Calcitonin Gene-related Peptide In Serum Research Articles

Expression of Calcitonin Gene-Related Peptide and Calcitonin Receptor-like Receptor in Colorectal Adenocarcinoma.

Colorectal cancer is one of the most widespread types of cancer that still causes many deaths worldwide. The development of new diagnostic and prognostic markers, as well as new therapeutic methods, is necessary. The calcitonin gene-related peptide (CGRP) neuropeptide alongside its receptor calcitonin receptor-like receptor (CRLR) could represent future biomarkers and a potential therapeutic target. Increased levels of CGRP have been demonstrated in thyroid, prostate, lung, and breast cancers and may also have a role in colorectal cancer. At the tumor level, it acts through different mechanisms, such as the angiogenesis, migration, and proliferation of tumor cells. The aim of this study was to measure the level of CGRP in colorectal cancer patients' serum by enzyme-linked immunosorbent assay (ELISA) and determine the level of CGRP and CRLR at the tumor level after histopathological (HP) and immunohistochemical (IHC) analysis, and then to correlate them with the TNM stage and with different tumoral characteristics. A total of 54 patients with newly diagnosed colorectal adenocarcinoma were evaluated. We showed that serum levels of CGRP, as well as CGRP and CRLR tumor level expression, correlate with the TNM stage, with local tumor extension, the presence of lymph node metastasis, and distant metastasis, and also with the tumor differentiation degree. CGRP is present in colorectal cancer from the incipient TNM stage, with levels increasing with the stage, and can be used as a diagnostic and prognostic marker and may also represent a potentially new therapeutic target.

Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1β, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(β-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1β, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and β-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1β, IL-6, CGRP, and NO in rat serum, increased VEGF and β-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and β-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1β. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing β-EP levels.

Antagonism of CGRP Receptor: Central and Peripheral Mechanisms and Mediators in an Animal Model of Chronic Migraine.

Calcitonin-gene-related peptide (CGRP) plays a key role in migraine pathophysiology and more specifically in the mechanisms underlying peripheral and central sensitization. Here, we explored the interaction of CGRP with other pain mediators relevant for neuronal sensitization in an animal model of chronic migraine. Male Sprague-Dawley rats were exposed to nitroglycerin (NTG, 5 mg/kg, i.p.) or vehicle co-administered with the CGRP receptor antagonist olcegepant (2 mg/kg i.p.), or its vehicle, every other day over a 9-day period. Twenty-four hours after the last injection of NTG (or vehicle), behavioral test and ex vivo analysis were performed. Olcegepant attenuated NTG-induced trigeminal hyperalgesia in the second phase of the orofacial formalin test. Interestingly, it also reduced gene expression and protein levels of CGRP, pro-inflammatory cytokines, inflammatory-associated miRNAs (miR-155-5p, miR-382-5p, and miR-34a-5p), and transient receptor potential ankyrin channels in the medulla–pons area, cervical spinal cord, and trigeminal ganglia. Similarly, olcegepant reduced the NTG-induced increase in CGRP and inflammatory cytokines in serum. The findings show that the activation of the CGRP pathway in a migraine animal model was associated to the persistent activation of inflammatory pathways, which was paralleled by a condition of hyperalgesia. These molecular events are relevant for informing us about the mechanisms underlying chronic migraine.

Protective effect of myocardial calcitonin gene-related peptide gene transduction on myocardial ischemia/reperfusion injury in diabetic rats

