Changes in CaMK Ⅱ α expression in spinal cord and dorsal root ganglia during remifentanil-induced hyperalgesia in a rat model of incisional pain / 中华麻醉学杂志

Objective To investigate the changes in the expression of Ca2+/calmodulin-dependent protein kinase Ⅱ α (CaMK Ⅱ α) in the spinal cord and dorsal root ganglia (DRG) during remifentanil-induced hyperalgesia in a rat model of incisional pain (IP).Methods Thirty-two male Sprague-Dawley rats in which caudal vein catheter was successfully placed,aged 260-280 g,were divided into 4 groups (n=8 each) using a random number table method:control group (group C),IP group,remifentanil group (group R) and remifentanil plus IP group (group RIP).Normal saline was infused via the caudal vein for 60 min at a rate of 0.1 ml · kg-1 · min-1 in group C.Normal saline was infused via the caudal vein for 60 min at a rate of 0.1 ml · kg-1 · min-1,and the model of IP was simultaneously established in group IP.Remifentanil was infused via the caudal vein for 60 min at a rate of 1.0 μg · kg-1 · min-1 in group R.Remifentanil was infused via the caudal vein for 60 min at a rate of 1.0 μg · kg-1 · min-1,and the model of IP was simultaneously established in group RIP.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion and 2,6,24 and 48 h after infusion (T0-4).The rats were sacrificed after the last behavioral test,and L4-6 segment of the spinal cord and DRGs were removed for determination of the expression of total and phosphorylated CaMK Ⅱ α (tCaMK Ⅱ α,pCaMK Ⅱ α) by Western blot.The ratio of pCaMK Ⅱ /tCaMK Ⅱ α was calculated.Results Compared with group C,MWT was significantly decreased,TWL was shortened,the expression of tCaMK Ⅱ α and pCaMK Ⅱ α in the spinal cord and DRGs was up-regulated,and the ratio of pCaMK Ⅱ α/tCaMK Ⅱ α was increased in I,R and RIP groups (P＜0.05 or 0.01).Compared with group IP and group R,MWT was significantly decreased,TWL was shortened,the expression of tCaMK Ⅱ α and pCaMK Ⅱ α in the spinal cord and DRGs was up-regulated,and the ratio of pCaMK Ⅱ α/tCaMK Ⅱ α was increased in group RIP (P＜0.05 or 0.01).Conclusion The mechanism by which remifentanil induces hyperalgesia may be related to upregulated expression of CaMK Ⅱ α in the spinal cord and DRGs in a rat model of IP.

Relationship between NR2B and CaMK Ⅱ α in spinal cord during remifentanil-induced hyperalgesia in a rat model of incisional pain / 中华麻醉学杂志

