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AIM: To investigate the effect of inhibiting Ca2+/calmodulin-dependent protein kinase Ⅱ(CAMKⅡ)expression in adult retinal pigment epithelial cell line-19(ARPE-19)cells on the migration, invasion, and tube formation of human umbilical vein endothelial cells(HUVECs)in a non-contact co-culture system.METHODS: RNA sequencing was performed on ARPE-19 cells overexpressing CAMKⅡ-δ, and bioinformatics was used to analyze the biological functions of the differentially expressed genes. Transwell inserts was used to construct a non-contact co-culture system of ARPE-19 and HUVECs. The experimental groups included: blank group: only HUVECs were inoculated without ARPE-19 cells; control group: ARPE-19 and HUVECs cells were co-cultured with complete medium; AIP group(CAMKⅡ inhibition group): ARPE-19 cells in AIP(160 nmol/L)were co-cultured with HUVECs in complete medium. The migration, invasion and tube formation abilities of HUVECs were detected. The protein expression levels of CAMKⅡ/AMPK/mTOR/VEGFA were detected by Western blotting.RESULTS:Bioinformatics analysis found that the differentially expressed genes could affect biological processes such as cell growth and death and cell movement. The scratch test and transwell migration test showed that the relative mobility of HUVECs in the AIP group was significantly lower than that in the control group(all P<0.05). However, the invasion and tube formation assay showed that the relative invasion rate and tube formation rate of the AIP group were not significantly different from those of the control group(both P>0.05). Western blotting results showed that the expression levels of CAMKⅡ, P-mTOR, and VEGFA proteins in the AIP group were significantly lower than those in the control group, while the expression level of the P-AMPK protein was significantly higher than that in the control group(all P<0.05).CONCLUSION:In the non-contact co-culture system, inhibition of CAMKⅡ expression in ARPE-19 cells significantly reduced the migration ability of HUVECs, but it cannot change the invasion and tube formation ability, which may be achieved by AMPK/mTOR/VEGFA.
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Objective To investigate the effects of therapeutic hypothermia (TH) on myocardial Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and cell autophagy after cardiopulmonary resuscitation (CPR) in swine.Methods Twenty healthy male domestic swine weighing 33-40 kg were randomly (random number) divided into 3 groups:sham group (n=4),CPR group (n=8) and TH group (n=8).Sham animals only underwent general preparation without experiencing cardiac arrest and resuscitation.The animal model was established by 8 min of electrically induced ventricular fibrillation and then 5 min CPR in the CPR and TH groups.Successful resuscitation was regarded as an organized rhythm with a mean arterial pressure of greater than 50 mmHg for 5 min or more.After successful resuscitation,body temperature was decreased to 33 ℃ by a cooling blanket and then maintained until 24 h post-resuscitation,and followed by a rewarming at a rate of 1 ℃/h for 5 h in the TH group.A normal temperature was maintained by the blanket throughout the experiment in the sham and CPR groups.At 6,12,24 and 30 h after resuscitation,the values of stroke volume (SV) and global ejection fraction (GEF) were measured by PiCCO,and meanwhile the serum concentrations of cardiac troponin Ⅰ (cTnI) were measured by ELISA assay and the serum activities of creatine kinase-MB (CK-MB) were evaluated by an automatic biochemical analyzer.At 30 h after resuscitation,the animals were sacrificed and left ventricular myocardium was obtained for the determination ofCaMK Ⅱ,microtubule-associated protein light chain 3 Ⅱ (LC3 Ⅱ) and p62 expressions by Western blot.The variables were compared with One way analysis of variance and then the Bonferroni test among the three groups.Results Compared with the sham group,myocardial dysfunction and injury after resuscitation were observed in the CPR and TH groups,which were indicated by decreased SV and GEF and also increased cTnI concentration and CK-MB activity in serum (all P＜0.05).Compared with the CPR group,the values of SV and GEF were significantly increased at 6 h after resuscitation,and serum cTnI concentration and CK-MB activity were significantly decreased starting 12 h after resuscitation in the TH group [SV (mL):25.0±6.9 vs 31.9±3.3 at 6 h,26.7±5.1 vs 34.6±3.7 at 12 h,28.8±3.3 vs 35.7±3.2 at 24 h,29.2±5.2 vs 36.7±3.3 at 30 h;GEF (％):17.1±2.7 vs 19.9±1.8 at 6 h,18.7±1.9 vs 21.6±1.8 at 12 h,19.3±2.3 vs 23.0±2.4 at 24 h,21.0±1.7 vs 23.7±1.7 at 30 h;cTnI (pg/mL):564±51 vs 466±56 at 12 h,534±38 vs 427±60 at 24 h,476±55 vs 375±46 at 30 h;CK-MB (U/L):803±164 vs 652±76 at 12 h,693±96 vs 557±54 at 24 h,633±91 vs 480±77 at 30 h,all P＜0.05].Tissue detection indicated that the expression of CaMK Ⅱ and LC3 Ⅱ were increased while the expression of p62 was decreased in post-resuscitation myocardium in the CPR and TH groups compared with the sham group (all P＜0.05).However,the expression of CaMK Ⅱ and LC3 Ⅱ were decreased and the expression of p62 was increased in postresuscitation myocardium in the TH group compared to the CPR group (CaMK Ⅱ:0.73±0.06 vs 0.58±0.05;LC3 Ⅱ:0.69±0.09 vs 0.50±0.07;p62:0.40±0.07 vs 0.68±0.14,all P＜0.05).Conclusion The mechanism of TH alleviating post-resuscitation myocardial dysfunction and injury may be related to the inhibition of CaMK Ⅱ expression and cell autophagy.