Objective To evaluate the feasibility of lentivirus (Lv) mediated calcitonin gene-related peptide(CGRP) gene transduction in diabetic rats by myocardial injection and whether this method has a protective effect on diabetic myocardium during ischemia/reperfusion(I/R) injury. Methods Sixty healthy male SD rats, weighing 200-220 g, were randomly divided into control group (group C), diabetes mellitus group (group DM), Sham transduction group (group Sham), myocardial injection control group (group MC) and myocardial injection experiment group(group ME), in accordance with a random number table. Rats in group Sham, group MC and group ME were given with normal saline, at a titer of 2×107 virus genomes(vg)/ml Lv-enhanced green fluorescent protein (eGFP) and Lv-CGRP-eGFP 50 μl respectively by myocardial injection. One week later, rats in group DM, group Sham, group MC and group ME received streptozocin(STZ) 50 mg/kg intraperitoneal injection to induce diabetes. Eight weeks after the injection of STZ, myocardial I/R was induced by occlusion and release of left anterior descending branch of coronary artery. The latency of tail flick reflection, the expression of eGFP, the transcription level of CGRP, the expression of CGRP in myocardium, spinal cord and dorsal root ganglion(DRG) and myocardial ischemia area, infarct size(IS) area were measured. Results Compared with group C, the latency of tail flick reflection in group DM, group Sham, group MC and group ME were significantly prolonged in the 4th week after diabetic model successfully manufactured(P<0.05). The transcription levels of CGRP in the ischemic myocardium, spinal cord T1-T5 and DRG T1-T5 in group DM, group Sham and group MC were lower than those in group C and group ME (P<0.05). The level of CGRP in ischemic myocardium and DRG T1-T5 of group ME was higher than that in group C(P<0.05). The content of CGRP in cord T1-T5, DRG T1-T5, ischemic myocardium and serum in group C and group ME were higher than those in group DM, group Sham and group MC (P<0.05). The content of CGRP in cord T1-T5, ischemic myocardium in group ME was higher than that in group C (P<0.05). The infarct size in group DM, group Sham, group MC were higher than that in group C and group ME(P<0.05), the infarct size of group ME was higher than that in group C(P<0.05). Conclusions Intramyocardial injection of CGRP gene can effectively enhance the expression of CGRP at I/R of diabetic rat′ heart. Increased CGRP may reduce cardiac I/R injury in diabetic rats. Key words: Calcitonin gene-related peptide; Diabetes mellitus; Myocardium; Ischemia-reperfusion

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.

Independent roles of CGRP in cardioprotection and hemodynamic regulation in ischemic postconditioning

Calcitonin gene-related peptide (CGRP) may participate in ischemic post-conditioning (IPost) attenuating myocardial ischemia/reperfusion injury. However, the molecular mechanism underlying the effect of CGRP is still elusive. In this study, we evaluated the difference in hemodynamics, infarct size and CGRP in myocardium and serum of rats after acute myocardial ischemia/reperfusion with and without IPost. A specific antagonist of CGRP receptor, CGRP8–37 was employed to testify the role of endogenous CGRP in IPost. The underlying mechanism was investigated by evaluation of the effect of CGRP on the changes in cytosolic reactive oxygen species, mitochondrial membrane potential and myocyte apoptosis induced by hypoxia/reoxygenation in cultured cardiomyocytes of neonatal rats. The results showed that IPost significantly upgraded CGRP in myocardium and serum with reduced infarct size, independent of the reductions in blood pressures observed in this study. The effects were significantly reversed by CGRP8–37, indicating an involvement of intrinsic CGRP. It was further demonstrated that CGRP effectively reversed the hypoxia/reoxygenation-induced reduction of mitochondrial membrane potential and attenuated the increases of cytosolic reactive oxygen species and cardiomyocyte apoptosis. The findings may demonstrate that CGRP mediates the cardioprotective and vasoactive effects of IPost. Homeostasis of cytosolic reactive oxygen species and mitochondrial membrane potential may be underlying the cardioprotective effect of CGRP.

The Relationship between Trypsin/Calcitonin Gene Related Peptide (CGRP) in Serum and Acute Pancreatitis (AP).

