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Abstract Background Dexmedetomidine (Dex) is widely used, and its most common side effect is bradycardia. The complete mechanism through which Dex induces bradycardia has not been elucidated. This research investigates the expression of gap junction proteins Connexin30.2 (Cx30.2) and Connexin40 (Cx40) within the sinoatrial node of rats with Dex-induced sinus bradycardia. Methods Eighty rats were randomly assigned to five groups. Saline was administered to rats in Group C. In the other four groups, the rats were administered Dex to induce bradycardia. In groups D1and D2, the rats were administered Dex at a loading dose of 30 μg.kg−1 and 100 μg.kg−1 for 10 min, then at 15 μg.kg−1.h−1 and 50 μg.kg−1.h−1 for 120 min separately. The rats in group D1A and D2A were administered Dex in the same way as in group D1and D2; however, immediately after the administration of the loading dose, 0.5 mg atropine was administered intravenously, and then at 0.5 mg.kg−1.h−1 for 120 min. The sinoatrial node was acquired after intravenous infusion was completed. Quantitative real-time polymerase chain reaction and western blot analyses were performed to measure mRNA and protein expression of Cx30.2 and Cx40, respectively. Results The expression of Cx30.2 increased, whereas the expression of Cx40 decreased within the sinoatrial node of rats with Dex-induced sinus bradycardia. Atropine reversed the effects of Dex on the expression of gap junction proteins. Conclusion Dex possibly altered the expression of gap junction proteins to slow down cardiac conduction velocity in the sinoatrial node.
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Animals , Rats , Sinoatrial Node/metabolism , Dexmedetomidine , Arrhythmias, Cardiac , Atropine Derivatives/metabolism , Bradycardia/chemically induced , Connexins/genetics , Connexins/metabolismABSTRACT
Objective:To evaluate the effects of different densities of rat cardiac fibroblasts (RCF) subjected to hypothermic hypoxia-reoxygenation on cardiomyocyte injury and intercellular coupling.Methods:RCF was cultured in vitro and divided into 3 groups ( n=12 each) using a random number table method: RCF density 0.5×10 5 cells/ml group (T 0.5 group), RCF density 1.0×10 5 cells/ml group (T 1.0 group), and RCF density 2.0×10 5 cells/ml group (T 2.0 group). The three groups were placed in an anoxic device, into which 95% N 2 + 5% CO 2 was continuously blown at the speed of 5 L/min for 15 min, and then placed in a 4 ℃ refrigerator for 1 h for low temperature treatment.After completion of culture, cells were placed in a incubator containing 95% air + 5% CO 2 at 37 ℃ for 4 h of reoxygenation.After the end of culture, RCF in three groups were indirectly co-cultured with cardiomyocytes of the same density (1.0×10 5 cells/ml) in a Transwell chamber for 16 h, cardiomyocytes were seeded in the lower chamber of Transwell, and RCF were seeded in the upper chamber of Transwell.After the end of co-culture, cardiomyocytes were collected for determination of the cell viability (by CCK8 method), apoptosis rate (by flow cytometry), expression of connexin 43 (Cx43) mRNA (by real-time fluorescence quantitative polymerase chain reaction), and expression of Cx43 and phosphorylated Cx43 (p-Cx43) (by Western blot). Results:Compared with T 0.5 group, the cell viability, apoptosis rate and expression of Cx43, p-Cx43 and Cx43 mRNA were significantly decreased in T 1.0 and T 2.0 groups ( P<0.01). Compared with T 1.0 group, the cell viability, apoptosis rate and expression of Cx43 and p-Cx43 were significantly decreased ( P<0.01), and no significant change was found in expression of Cx43 mRNA in cardiomyocytes in T 2.0 group ( P>0.05). Conclusions:RCF subjected to hypothermic hypoxia-reoxygenation induces cardiomyocyte injury in a density-dependent manner in a certain range, and the mechanism may be related to down-regulation of the expression of Cx43 and reduction of the activity of Cx43.