Objective To evaluate the relationship between NR2B subunit-containing N-methyl-D-aspartate (NMDA) receptors (NR2B receptors) and Ca2+/calmodulin-dependent protein kinase Ⅱ α (CaMK Ⅱ α) in the spinal cord during remifentanil-induced hyperalgesia in a rat model of incisional pain (IP).Methods Forty male Sprague-Dawley rats in which intrathecal and caudal catheters were successfully placed,weighing 260-280 g,aged 2-3 months,were divided into 4 groups (n=10 each) using a random number table method:control group (group C),remifentanil plus IP group (group RI),NR2B antagonist Ro 25-6981 group (group Ro) and remifentanil plus IP plus Ro 25-6981 group (group RI+Ro).In group C,normal saline 0.1 ml was intrathecally injected,and 10 min later normal saline was infused for 60 min via the tail vein at a rate of0.1 ml · kg-1 · min-1.In group RI,normal saline 0.1 ml was intrathecally injected,and 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1.0 μg · kg-1 · min-1,and IP was established immediately after onset of remifentanil infusion.In group Ro,Ro 25-6981 (0.1 ml) 10 μg was intrathecally injected,and 10 min later normal saline was infused for 60 min via the tail vein at a rate of 0.1 ml · kg-1 · min-1.In group RI+Ro,Ro 25-6981 (0.1 ml) 10 μg was intrathecally injected,and 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1.0 μg · kg-1 · min-1,and IP was established immediately after onset of remifentanil infusion.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before intravenously infusing normal saline or remifentanil and at 2,6,24 and 48 h after the end of infusion (T0-4).The rats were sacrificed after the last behavioral test,and the L4-6 segment of the spinal cord was removed for determination of the expression of NR2B in total and membrane protein (tNR2B and mNR2B) and expression of CaMK Ⅱ α in total protein (tCaMK Ⅱ α) and phosphorylated CaMK Ⅱ α (pCaMKⅡα).The ratios of mNR2B/tNR2B and pCaMKⅡα/tCaMK Ⅱα were calculated.Results Compared with group C,the MWT was significantly decreased,TWL was shortened,the expression of tNR2B,mNR2B,tCaMKⅡα and pCaMKⅡα was up-regulated,and the ratios of mNR2B/tNR2B and pCaMK Ⅱ α/tCaMK Ⅱ α were increased in group RI (P＜0.05 or 0.01).Compared with group RI,the MWT was significantly increased,TWL was prolonged,the expression of tNR2B,mNR2B,tCaMKⅡα and pCaMKⅡα was down-regulated,and the ratios of mNR2B/tNR2B and pCaMK Ⅱ α/tCaMK Ⅱ α were decreased in group RI+ Ro (P＜0.05 or 0.01).Conclusion Enhanced function of NR2B can activate CaMKⅡα during remifentanil-induced hyperalgesia,which may be involved in the mechanism of remifentanil-induced hyperalgesia in a rat model of IP.

Role of calcium/calmodulin-dependent kinase Ⅱ alpha in central nucleus of amygdale in fentanyl-induced hyperalgesia in rats: the relationship with mEPSCs / 中华麻醉学杂志

Objective To evaluate the role of calcium/calmodulin-dependent kinase Ⅱ alpha (CaMK Ⅱα) in the central nucleus of the amygdale (CeA) in fentanyl-induced hyperalgesia in rats and the relationship with miniature excitatory postsynaptic currents (mEPSCs).Methods Thirty-two male Sprague-Dawley rats,weighing 50-80 g,in which the CeA was successfully cannulated,were randomly divided into 4 groups (n=8 each) using a random number table:control 1 group (group C1),fentanylinduced hyperalgesia 1 group (group FIH1),KN92 group,and KN93 group.Normal saline was injected subcutaneously,and dimethyl sulfoxide (DMSO) was given into the amygdale in group C1.In group FIH1,fentanyl was injected subcutaneously (60 μg/kg per time,4 times in total,15-min interval,cumulative dose of 240 μg/kg) to establish the model of hyperalgesia.In KN92 and KN93 groups,KN92 and KN93 10 nmol were given into the CeA after establishing the model.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal threshold (TWT) were measured at 6 and 7 h after fentanyl or normal saline injection.Another 12 Sprague-Dawley rats were selected and randomly divided into either control 2 group (group C2) or fentanyl-induced hyperalgesia 2 group (group FIH2) using a random number table with 6 rats in each group.The brains were removed and sliced 12 h later,and the frequency and amplitude of mEPSCs were recorded.KN93 10 nmol was then added to the artificial cerebral spinal fluid,and the frequency and amplitude of mEPSCs were recorded by whole cell patch-clamp technique.Results Compared with group C 1,the MWT and TWT were significantly decreased at 6 h after fentanyl or normal saline injection in FIH1,KN92 and KN93 groups,and at 7 h after fentanyl or normal saline injection in FIH and KN92 groups (P＜0.05).Compared with group FIH1,the MWT and TWT were significantly increased at 7 h after fentanyl or normal saline injection in group KN93 (P＜0.05),and no significant change was found in group KN92 (P＞0.05).Compared with group C2,the frequency and amplitude of mEPSCs were significantly increased before administration of KN93 (P ＜ 0.05),and no significant change was found in the frequency and amplitude of mEPSCs after administration of KN93 in group FIH2 (P＞0.05).Compared with the value before KN93 administration,no significant change was found in the frequency and amplitude of mEPSCs after administration of KN93 in group C2 (P＞0.05),and the frequency and amplitude of mEPSCs were significantly decreased after administration of KN93 in group FIH2 (P＜ 0.05).Conclusion Activation of CaMK Ⅱ α in the CeA enhances synaptic excitation in neurons,which is involved in fentanyl-induced hyperalgesia in rats.

