Calcitonin Gene-related Peptide Group Research Articles

Effectiveness of Anti-Calcitonin Gene-Related Peptide Medication in Vestibular Migraine: A Retrospective Cohort Study in an Asian Population.

Migraine and dizziness often coexist, with vestibular migraine (VM) presenting with vestibular symptoms and headaches. Calcitonin gene-related peptide (CGRP) may be involved in motion-induced symptoms; however, studies on the use of anti-CGRP monoclonal antibodies (mAbs) for the treatment of VM have yielded conflicting results. This study aimed to clarify the effectiveness of anti-CGRP mAbs in VM treatment. This retrospective observational cohort study, conducted between 1 January 2021 and 31 March 2023, assessed 12 Japanese patients with VM who were treated with anti-CGRP mAbs (CGRP group) for 6 months and 11 Japanese patients who received standard of care for VM and served as controls. Clinical questionnaires and equilibrium tests were administered, with primary outcomes including changes in Dizziness Handicap Inventory (DHI) scores compared with baseline values. Objective variables included the DHI score and explanatory variables included demographic data, balance test results, head-up tilt (HUT) test results, vestibular test results and questionnaire survey results. Analysis of variance was used to assess the treatment effects of anti-CGRP mAbs, and multivariate regression analysis was performed to identify mAb responders. After 6 months, the CGRP group showed significant improvements in DHI scores [0 versus 6 months, odds ratio (95% confidence interval): 22.01 (0.13-43.88)] and number of vertigo/dizziness attacks per month [0 versus 6 months: 10.28 (2.80-17.76)]. No significant difference was observed in the control group [DHI scores, 0 versus 6 months: 0.65 (-26.84 to 28.14); number of vertigo/dizziness attacks per month, 0 versus 6 months: - 8.07 (- 23.77 to 7.62)]. Multivariate regression analysis showed that autonomic function at baseline was associated with mAb response in patients [β estimates (95% confidence interval): 3.63 (0.21-7.06)]. Treatment with anti-CGRP mAbs was more effective than conventional treatment in preventing migraine in patients with VM. While the identified factors associated with treatment responsiveness offer valuable insights into personalised treatment approaches, further prospective studies are warranted to validate the findings due to our study's retrospective design and limited sample size.

Effects of intracerebroventricular administration of calcitonin gene-related peptide (CGRP) on sex hormones and sperm quality in rats.

Therapeutic strategies with calcitonin gene-related peptide (CGRP) or its receptor have been investigated, but there are few studies regarding the possible harmful effects of CGRP in other body organs. This study aimed to investigate the effect of intracerebroventricular (ICV) injection of CGRP on sex hormones and sperm quality in rats. Twelve male rats were divided into two groups (n=6 per group). The first group (control) rats were injected with 5µl artificial cerebrospinal fluid intra-ICV; the second group rats, 5µl (1.5nmol) CGRP. The levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were measured. Epididymal sperms were used to determine the sperm parameters. The levels of testosterone, LH and FSH in CGRP group was significantly lower than in artificial cerebrospinal fluid (ACSF) group (P<0.05). The concentration and motility of sperm in CGRP group was significantly lower than in ACSF group (P<0.05). In CGRP group live spermatozoa and intact acrosome significantly reduced compared to the ACSF group (P<0.05). In addition, in CGRP group dead spermatozoa and lose acrosome significantly increased compared to the ACSF group (P<0.05). ICV injection of CGRP may reduce sperm quality, probably through induction of an imbalance in FSH and LH production as well as testosterone.

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

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

Low Serum Calcitonin Gene-Related Peptide Level is Associated with Severity of Coronary Stenosis.

