Rat Calcitonin Gene-related Peptide Research Articles

Study of the Central Injection Effects of Chrysin on Behavioral and Intra Hypothalamic Gene Expression Levels of CRH and CGRP in Male Rats

Background: Chrysin is a natural flavonoid with several demonstrated neuro-pharmacological effects in brain areas related to anxiety. However, the intra-hypothalamic molecular mechanisms underlying the anxiolytic effects of chrysin remain unclear. Objectives: The current study revealed the effects of chrysin on hypothalamic corticotrophin-releasing hormone (CRH) and calcitonin gene-related peptide (CGRP) gene expression levels in a rat model of stress. Methods: Thirty male Wistar rats weighing 200 ± 10 g were divided into six groups for this investigation. Acute restraint stress was induced in the animals for 2 hours. Intact or stress-induced rats received 20 or 40 µg of chrysin via the third cerebral ventricle, respectively. Open-field and forced swimming tests were performed to evaluate stress-related behaviors. Hypothalamic samples were then removed, and real-time polymerase chain reaction (PCR) was used to measure relative gene expression. Results: The results showed that in the rats receiving chrysin, CRH and CGRP gene expression levels were significantly decreased compared to the stress group. Additionally, chrysin injection reduced anxiogenic behaviors. Conclusions: Chrysin decreased the expression of hypothalamic CRH and CGRP genes in stressed rats.

Aryl hydrocarbon receptors improve migraine-like pain behaviors in rats through the regulation of regulatory T cell/T-helper 17 cell-related homeostasis.

To investigate the effect of the aryl hydrocarbon receptor (AHR)/regulatory T cell (Treg)/T-helper 17 (Th17) cell pathway on the pathogenesis of migraine. Migraine is a disabling neurovascular disease that imposes an enormous burden on both individuals and society. The pathophysiological mechanisms of migraine remain controversial. Recent studies have suggested that immune dysfunction may be involved in the pathogenesis of migraine. The AHR, a receptor expressed on most immune cells, has been implicated in the occurrence of many autoimmune diseases; however, whether it is involved in the pathogenesis of migraine is unclear. A chronic migraine rat model was established through repeated intraperitoneal injection of nitroglycerin (NTG). The mechanical and thermal pain thresholds were assessed using von Frey filaments and radiant heat. Next, the protein expression levels of AHR in the trigeminal nucleus caudalis (TNC) region of chronic migraine (CM)-like rats were quantified and the changes in Treg/Th17-related transcription factors and inflammatory factors in the TNC were explored. To determine the role of AHR in CM, we examined the effects of the AHR agonist 2-(1'-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and AHR antagonist CH-223191 on pain behavior, c-Fos, calcitonin gene-related peptide (CGRP), AHR, and Treg/Th17-related factor expression in CM-like rats. Repeated administration of NTG significantly enhanced nociceptive hypersensitivity and increased expression of c-Fos and CGRP in rats, while AHR was significantly decreased in the TNC. In addition, the expression of the transcription factor forkhead box protein P3 and the signal transducer and activator of transcription 5 decreased significantly. In contrast, the expression of the transcription factor retinoic acid receptor-related orphan receptor γ t and signal transducer and activator of transcription 3 were significantly increased. Moreover, the mRNA level of transforming growth factor beta-1 was decreased, while that of interleukin (IL)-10 and IL-22 was increased in the TNC. The AHR agonist ITE alleviated migraine-like pain behaviors in rats, activated the AHR signaling pathway, and improved the imbalance of Treg/Th17-related transcription factors and inflammatory factors. Conversely, the AHR antagonist CH-223191 did not alleviate migraine-like pain behaviors in rats; and even exacerbated them. The AHR participates in the development of CM by regulating Treg/Th17-related homeostasis. Therefore, treatments targeting the AHR/Treg/Th17 signaling pathway could be new effective interventions for CM treatment.