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Objective:To evaluate the effect of mild hypothermia on inositol requiring enzyme 1-X-box binding protein 1 (IRE1-XBP1) signaling pathway in endoplasmic reticulum in cortex in a rat model of focal cerebral ischemia-reperfusion (I/R).Methods:Fifty-four clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 200-230 g, were divided into 3 groups ( n=18 each) using a random number table method: sham operation group (group S), cerebral I/R group (group I) and mild hypothermia group (group T). Cerebral I/R was induced by inserting a nylon thread with rounded tip into the internal carotid artery which was occluded for 2 h and then released for reperfusion.The surface cooling was started immediately after reperfusion, and the rectal temperature was maintained at 32-34 ℃ for 3 h in group T. Blood vessels were only exposed, without occlusion in group S. The neurologic deficit was assessed and scored at 24 h of reperfusion.The animals were then sacrificed and the ischemic area of the cerebral cortex was removed for examination of the ultrastructure of the cells (with a transmission electron microscope), for determination of nerve cell apoptosis (using TUNEL), for detection of the expression of IRE1 and XBP1 (by Western blot) and for determination of the expression of IRE1 and XBP1 protein mRNA (using quantitative real-time polymerase chain reaction). Results:Compared with group S, the neurologic deficit scores were significantly increased, nerve cell apoptosis in the ischemic area of the cerebral cortex was increased, the expression of IRE1, XBP1 protein and mRNA was up-regulated ( P<0.05), the neuronal nuclei was degenerated and swollen, the nuclear membrane was fragmented and defective, the chromatin was pyknotic and marginalized, and the endoplasmic reticulum was dilated and cisternal in group I and group T. Compared with group I, the neurologic deficit scores were significantly decreased, nerve cell apoptosis in the ischemic area of the cerebral cortex was decreased, the expression of IRE1, XBP1 protein and mRNA was up-regulated ( P<0.05), and the damage to the ultrastructure of nerve cells was reduced in group T. Conclusion:The mechanism by which mild hypothermia alleviates focal cerebral I/R injury is associated with further activation of neuronal IRE1-XBP1 signaling pathway and alleviation of endoplasmic reticulum stress response in rats.
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Objective:To evaluate the relationship between spinal kindlin-1/Wnt3a signaling pathway and inflammatory response in a rat model of neuropathic pain (NP).Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (SH group), NP group, kindlin-1 shRNA group (K group) and Wnt3a inhibition group (W group). NP was induced by chronic constrictive injury in anesthetized animals.At 21 days before operation, kindlin-1 shRNA adenovirus vector 10 μl was intrathecally injected in group K, and empty viral vector 10 μl was intrathecally injected in SH, NP and W groups.Wnt inhibitor IWP-2 10 μl was intrathecally injected in group W, and artificial cerebrospinal fluid 10 μl was intrathecally injected in SH, NP and K groups at 1-3 days after operation.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 4 and 7 days after operation, respectively.At the end of pain threshold measurement at 7 days after operation, the animals were sacrificed and the lumbar segments (L 4-6) of the spinal cord were obtained for determination of the contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (IL-1β) (using enzyme-linked immunosorbent assay) and the expression of kindlin-1 and Wnt3a (by Western blot). Results:Compared with group SH, MWT was significantly decreased, TWL was shortened at 4 and 7 days after operation, and the contents of TNF-α and IL-1β in spinal cord were increased in NP, K, and W groups, the expression of kindlin-1 and Wnt3a was up-regulated in NP and W groups, and expression of Wnt3a was up-regulated in group K ( P<0.05). Compared with group NP, MWT was significantly increased and TWL was prolonged at 4 and 7 days after operation in K and W groups, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of kindlin-1 and Wnt3a was down-regulated in group K, the contents of TNF-α and IL-1β in spinal cord were decreased, and the expression of Wnt3a was down-regulated in group W ( P<0.05), and no significant change was found in kindlin-1 expression ( P>0.05). Conclusion:Spinal kindlin-1 regulates the inflammatory response by up-regulating the expression of Wnt3a, and it is involved in the maintenance of NP in rats.
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Objective:To evaluate the effects of different density rat fibroblasts on the expression of conjunctin 43 (Cx43) in cardiomyocytes and cell viability.Methods:Cardiomyocytes and fibroblasts were co-cultured using Transwell, cardiomyocytes were inoculated into the lower chamber of Transwell and fibroblasts into the upper chamber of Transwell.The cells were divided into 3 groups ( n=12 each) by a random number table method: fibroblast density 0.5×10 5 cells/ml group (group C 0.5), fibroblast density 1×10 5 cells/ml group (group C 1), and fibroblast density 2×10 5 cells/ml group (group C 2), with the density of cardiomyocytes 1×10 5 cells/ml in three groups.Cardiomyocytes and fibroblasts were co-cultured for 20 h in three groups.Cardiomyocytes were collected after co-culture for determination of cell viability (by CCK8 method), apoptosis rate (by flow cytometry), and expression of Cx43 mRNA (by quantitative real-time polymerase chain reaction) and expression of Cx43 and phosphorylated Cx43 (p-Cx43) (by Western blot). Results:There was no significant difference in the apoptosis rate of cardiomyocytes among the three groups ( P>0.05). Compared with group C 0.5, the expression of Cx43 protein and mRNA and p-Cx43 was significantly up-regulated in group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Compared with group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Conclusion:Rat fibroblasts up-regulate the expression of Cx43 and enhance the activity of Cx43 in cardiomyocytes and enhance cell viability in a density-dependent manner in a certain range.