To evaluate the relationship between the serum calcitonin gene-related peptide (CGRP) level and severity of coronary stenosis. A total of 233 eligible patients who underwent coronary angiography were divided into two groups: a control and a coronary heart disease (CHD) group. The angiographic severity of coronary stenosis was evaluated by SYNTAX and Gensini scores. The incidence of major adverse cardiovascular events within two years was collected. A negative correlation between serum CGRP levels and Gensini scores was observed in all patients (r=-0.352, p<0.001), the control group (r=-0.422, p<0.001) and the CHD group (r=-0.393, p<0.001). Serum CGRP levels were negatively associated with SYNTAX scores in the CHD group (r=-0.522, p<0.001). The area under the curve of CGRP for identifying high SYNTAX scores (>22) was 0.772 [95% confidence interval (CI): 0.673-0.870, p<0.001], and for identifying high Gensini scores was 0.744 (95% CI: 0.646-0.842, p<0.001). A CGRP concentration of 25.05 pg/ml was selected as the cutoff point. A low CGRP level (<25.05 pg/ml) was an independent predictor of severe coronary stenosis, a SYNTAX score >22 [odds ratio (OR) =5.819, 95% CI: 2.240-15.116; p<0.001] and a high Gensini score (>64) (OR=4.943, 95% CI: 2.020-12.095; p<0.001). The low CGRP group had a higher incidence of major adverse cardiovascular events within two years (11.1 vs. 3.1%, p=0.031). In coronary atherosclerosis patients without acute myocardial injury, serum CGRP levels were negatively associated with the severity of coronary stenosis.

Calcitonin Gene-Related Peptide Influences Bone-Tendon Interface Healing Through Osteogenesis: Investigation in a Rabbit Partial Patellectomy Model.

Background:Calcitonin gene-related peptide (CGRP), which has been shown to play an important role in osteogenesis during fracture repair, is also widely distributed throughout the tendon and ligament. Few studies have focused on the role of CGRP in repair of the bone-tendon interface (BTI).Purpose:To explore the effect of CGRP expression on BTI healing in a rabbit partial patellectomy model.Study Design:Controlled laboratory study.Methods:A total of 60 mature rabbits were subjected to a partial patellectomy and then randomly assigned to CGRP, CGRP-antagonist, and control groups. In the CGRP-antagonist group, the CGRP receptor antagonist BIBN4096BS was administered to block CGRP receptors. The patella–patellar tendon complex was harvested at 8 and 16 weeks postoperatively and subjected to radiographic, microlaser Raman spectroscopy, histologic, and biomechanical evaluation.Results:Radiographic data showed that local CGRP expression improved the growth parameters of newly formed bone, including area and volumetric bone mineral density (P < .05 for both). Raman spectroscopy revealed that the relative bone mineral composition increased in the CGRP group compared with in the control group and the CGRP-antagonist group (P < .05 for both). Histologic testing revealed that the CGRP group demonstrated better integration, characterized by well-developed trabecular bone expansion from the residual patella and marrow cavity formation, at the 8- and 16-week time points. Mechanical testing demonstrated that the failure load, ultimate strength, and stiffness in the CGRP group were significantly higher than those in the control group (P < .05 for all), whereas these parameters in the CGRP-antagonist group were significantly lower compared with those in the control group at 16 weeks after surgery (P < .05 for all).Conclusion:Increasing the local concentration of CGRP in the early stages of BTI healing enhanced osteogenesis in a rabbit partial patellectomy model and promoted healing of the BTI injury, whereas treatment using a CGRP antagonist had the opposite effect. However, exogenous CGRP expression did not induce novel bone remolding.Clinical Relevance:CGRP may have potential as a new therapy for BTI injuries or may be added to postoperative regimens to facilitate healing.

CGRP protects bladder smooth muscle cells stimulated by high glucose through inhibiting p38 MAPK pathway in vitro