Adolescent binge-like alcohol exposure dysregulates NPY and CGRP in rats: Behavioural and immunochemical evidence

Alcohol binge drinking during adolescence impacts affective behaviour, possibly impinging on developing neural substrates processing affective states, including calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY). Here, we modelled binge-like alcohol exposure in adolescence, by administering 3.5 g/kg alcohol per os, within 1 h, to male adolescent rats every other day, from postnatal day 35 to 54. The effects on positive and negative affective behaviour during abstinence were explored including: consummatory behaviour and weight gain; social behaviour in the modified social interaction test; thermal nociception in the tail-flick test; psychosocial stress coping in the resident-intruder paradigm. Moreover, CGRP and NPY levels were evaluated in functionally relevant brain regions. Our data shows that binge-like intermittent alcohol administration during adolescence decreased weight gain, social preference and motivation, nociception, and active psychosocial stress coping during abstinence. In addition, intermittent alcohol-exposed rats displayed increased expression of CGRP and NPY in the prefrontal cortex and nucleus accumbens; decreased NPY levels in the amygdala; opposite changes in CGRP levels in the hypothalamus and brainstem. Overall, our data shows that adolescent binge-like alcohol exposure, through the allostatic load of alternate intoxication and withdrawal, produces long-term consequences in sensory and affective processes and dysregulated complementary neuropeptidergic systems. Thus, neuropeptide-targeted interventions hold promising potential for addressing negative affect during prolonged withdrawal in young subjects.

Emodin Interferes With Nitroglycerin-Induced Migraine in Rats Through CGMP-PKG Pathway.

The purpose of this research was to explore the effect and mechanism of emodin in interfering with nitroglycerin-induced migraine rats. We carried out behavioral research within 2 h post-nitroglycerin (NTG) injection, and blood samples were collected through the abdominal aorta for measurements of nitric oxide (NO), calcitonin gene-related peptide (CGRP), substance P (SP), tumor necrosis factor (TNF-α) and cyclic guanosine monophosphate (cGMP) levels. Immunohistochemistry was adopted to detect the activation of c-Fos immunoreactive neurons in brain tissues. The number and integrated optical density (IOD) of c-Fos positive cells were measured using Image-Pro Plus. Western blotting was applied to detect the levels of PKG protein in rat brain tissues. The results showed that emodin can alleviate the pain response of migraine rats and significantly reduce the levels of NO, CGRP, SP, TNF-α and cGMP in migraine rats. In addition, emodin can significantly reduce the number of c-Fos positive cells and the IOD value. Moreover, the expression of PKG protein was significantly inhibited by emodin. Therefore, it is inferred that emodin can relieve migraine induced by NTG through the cGMP-PKG pathway, and can be used as a potential botanical medicine for the treatment of migraine.

Effect of Cholecystokinin-8 (CCK-8) on Blood Pressure and Blood Content of Calcitonin-Gene-Related Peptide (CGRP) in Rats with Hypertension Caused by Fructose or Inhibition of Nitric Oxide Synthesis

We studied the effect of CCK-8 on BP and blood content of CGRP in rats with hypertension caused by fructose or inhibition of NO synthase with L-NAME. The decrease in the CGRP content was found during the development of fructose-induced hypertension, but not L-NAME-caused hypertension. Administration of CCK-8 to fructose-fed animals reduced BP and increased the content of CGRP. In rats with hypertension caused by NO deficit, CCK-8 lowered BP, but did not affect the content of CGRP. These findings suggest that CGRP mediates the hypotensive effect of CCK-8 in fructose-induced hypertension, but not in NO-deficient hypertension.

Effect of electroacupuncture of "Zhiyang" (GV9) and "Jizhong" (GV6) on expression of CGRP and NLRP3 in rats with spinal cord injury

To observe the effect of electroacupuncture (EA) at "Zhiyang" (GV9) and "Jizhong" (GV6) of the Governor Vessel on the expression of calcitonin gene related peptide (CGRP)and NOD-like receptor pyrin containing 3 (NLRP3) in the injured anterior horn (AH) of rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI. Thirty-six male SD rats were randomly divided into sham operation (sham) group, model group and EA group which were further randomly allocated to 7 day (d) and 14 d subgroups (n=6 per subgroup). The SCI model was established by clamping the exposed spinal cord with an aneurysm clip. Rats of the EA group received EA of GV6 and GV9 for 30 min, once daily for 7 and 14 days, respectively. The Basso, Beattie and Bresnahan (BBB) was used to assess changes of the locomotor function on the 1st, 3rd, 7th and 14th day after SCI. The protein expression levels of CGRP, NLRP3, apoptosis-associated speck-like protein (ASC) and cysteinyl aspartate specific protease-1 (caspase-1) in the anterior horn region of the spinal cord on the 7th and 14th after SCI were detected by Western blot. The BBB scores on the 1st, 3rd, 7th and 14th d after SCI were significantly lower in the model group than in the sham group (P<0.05), whereas the expression levels of NLRP3, ASC and caspase-1 proteins on day 7 and 14 were markedly higher in the model group than in the sham group (P<0.05, P<0.01). Compared with the model group, the BBB score and CGRP expression on the 7th and 14th d were significantly increased in the EA group (P<0.05,P<0.01), while the expression levels of NLRP3, ASC and caspase-1 were obviously decreased in the EA group (P<0.01). EA of GV6 and GV9 can improve the locomotion of SCI rats, which may be associated with its function in up-regulating the expression of CGRP and down-regulating the expression of NLRP3, ASC and caspase-1 proteins in the spinal anterior horn tissue.