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Objective@#To evaluate the effect of sevoflurane on phosphorylation of connexin 43 (Cx43) at Ser368 (Cx43 Ser368) in ventricular myocardium in isolated hearts of diabetic rats.@*Methods@#Twenty-four clean-grade healthy adult male Sprague-Dawley rats, aged 3 months, weighing 180-220 g, were used in this study.The diabetic model was established by intraperitoneally injecting streptozotocin 60 mg/kg.The hearts were rapidly excised and retrogradely perfused with K-H solution saturated with 95% O2-5% CO2 at 37 ℃ in a Langendorff apparatus.Sixteen Langendorff-perfused diabetic hearts were divided into 3 groups (n=8 each) using a random number table method: diabetes mellitus group (group D) and sevoflurane group (group DS). Another 8 Langendorff-perfused normal hearts of rats were selected and served as control group (group C). After 15 min equilibration with K-H solution, the hearts were continuously perfused for 30 min with K-H solution in group C and group D and with K-H solution saturated with 2.5% sevoflurane in group DS.S1S2 program-controlled stimulation was performed at the end of perfusion, the occurrence of induced ventricular arrhythmia (VA) and the maximal pacing cycle length (PCL) of induced VA were recorded, and conduction velocity (CV) was calculated.The expression of phosphorylated Cx43 at Ser368 (p-Cx43 Ser368) in ventricular myocytes was determined by Western blot.@*Results@#Compared with group C, the incidence of induced VA was significantly increased, the maximal PCL of induced VA was prolonged, the CV was decreased, and the expression of p-Cx43 Ser368 was up-regulated in group D (P<0.05). Compared with the incidence of induced VA was significantly decreased, the maximal PCL of induced VA was shortened, the CV was increased, and the expression of p-Cx43 Ser368 was down-regulated in group DS (P<0.05).@*Conclusion@#The mechanism by which sevoflurane stabilizes the ventricular electrical conduction is associated with decreasing the phosphorylation of Cx43 at Ser368 in diabetic rats.
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Objective@#To investigate the protective effects of pretreatment with a Shenfu(SF)injection on arrhythmias induced by myocardial ischemia-reperfusion(IR)injury in aged mice.@*Methods@#Thirty 18-month-old C57BL/6J mice were randomly divided into 3 groups(n=10, each group): the sham treatment group(receiving sham operation without thoracotomy), the IR group(undergoing the ligation of the left anterior descending coronary artery with ischemia for 30 min and reperfusion for 2 h)and the SF group(receiving SF intraperitoneal injection of 10 ml/kg 30 min before thoracotomy and the same treatment as the IR group). Arrhythmias were monitored, and serum levels of creatine kinase-isoenzyme(CK-MB), troponin(cTnI)and tumor necrosis factor α(TNFα), and the ratio of myocardial connexin 43(Cx43)phosphorylation(ser368)to total protein(p-Cx43/t-Cx43)were detected in the three groups.@*Results@#Ventricular arrhythmias occurred in the IR and SF groups.Compared with the IR group, ventricular arrhythmias in the SF group were alleviated, the frequency of ventricular premature systolic episodes was reduced(36.6±13.5 times vs. 48.4±22.1 times), the frequency of ventricular tachycardia/fibrillation decreased(3.4±1.8 times vs. 7.6±3.5 times), and the total duration of ventricular tachycardia/fibrillation episodes was shortened(8.9±4.5 times vs. 17.7±5.1 times)in the SF group(P<0.05), but there was no significant difference in arrhythmia scores(1.8±1.2 points vs. 1.9±1.7 points, P>0.05)between the two groups.Compared with the sham treatment group, serum levels of CK-MB, cTnI and myocardial TNF(increased in the IR and SF groups(P<0.05), and their levels were lower in the SF group than in the IR group(P<0.05). Compared with the sham treatment group, the ratio of Cx43 ser368/total protein was lower in the IR and SF groups, but was higher in the SF group than in the IR group(P<0.05).@*Conclusions@#SF pretreatment can significantly reduce IR-induced arrhythmias in aged mice possibly by reducing TNF(and up-regulating the phosphorylation activity of Cx43.