This study aimed to explore the effect of calcitonin gene-related peptide (CGRP) on bladder smooth muscle cells (BSMCs) under high glucose (HG) treatment in vitro. BSMCs from Sprague–Dawley rat bladders were cultured and passaged in vitro. The third-generation cells were cultured and divided into control group, HG group, HG + CGRP group, HG + CGRP + asiatic acid (AA, p-p38 activator) group, CGRP group, AA group, HG + CGRP + CGRP-8-37 (CGRP receptor antagonist) group and HG + LY2228820 (p38 MAPK inhibitor) group. The cell viability, apoptosis, malondialdehyde (MDA) and superoxide dismutase (SOD) levels of BSMCs were observed by the relevant detection kits. The expressions of α-SM-actin, p38 and p-p38 were detected by qRT-PCR or Western blot analysis. Compared with the control group, the cell viability, SOD and α-SM-actin levels of BSMCs were decreased and apoptotic cells, MDA and p-p38 levels were increased after HG treatment, while these changes could be partly reversed when BSMCs were treated with HG and CGRP or LY2228820 together. Moreover, AA or CGRP-8-37 could suppress the effect of CGRP on BSMCs under HG condition. Our data indicate that CGRP protects BSMCs from oxidative stress induced by HG in vitro, and inhibit the α-SM-actin expression decrease through inhibiting the intracellular p38 MAPK signaling pathway.

Calcitonin gene-related peptide enhances osteogenic differentiation and recruitment of bone marrow mesenchymal stem cells in rats.

The present study evaluated the effects of calcitonin gene-related peptide (CGRP) on bone marrow mesenchymal stem cells (BMMSCs) in vitro and in a rat model of mandibular distraction osteogenesis (MDO). Rat BMMSCs were isolated then treated with CGRP or CGRP antagonist (CGRP8-37). The proliferation and migration ability of BMMSCs was determined using 5-bromo-2′-deoxyuridine and Transwell assays, respectively. Osteogenic-related gene expression was analyzed with reverse transcription-quantitative polymerase chain reaction. For the in vivo analysis, thirty MDO rats were randomly assigned to control, CGRP or CGRP8-37 groups. To evaluate the mobilization of BMMSCs, nestin and stromal cell-derived factor 1 (SDF-1) were detected by immunohistochemistry and ELISA. Rats were sacrificed following 14 days and new bone formation was assessed by histological and micro-computed tomography analysis. In the in vitro results, the CGRP group demonstrated significantly higher migration and proliferation, as well as enhanced alkaline phosphatase and runt-related transcription factor 2 expression compared with the control. In the in vivo experiments, bone mineral density of the newly formed bone in the CGRP group was significantly higher than controls. The nestin and SDF-1 expression in the CGRP group was also significantly upregulated. In conclusion, the present study demonstrated that CGRP administration increased new bone formation, possibly via enhancing BMMSC migration and differentiation in MDO rats.

Expression of calcitonin gene-related peptide and pulp sensitivity tests in irreversible pulpitis.

The aim of the present study was to identify the relationship between the expression of calcitonin gene-related peptide (CGRP) and the responses of pulp sensitivity tests in healthy pulps and irreversible pulps by performing a cross-sectional study on patients. Two hundred subjects were evaluated. A total of 75 subjects complied with the criteria. The participants were divided into two groups: a) Healthy pulp (subjects [n = 35] having posterior teeth with clinically normal pulp tissue), and b) Irreversible pulpitis (subjects [n = 40] having posterior teeth with irreversible pulpitis). All participants were evaluated using the following variables: a) medical and dental history, b) pulp sensitivity tests, c) expression of CGRP by the enzyme-linked immunosorbent assay (ELISA), and d) expression levels of mRNA CGRP and mRNA CGRP receptor genes. We determined that the responses of the cold test between 4 and ≥12 s presented a higher average of the expression of CGRP in the group having irreversible pulpitis (p = 0.0001). When we compared the groups with the value of the electrical impulse, we found statistically significant differences (p = 0.0001), observing positive responses to the test with electrical impulses of 7 to 10, with an average of 72.15 ng/mL of CGRP in the irreversible pulpitis group. High values of CGRP expression were observed in that group in the responses of pulp sensitivity.

Protective Effects of Calcitonin Gene-Related Peptide-Mediated p38 Mitogen-Activated Protein Kinase Pathway on Severe Acute Pancreatitis in Rats.