Effects of manual loading on calcitonin gene-related peptide and nerve growth factor in rats with chronic low back pain

To observe the analgesic effect of manipulation loading on chronic low back pain (CLBP) model rats and the expression of inflammatory factors in psoas major muscle tissue, and to explore the improvement of manipulation on local inflammatory microenvironment. Thirty two SPF male SD rats weighing 340-360g were randomly divided into blank group, sham operation group, chronic low back pain model group and treatment group, with 8 rats in each group. In the model group, L4-L6 lumbar vertebrae were implanted with external link fixation system (ELFS). After implantation of ELFS, the treatment group received manualintervention with 5N force and 2Hz frequency on both sides of the spine, 15 min / time, once a day, for 14 consecutive days. Paw with drawl threshold (PWT) and paw withdrawl latency (PWL) was measured before modeling and on the 1st, 3rd, 7th, 10th and 14th day after intervention. At the end of the treatment cycle, the concentrations of calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) in psoas muscle were detected by enzyme-linked immunosorbent assay (ELISA). There was no significant difference in PWT and PWL between the blank group and the sham operation group after modeling (P>0.05);after modeling, PWT and PWL in the CLBP model group and the treatment group were significantly decreased(P<0.01);PWT in the treatment group was not significantly improved than that in the CLBP model group on the 1st and 3rd day after manual loading(P>0.05);on the 7th day after manual loading, the pain threshold value in the treatment group was higher than that in the CLBP model group, but there was no significant difference There was no significant difference between the two groups (P=0.056>0.05). On the 10th and 14th day of treatment, the mechanical pain threshold of the treatment group began to rise, and it was statistically significant compared with CLBP model rats (P<0.05, P< 0.01);on the 1st and 3rd day after manual treatment, the PWL of the treatment group was not significantly improved compared with CLBP model group (P>0.05);on the 7th day, the PWL of the treatment group was significantly higher than that of CLBP model group, there was statistical significance (P=0.016<0.05). Manual loading improved thermal hyperalgesia in CLBP rats until the end of the experiment. The contents of CGRP and NGF in psoas major muscle of CLBP model group were higher than those of blank group and sham operation group (P<0.01). After treatment, the contents of CGRP and NGF decreased significantly(P<0.01). Local massage loading has analgesic effect on CLBP rats, at the same time, it can inhibit the content of CGRP and NGF in psoas muscle tissue of CLBP rats, and improve the local inflammatory microenvironment.

Measuring protein biomarker concentrations using antibody tagged magnetic nanoparticles

Under physiological conditions biomarker concentrations tend to rise and fall over time e.g. for inflammation. Ex vivo measurements provide a snapshot in time of biomarker concentrations, which is useful, but limited. Approaching real time monitoring of biomarker concentration(s) using a wearable, implantable or injectable in vivo sensor is therefore an appealing target. As an early step towards developing an in vivo biomarker sensor, antibody (AB) tagged magnetic nanoparticles (NPs) are used here to demonstrate the in vitro measurement of ∼ 5 distinct biomarkers with high specificity and sensitivity. In previous work, aptamers were used to target a given biomarker in vitro and generate magnetic clusters that exhibit a characteristic rotational signature quite different from free NPs. Here the method is expanded to detect a much wider range of biomarkers using polyclonal ABs attached to the surface of the NPs. Commercial ABs exist for a wide range of targets allowing accurate and specific concentration measurements for most significant biomarkers. We show sufficient detection sensitivity, using an in-house spectrometer to measure the rotational signatures of the NPs, to assess physiological concentrations of hormones, cytokines and other signaling molecules. Detection limits for biomarkers drawn mainly from pain and inflammation targets were: 10 pM for mouse Granzyme B (mGZM-B), 40 pM for mouse interferon-gamma (mIFN-γ), 7 pM for mouse interleukin-6 (mIL-6), 40 pM for rat interleukin-6 (rIL-6), 40 pM for mouse vascular endothelial growth factor (mVEGF) and 250 pM for rat calcitonin gene related peptide (rCGRP). Much lower detection limits are certainly possible using improved spectrometers and nanoparticles.