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Objective To investigate the protective effects of pretreatment with a Shenfu(SF) injection on arrhythmias induced by myocardial ischemia-reperfusion(IR)injury in aged mice.Methods Thirty 18-month-old C57BL/6J mice were randomly divided into 3 groups (n=10,each group):the sham treatment group(receiving sham operation without thoracotomy),the IR group (undergoing the ligation of the left anterior descending coronary artery with ischemia for 30 min and reperfusion for 2 h)and the SF group(receiving SF intraperitoneal injection of 10 ml/kg 30 min before thoracotomy and the same treatment as the IR group).Arrhythmias were monitored,and serum levels of creatine kinase-isoenzyme (CK-MB),troponin (cTnI) and tumor necrosis factor α (TNFα),and the ratio of myocardial connexin 43 (Cx43) phosphorylation (ser368) to total protein (p-Cx43/t-Cx43) were detected in the three groups.Results Ventricular arrhythmias occurred in the IR and SF groups.Compared with the IR group,ventricular arrhythmias in the SF group were alleviated,the frequency of ventricular premature systolic episodes was reduced(36.6 ± 13.5 times vs.48.4 ± 22.1 times),the frequency of ventricular tachycardia/fibrillation decreased(3.4 ± 1.8 times vs.7.6 ± 3.5 times),and the total duration of ventricular tachycardia/fibrillation episodes was shortened(8.9± 4.5 times vs.17.7± 5.1 times)in the SF group(P＜0.05),but there was no significant difference in arrhythmia scores(1.8± 1.2 points vs.1.9 ± 1.7 points,P ＞0.05) between the two groups.Compared with the sham treatment group,serum levels of CK-MB,cTnI and myocardial TNF(increased in the IR and SF groups (P ＜ 0.05),and their levels were lower in the SF group than in the IR group (P＜0.05).Compared with the sham treatment group,the ratio of Cx43 ser368/total protein was lower in the IR and SF groups,but was higher in the SF group than in the IR group(P＜0.05).Conclusions SF pretreatment can significantly reduce IR-induced arrhythmias in aged mice possibly by reducing TNF(and up-regulating the phosphorylation activity of Cx43.
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Objective To determine the distribution of common deafness gene mutations by microarray-PCR in pregnant women of Beijing, then explore their prevalence and clinical significance. Methods Totally 1709 pregnant women were prospectively enrolled from the outpatients of Obstetrical Clinical from Peking University Peoples' Hospital in this study from June 2016 to April 2018. Peripheral blood samples were obtained and DNA templates were extracted from all subjects. The coding region of the GJB2 gene(35del G, 235delC, 176-191del16, 299-300delAT), SLC26A4 gene(IVS7-2 A>G, 2168A>G), 12sRNA gene(1494C>T,1555A>G) and GJB3 gene(538 C>T)were detected by microarray-PCR. Meanwhile,256 cases firstly recruited in this study were confirmed by Sanger sequencing. The analysis was performed to explore the distribution of deafness gene mutations and the subjects detected to be positive were further followed up until they had given birth to their babies.Results Among 1709 pregnant women, 89 cases were found to be carrying at least one mutation sites(5.21％).Among them,83 cases were heterozygous mutation(35 cases of GJB2235delC, 12 cases of GJB2299-300delAT mutation, one case of GJB2176-191del 16, 30 cases of SLC26A4 IVS 7-2 A>G, 3 cases of SLC26A42168 A>G and two cases of GJB3538 C>T). There were one case of 235delC homozygous mutation and 2 cases of double mutations(IVS7-2 A>G /GJB2299-300delAT, IVS7-2 A>G/GJB2235delC). The positive rate of 235delC, 299-300delAT and 176-191del16 was 2.17％, 0.76％and 0.06％, respectively. As to SLC26A4, 1.87％of the pregnant women were carrying IVS 7-2 A>G and 0.18％for 2168 A>G. Two cases were detected carrying GJB3 C>T mutation and three for 12s RNA1555 A>G mutation, respectively. The results of microarray-PCR were identical to those of Sanger sequencing, with the coincidence of 100％.26 spouses of the 56 cases were followed up. Two proved to be carrier of the same gene mutation. One of their babies was born with normal hearing until the end of this research, while another baby was born deafness and implant cochlear when three month old. Conclusions GJB2235delC and SLC26A4 IVS 7-2 A>G were the most prevalent mutations in pregnant women of Beijing, and it may provide guidance to eugenics and early clinical intervention.
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Objective@#To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.@*Methods@#Forty SPF healthy male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 5 groups (n=8 each) using a random number table method: control group (group C), NL1-shRNA plasmid group (group NL), incisional pain plus remifentanil group (group I+ R), incisional pain plus remifentanil plus blank vector group (group I+ R+ B), and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+ R+ NL). Negative lentivirus was intrathecally injected in group I+ R+ B.In NL and I+ R+ NL groups, 10 μl NL-1-shRNA lentivirus at 1×108 IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+ R groups.After transfection was stable, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+ R, I+ R+ B and I+ R+ NL groups, 0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals, and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3, 6, 24 and 48 h after the end of administration (T1-4). The animals were sacrificed after measurement of pain threshold at T4, and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors, and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.@*Results@#Compared with group C, the MWT was significantly decreased, and TFL was shortened at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated, and m/t ratio was increased in I+ R and I+ R+ B groups (P<0.05). Compared with I+ R and I+ R+ B groups, the MWT was significantly increased and TFL was prolonged at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated, and m/t ratio was decreased in group I+ R+ NL (P<0.05).@*Conclusion@#NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane, which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.