Calcitonin gene-related peptide (CGRP) has antioxidant and anti-inflammatory activities on the pathological damage of acute pancreatitis. However, its molecular mechanism on severe acute pancreatitis (SAP) remains unknown. To evaluate the influence of CGRP-mediated p38MAPK signaling pathway in rats with SAP. SD rats were randomly divided into Sham group, SAP group, CGRP group (SAP rats injected with CGRP), SB203580 group (rats injected with p38MAPK pathway inhibitor SB203580), and CGRP8-37 group (SAP rats injected with CGRP8-37). Serum amylase and lipase activities were determined. Histopathological observations were evaluated, and the expression of inflammatory cytokines and oxidative stress-related indexes were measured. Compared with Sham group, SAP rats were increased in the activities of serum amylase and lipase, the pathologic assessment of pancreatic tissue, the levels of TNF-α, IL-1β, IL-6, and IL-8, the content of MDA and MPO, and the expressions of CGRP, and p-p38MAPK protein, but they were decreased in SOD activity and GSH content. The above alterations were aggravated in the CGRP8-37 group when compared with SAP group. Besides, in comparison with SAP group, rats in the CGRP and SB203580 groups presented a reduction in the activities of serum amylase and lipase, the levels of inflammatory cytokines, the content of MDA and MPO, and the expressions of p-p38MAPK protein, while showed an elevation in SOD activity and GSH content. Pretreatment with CGRP alleviated oxidative stress and inflammatory response of SAP rats possibly by suppressing the activity of p38MAPK pathway, and thereby postponing the disease progression.

CGRP gene-modified rBMSCs show better osteogenic differentiation capacity in vitro.

Calcitonin gene-related peptide (CGRP) is a marked and important neuropeptide expressed in nerve fibers during bone repair. This study investigated the role of CGRP overexpression on osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs). rBMSCs were infected with viral stocks of pLenO-DCE-CGRP (CGRP group) or pLenO-DCE (Vector group), while normal rBMSCs were used as a control. Transfection efficiency of rBMSCs was analyzed by flow cytometry. Cell proliferation was examined using a Cell Counting Kit-8 and flow cytometry. Expressions of alkaline phosphatase(ALP), bone sialoprotein (BSP) and Runt-related transcription factor 2(Runx2) in rBMSCs were detected at 1 and 2weeks after mineral induction by real-time PCR and western blotting. Alizarin Red staining was applied at 28days. The ratio of osteoprotegerin (OPG) to receptor activator of nuclear factor kappa B ligand (RANKL) was also detected to determine the underlying mechanism. pLenO-DCE-CGRP-induced rBMSCs stably overexpressing CGRP were successfully established. Overexpression of the CGRP gene significantly promoted rBMSC proliferation (p < 0.05). In addition, expressions of osteogenesis-related indexes were upregulated in the CGRP group (p < 0.05) compared with vector and control groups, and more mineralization nodules were observed in the CGRP group (p < 0.05). CGRP gene increased OPG and reduced RANKL in rBMSCs. Hence, the OPG/ RANKL ratio was increased in the CGRP group compared with the other two groups. CGRP gene-modified rBMSCs show better osteogenic differentiation capacity compared with rBMSCs in vitro.

Effects of calcitonin gene-related peptide on aquaporin 1 and aquaporin 5 expressions in young rats with acute lung injury

Objective To explore the effect of calcitonin gene-related peptide (CGRP) on expressions of aquaporin (AQP)1 and AQP 5 in young rats with acute lung injury(ALI) caused by lipopolysaccharide(LPS). Methods Eighty-four young rats were randomly divided into control group, ALI model group and CGRP group.The rats in ALI model group were given intraperitoneal injection of LPS (5 mg/kg)for 2, 6, 12, 24 hours; while the rats in CGRP group were given intraperitoneal CGRP (1 mg/kg) after 1 h injection of LPS.At 2 h, 6 h, 12 h and 24 h, all rats were sacrificed and lung tissues were obtained.The histopathological changes in lung tissues were evaluated by adopting hematoxylin-eosin staining, and wet/dry(W/D) was measured.The mRNA and protein levels of AQP1 and AQP5 in lung tissues were detected by adopting fluorogenic quantitative PCR(qPCR) and Western blot. Results Pathological stain showed that rats in control group had a normal lung tissue structure, and LPS made lung tissue edema, narrowing the alveolar cavity and inflammatory cell infiltration.CGRP attenuated the effect of LPS on rat′s lung.The W/D ratio of lung tissue was significantly higher than that in the control group, and CGRP reduced the W/D ratio of lung tissue.qPCR showed that the mRNA levels of AQP1 and AQP5 from rats in ALI group (0.009±0.001 and 0.055±0.006) decreased compared with those in the control group (0.035±0.002 and 0.167±0.006) and CGRP group (0.024±0.002 and 0.134±0.012) (all P<0.001). Western blot results showed after 24 h injection of LPS, both AQP1 and AQP5 levels from ALI group (0.397±0.041 and 0.215±0.029) were significantly lower than those in the control group (0.850±0.020 and 0.741±0.032) (all P<0.001), and their levels in CGRP group (0.593±0.065 and 0.461±0.039) were also lower than those in the control group, but higher than those in ALI group (all P<0.001). Conclusion CGRP can enhance AQP1 and AQP5 levels and reduce pulmonary edema, and it has a protective effect on rats with acute lung injury. Key words: Acute lung injury; Calcitonin gene-related peptide; Aquaporin