Влияние активации ванилоидных рецепторов эводиамином на содержание кальцитонин-ген-родственного пептида в крови крыс в норме и при гипертензии, вызванной потреблением фруктозы

Background: Calcitonin-gene-related peptide (CGRP) is a neurotransmitter of capsaicin-sensitive nerves, one of the most powerful vasodilators, in mediation of vasoactive action of which the nitrogen oxide (NO) takes part. The aim of this study was to evaluate the CGRP and NO blood levels in normal rats and rats with fructose-induced hypertension, with unaffected transient receptor potential vanilloid 1 (TRPV1) receptors, and after the administration of their agonist evodiamine. Male Wistar Rats were given to drink either 12.5% fructose solution or tap water (control) for 10 weeks. Evodiamine (10 mg/kg), a TRPV1 agonist, was administered intraperitoneally every other day, for 2 weeks, starting 8 weeks after the start of the fructose intake. The systolic blood pressure was measured using a noninvasive technuque. The glucose levels, fasting and 1 hour after the glucose load (2g/kg, i.p), were measured by a glucometer in the blood sampled from the caudal vein. Concentrations of CGRP, NO metabolites (NOx), triglycerides (TG) and lipid peroxidation products (LPO) were measured in the blood serum of rats. The fructose consumption led to hypertension, impaired glucose tolerance, increased the blood levels of LPO products and TG, and decreased the CGRP blood content. The evodiamine introduction to fructose-consuming rats restored the blood CGRP content to the control values, reduced the blood pressure, LPO products and TG levels, and improved the glucose tolerance. The NOx levels did not differ from the control in all groups. We suggest that CGRP is involved in the development of the fructose-induced hypertension and in mediating the therapeutic effect of the evodiamine.

The Presence of Calcitonin Gene-Related Peptide and Its Receptors in Rat, Pig and Human Brain: Species Differences in Calcitonin Gene-Related Peptide Pharmacology

Aim: The aim of present study is to investigate the binding characteristics of non-peptide calcitonin gene-related peptide (CGRP) receptor antagonists (i.e., gepants) in the brain membranes of rat, pig and human. Methods: The interaction of available gepants with the CGRP receptor was studied in the brain membranes of 3 different species using a radioligand competitive binding assay. In addition, the distribution of CGRP and its receptor component receptor activity modifying protein 1 (RAMP1) in rat cerebellum and cortex was explored using immunohistochemistry. Results: All gepants, except SB268262, displaced 100% of the radioligand specific binding in the brain tissue of all 3 species and showed highest affinity for CGRP receptors in human brain as compared to rat and pig brain membranes. Furthermore, radioligand binding studies revealed the presence of higher CGRP receptor density in human cerebellum compared to human cortex. The morphology, size and density of CGRP immunoreactive cells suggest that all cerebral cortical neurons were positive for CGRP. Slender receptor immunoreactive fibres were found spanning through the entire cortex. CGRP immunoreactivity was displayed in the cell soma of cerebellar Purkinje cells and in large neurons in the medial cerebellar nucleus. RAMP1 was found on the surface of the Purkinje cells and in parallel fibres, indicating presence in the granule cell axons. Conclusion: Cerebellum and cerebral cortex are rich in CGRP and CGRP receptors, which can be antagonized by gepants. However, all gepants display higher affinity for human CGRP receptors as compared to rat and pig CGRP receptors. Furthermore, human cerebellum seems to express higher density of CGRP receptors.

CGRP derived from cardiac fibroblasts is an endogenous suppressor of cardiac fibrosis.