Activation of trypsin from proteolytic cleavage of trypsinogen in the pancreas can lead to acute pancreatitis. Calcitonin gene related peptide (CGRP) from both peripheral and central neurons is involved in a variety of physiological/pathophysiological processes, especially sensory (nociceptive) and efferent (effector) functions. To better understand the change of trypsin/CGRP in acute pancreatitis, the study investigated the serum level of trypsin/CGRP in patients with acute pancreatitis. The study investigated 140 patients with acute pancreatitis, including 72 cases of biliary acute pancreatitis, 60 cases of hyperlipidemic acute pancreatitis, and 8 cases of idiopathic acute pancreatitis. Sixty volunteers acted as the normal control group. The levels of trypsin and CGRP in serum were analyzed. The serum levels of trypsin and CGRP in males with acute pancreatitis were higher than in females, but there was no statistical difference (p > 0.05). However, the serum levels of trypsin and CGRP in different types of acute pancreatitis were significantly higher than controls (p < 0.001), and the level of trypsin and CGRP in serum of patients with inflammation effusion was significantly higher than patients without inflammation effusion (p < 0.001). In addition, the serum levels of trypsin and CGRP in patients with I-II, III, IVA and IVB acute pancreatitis were higher than controls (p < 0.001). According to the results, we concluded that the trypsin and CGRP in serum can act as a new detection index of acute pancreatitis occurring. The serum levels of trypsin and CGRP in patients with acute pancreatitis is able to determine whether inflammation effusion happens.

Association of down-regulation of calcitonin gene-related peptide and substance P with increase of myocardial vulnerability in diabetic neuropathic rats

Diabetic patients present high co-morbidities of neuropathy and severer consequences of coronary heart disease. But the pathological mechanism is still unclear. Here we investigated a potential association of diabetic impairment of sensory nerves with increase of vulnerability of myocardium in acute myocardial ischemia/reperfusion. A rat model of diabetes mellitus was induced by high fat and sugar diet plus a small dose of streptozotocin. Impairment of sensory nerves was evaluated by measurement of changes in tail flick latency to noxious thermal stimulation and calcitonin gene-related peptide (CGRP) and substance P (SP) in the dorsal root ganglia (DRG) and the myocardium of the heart were examined. The myocardial injury was examined by infarct size, apoptosis ratio of cardiomyocytes and cardiac troponin I in the animals underwent acute myocardial ischemia (for 30min) and reperfusion (for 120min). The effects of CGRP and SP on cardiomyocyte injury induced by high glucose and hypoxia/reoxygenation were tested in cultured myocytes. The diabetic animals presented significant elevation of noxious thermal threshold with obvious reduction of the contents of CGRP and SP in the DRG and the myocardium. Importantly, the diabetic animals showed significant increases of infarct size, myocyte apoptosis and serum cardiac troponin I after acute myocardial ischemia/reperfusion, compared to the non-diabetic control. Furthermore, exogenously administered CGRP and SP attenuated the myocyte injury induced by the high concentration of glucose and hypoxia/reoxygenation. These findings suggested that impairment of sensory nerves with significant reduction of CGRP and SP in DRG, ventricular myocardium and serum may be associated with increase of myocardial vulnerability in acute myocardial ischemia/reperfusion in streptozotocin-induced diabetic rats.

Calcitonin gene-related peptide can be selected as a predictive biomarker on progression and prognosis of knee osteoarthritis.

The purpose of this study was to examine calcitonin gene-related peptide (CGRP) concentrations in serum and synovial fluid of patients with primary knee osteoarthritis (OA) and healthy controls and to explore their relationship with clinical and radiographic severity of OA. Sixty-five patients with primary knee OA and 21 healthy controls were recruited. CGRP concentrations in the serum and synovial fluid were measured using enzyme-linked immunosorbent assays. The radiographic severity of OA was evaluated using the Kellgren and Lawrence (KL) classification. The Western Ontario and McMaster University Osteoarthritis Index (WOMAC) was used to assess pain, stiffness and physical function. Serum and synovial fluid CGRP concentrations tended to be higher with the increase in KL grades (r = 0.565 and r = 0.441, P < 0.001, respectively), and were significantly positively correlated with KL grades, total WOMAC score and each subscale (pain, stiffness and physical function). The result demonstrated that CGRP in serum and synovial fluid was related to progressive joint damage in knee OA. CGRP can be selected as a biomarker for monitoring disease severity and could be a predictive role on prognosis and progression of knee OA.