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

CGRP attenuates hyperoxia-induced oxidative stress-related injury to alveolar epithelial typeII cells via the activation of the Sonic hedgehog pathway.

The aim of this study was to examine the effect of calcitonin gene-related peptide(CGRP) on primary alveolar epithelial typeII(AECII) cells and expression of Sonic hedgehog(SHH) signaling pathway components following exposure to hyperoxia. The AECII cells were isolated and purified from premature rats and exposed to air(21%oxygen), air+CGRP, hyperoxia(95%oxygen) or hyperoxia+CGRP. The production of intracellular reactive oxygen species(ROS) was determined using the 2',7'-dichlorofluorescin diacetate molecular probe. The levels of malondialdehyde(MDA) and superoxide dismutase(SOD) in the culture supernatant were detected by spectrophotometry. The apoptosis of AECII cells was assayed by flow cytometry, and the mRNA and protein expression levels of Shh and Ptc1 in the AECII cells were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR), western blot analysis and immunofluorescence, respectively. The cellular pathological changes partly improved and apoptosis was markedly decreased upon treatment with CGRP under hyperoxic conditions. The levels of ROS in the hyperoxia+CGRP group were significantly lower than thoe in the hyperoxia group. In addition, the hyperoxia-induced increase in MDA levels and the decrease in SOD activity in the culture supernatant of the AECII cells were attenuated by CGRP. Compared with the cells exposed to air, hyperoxia markedly inhibited the mRNA and protein expression levels of Shh and Ptc1 in the AECII cells; however, this inhibition was partly attenuated by treatment with CGRP. On the whole, our data suggest that CGRP can partly protect AECII cells from hyperoxia-induced injury, and the upregulation of CGRP may be a potential therapeutic approach with which to combat hyperoxia-induced lung injury, which may be associated with the activation of the SHH signaling pathway.

Effect of calcitonin gene-related peptide on osteoporotic fracture healing in vitro

Objective To explore the effects of calcitonin gene-related peptide (CGRP) on proliferation of vascular endothelial cells (VECs) and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) of osteoporotic rats in vitro, and cell/molecular mechanism of promoting osteoporotic fracture healing. Methods 10 3-month-old SD rats were employed to make osteoporosis model by taking bilateral ovariectomy. Micro-CT analysis was used to determine the success of modeling at 6 months of postoperation. BMSCs and VECs were isolated from osteoporotic rats by blood adherence method. 10-7-10-10 mol/L CGRP were added to the culture medium. CCK-8 method was used to examine the cell proliferation. Effect of CGRP on osteogenic differentiation of BMSCs: Alkaline phosphatase (ALP) activity, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of ALP, CollagenⅠ, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs: Vascular cell endothelial growth factor (VEGF) protein secretion and gene expression level were tested by ELISA and RT-PCR. Results BMSCs and VECs of osteoporotic rats were isolated successfully.The optical density (OD) values of CCK-8 method increased in a dose-dependent manner, for BMSCs at 7 d, the control group of OD value was 0.58±0.02, the CGRP group 10-7-10-10 mol/L was to 0.80±0.03, 0.79±0.03, 0.74±0.03, 0.69±0.03, difference was significant; for VECs, the control group of OD value was 2.20±0.01, the CGRP group 10-7-10-10 mol/L was to 4.12±0.04, 3.90±0.02, 3.56±0.04, 3.12±0.04, difference was significant. Effect of CGRP on osteogenic differentiation of BMSCs: Alkaline phosphatase (ALP) activity, and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of ALP, CollagenⅠ, BMP-2, Osteonectin and RunX2 gene. Effect of CGRP on vascularization of VECs: Vascular cell endothelial growth factor (VEGF) protein secretion and gene expression level were tested by ELISA and RT-PCR, difference between the control group and CGRP group were all significant. Conclusion 10-7 mol/L, 10-10 mol/L of CGRP can directly promote the proliferation of the BMSCs and VECs of osteoporotic rats, promote the osteogenic differentiation of BMSCs, improve the expression of VEGF gene and protein secretion, promote angiogenesis. Key words: Calcitonin gene-related peptide; Osteoporosis; Fractures, bone