Aberrant activation of cardiac fibroblasts leads to cardiac fibrosis, and evolving evidences suggest that endogenous bioactive substances derived from cardiac fibroblasts regulate cardiac fibroblasts activation in an autocrine/paracrine manner. Here we first presented evidence that cardiac fibroblasts can synthesize and secrete calcitonin gene-related peptide (CGRP), therefore, this study aimed to investigate the role of cardiac fibroblasts-derived CGRP in cardiac fibroblasts activation and its regulative mechanism. The abundantly expression of CGRP in rat, mouse, and human myocardium allowed us to explore the cellular origin of CGRP, and found that the cardiac CGRP was mainly derived from cardiac fibroblasts. Activating TRPA1 with a specific agonist allyl isothiocyanate promoted the synthesis and secretion of CGRP, as well as intracellular Ca2+. These effects were reversed by TRPA1-specific antagonist HC030031 and Ca2+ chelator BAPTA-AM. TGF-β1 was applied to induce the activation of cardiac fibroblasts, and found that TGF-β1 can increase the mRNA expression and secretion levels of CGRP in cardiac fibroblasts. Either CGRP8-37 (CGRP receptor antagonist) or α-CGRP small interfering RNA (siRNA) aggravated TGF-β1-induced proliferation, differentiation, collagen production, and instigated inflammation in cardiac fibroblasts. Moreover, TGF-β1-induced NF-κB activation including IκBα phosphorylation and p65 nuclear translocation were also promoted by CGRP8-37 and α-CGRP siRNA. NF-κB inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) reversed the effects of CGRP8-37 on NF-κB activation. The promotive effects of CGRP8-37 on TGF-β1-induced activation of cardiac fibroblasts were all reversed by PDTC. Monocrotaline (MCT) induces pulmonary arterial hypertension, progressively leading to right ventricular fibrosis. This model of cardiac fibrosis was developed here to test the potentially beneficial effects of TRPA1 activation in vivo. The non-toxic TRPA1 agonist Cinnamaldehyde (CA) inhibited MCT-induced elevation in right ventricle systolic pressure, RV/LV + S, and right ventricular collagen accumulation, as well as down-regulation of CGRP. CA increased the synthesis and secretion of CGRP, and inhibited TGF-β1-induced activation in cardiac fibroblasts. Our data suggested an autocrine role for cardiac fibroblasts-derived CGRP in suppressing activation of cardiac fibroblasts through inhibiting NF-κB activation. Increasing autocrine CGRP by activating TRPA1 can ameliorate cardiac fibrosis. These findings support the notion that CGRP derived from cardiac fibroblasts is an endogenous suppressor of cardiac fibrosis.

Effect of Transcutaneous Electrical Stimulation of Nerves on Blood Pressure and Blood Content of Neuropeptide CGRP and Nitric Oxide in Hypertensive Rats with Metabolic Disturbances.

The development of arterial hypertension in male Wistar rats with fructose-induced metabolic syndrome (12.5% of fructose solution as the only drinking source for 10 weeks) along with impaired glucose tolerance and increased serum concentration of triglycerides and LPO products caused a decrease in the content of serum blood calcitonin gene-related peptide (CGRP). Low-frequency transcutaneous electrical nerve stimulation (1 mA, 2 Hz, 10 min daily for 2 weeks) performed in 8 weeks after the beginning of fructose treatment reduced systolic BP and serum concentration of triglycerides and LPO produces and improved glucose tolerance. After stimulation, CGRP content in rats maintained on fructose diet returned to normal values and the content of nitric oxide metabolites increased. We hypothesize that CGRP and nitric oxide are involved in mechanisms mediating the therapeutic effect of low-frequency transcutaneous electrical nerve stimulation on arterial hypertension developing in metabolic syndrome.

Intranasal Calcitonin Gene-Related Peptide Protects Against Focal Cerebral Ischemic Injury in Rats Through the Wnt/β-Catenin Pathway.