Effects of luteolin on CaM-CaMPK signaling pathway in hippocampus in epileptic rats / 国际中医中药杂志

Objective To explore the effects of luteolin on cognition function in pentylenetetrazol (PTZ)-induced epileptic rats and related mechanism.Methods Fifty male SD rats were randomly divided into a normal control group(n=8), a model group(n=12), and groups of 25, 50 mg/kg luteolin(both ofn=11), as well as 100 mg/kg luteolin group(n=8). Those rats were given different doses of luteolin (25, 50 and 100 mg/kg, daily, intragastric administration) for 36 consecutive days. Similarly, rats of the normal control group and the model group were given 0.5% sodium carboxymethyl cellulose suspension liquid via intragastric administration. Thirty minutes later, a model of epilepsy was induced using PTZ (40 mg/kg, daily) via intraperitoneal injection except the control group. Learning and memory of rats were evaluated by Morris water maze and novel objective recognition trials(including escape latency and recognition index). The levels of CaM and CaMPK were determined by ELISA methods, and expression of Ras proteins in the hippocampus were detected by Western Blot.Results Compared with the model group, luteolin treatment groups significantly shorten the escape latency(28.51 ± 3.84 s, 19.77 ± 5.41 s, 14.86 ± 2.76 svs. 37.08 ± 5.18 s) in the Morris water maze, and increased recognition index(18.77% ± 2.02%, 25.06% ± 4.32%, 31.92% ± 2.65%vs. 13.87% ± 2.14%) in the novel objection trial(P<0.05 orP<0.01). Meanwhile, CaM(140.33 ± 13.52 ng/L, 124.26 ± 9.97 ng/L, 113.52 ± 11.57 ng/Lvs. 158.36 ± 10.68 ng/L) and CaMPK(8.25 ± 1.37 ng/ml, 7.69 ± 0.84 ng/ml, 6.74 ± 0.93 ng/mlvs. 9.87 ± 1.02 ng/ml) were significantly decreased(P<0.05 orP<0.01). What’s more, the expression of Ras proteins(0.99 ± 0.08, 0.76 ± 0.07, 0.52 ± 0.07vs. 1.58 ± 0.12) was obviously decreased compared with the model group(P<0.05 orP<0.01).Conclusion Luteolin could effectively improve the cognition dysfunction of epileptic rats, and the mechanism might be relevant to regulate the CaM-CaMPK signaling pathway via down-regulation of CaM, CaMPK, as well as Ras protein.

Static compression regulates OPG expression in periodontal ligament cells via the CAMK II pathway

ABSTRACT Objective This study aimed to investigate the potential role of CAMK II pathway in the compression-regulated OPG expression in periodontal ligament cells (PDLCs). Material and Methods The PDL tissue model was developed by 3-D culturing human PDLCs in a thin sheet of poly lactic-co-glycolic acid (PLGA) scaffolds, which was subjected to static compression of 25 g/cm2 for 3, 6 and 12 h, with or without treatment of KN-93. After that, the expression of OPG, RANKL and NFATC2 was investigated through real-time PCR and western blot analysis. Results After static compression, the NFATC2 and RANKL expression was significantly up-regulated, while partially suppressed by KN-93 for 6 and 12 h respectively. The OPG expression was significantly down-regulated by compression in 3 h, started to elevate in 6 h, and significantly up-regulated in 12 h. The up-regulation after 12 h was significantly suppressed by KN-93. Conclusions Long-term static compression increases OPG expression in PDLCs, at least partially, via the CAMK II pathway.