Effects of calcitonin gene-related peptide on eIF3a and p27 expression in bleomycin-induced pulmonary fibrosis of rats

To observe the effects of calcitonin gene-related peptide (CGRP) on eukaryotic translation initiation factor 3a (eIF3a) and p27 expression in bleomycin-induced pulmonary fibrosis of rats and its possible mechanism. Twenty-four male SD rats weighing 180~220 g were randomly divided into three groups (n=8):control group, bleomycin group, bleomycin plus capsaicin group. Bleomycin (5 mg/kg) was used to induce pulmonary fibrosis rat model. Rats were given capsaicin (50 mg/kg·d) by subcutaneous injections 4 days before to deplete endogenous CGRP. At the end of experiments, blood samples were collected from carotid artery to determinate the plasma levels of CGRP by ELISA. The cells were divided into 6 groups as follows:control group, transforming growth factor-β1 (TGF-β1) group, +CGRP (1, 10, 100 nmol/L) group, +CGRP 100 nmol/L and CGRP8-37 1 μmol/L group respectively(n=9). TGF-β1 (5 ng/ml) stimulated proliferation of pulmonary fibroblasts and proliferation was measured by BrdU marking. The expression levels of eIF3a, p27, α-smooth muscle actin (α-SMA) and collagen Ⅰ were detected by immunohistochemisty, real-time PCR or Western blot. The expressions of eIF3a, α-SMA, and collagen I were increased and the expression of p27 was decreasing in pulmonary fibrosis rats induced by bleomycin. Exogenous application of CGRP significantly inhibited TGF-β1-induced proliferation and differentiation of pulmonary fibroblasts and the expressions of α-SMA, collagen I and eIF3a, and upregulated the expression of p27. All these effects of CGRP were abolished in the presence of CGRP8-37. These results suggest that endogenous CGRP is related to the development of pulmonary fibrosis induced by bleomycin, and the inhibitory effect of CGRP on proliferation of lung fibroblasts involves the eIF3a/p27 signaling pathway.

Effect of pentazocine on the alteration of cardiac troponin I in leg ischemia/reperfusion induced by tourniquet