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Objective To evaluate the effects of hypothermia on Ca2+∕calmodulin-dependent pro-tein kinase Ⅱ ( CaMKⅡ) and cell autophagy in brain tissues after cardiac arrest and cardiopulmonary re-suscitation ( CA-CPR) in swine. Methods Twenty-one healthy male white swine, weighing 33-40 kg, were divided into 3 groups using a random number table method: sham operation group ( group S, n=5) , CA-CPR group ( n=8) and hypothermia group ( group H, n=8) . The experimental model of CA-CPR was established in CA-CPR and H groups. The Swan-Ganz catheters were placed in the right femoral artery and vein to monitor the pressure of thoracic aorta and right atrium and body temperature and to collect blood sam-ples. A pacing catheter was advanced from the right external jugular vein into the right ventricle. Ventricu-lar fibrillation was induced by using a 1 mA alternating current through the pacing catheter. Once ventricular fibrillation was successfully induced, mechanical ventilation was discontinued for 8 min, and then CPR was initiated. Epinephrine 20 μg∕kg was intravenously injected at 2. 5 min of CPR, followed by repetition once every 3 min. Defibrillation was delivered at 5 min of CPR, and then spontaneous circulation was evaluated. If the spontaneous circulation was not restored, CPR was immediately resumed for 2 min, and then defibril-lation was delivered again. Mechanical ventilation was continued for 30 h after successful CPR. At 5 min af-ter successful resuscitation, body temperature was decreased to 33 ℃ by using a cooling blanket, then maintained at 33 ℃ until 24 h after resuscitation, and finally increased at a rate of 1℃∕h for 5 h in group H. The temperature was maintained at a normal level of 37. 5-38. 5 ℃ with the aid of a cooling blanket in S and CA-CPR groups. At 1, 6, 12, 24 and 30 h after resuscitation (T1-5), blood samples were collected from the femoral vein for measurement of the concentration of neuron specific enolase ( NSE) and S100βprotein in serum by enzyme-linked immunosorbent assay. Five animals in each group were then sacrificed, and brains were removed to determine the expression of CaMKⅡ, microtubule-associated protein 1 light chain 3 Ⅱ( LC3Ⅱ) and p62 in cerebral cortex by Western blot. Neurological deficit score was evaluated in the remaining three swine at 48, 72 and 96 h after resuscitation (T6-8) in CA-CPR and H groups. Results Compared with group S, the concentrations of NSE and S100β protein in serum were significantly in-creased at T1-5 , the expression of CaMKⅡand LC3Ⅱin cerebral cortex was up-regulated, and the expres-sion of p62 in cerebral cortex was down-regulated in CA-CPR and H groups (P<0. 05). Compared with group CA-CPR, the concentrations of NSE and S100βprotein in serum were significantly decreased at T3-5, the neurological deficit score was decreased at T6-8 , the expression of CaMKⅡand LC3Ⅱin cerebral cortex was down-regulated, and the expression of p62 in cerebral cortex was up-regulated in group H ( P<0. 05) . Conclusion The mechanism by which hypothermia alleviates brain injury after CA-CPR may be related to inhibiting CaMKⅡ activation and reducing cell autophagy in brain tissues of swine.
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Objective:To investigate the role of Na+/Ca2+ exchanger (NCX) in myocardial ischemiareperfusion injury and the underlying mechanisms.Methods:Forty Sprague-Dawley rats were divided into 4 groups randomly:a control group,a KBR7943 group,an ischemia-reperfusion group (IR group),and an IR plus KB-R7943 group (KB-R7943+IR group).Isolated Sprague Dawley male rat hearts underwent Langendorffperfusion.The ratio of left ventricular developed pressure (LVDP),left ventricular end-diastolic pressure (LVEDP),the infarct size of myocardium,and the lactate dehydrogenase (LDH) activity in the coronary flow was determined.HE staining was used to assess the change of myocardial morphology.Western blot was used to determine the levels of cleaved caspase-3,cytochrome c and the phosphorylation of Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) and the Thr17 site ofphospholamban.Results:Compared with the control group,IR group significantly induced an enlarged infarct size,reduction of the ratio of LVDP,up-regulation of cytochrome c,cleaved caspase-3,p-CaMKⅡ and p-phospholamban,and increased in the activity of LDH,the level of LVEDP (P＜0.01) and the disordered myocardial morphology.These effects were significantly attenuated in the presence of KB-R7943 treatment (10 μmol/L).Conclusion:NCX mediates myocardial ischemia-reperfusion-induced cell apoptosis and necrosis through activation of CaMKⅡ.