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Objective To evaluate the effect of sevoflurane on phosphorylation of connexin 43 (Cx43) at Ser368 (Cx43 Ser368) in ventricular myocardium in isolated hearts of diabetic rats.Methods Twenty-four clean-grade healthy adult male Sprague-Dawley rats,aged 3 months,weighing 180-220 g,were used in this study.The diabetic model was established by intraperitoneally injecting streptozotocin 60 mg/kg.The hearts were rapidly excised and retrogradely perfused with K-H solution saturated with 95％ O2-5％ CO2 at 37 ℃ in a Langendorff apparatus.Sixteen Langendorff-perfused diabetic hearts were divided into 3 groups (n =8 each) using a random number table method:diabetes mellitus group (group D) and sevoflurane group (group DS).Another 8 Langendorff-perfused normal hearts of rats were selected and served as control group (group C).After 15 min equilibration with K-H solution,the hearts were continuously perfused for 30 min with K-H solution in group C and group D and with K-H solution saturated with 2.5％ sevoflurane in group DS.S1S2 program-controlled stimulation was performed at the end of perfusion,the occurrence of induced ventricular arrhythmia (VA) and the maximal pacing cycle length (PCL) of induced VA were recorded,and conduction velocity (CV) was calculated.The expression of phosphorylated Cx43 at Ser368 (p-Cx43 Ser368) in ventricular myocytes was determined by Western blot.Results Compared with group C,the incidence of induced VA was significantly increased,the maximal PCL of induced VA was prolonged,the CV was decreased,and the expression of p-Cx43 Ser368 was up-regulated in group D (P＜0.05).Compared with the incidence of induced VA was significantly decreased,the maximal PCL of induced VA was shortened,the CV was increased,and the expression of p-Cx43 Ser368 was down-regulated in group DS (P＜0.05).Conclusion The mechanism by which sevoflurane stabilizes the ventricular electrical conduction is associated with decreasing the phosphorylation of Cx43 at Ser368 in diabetic rats.
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Objective To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.Methods Forty SPF healthy male C57BL/6J mice,aged 8-10 weeks,weighing 18-22 g,were divided into 5 groups (n=8 each) using a random number table method:control group (group C),NL1-shRNA plasmid group (group NL),incisional pain plus remifentanil group (group I+R),incisional pain plus remifentanil plus blank vector group (group I+R+B),and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+R+NL).Negative lentivirus was intrathecally injected in group I+R+B.In NL and I+R+NL groups,10 μl NL-1-shRNA lentivirus at 1×10s IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+R groups.After transfection was stable,normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+R,I+R+B and I+R+NL groups,0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals,and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3,6,24 and 48 h after the end of administration (T1-4).The animals were sacrificed after measurement of pain threshold at T4,and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors,and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated.Results Compared with group C,the MWT was significantly decreased,and TFL was shortened at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated,and m/t ratio was increased in I+R and I+R+B groups (P＜0.05).Compared with I+R and I+R+B groups,the MWT was significantly increased and TFL was prolonged at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated,and m/t ratio was decreased in group I+R+NL (P＜0.05).Conclusion NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane,which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.
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Objective To evaluate the changes in the expression of Cx45 and Cx40 in the sinoatrial node during dexmedetomidine-induced sinus bradycardia in rats. Methods Forty healthy Sprague-Dawley rats, weighing 250-300 g, were divided into 5 groups ( n=8 each) using a random number table method:control group ( group C) , low-dose dexmedetomidine group ( group D1 ) , high-dose dexmedetomidine group ( group D2 ) , low-dose dexmedetomidine plus atropin group ( group D1 A) , and high-dose dexmedetomidine plus atropin group (group D2A). Normal saline was intravenously infused in group C. Dexmedetomidine was intravenously infused for 10 min as a loading dose of 20 and 120 μg∕kg, followed by an infusion of 10 and 60 μg·kg-1 ·h-1 for 110 min in D1 and D2 groups, respectively. In D1 A and D2 A groups, dexme-detomidine was correspondingly given according to the method previously described in D1 and D2 groups, and in addition atropin 0. 5 mg was intravenously injected at the end of infusing the loading dose of dexme-detomidine. Heart rate ( HR ) , mean arterial pressure ( MAP ) and SpO2 were recorded before giving dexmedetomidine and at 10, 60 and 120 min after giving dexmedetomidine, and the development of brady-cardia was recorded. The sinoatrial node tissues were obtained at the end of administration for determination of the expression of Cx45 and Cx40 protein and mRNA by Western blot and real-time polymerase chain reac-tion, respectively. Results The incidence of bradycardia was 100％ in D1 and D2 groups and 0 after using atropin in D1 A and D2 A groups. Compared with group C, HR and MAP were significantly decreased in the other four groups, the expression of Cx45 protein and mRNA was up-regulated, and the expression of Cx40 protein and mRNA was down-regulated in D1 and D2 groups (P<0. 05), and no significant change was found in the expression of Cx45 and Cx40 protein and mRNA in D1A and D2A groups (P>0. 05). Com-pared with group D1 , HR and MAP were significantly increased, the expression of Cx45 protein and mRNA was down-regulated, and the expression of Cx40 protein and mRNA was up-regulated in group D1 A ( P<0. 05) . Compared with group D2 , HR and MAP were significantly increased, the expression of Cx45 pro-tein and mRNA was down-regulated, and the expression of Cx40 protein and mRNA was up-regulated in group D2 A ( P<0. 05) . Conclusion The mechanism by which dexmedetomidine induces sinus bradycardia may be related to up-regulated expression of Cx45 and down-regulated expression of Cx40, and the auto-nomic nervous activity is involved in dexmedetomidine-induced regulation of Cx45 and Cx40 expression in rats.