Objective To explore the effect of pentazocine on the changes in cardiac troponin I(cTnI) and its association with calcitonin gene-related peptide(CGRP) in tourniquet-induced leg ischemia/reperfusion(I/R). Methods Sixty patients, undergoing elective unilateral leg surgery using tourniquet, under spinal anesthesia, were enrolled and randomly assigned to control group (group C) and pentazocine group(group P)(n=30), in accordance with a random number table. Patients in group P pretreated with pentazocine 0.5 mg/kg before surgery, while only the same volume of saline was infused intravenously in group C at the same schedule. 5 ml of blood samples were collected from cubital vein in a not transfusion arm immediately before inflating tourniquet (T0), 0.5 h(T1) and 24 h(T2) after tourniquet deflation. Serum concentration of cTnI and CGRP were measured by ELISA. Results There was no statistical difference detected in the general information in two groups(P>0.05). Compared with the cTnI at the time of T0 [(0.21±0.06) μg/L] the concentration of cTnI in group C at the time of T1 was not obvious changed(P>0.05), while significantly increased at the time of T2 [(0.28±0.10) μg/L] (P 0.05). No significant change in the concentration of CGRP of group C was detected at the time of T1 and T2(P>0.05), while CGRP in group P was increased significantly at the time of T1[(17.9±2.7) ng/L] and T2[(18.8±2.8) ng/L](P 0.05), cTnI in group P was decreased significantly at the time of T2[(0.22±0.06) μg/L vs (0.28±0.10) μg/L] (P<0.05), CGRP in group P was increased obviously at T1 and at T2(P<0.05). Conclusions The results of this study indicates that pentazocine may attenuate myocardial injury induced by leg I/R tourniquet, in which endogenous CGRP may be associated. Key words: Pentazocine; Tourniquets; Ischemia/reperfusion, Leg; Cardiac troponin I; Calcitonin gene-related peptide

Calcitonin Gene-Related Peptide Inhibits Cardiomyocyte Injury Induced by Hypoxia/Reoxygenation via Restoration of Cytosolic and Mitochondrial Calcium

Background: Calcium overload plays an important role in myocardial ischemia and reperfusion injury. Calcitonin gene-related peptide (CGRP) presents a property of cardioprotection, underlying which the mechanism is still elusive. In this study, we investigated the association of CGRP in attenuation of myocyte injury induced by hypoxia/reoxygenation (H/R) with modulation of cytosolic and mitochondrial calcium.Methods: Cultured cardiomyocytes were assigned to four groups, 1) control group, without any treatment with any test agent, 2) H/R group, cells exposed to H/R, 3) H/R+CGRP group, cells treated with CGRP (at 10-8 mol/L) at the beginning of reoxygenation, and 4) H/R+CGRP+CGRP8-37 group, cells treated with CGRP (10-8 mol/l) and CGRP8-37, a special antagonist of CGRP receptor (at 10-7 mol/l), 1 hour before start of the hypoxia. The changes in calcium concentration, cell apoptosis, lactate dehydrogenase (LDH) and caspase-3 were analyzed using immunocytochemical, terminal deoxyuridine triphosphate (dUTP) deoxynucleotidyltransferase nick end-labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) respectively.Results: The greater levels of the apoptosis ratio, LDH, caspase-3, the cytosolic and mitochondrial calcium in H/R group were detected (all P<0.05), compared to those in the control group, respectively. CGRP significantly attenuated the increases of the LDH, caspase-3 and myocyte apoptosis, with reduction of cytosolic and mitochondrial calcium in the myocytes treated with H/R. The effect of CGRP was reversed by CGRP8-37.Conclusions: The findings in this study suggested that CGRP prevents myocyte injury induced by H/R in cultured cardiomyocytes, which may be associated with homeostasis of cytosolic and mitochondrial calcium. Citation: Mu-Rong Li, Zheng Guo, Lu Chen. Calcitonin gene-related peptide inhibits cardiomyocyte injury induced by hypoxia/reoxygenation via restoration of cytosolic and mitochondrial calcium. J Anesth Perioper Med 2016; 3: 125-131. doi: 10.24015/JAPM.2016.0017This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Effect of calcitonin gene-related peptide on anoxia-reoxygenation induced injury to neonatal rat cardiomyocytes incubated in high glucose medium