BackgroundIntranasal calcitonin gene-related peptide (CGRP) delivery offers a noninvasive method of bypassing the blood-brain barrier for the delivery of CGRP to the brain. Here, we first reported the therapeutic benefits of intranasal CGRP delivery in rats following middle cerebral artery occlusion (MCAO).Material/MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) assay, enzyme-linked immunosorbent assay (ELISA), rat MCAO model, TTC (2, 3, 5-triphenyltetrazolium chloride) staining, hematoxylin and eosin (H & E) staining, Morris water maze test, TUNEL assay, immunofluorescence, and western blot assay were used to investigate the role of CGRP in rats. Cell Counting Kit-8 assay, colony formation assay, cell cycle assay, apoptosis assay, western blot assay, and TOP/FOP assay were used to investigate the role of CGRP in normal human astrocytes (NHA) cells.ResultsThe CGRP-MCAO-NDDS (nasal drug delivery system) group showed a significant reduction in the infarct volume and improvement in neurologic deficit tests of motor, sensory, reflex and vestibulo-motor functions compared to those rats in the CGRP-MCAO-IV group. CGRP markedly inhibited apoptosis and increased the expression of vascular endothelial growth factor (VEGF) and bFGF and decreased the expression of GAP43 in the cortex of MCAO rats. CGRP promoted cell proliferation and cell cycle process and inhibited cell apoptosis through the Wnt/β-catenin pathway in NHA cells.ConclusionsThis noninvasive, simple, and cost-effective method is a potential treatment strategy for focal cerebral ischemic injury.

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.

Effect of Manual Acupuncture Preconditioning on Behavior and Contents of Serum CGRP, SP, IL-1 β and TNF-α Levels in Migraine Rats

To observe the effect of liver-soothing and mental-activity-regulating (LSMAR) needling on behavior reactions and contents of serum calcitonin gene-related peptide (CGRP), substance P (SP), interleukin-1 β (IL-1 β) and tumor necrosis factor-α (TNF-α) in migraine rats, so as to explore its mechanism underlying relief of migraine. Thirty-two male Wistar rats were randomly divided into control, model, LSMAR needling and conventional needling groups (n=8 rats in each). The migraine model was established by hypodermic injection of glyceryl trinitrate (5 mg/kg) at the napex. Acupuncture preconditioning was conducted once daily for 8 days before modeling. Acupuncture needles were inserted into "Baihui" (GV 20), and bilateral "Fengchi" (GB 20), "Neiguan" (PC 6) and "Taichong" (LR 3), manipulated for a while and retained for 30 min. The rats' behavioral changes (times of head scratching, tail-biting, cage-crawling, and to- and fro-movement in 30 min) were scored (one symptom = one point) before modeling, after modeling, and after intervention. The concentrations of CGRP, SP, IL-1 β, TNF-α in serum were assayed by enzyme-linked immunosorbent assay (ELISA). After modeling, the behavioral scores were significantly increased in the migraine model rats relevant to the control group (P<0.05). Whereas after the treatment, the behavior scores were considerably decreased during 60-90 min and 120-150 min of the measuring period in both LSMAR and conventional needling groups compared with the model group (P<0.05). The concentrations of CGRP, SP, IL-1 β and TNF-α were significantly higher in the model group than in the control group (P<0.05), and notably lower in both LSMAR and conventional needling groups than in the model group (P<0.05). The therapeutic effect of LSMAR was markedly superior to that of conventional needling in down-regulating the concentrations of CGRP, SP, IL-1 β and TNF-α (P<0.05). Acupuncture preconditioning can effectively relieve pain in migraine rats possibly by decreasing the concentrations of CGRP, SP, IL-1 β and TNF-α in the peripheral blood, and the therapeutic effect of LSMAR is evidently superior to that of conventional needling.

Serum calcitonin gene-related peptide facilitates adipose tissue lipolysis during exercise via PIPLC/IP3 pathways.

Calcitonin gene-related peptide (CGRP) is formed by alternative transcription of the calcitonin/α-CGRP gene, which also gives rise to calcitonin (CT). Recently, CGRP has been the focus of research for its metabolic effects in vitro. In the present study, the in vivo effects of CGRP on epididymal fat pads lipolysis at rest and during exercise were investigated in trained male Wistar rats. Male Wistar rats were assigned to control and trained groups, which underwent endurance training for 12 weeks. The control (at rest) and trained (during acute exercise) animals were subjected to an intravenous injection of rat recombinant CGRP (2 µg kg-1) and CGRP-(8-37), a competitive CGRP receptors antagonist, to evaluate if and how CGRP can affect adipose tissue lipolysis at rest and during exercise. Intravenous injection of rat CGRP recombinant at rest upregulated major lipolysis pathways (cyclic AMP (cAMP), AMP-activated protein kinase (AMPK), and phospholipase C (PIPLC/IP3)) in fat pads, causing an elevation in plasma-free fatty acid (FFA) and a decrease in plasma triglyceride (TG). All the effects were eliminated by pretreating the animals with CGRP-(8-37), suggesting that CGRP receptors were necessary for lipolytic effects of CGRP in fat pads. In trained animals, acute exercise augmented CGRP in serum, cerebrospinal fluid (CSF), and the cortex. Pretreating the animals with CGRP-(8-37) attenuated PIPLC/IP3 pathway in fat pads and had no effect on cAMP and AMPK pathways. Epididymal fat pads is a metabolic target for CGRP during exercise and CGRP effects on adipose tissue metabolism during exercise could be related to PIPLC/IP3 pathway.