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Objective To investigate the expression and clinical significance of epithelial cell adhesion molecule(EpCAM) and Claudin-18 in gastric cancer.Methods Surgical specimens of gastric cancer were taken from 64 patients.The histological diagnostic criteria were based on WHO standards.Immunohistochemical staining was used to detect expression levels of EpCAM and Claudin-18 in all samples.The relationship between the expression of EpCAM and Claudin-18 and clinicopathological parameters of gastric cancer was analyzed.Correlations of the prognosis of gastric cancer patients with EpCAM and Claudin-18 expression were analyzed using the Cox proportional hazards regression model.Results The positive expression rate of EpCAM was elevated with the increase in tumor size and the occurrence of distant metastasis(x2 =4.526 and 36.090,P=0.033 and 0.000).There were significant differences in Claudin-18 protein expression among different age,TNM stage,growth pattern,depth of invasion and distant metastasis patients(all P＜0.05).Cox regression model analysis showed that tumor size and distant metastasis were the main risk factors affecting the survival of patients with gastric cancer (RR =50.076 and 1.617,P =0.016 and 0.032),while levels of EpCAM and Claudin-18 were not independent risk factors (both P ＞ 0.05).Conclusions EpCAM and Claudin-18 are closely related to the invasiveness of gastric cancer,but abnormally high levels of EpCAM and Claudin-18 are not independent risk factors for the prognosis of gastric cancer patients.
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Objective To evaluate the role of spinal kindlin-1 in neuropathic pain in rats and the relationship with Wnt3a.Methods Eighteen clean-grade healthy male Sprague-Dawley rats,weighing 250-280 g,aged 10-12 weeks,were divided into 3 groups (n =6 each) using a random number table:sham operation group (group S),neuropathic pain group (group NP) and kindlin-1 inhibitor group (group K).Neuropathic pain was induced by chronic compression of the sciatic nerve.The sciatic nerve was only exposed but not ligated in group S.In group K,shRNA was intrathecally injected at 21 days before operation to inhibit the expression of kindlin-1.Vector virus was intrathecally injected at 21 days before operation in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before operation and 1,4,7,10 and 13 days after operation.Rats were sacrificed at 13 days after measurement of pain threshold and the spinal cord was removed for determination of the expression of kindlin-1 and Wnt3a (by Western blot) and expression of Wnt3a mRNA (by real-time polymerase chain reaction).Results Compared with group S,the MWT was significantly decreased and the TWL was shortened at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was up-regulated in group NP (P＜0.05).Compared with group NP,the MWT was significantly increased and the TWL was prolonged at 4,7,10 and 13 days,and the expression of Wnt3a protein and mRNA and kindlin-1 was down-regulated in group K (P＜0.05).Conclusion Kindlin-1 is involved in the development of neuropathic pain by up-regulating the expression of Wnt3a in rats.
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X-linked Charcot-Marie-Tooth disease type 1 (CMTX1) is caused by the mutation in GJB1 gene, characterized by the transient central nervous system involvement and long standing peripheral polyneuropathy which does not fulfill the criteria of demyelination or axonopathy. We describe a 37-year-old man with progressive bilateral leg weakness since his early teen. He suffered transient right hemiparesis, followed by quadriparesis at 14 years of age. When we examined him at 37 years of age, he presented a distal muscle weakness on lower extremities with a sensory symptom. The nerve conduction study demonstrated a motor conduction velocity between 26 and 49 m/s. The whole exome sequencing revealed a novel variant c.136 G>A in GJB1. This report will raise awareness in this rare disease, which is frequently misdiagnosed early in its course.
