The Experimental Study on the Effect Calcitonin Gene related Peptide on Bone Resorption Mediated by Interleukin-1

To investigate the effect of calcitonin gene related peptide (CGRP) on bone resorption mediated by interleukin 1β(IL 1β) in vitro , the osteoclasts isolated from the long bones of newborn SD rats were co cultured with osteoblasts on ivory slices placed in 24 well plates . 24 h later, conditioned media containing CGRP and/or IL 1β were added to the wells respectively, and continued culturing for 48 h. After the cells were stripped off by ultrasonication, the ivory slices were stained in toludine blue. The number and the total area of resorption lacunae on each slice were measured by computer imaging analysis system. Our results showed that IL 1β significantly stimulated bone resorption, but CGRP inhibited the effect mediated by IL 1β in a dose dependent manner. It is suggested that CGRP may inhibit osteoclastic bone resorption through two ways: One is that CGRP functions directly on osteoclasts to block their activation; the other is that CGRP regulates the release of cytokines by osteoblasts and indirectly affects the function of osteoclasts.

RELATIONSHIP BETWEEN THE PATTERN OF METHYLATION OF CALCITONIN GENE AND ACTIVITY OF METHYLTRANSFERASEIN 8 TUMOR CELL LINES

Objective: The current study was designed to investigate the relationship between the pattern of methylation of calcitonin (CT) gene and the activity of DNA methyltranferase (MTase). Methods: The methylation rate of the 5′ region of the CT gene (CTMR) and the activity of MTase in 6 solid tumor cell lines and 2 leukemia cell lines were determined. CTMR was detected by polymerase chain reaction (PCR) with inter and external references in combination with restriction endonuclease and laser scan technique. MTase activity was examined by isotope labeled microassay. Results: Both CTMR and MTase activity in all tumor cell lines were significantly higher than that of control cells. The increased MTase activity was relative to elevated CTMR. Conclusion: There is prevalence of hypermethylation of CT gene and elevated activity of MTase in malignant cells. The increased MTase activity is one of the possible reasons for CT gene hypermethylation in tumor cells.

Controlling N-methyl-D-aspartate receptor subunit 1 with calcitonin gene related peptide after cerebral ischemic injury

BACKGROUND: Activation of N-methyl-D-aspartate receptor (NMDAR) is a key link of exitotoxicity at the phase of cerebral ischemic injury. Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid receptors, over-excitation can cause neuronal apoptosis. Calcitonin gene related peptide has a strongly biological activity. On one hand, it can protect ischemic neurons through inhibiting the expression of NMDAR1 mRNA; on the other hand, it can play the protective effect through down-regulating the expression of NMDAR1 mRNA by exogenous calcitonin gene related peptide. OBJECTIVE: To observe the expression of NMDAR1 and the regulatory effect of calcitonin gene related peptide on the expression of NMDAR1 mRNA and protein in the cerebral cortex of rats with focal cerebral ischemia/reperfusion (I/R). DESIGN: Randomized controlled animal study. SETTING: China Medical University. MATERIALS: A total of 216 healthy male Wistar rats, general grade, weighing 250-280 g, were selected in this study. Twelve rats were randomly selected to regard as control group; meanwhile, other 204 rats were used to establish middle cerebral artery occlusion/reperfusion (MACO) models. The main reagents were detailed as follows: calcitonin gene related peptide (Sigma Company); calcitonin gene related peptide kit (Boster Company); antibody Ⅰ, Ⅱ and antibody β-actin Ⅰ, Ⅱ of NMDAR1 mRNA and chemiluminescence reagent (Santa Cruz Company, USA). METHODS: The experiment was carried out in the Laboratory of Neurobiology of China Medical University from August 2005 to June 2006. ① Right MCAO models of rats were established to cause focal ischemia and scored based on Zea Longa five-grade scale. If the scores were 1, 2 and 3 after wakefulness, the MACO models were established successfully and involved in the experiment. A total of 120 rats with successful modeling were randomly divided into I/R group and administration group with 60 in each group. All rats in the both groups were observed at five time points, including 6, 12, 24, 48 and 72 hours after reperfusion and after 2-hour ischemia, with 12 experimental animals at each time point. Six rats were prepared for detection of hybridization in situ, and the other 6 were used for Western blotting histochemical detection. Rats in the control group were opened their skin to separate common carotid artery and not treated with line and drugs. In addition, rats in the I/R group were treated with 1 mL saline at 2 hours after focal cerebral ischemia, and then, rats in the administration group were treated with 1 mL (1 g/L) calcitonin gene related peptide at 2 hours after focal cerebral ischemia. ② The expression of NMDAR1 mRNA was detected with hybridization in situ at various time points; moreover, the expression of NMDAR1 protein was measured with Western blotting method at various time points. The results were analyzed with Metamoph imaging analytical system. MAIN OUTCOME MEASURES: The expression of NMDAR1 mRNA and its protein in cortical neurons of rats at various time points. RESULTS: A total of 84 rats were excluded because of non-symptoms, exanimation or death; and then, 132 rats were involved in the final analysis. The expression of NMDAR1 mRNA and its protein in cortical neurons of rats in the control group was 0.205±0.001 and 0.184±0.001, respectively; after I/R, expression of NMDAR1 mRNA and its protein was up-regulated, especially, expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.245±0.003, 0.287±0.004, 0.354±0.008, 0.284±0.002 and 0.217±0.006, respectively; moreover, expression of protein at 6, 12, 24, 48 and 72 hours was 0.222±0.003, 0.261±0.028, 0.311±0.004, 0.259±0.013 and 0.210±0.008, respectively. There was significant difference between the two groups (0.205±0.001, P < 0.01). The expression was up-related in the former 24 hours, reached peak at 24 hours, down-regulated, and decreased to the level of control group at 72 hours. Except 72 hours, the expression of NMDAR1 mRNA and its protein was lower in administration group than that in I/R group at other four time points. In addition, the expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.223±0.005, 0.243±0.001, 0.292±0.002, 0.250±0.003 and 0.213±0.003, respectively; moreover, the expression of protein at 6, 12, 24, 48 and 72 hours was 0.216±0.006, 0.245±0.025, 0.276±0.003, 0.241±0.045 and 0.202±0.013, respectively. There was significant difference at various time points (P < 0.05). CONCLUSION: The expressions of NMDAR1 mRNA and its protein of peripheral cortical neurons are up-related in ischemic area after focal cerebral I/R. Meanwhile, exogenous calcitonin gene related peptide can protect cortical neurons through inhibiting expression of NMDAR1 mRNA and its protein after focal cerebral I/R.