Blocking Mammalian Target of Rapamycin (mTOR) Alleviates Neuropathic Pain Induced by Chemotherapeutic Bortezomib

Bortezomib (BTZ) is largely used as a chemotherapeutic agent for the treatment of cancer. However, one of the significant limiting complications of BTZ is painful peripheral neuropathy during BTZ therapy. Drugs preventing and/or treating the painful symptoms induced by BTZ are lacking since the underlying mechanisms leading to neuropathic pain remain largely unclear. The purposes of this study were to examine 1) the effects of blocking mammalian target of rapamycin (mTOR) on mechanical pain and cold hypersensitivity evoked by BTZ and 2) the underlying mechanisms responsible for the role of mTOR in regulating BTZ-induced neuropathic pain. Behavioral test was performed to determine mechanical pain and cold sensitivity in a rat model. Western blot analysis and ELISA were used to examine expression of mTOR and phosphatidylinositide 3-kinase (p-PI3K) signals, and the levels of substance P and calcitonin gene-related peptide (CGRP). Systemic injection of BTZ significantly increased mechanical pain and cold sensitivity as compared with control animals (P< 0.05 vs. control rats). The expression of p-mTOR, mTOR-mediated phosphorylation of p70 ribosomal S6 protein kinase 1 (p-S6K1), 4E-binding protein 4 (p-4E-BP1) as well as p-PI3K was amplified in the dorsal horn of spinal cord of BTZ rats as compared with control rats. Blocking mTOR by intrathecal infusion of rapamycin attenuated mechanical pain and cold hypersensitivity. Blocking PI3K signal also attenuated activities of mTOR, which was accompanied with decreasing neuropathic pain. Inhibition of either mTOR or PI3K blunted enhancement of the spinal substance P and CGRP in BTZ rats. The data for the first time revealed specific signaling pathways leading to BTZ-induced peripheral neuropathic pain, including the activation of mTOR and PI3K. Inhibition of these signal pathways alleviates pain. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of peripheral painful neuropathy observed during chemotherapeutic application of BTZ.

Amygdalin improves microcirculatory disturbance and attenuates pancreatic fibrosis by regulating the expression of endothelin-1 and calcitonin gene-related peptide in rats

BackgroundThe pathogenesis of chronic pancreatitis (CP) is a complex process of interaction between tissue injury and repair, which involves microcirculatory disturbance. Amygdalin, an effective component extracted from Semen Persicae (a kind of Chinese herbal medicine), can decrease blood viscosity and improve microcirculation. In this study, we investigated the therapeutic effects of amygdalin on pancreatic fibrosis in rats with CP. MethodsThe rat CP model was induced by injecting dibutyltin dichloride (DBTC) into the right caudal vein. Amygdalin was administrated via the penile vein at a dose of 10 mg/(kg d) from the next day, after the induction of CP, once a day for the previous 3 days, and then once every 2 days, until the end of the experiment. Body weight was observed every 7 days. Pancreatic blood flow and histopathological changes were assessed at 28 days. The activation of pancreatic stellate cells (PSCs) was estimated by the expression of α-smooth muscle actin (α-SMA). At the same time, the expression of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor β-1 (TGFβ-1), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP) of pancreatic tissues were detected. ResultsTreatment of CP rats with amygdalin improved body weight and pancreatic blood flow, as well as alleviated pancreatic fibrosis and acinar destruction, accompanied by the down-regulation of the expressions of α-SMA, PDGF-BB, TGFβ-1, and ET-1, and the up-regulation of the CGRP's expression. ConclusionAmygdalin could reduce the production of pro-fibrotic cytokines, inhibit the activation of PSCs, and attenuate pancreatic fibrosis in a rat with CP. The mechanism probably includes improving microcirculatory disturbance by regulating the production of ET-1 and CGRP.