Subject(s)
Adolescent , Adult , Humans , Central Nervous System , Charcot-Marie-Tooth Disease , Connexins , Demyelinating Diseases , Exome , Leg , Lower Extremity , Muscle Weakness , Mutation, Missense , Neural Conduction , Paresis , Polyneuropathies , Quadriplegia , Rare DiseasesABSTRACT
Connexins (Cxs) are a family of transmembrane proteins that form gap junctions and hemi-channels, which mediate cell-cell communication between neighboring cells and the respective extracellular milieu in different tissues. Most tissues and cell types throughout the body express one or more Cx proteins, highlighting its importance in regulating cell growth, differentiation, adhesion, migration, cell death and others. Moreover, Cx can propagate intracellular signals through its C-terminus domain, and thus function beyond a mere channel. Cx43 is the most highly expressed and most well studied Cx in bone and musculoskeletal tissues, although Cx40, Cx45, Cx46 and more recently, the Cx37 have been described in bone tissue, along with Cx26, Cx32 and Cx39 in other musculoskeletal tissues. Here, we discuss the basic structure of gap junctions and the role of the Cxs in musculoskeletal tissue, with special focus on Cx37. (AU)
Las conexinas (Cxs) son una familia de proteínas transmembrana que forman uniones en hendidura y hemicanales encargados de mediar la comunicación entre células vecinas y el respectivo medio extracelular en diferentes tejidos. La mayoría de los tejidos y células expresan una o más proteínas conexina, jugando un papel importante en la regulación de la proliferación celular, diferenciación, adhesión, migración y muerte celular, entre otras funciones. Además de actuar como un canal, las conexinas pueden propagar señales intracelulares a través del dominio C-terminal. La Cx43 es la conexina mas expresada y mejor estudiada en el tejido óseo y el músculo, aunque las Cx40, Cx45, Cx46, y mas recientemente Cx37, son también detectadas en el hueso. A su vez la expresión de la Cx26, Cx32 y Cx39 ha sido observada en otros tejidos músculoesqueléticos. En este manuscrito describimos la estructura básica de las uniones tipo gap y el papel que las Cxs, y en especial la Cx37, tienen en tejidos músculo-esqueléticos. (AU)
Subject(s)
Humans , Bone and Bones/metabolism , Bone Resorption/prevention & control , Connexins/physiology , Osteoblasts/metabolism , Osteocytes/metabolism , Tendons/metabolism , Signal Transduction/physiology , Cartilage/metabolism , Cell Communication/physiology , Cell Physiological Phenomena , Gap Junctions/drug effects , Gap Junctions/physiology , Connexin 43/physiology , Muscle, Skeletal/metabolism , Bone Density Conservation Agents/therapeutic use , Ligaments/metabolism , Anti-Arrhythmia Agents/adverse effectsABSTRACT
ABSTRACT Objective Complexes like conjugated linoleic acid (CLA) reduce the percentage of body fat by increasing energy expenditure, fat oxidation, or both. The aim of this study was to verify if CLA is able to mimic caloric restriction (CR), and determine the effects of CLA on liver metabolic profile of young adult male Wistar rats. Materials and methods We divided 36 animals into the following groups: 1) Control; 2) CLA (1% of daily food intake, 21 days, orogastric intubation); 3) Restr (fed 60% of the diet offered to controls); and 4) CLA Restr. Liver tissues were processed for biochemical and molecular or mitochondrial isolation (differential centrifugation) and blood samples were collected for biochemical analyses. Results Treatment of the animals for 21 days with 1% CLA alone or combined with CR increased liver weight and respiration rates of liver mitochondria suggesting significant mitochondrial uncoupling. We observed a decrease in adipose tissue leading to insulin resistance, hyperinsulinemia, and hepatic steatosis due to increased liver cholesterol and triacylglycerol levels, but no significant effects on body mass. The expression of hepatic cellular connexins (43 and 26) was significantly higher in the CLA group compared with the Control or Restr groups. Conclusion CLA does not seem to be a safe compound to induce mass loss because it upregulates the mRNA expression of connexins and induces hepatic mitochondrial changes and lipids disorders.