Therapeutic effects of Clostridium butyricum on experimental colitis induced by oxazolone in rats

AIM： To evaluate the therapeutic effects of a probiotic supplement （Clostridium butyricum, CGMCC0313 ） in a chemically-induced rat model of experimental colitis. METHODS： An experimental ulcerative colitis model was established by rectal injection of oxazolone into the colon of 40 Wistar rats randomly divided into four groups. The positive control group was sacrificed 3 d after colitis onset. The remaining groups were fed daily with either 2 mL of C. butyricum （2.3 × 10^11 CFU/L）, 2 mL of mesalamine （100 g/L）, or 1 mL of sodium butyrate （50 mmol/L） for 21 d. The animals＇ body weight, behavior, and bowel movements were recorded weekly. After sacrifice, visual and microscopic observations of pathological changes of colon tissue were made, body weight and wet colon mass index were measured and recorded, and serum levels of interleukin-23 （IL-23） and TNF-α were measured using ELISA. Expression of calcitonin gene-related peptide in colon tissue was measured by RT-PCR. Finally, changes in rat intestinal microflora status were measured in all groups.lowered the serum levels of both IL-23 and tumor necrosis factor-α （TNF-α） with similar or even better efficiency than that of mesalamine or sodium butyrate. The rat intestinal flora appeared to recover more quickly in the group treated with C. butyricum than in the mesalamine and sodium butyrate groups. Finally, we found that the expression level of calcitonin gene related peptide was elevated in colon tissue in the sodium butyrate treated group but not in the C. butyricum or mesalamine treated groups, indicating a sensitization of colon following sodium butyrate treatment. CONCLUSION： In our experimental colitis model, treatment with C. butyricum CGMCC0313, a probiotic supplement, is at least as efficient as treatment with mesalamine.

A novel large animal model of recurrent migraine established by repeated administration of inflammatory soup into the dura mater of the rhesus monkey

Several animal models of migraine have been established, and those based on trigeminovascular system activation are widely accepted. How- ever, most of these models have been established on lower animals, such as rodents, and involve only a single administration of a noxious stimulus. In this study, an inflammatory soup （10 μL）, consisting of prostaglandin E2 （0.2 mM）, serotonin （2 mM）, bradykinin （2 raM） and histamine （2 raM）, was injected into the dura mater of conscious rhesus monkeys through an indwelling catheter. The infusion started on day 8 and was repeated every 3 days, for a total of six administrations, to induce neurogenic inflammation. We performed behavioral assessments and measured the expression of the oncogene c-fos, neuronal nitric oxide synthase （nNOS） and calcitonin gene related peptide （CGRP） ill the trigeminal system and in multiple brain regions involved in pain processing by immunohistochemical staining. Compared with monkeys in the control group, three of the four animals in the inflammatory soup group displayed decreased motor behaviors, and two showed increased ipsilateral nose and mouth secretions during the stimulus period. Higher expression levels of c-fos, nNOS and CGRP were found in various brain areas of experimental animals compared with controls, including the trigeminal nucleus caudalis, thalamus, hypothalamus, midbrain, pons and other areas involved in pain perception. These results suggest that repeated inflammatory soup stimulation of the dura activates the trigeminovascular system and produces migraine-like pathological changes and abnormal behaviors in conscious rhesus monkeys.