Role of mitochondrial ATP-sensitive potassium channel in reduction of myocardial ischemia-reperfusion injury by calcitonin gene-related peptide in rats: an in vitro experiment

Objective To evaluate the role of the mitochondrial ATP-sensitive potassium (mito-KATP) channel in reduction of myocardial ischemia-reperfusion (I/R) injury by calcitonin gene-related peptide (CGRP) in rats in an in vitro experiment. Methods Healthy adult male Sprague-Dawley rats, weighing 250-300 g, were used in this study.After the animals were anesthetized, their hearts were immediately removed and retrogradely perfused with oxygenated K-H solution at 37 ℃ in a Langendorff apparatus.Twenty-four isolated rat hearts were assigned into 4 groups (n=6 each) using a random number table: control group (C group), I/R group, CGRP group and 5-hydroxydecanoate (5-HD) group.The hearts were first perfused with K-H solution for 30 min in the three groups.The hearts were continuously perfused with K-H solution for 150 min in group C. The hearts were subjected to ischemia for 30 min followed by 120 min of reperfusion to establish the model of myocardial I/R injury.In group CGRP, after the hearts were perfused with K-H solution for 10 min, 10-8 mol/L CGRP was infused for 20 min at a rate of 0.5 ml/min via the aorta, and then the model of myocardial I/R injury was established.In 5-HD group, specific mito-KATP channel blocker 5-HD 100 μmol/L was infused for 10 min at a rate of 0.5 ml/min via the aorta, and the other treatments were similar to those previously described in CGRP group.At the end of equilibration and 30, 60, 90 and 120 min of reperfusion, heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and the maximum rate of increase or decrease in left ventricular pressure (±dp/dtmax) were recorded.The myocardial infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride staining at 120 min of reperfusion. Results Compared with C group, HR, LVSP and ±dp/dtmax were significantly decreased and LVEDP was increased during reperfusion, and the percentage of myocardial infarct size was increased at 120 min of reperfusion in the other three groups (P<0.05). Compared with I/R group, HR, LVSP and ±dp/dtmax were significantly increased and LVEDP was decreased during reperfusion, and the percentage of myocardial infarct size was decreased at 120 min of reperfusion in CGRP group (P<0.05). Compared with CGRP group, HR, LVSP and ±dp/dtmax were significantly decreased and LVEDP was increased during reperfusion, and the percentage of myocardial infarct size was increased at 120 min of reperfusion in 5-HD group (P<0.05). Conclusion Opening of mito-KATP channels is involved in CGRP-induced reduction of myocardial I/R injury in rats in an in vitro experiment. Key words: Calcitonin gene-related peptide; Myocardial reperfusion injury; Mitochondria; KATP channels

Evaluation of Histological Changes in Back Muscle Injuries in Rats over Time.

Study DesignAnimal model study.PurposeThe purpose of this study was to evaluate the histological variation in the injured muscle and production of calcitonin gene-related peptide in rats over time.Overview of LiteratureVertebral surgery has been reported to cause atrophy of the back muscles, which may result in pain. However, few reports have described the time series histological variation in the injured muscle and changes in the dominant nerve.MethodsWe used 30 male, 8-week-old Sprague-Dawley rats. The right and left sides of the paravertebral muscle were considered as the injured and uninjured sides, respectively. A 115 g weight was dropped from a height of 1 m on the right paravertebral muscle. Hematoxylin and eosin (H&E) staining of the muscle was performed 1–3 weeks after injury for histological evaluation. Fluoro-Gold (FG) was injected into the paravertebral muscle. The L2 dorsal root ganglia on both sides were resected 1, 2, and 3 weeks after injury, and immunohistochemical staining for calcitonin gene-related peptide was performed.ResultsH&E staining of the paravertebral muscle showed infiltration of inflammatory cells and the presence of granulation tissue in the injured part on the ipsilateral side 1 week after injury. Muscle atrophy occurred 3 weeks after injury, but was repaired via spontaneous replacement of muscle cells/fibers. In contrast, compared with the uninjured side, the percentage of cells double-labeled with FG and calcitonin gene-related peptide in FG-positive cells in the dorsal root ganglia of the injured side was significantly increased at each time point throughout the study period.ConclusionsThese results suggest that sensitization of the dominant nerve in the dorsal root ganglia, which may be caused by cicatrix formation, can protract injured muscle pain. This information may be helpful in elucidating the underlying mechanism of persistent pain after back muscle injury.