Subject(s)
Animals , Male , Rats , Caloric Restriction , Linoleic Acids, Conjugated/administration & dosage , Energy Metabolism , Fatty Liver/prevention & control , Liver/metabolism , Time Factors , Rats, Wistar , Lipid MetabolismABSTRACT
Objective@#To investigate the early intervention effects of metoprolol on connexin 43(Cx43) and phosphorylated Cx43 (p-Cx43) expression in rabbits with post myocardial infarction.@*Methods@#A total of 24 adult male New Zealand white rabbits were divided into sham group (n=6), early treatment group(n=6), routine treatment group(n=6), and myocardial infarction group(n=6) with a randomized block design blocked by weight. Myocardial infarction was induced by left anterior descending coronary artery (LAD) ligation. Rabbits in sham group received similar surgical procedure without LAD ligation. Metoprolol (12.5 mg/kg dissolved in 2 ml distilled water) was applied to rabbits in early treatment group and routine treatment group per gavage immediately after recovery from anesthesia and at 24 hours after myocardial infarction, respectively, then treated daily for 40 days. Rabbits in sham group and myocardial infarction group received 2 ml distilled water per gavage daily for 40 days. Plasma lactate dehydrogenase (LDH) and creatine kinase (CK) level were detected by automatic biochemistry analyzer after 6 hours in all rabbits. Ventricular fibrillation threshold (VFT) was measured in vivo by bipolar pacing electrodes at 40 days. Cx43 and p-Cx43 distribution in ventricular tissue was detected by immunofluorescence analyses. Cx43 and p-Cx43 protein level in ventricular tissue was determined by Western blot.@*Results@#(1) Plasma LDH ((851.7±85.9)U/L vs. (332.3±39.6)U/L, P<0.01) and CK ((1 192.7±105.3)U/L vs. (462.3±65.6)U/L, P<0.01) were significantly higher in myocardial infarction group than in sham group (both P<0.01). (2) VFT was significantly lower in myocardial infarction group than that in sham group ((470.0±91.0) beats per minute vs. (683.3±60.9) beats per minute, P<0.05), and VFT was significantly higher in early treatment group ((633.3±43.2) beats per minute) and routine treatment group ((645.0±30.8) beats per minute) than in the myocardial infarction group (both P<0.05). (3) Immunofluorescence analyses showed that Cx43 was mainly localized in the intercalated disk, which was perpendicular to the cell long axis with linear arrangement, and less lateral distribution in sham group, early treatment group and routine treatment group, which was significantly different as the case in the myocardial infarction group. The expression of p-Cx43 in myocardial infarction group was less than in sham group, which was significantly upregulated in in early treatment group and routine treatment group when compared with myocardial infarction group, and expression of p-Cx43 was significantly higher in early treatment group than in routine treatment group. (4)The p-Cx43/Cx43 ratio of protein was significantly lower in myocardial infarction group than in sham group (0.165±0.011 vs. 0.363±0.046, P<0.05), and significantly higher in early treatment group (0.720±0.063) and routine treatment group (0.364±0.030) than in myocardial infarction group (both P<0.05), and this ratio was significantly higher in early treatment group than in routine treatment group (P<0.05).@*Conclusion@#Metoprolol treatment, especially the early metoprolol treatment (within 24 hours after LAD ligation), could significantly improve VFT by ameliorating the distribution and dephosphorylation of myocardial Cx43 in rabbits with experimental myocardial infarction.
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Objective@#To investigate the effect and mechanism of hydroxyfasudi (HF), a specific Rho kinase inhibitor, on lipopolysaccharide(LPS)induced endothelial dysfunction.@*Methods@#A total of 24 male Sprague Dawley rats were randomly divided into control group(n=6), HF group(n=6), LPS group(n=6) and LPS + HF group(n=6) with random number table method. There was no special treatment in control group. HF (30 mg/kg) was injected intraperitoneally in HF group. LPS (1 mg/kg) were injected intravenously in LPS group. In LPS+ HF group, HF (30 mg/kg) was injected intraperitoneally, followed by intravenous LPS injection (1 mg/kg) 30 minutes later. All rats were sacrificed after 8 hours, and aortic tissue was extracted. RT-PCR was performed to detect mRNA levels of Rho-associated coiled-coil protein kinase (ROCK)1, connexin (Cx)43 and caveolin (Cav)1. The protein levers of ROCK1, Cx43 and Cav-1 were assessed by Western blot and immunohistochemical staining respectively.@*Results@#(1) RT-PCR experiments showed that mRNA levels of ROCK1(2.67±0.03 vs. 1.00±0.04), Cx43(1.73±0.03 vs. 1.00±0.08), and Cav1(1.85±0.04 vs. 1.0±0.03) in LPS group were significantly higher than in control group(all P<0.05). mRNA levels of ROCK1(0.38±0.02), Cx43(0.58±0.02), and Cav1(0.27±0.01) in LPS + HF group were significantly lower than in LPS group(all P<0.05). (2)Western blot analysis showed that protein levels of ROCK1(3.46±0.82 vs. 2.19±0.56), Cx43(0.33±0.09 vs.0.11±0.06), and Cav1(3.45±0.74 vs. 2.25±0.91) in LPS group were significantly higher than in control group(all P<0.05). Protein levels of ROCK1(1.09±0.52), Cx43(0.01±0.06), and Cav1(2.06±0.40) in LPS + HF group were significantly lower than in LPS group(all P<0.05). (3) Immunohistochemical staining showed that protein levels of ROCK1(84.1±0.9比53.7±2.9), Cx43(99.1±2.1 vs. 46.2±0.8), and Cav1(167.0±6.4 vs. 84.9±1.0) in LPS group were significantly higher than in control group(all P<0.05). Protein levels of ROCK1(30.4±0.6), Cx43(21.4±1.3), and Cav1(55.8±2.8) in LPS + HF group were significantly lower than in LPS group(all P<0.05).@*Conclusion@#HF attenuates LPS induced endothelial dysfunction probably via suppressing the expression of ROCK1, Cx43 and Cav1.