CALCITONIN GENE-RELATED PEPTIDE IN SERUM AFTER SPONTANEOUS SUBARACHNOID HEMORRHAGE

Endogenous Calcitonin Gene-Related Peptide (CGRP) stored in perivascular nerve fibers around the cerebral arteries mediates vasodilation and regulates cerebral blood flow under physiological conditions. Increased levels of CGRP have been continually reported in patients with migraine, but the role of CGRP in the pathophysiology of Subarachnoid Hemorrhage (SAH) has not yet been sufficiently evaluated. Over 10 days, serum was prospectively collected from 33 consecutive patients (17 women, 16 men; mean age 52.8 years) with spontaneous non-traumatic SAH. All patients were graded III or IV according to the Fisher Score. CGRP levels were determined by means of an Enzyme-Immunosorbent Assay (EIA). CGRP concentrations were correlated to (1) the anatomical localization of the aneurysm (anterior circulation or posterior circulation), (2) the treatment modality (clipped group, coiled group, or untreated group) and (3) the rate of ischemia determined by computed tomography (ischemia group, non-ischemia group, or iatrogenic ischemia group). (1) Anatomical localization: CGRP levels were significantly higher in patients with a ruptured aneurysm of the anterior circulation (p<0.001). (2) Treatment modality: CGRP levels differed significantly between the groups (coil group, clip group, or untreated group; p<0.001). The highest levels of CGRP were detected in the coil group and the lowest levels in the untreated group. About (3) Ischemia: CGRP levels differed significantly between the three groups (ischemia group, non-ischemia group, or iatrogenic ischemia group; p = 0.038). The highest levels of CGRP were found in the ischemia group. The maximum levels of CGRP in serum were found for patients with a ruptured aneurysm of the anterior circulation and subsequent ischemia treated by endovascular coiling. These findings support the theory that CGRP is produced and excessively released to counteract cerebral vasospasm and subsequent ischemia. Presumably, the serum levels of CGRP are affected by the anatomical localization of the aneurysm and can be stimulated by any endoluminal manipulation of the parent artery.

Increased levels of calcitonin gene-related peptide in serum accelerate fracture healing following traumatic brain injury

The mechanisms responsible for the phenomenon of an accelerated speed of fracture healing in patients with traumatic brain injury (TBI) remain unclear. The present study was performed to test the hypothesis that TBI causes changes in calcitonin gene-related peptide (CGRP) levels in sera that enhance fracture healing. A standard closed femoral fracture was produced in rats, which were subjected to additional closed head trauma. The fracture healing was assessed 4 and 8 weeks later using micro-CT. Sera, brain tissues and muscles surrounding the fracture sites collected at 24, 48, 72 and 168 h after injury were used to detect the expression of CGRP using ELISA, immunohistochemistry and RT-PCR. Micro-CT demonstrated that fracture healing and mineralization in the TBI-fracture group occurred earlier compared to the fracture-only group. ELISA analysis revealed a high concentration of CGRP in the TBI-fracture group (P<0.05), and immunohistochemistry assay and RT-PCR analysis revealed a significant increase in CGRP in the brain and muscle of the TBI-fracture group at 168 h after fracture (P<0.001). Our results indicate that the mechanism for the enhancement of fracture-healing secondary to traumatic brain injury is correlated to the high levels of CGRP, which may be released from the brain tissue into the serum.

Presence of VIP, CGRP, SP neuropeptides and PAF-AH enzyme in porcine colostrum

The aim of this study was to examine levels of vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP) and platelet activating factor-acetylhydrolase (PAF-AH) in porcine serum and colostrum. Samples of porcine blood were obtained from six multiparous sows during farrowing and post-lactation. Porcine colostrum was obtained from lactating sows at parturition. Neuropeptides were measured by competitive enzyme immunoassay using streptavidin-conjugated horseradish peroxidase (VIP) and acetyl cholinesterase-peptide conjugates (SP and CGRP) as tracers. PAF-AH activity was measured by a continuous kinetic assay using 2-thio PAF as substrate. The presence of VIP, CGRP, SP and PAF-AH activity in both porcine serum and colostrum was confirmed. The concentrations of VIP, CGRP and PAF-AH in serum and colostrum were comparable, suggesting passive rather than active secretion in the colostral milk. A significant higher concentration of SP was found in porcine colostrum. This could be explained by active secretion into colostrum or production of SP in the mammary gland. The presence of these factors in porcine colostrum may be important for the maturation of the gastrointestinal system in neonatal piglets. However, further research is necessary in order to elucidate the role of these factors in porcine colostrum.