Clinical Study on Influence of Buyang Huanwu Decoction (补阳还五汤) on the Metabolic Imbalance of Endothelin and Calcitonin Gene Related Peptide in Patients with Early Cerebral Infarction

Objective: To explore the mechanism of Buyang Huanwu Decoction (BHD) in treating early cerebral infarction. Methods: Seventy cases with early cerebral infarction were random ly divided into two groups. Chinese medicine group (CMG, n=35) was treated with BHD; western medicine group (WMG, n=35) was treated with hydroxyethyl starch injection and enteric coated aspirin tablets. The levels of endothelin (ET) and calcitonin gene related peptide (CGRP) in plasma before and after treatment and the results of clinical treatment were observed.Al so healthy subjects were used as the normal control. Results: The levels of ET before treatment in the two treated groups were significantly higher than that of the healthy subjects (P<0.001), and the levels of CGRP were significantly lower (P<0.001). After treatment the metabolic imbalance of ET and CGRP improved significantly in the two treated groups (P<0.001), but the ET and CGRP in CMG improved more obviously than those in WMG (P<0.01, P<0.05). The markedly effective and curative rate of CMG was higher than that of WMG (68.6% vs 31.4%; χ2=9.65, P<0.01). Conclusion: BHD could improve the metabolic imbalance of ET and CGRP in patients with early cerebral infarction and on the virtue of this m echanism it could be used to treat cerebral infarction

Effect of Buyang Huanwu Decoction (补阳还五汤) on Plasma Thromboxane B_2, 6-Keto-Prostaglandin F_(1α), Endothelin and Calcitonin Gene Related Peptide in Primary Nephrotic Syndrome Patients

Objective: To explore the mechanism of Buyang Huanwu Decoction (BHD) in treating early cerebral infarction. Methods: Seventy cases with early cerebral infarction were randomly divided into two groups. Chinese medicine group (CMG, n=35) was treated with BHD; western medicine group (WMG, n=35) was treated with hydroxyethyl starch injection and enteric coated aspirin tablets. The levels of endothelin (ET) and calcitonin gene related peptide (CGRP) in plasma before and after treatment and the results of clinical treatment were observed.Also healthy subjects were used as the normal control. Results: The levels of ET before treatment in the two treated groups were significantly higher than that of the healthy subjects ( P <0.001), and the levels of CGRP were significantly lower ( P <0.001). After treatment the metabolic imbalance of ET and CGRP improved significantly in the two treated groups ( P <0.001), but the ET and CGRP in CMG improved more obviously than those in WMG ( P <0.01, P <0.05). The markedly effective and curative rate of CMG was higher than that of WMG (68.6% vs 31.4%; χ 2=9.65, P <0.01). Conclusion: BHD could improve the metabolic imbalance of ET and CGRP in patients with early cerebral infarction and on the virtue of this mechanism it could be used to treat cerebral infarction.

Clinical Study on Protective Effect of Xinmaitong Capsule(心脉通胶囊) on Damage of Vascular Endothelial Cells

Objective: To assess the effect of Xinmaitong (XMT) capsule in treating coronary heart disease (CHD).Methods: The 38 patients of coronary heart disease with myocardial ischemia were divided randomly into XMT group (20 cases) and control group (18 cases). Conventional western medical therapy was given to both groups and the XMT group received XMT treatment in addition. The changes of endothelin (ET) and calcitonin gene related peptide (CGRP) levels, ST segment of ECG and clinical symptoms after treatment in all the patients were observed. 14 healthy persons were taken as normal control.Results: The ET level of all patients was significantly higher than that of normal control ( P <0.001), and level of CGRP in patients was not significantly different from normal ( P >0.05). After treatment, results showed that: (1) The ET levels and the scores of clinical symptoms of both groups decreased significantly ( P <0.01), and the depressed ST segment elevated markedly ( P <0.01) as compared with that before treatment, and the changes revealed more evidently in XMT group in comparison with those in the control group ( P <0.05～0.01). (2) The level of CGRP was significantly increased in XMT group ( P <0.01) while it was unchanged in the control group ( P >0.05).Conclusion: There is a severe damage of vascular endothelial cells (VEC) in patients of coronary heart disease. XMT could not only reduce significantly the plasma ET content, but also enhance markedly the production and release of CGRP, so it has a good anti ischemic effect, which may be closely correlated with its action on improving the function of VEC and regulating metabolism of ET and CGRP.