Objective To evaluate the effect of calcitonin gene-related peptide (CGRP) on anoxiareoxygenation (A/R)-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium.Methods Cardiomyocytes were obtained from 1-3-day old Sprague-Dawley rats and cultured in the culture medium containing 15％ bovine calf serum and then seeded onto 6-well plates at a density of 10 × 105/ml (3 ml/well).The cells were randomly divided into 5 groups (n =9 each):normal glucose medium control group (NG group),high glucose medium group (HG group),high glucose medium + A/R group (HG+ A/R group),high glucose medium + A/R + CGRP group (HG + A/R + CGRP group),and high glucose medium + A/R + CGRP+ CGRP8-37 group (HG + A/R + CGRP + CGRP8-37 group).The cells were incubated in normal glucose (5.5 mmol/L) medium for 72 h in NG group.In HG group,the cells were incubated in high glucose (25.0 mmol/L) medium for 72 h.In HG + A/ R group,the cells were incubated in high glucose medium for 72 h and then exposed to 3 h of anoxia followed by 2 h of reoxygenation.In group HG + A/R + CGRP,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10-8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP (final concentration 10 8 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.In HG + A/R + CGRP + CGRP8-37 group,the cells were incubated in high glucose medium for 72 h,CGRP8-37 (final concentration 10-8 mol/L) and CGRP8-37 (CGRP receptor antagonist,final concentration 10-7 mol/L) was then added to the culture media and the cells were incubated for 1 h and then underwent A/R.Apoptosis in cardiomyocytes was detected using TUNEL and apoptosis index (AI) was calculated.The lactate dehydrogenase (LDH) activity in the culture medium was analyzed.Results AI and LDH activity were significantly higher in HG group than in NG group,and in HG + A/R group than in HG group.Compared with HG + A/R group,AI and LDH activity were significantly decreased in HG + A/R + CGRP group,while no significant changes were found in HG + A/R + CGRP + CGRP8-37 group.Compared with HG + A/R + CGRP group,AI and LDH activity were significantly increased in HG + A/R + CGRP + CGRP8-37 group.Conclusion CGRP attenuates A/R-induced injury to neonatal rat cardiomyocytes incubated in high glucose medium via combing with CGRP receptor. Key words: Calcitonin gene-related peptide; Diabetes mellitus; Cell hypoxia; Myocytes, cardiac ; Infant, newborn

The changes of Proteome in MG-63 cells after induced by calcitonin gene-related peptide

The aim of this study was to elucidate the changes of Proteomics in MG-63 cells after induced by calcitonin gene-related peptide (CGRP). The subcultured MG63 cells were randomly divided into CGRP group and the control group. Two-dimensional electrophoresis (2-DE), coomassie brilliant blue gel staining and mass spectrometry were used to analyze the changes of protein extracted from the two groups. Six protein spots with significant differences were selected to take in-gel digestion, peptide mass fingerprint analysis and IPI database search. There were more than 967±17 protein spots separated by Two-dimensional gel electrophoresis and the matching rate was (85.1±1.4%). Compared with the control group, six protein spots were significantly different in CGRP-induced group and expression of the 6 differently proteins was downregulated. Mass spectrometry analysis identified 5 proteins, including ribosome binding protein p180 which is related to new synthetic protein’s translocation and calcium-binding protein (HRNR Hornerin) that can affect intracellular calcium concentration to regulate cell activity. These changes of proteome, including several groups of proteins that influence calcium ion concentration and new protein translocation, suggested that CGRP may regulate the cells by the second messengers and cytokines. This study contributes to a better understanding of the molecular mechanisms in CGRP-induced MG-63 cells.

Effects of subcutaneous carbon dioxide on calcitonin gene related peptide and substance P secretion in rat skin.

To investigate the subcutaneous injection of carbon dioxide (CO2) on neuropeptides Calcitonin Gene-Related Peptide (CGRP) and Substance P (SP) secretion in rat skin. Fifty-six Wistar-EPM rats were distributed in two groups: one for CGRP analysis, the other for SP analysis. Each group was subdivided into four subgroups: control (Cont), control with needle (ContNd), CO2 injection (CO2Inj) and atmospheric air injection (AirInj) - with seven animals each. Sample analyses of partial skin were conducted by Western Blotting (WB). In SP group, there was a decrease in the amount of neuropeptides in subgroups CO2Inj and AirInj. Similarly, in CGRP group, there was a decrease in the amount of pro-CGRP neuropeptides (15 kDa) in subgroups CO2Inj and AirInj; Nevertheless, there was no decrease in the amount of CGRP (5 kDa) in any subgroups. Subcutaneous injection of CO2 and atmospheric air decreased the amount of Substance P and pro-Calcitonin Gene-Related Peptide (15 kDa) neuropeptides in rat skin.