Calcitonin gene-related peptide inhibits early B cell development in vivo

Recent in vitro studies suggest that calcitonin gene-related peptide (CGRP) inhibits early B cell differentiation; however, there is no evidence in the intact animal for a role for CGRP in B cell development. Here, we show that in vivo treatment of mice with CGRP reduces the number of IL-7 responsive B cell progenitors in bone marrow. A single CGRP treatment reduces IL-7-responsive B cell progenitors by up to 40% for up to 72 h. The reduction is dose-dependent and can be blocked by a CGRP receptor antagonist, CGRP(8-37). CGRP in serum following injection is highly elevated at 30 min but returns to basal levels by 4 h, suggesting that a single injection of CGRP has long-lasting effects on B cell development. This report provides the first direct in vivo evidence that CGRP, a neuropeptide with multiple effects on mature lymphocytes, also plays a regulatory role in early B cell development in the bone marrow.

Radioimmunoassay for Calcitonin Gene-Related Peptide and Its Measurement in Sera of Patients with Thyroid Disease.

To investigate serum levels of calcitonin gene-related peptide (CGRP), we developed a sensitive radioimmunoassay (RIA). RIA for CGRP in serum can present some problems: the serum may degradate the tracer during incubation and suppress the antigen-antibody reaction. We avoided these problems by using aprotinin and CGRP-free serum instead of a buffer for the standard curve. We detected serum CGRP in all 39 healthy subjects when CGRP-free serum was not used for the standard curve, but 34 of these subjects had serum CGRP levels below the detection limit (less than 80 pmol/l) when CGRP-free serum was used for the standard curve. We defined the normal range for serum CGRP as below 100.8 pmol/l, which was the maximum level found in the healthy subjects. We studied serum levels of this peptide in patients with thyroid diseases, because the thyroid may be one origin of circulating CGRP. Four of 10 patients with medullary thyroid carcinoma had elevated serum levels of CGRP. Seven of 24 patients with subacute thyroiditis had elevated serum levels of CGRP, but at least one year after clinical recovery, CGRP was undetectable in all. Seven of the 37 patients with hypothyroidism had elevated serum levels of CGRP. None of the patients with hyperthyroidism, adenomatous goiter, thyroid adenoma, or thyroid carcinoma had elevated serum CGRP levels. It is necessary to use a standard curve obtained by the addition of aprotinin and CGRP-free serum to the assay standards to measure serum CGRP levels. Some patients with subacute thyroiditis, hypothyroidism, or medullary thyroid carcinoma had elevated serum CGRP levels.

Calcitonin gene-related peptide and calcitonin in medullary thyroid carcinoma.

We have investigated the levels of serum calcitonin and calcitonin-gene related peptide (CGRP) in 35 patients with well-documented medullary thyroid carcinoma (MTC). Immunohistochemical investigations for calcitonin and CGRP have been performed on tumour tissue from 9 patients to clarify the cellular pattern of production. In four patients with aggressive disease, serum calcitonin and CGRP values have been monitored in relation to progression of disease after surgery. All 35 patients with MTC have elevated calcitonin and 26 elevated CGRP levels. Generally calcitonin values were found to be higher than those of CGRP, although the ratio of the two peptides varied from patient to patient. The immunohistochemical investigations corresponded with these findings, generally showing diffuse staining for calcitonin in MTC tumour-cells and only a small number of CGRP positive cells. Calcitonin and CGRP are produced by alternative processing of the common precursor gene transcript. Our results suggest that absolute values of either calcitonin or CGRP in serum have no direct relationship to aggressiveness of disease. Thus whilst serum CGRP measurements appear to be a useful additional marker for the disease, they can be considered to be only a useful adjunct to serum calcitonin as a marker for tumour progression.