs. 45.5 ± 3.4; CGRP OD: vehicle vs. CM = 0.1 ± 0.0 vs. 0.2 ± 0.0) and VN (c-fos: vehicle vs. CM = 2.3 ± 0.8 vs. 54.0 ± 2.1; CGRP mRNA: vehicle vs. CM = 1.0 ± 0.1 vs. 2.4 ± 0.1). Furthermore, FG-positive neurons was accumulated in the superficial layer of the TNC, and the number of c-fos+/FG+ neurons were significantly increased in rats with CM compared to the vehicle group (vehicle vs. CM = 25.3 ± 2.2 vs. 83.9 ± 3.0). Meanwhile, CTB-555+ neurons dispersed throughout the VN. The structure of vestibular afferent terminals was less pronounced after CM compared with the peripheral vestibular dysfunction model. In vivo knockdown of CGRP in the trigeminal ganglion significantly reduced the number of c-fos labeling neurons (LV-CGRP vs. LV-NC = 9.9 ± 3.0 vs. 60.0 ± 4.5) and CGRP mRNA (LV-CGRP vs. LV-NC = 1.0 ± 0.1 vs. 2.1 ± 0.2) in the VN, further attenuating vestibular dysfunction after CM.ConclusionsThese data demonstrates the possibility of sensitization of vestibular nucleus neurons to impair vestibular function after CM, and anti-CGRP treatment to restore vestibular dysfunction in patients with CM.

Immunomodulatory and anti-oxidative effect of the direct TRPV1 receptor agonist capsaicin on Schwann cells

BackgroundOnly few studies describe the impact of nutritive factors on chronic inflammatory demyelinating polyneuropathy (CIDP), an inflammatory disease of the peripheral nervous system. The active component of chili pepper, capsaicin, is the direct agonist of the transient receptor potential channel vanilloid subfamily member 1. Its anti-inflammatory effect in the animal model experimental autoimmune neuritis (EAN) has been previously demonstrated.MethodsIn the present study, we describe the anti-inflammatory and anti-oxidative influence of capsaicin on Schwann cells (SCs) in an in vitro setting. Hereby, we analyze the effect of capsaicin on Schwann cells’ gene expression pattern, major histocompatibility complex class II (MHC-II) presentation, and H2O2-induced oxidative stress. Furthermore, the effect of capsaicin on myelination was examined in a SC-dorsal root ganglia (DRG) coculture by myelin basic protein staining. Finally, in order to investigate the isolated effect of capsaicin on SCs in EAN pathology, we transplant naïve and capsaicin pre-treated SCs intrathecally in EAN immunized rats and analyzed clinical presentation, electrophysiological parameters, and cytokine expression in the sciatic nerve.ResultsIn SC monoculture, incubation with capsaicin significantly reduces interferon gamma-induced MHC-II production as well as toll-like receptor 4 and intercellular adhesion molecule 1 mRNA expression. Calcitonin gene-related peptide mRNA production is significantly upregulated after capsaicin treatment. Capsaicin reduces H2O2-induced oxidative stress in SC in a preventive, but not therapeutic setting. In a SC-DRG coculture, capsaicin does not affect myelination rate. After intrathecal transplantation of naïve and capsaicin pre-treated SCs in EAN-immunized rats, naïve, but not capsaicin pre-treated intrathecal SCs, ameliorated EAN pathology in rats.ConclusionsIn conclusion, we were able to demonstrate a direct immunomodulatory and anti-oxidative effect of capsaicin in a SC culture by reduced antigen presentation and expression of an anti-inflammatory profile. Furthermore, capsaicin increases the resistance of SCs against oxidative stress. A primary effect of capsaicin on myelination was not proven. These results are in concordance with previous data showing an anti-inflammatory effect of capsaicin, which might be highly relevant for CIDP patients.

Majie cataplasm provides a shield against asthmatic punch from the neuroimmune system

Asthma poses a threat to human health, and its pathogenesis is closely related to the neuroimmune system. Majie cataplasm can not only regulate the immune system but also the nervous system in asthma patients for its components. We speculate that Majie cataplasm may relieve asthmatic patients with sensitivity to hormone or not by regulating the body's neuroimmune system. MethodsIn this experiment, a mouse model of asthma was well established by ovalbumin. The lung function of animals was examined and pathological changes in the lung tissue were assessed by hematoxylin-eosin staining. Serum immunoglobulin E (IgE), calcitonin gene-related peptide (CGRP) and neurokinin A (NKA) were measured by ELISA. The location of CGRP, CD3 and neutrophil in lung tissue and their expressions were detected by immunofluorescence staining. In addition, contents of CGRP mRNA, Substance P (SP) mRNA, interleukin (IL)-17 mRNA and interleukin(IL)-13 mRNA were detected by quantitative polymerase chain reaction. ResultsCompared with the asthma model group, Majie cataplasm and dexamethasone can not only equivalently relieve airway hyperresponsiveness, but also make the content of serum IgE reduced. In addition, they can lower the content of serum CGRP and NKA after OVA stimulation, and this effect was more obvious for Majie cataplasm. Our results also showed that Majie Cataplasm and dexamethasone could inhibit the secretion of CGRP and the infiltration of T lymphocytes together with neutrophils in lung tissue and reduce expressions of CGRP mRNA, SP mRNA, IL-17 mRNA and IL-13 mRNA in lung tissue. ConclusionMajie cataplasm effectively relieves expressions of neuropeptides such as CGRP, reduces the infiltration of immune cells in lung tissue, regulates the body's neuroimmune system, and has a therapeutic potential for both Th2 asthma and neutrophilic asthma.

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.

Leukocyte Apheresis Using a Fiber Filter Suppresses Colonic Injury Through Calcitonin Gene-Related Peptide Induction.

The aim of this study was to address whether the therapeutic effect of leukocytapheresis (LCAP) depends on calcitonin gene- related peptide (CGRP) induction. An HLA-B27 transgenic rat model was treated with an LCAP column. The effects of LCAP on clinical, endoscopic, and histologic disease activity, the colony-forming ability of colony-forming unit (CFU)-granulocyte macrophages (GMs), colonic blood flow, and tissue expression of tumor necrosis factor (TNF)-α and CGRP were examined. Changes in the effects of LCAP after pretreatment with the CGRP antagonist CGRP8-37 were also observed. A dextran sulfate sodium-induced colitis rat model included treatment with CGRP, and the effect was assessed based on clinical, endoscopic, and histologic disease activity, colonic blood flow, the colony-forming ability of CFU-GMs, and tissue expression of inflammatory cytokines and CGRP receptor families. LCAP improved disease activity, enhanced colonic blood flow, and induced the bone marrow colony-forming ability of CFU-GMs with an increase in CGRP mRNA levels. These effects were abolished by pretreatment with CGRP8-37. The administration of CGRP suppressed colitis, promoting colonic blood flow, inducing bone marrow-derived cells, downregulating inflammatory cytokines, and upregulating receptor activity-modifying protein-1. The mRNA and protein levels of inflammatory cytokines in lipopolysaccharide-stimulated mononuclear cells were also decreased after CGRP treatment. The therapeutic effects of LCAP depend on CGRP induction. CGRP can effectively suppress colitis through the downregulation of inflammatory events and upregulation of protective events.