Promoter methylation of DAPK1, RAR-β and MGMT in exfoliated cervical cytology and its clinicalapplication / 中华妇产科杂志

Objective To assess the correlation of promoter methylation of DAPK1,RAR-β and MGMT with cervical lesions from cytology to histology,and to reveal the clinical value of DNA methylation in diagnosis of cervical intraepithelial neoplasia (CIN).Methods A total of 103 random-selected cervical samples were collected from residual liquid-based cytology specimens after clinical use in cytopathological diagnosis in outpatient clinic of obstetrics and gynecology,Peking Union Medical Collage Hospital from March 2010 to October 2010.Informed consent was obtained from each woman before the initiation of the study.The methylation seusitive-high resolution melt (MS-HRM) assay was used to evaluate promoter methylation of three genes ( DAPKI,RAR-β and MGMT) in 103 biopsy-confirmed liquid-based cervical cytology samples.Methylation levels and high-risk HPV DNA loading ( HC Ⅱ values) were analyzed in relation to both cytological and histological diagnosis.Results The methylation level of all three genes showed significant difference among the different cytological groups ( P =0.000,0.011 and 0.002,respectively).The methylation level of DAPK1 and RAR-β showed significant difference among the different histological groups ( P =0.000 and 0.021 ),while there was no significant difference for MGMT.DAPK1 methylation levels was 1.47％ in the CIN Ⅱ/high-grade precancerous lesions group,and 20.98％ in the normal/CIN I groups ( P =0.000 ),but there was no significant difference between CIN I/high-grade precancerous lesions and normal/CIN Ⅰ groups for RAR-β and MGMT.The combination of DAPK1/HR-HPV loading showed a sensitivity of 0.825 and an area under the receiver operating characteristic curve (ROC) curve (AUC) of 0.695 as diagnostic methods for detecting CIN Ⅱ/high-grade precancerous lesions.Conclusions DNA methylation such as DAPK1 and RAR-β,in combination with HR-HPV detection,may serve as biomarkers to detect CIN Ⅱ/high-grade precancerous lesions.Detection of methylated DNA from liquid-based cervical cytology specimens is technically feasible with the MS-HRM assay.

Effects of sevoflurane or ischemic preconditioning on the expression of ERK and CaM during lung ischemia-reperfusion in rats / 中华麻醉学杂志

Objectlve To investigate the effects of sevoflurane or ischemic preconditioning on the expression of extracellular signal-regulated kinase (ERK) and calmodulin (CaM) during lung ischemia-reperfusion (I/R)in rats. Methods Twenty-four male SD rats weighing 270-320 g were randomly divided into 4 groups ( n = 6each): sham operation group (group S), group I/R, ischemic preconditioning group (group IP), and sevoflurane preconditioning group (group SP). In group S, the hilum of the left lung was dissociated after thoracotomy but not occluded. In group I/R, lung I/R was produced by occlusion of the hilum of the left lung for 45 min followed by 120 min of reperfusion. In group IP, the hilum of the left lung was occluded for 5 min and unclamped for 5 min for 2 times before the model was established. In group SP, sevoflurane was inhaled for 30 min at the end-tidal concentration of 2.1% before lung ischemia. All rats were sacrificed at 120 min of reperfusion and the lung tissues were taken for determination of the contents of TNF-α and IL-6 by ELISA and the expression of ERK mRNA and CaM mRNA by RT-PCR. Results Compared with group S, the contents of TNF-α and IL-6 and the expression of ERK mRNA and CaM mRNA were significantly increased in group I/R, IP and SP ( P ＜ 0.05). Compared with group I/R, the contents of TNF-α and IL-6 and the CaM mRNA expression were significantly decreased, while the expression of ERK mRNA was significantly increased in group IP and SP ( P ＜ 0.05). There was no significant difference in the each index metioned above between group IP and SP ( P ＞ 0.05 ). Conclusion Sevoflurane preconditioning and ischemic preconditioning can protect the lung from I/R injury through down-regulating the expression of CaM and up-regulating the expression of ERK.