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Objective To investigate the effects of N-butylphthalide (NBP) on cognitive function in acute severe carbon monoxide (CO) poisoning rats and its mechanism. Methods 120 health Sprague-Dawley (SD) rats were randomly divided into three groups (n = 40): normal control group (NC group), CO poisoning group (CO group) and NBP treatment group (NBP group). The acute severe CO poisoning model was established in a hyperbaric oxygen chamber by intoxicated with 1 000 ×10-6CO for 40 minutes, followed with 3 000 ×10-6CO for another 20 minutes, and then received hyperbaric oxygen therapy 1.5 hours once a day until sacrificed. Rats in NBP group were administered orally NBP 60 mg/kg for 2 times daily until death. NC group and CO group were treated with equal amount of pure olive oil. Four rats in each group were taken from 1, 3, 7, 14, 30 days after model setup, respectively. The cognitive function score was assessed by Morris water maze test. The changes in ultrastructure of hippocampus were observed under transmission electron microscope. The expressions of calpain 1 and Ca2+/calmodulin dependent protein kinase Ⅱ(CaMK Ⅱ) in hippocampus of brain tissue were detected by immunofluorescence staining, and the localization of the two target proteins in neurons was observed by immunofluorescence double staining. Results Compared with NC group, the escape latency at 1 day after poisoning in CO group was significantly prolonged (s: 55.6±3.2 vs. 44.5±3.5, P < 0.05), and the times of the platform crossing was significantly decreased (times: 1.3±0.8 vs. 6.6±1.2, P < 0.05);the ultrastructure of hippocampus was obviously injured; the protein expressions of calpain 1 and CaMK Ⅱ in brain tissue were significantly increased at 1 day after CO poisoning [calpain 1 (A value): 41.24±5.21 vs. 6.44±1.13, CaMK Ⅱ (A value): 56.19±5.04 vs. 9.84±1.53, both P < 0.05], and the protein expression of calpain 1 reached the peak at 3 days (A value: 59.34±6.11), the protein expression of CaMK Ⅱ reached the peak at 1 day (A value:56.19±5.04). Compared with CO group, the cognitive function was significantly improved in NBP group in the late stage of poisoning [7-30 days, escape latency (s): 40.3±1.9 vs. 49.1±3.1 at 7 days, 30.1±2.9 vs. 39.4±3.1 at 30 days;times of the platform crossing (times): 2.8±1.0 vs. 1.0±0.9 at 14 days, 3.2±0.8 vs. 1.0±0.9 at 30 days, all P < 0.05];the degree of injury of hippocampal neuron was relatively slight; the protein expression of calpain 1 in brain tissue was significantly decreased from 3 days after CO poisoning (A value: 39.63±3.03 vs. 59.34±6.11, P < 0.05), and the protein expression of CaMK Ⅱ was significantly decreased from 1 day after CO poisoning (A value: 42.22±3.84 vs. 56.19±5.04, P < 0.05). Immunofluorescence double staining suggested that calpain 1 and CaMK Ⅱ protein could not only coexist in the same cell, but also could be expressed separately in different cells. Linear regression analysis showed that the expression of calpain 1 and CaMK Ⅱ was positively correlated (R 2= 0.852, P = 0.002). Conclusions NBP treatment could maintain ultrastructure integrity of hippocampus, balance the expression levels of calpain 1 and CaMK Ⅱproteins, and significantly improve cognitive impairment induced by CO poisoning, thus play a protective role against hippocampus damage in rats with acute severe CO poisoning.
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OBJECTIVE To determine the functional role of hydrogen sulfide (H2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca2 +/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) using wild type and CSE knockout mouse models. METHODS Continuous subcutaneous injection isoprenaline (7.5 mg·kg-1·d-1), once a day for 4 weeks to induce heart failure in Male C57BL/6 (6-8 weeks old) mice and CSE-/- mice. 150 μmol·L-1 H2O2 was used to induce oxidative stress in H9c2 cells. Echocardiograph was used to detect cardiac parameters. H&E stain and Masson stain was to observation histopathological changes. Western blot was used to detect protein expression and activity. The siRNA was used to silence protein expression. HPLC was used to detect H2S level. Biotin assay was used to detect the level of S- sulfhydration protein. RESULTS Treatment with S-propyl-L-cysteine (SPRC) or sodium hydrosulfide (NaHS), modulators of blood H2S levels, attenuated the development of heart failure in animals, reduced lipid peroxidation, and preserved mitochondrial function. The inhibition CaMKⅡ phosphorylation by SPRC and NaHS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds. Interestingly, CaMKⅡ activity was found to be elevated in CSE-/- mice as compared to wild type animals and the phosphorylation status of CaMKⅡ appeared to relate to the severity of heart failure. Importantly, in wild type mice SPRC was found to promote S-sulfhydration of CaMKII leading to reduced activity of this protein however, in CSE-/- mice S-sulfhydration was abolished following SPRC treatment. CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of CaMKⅡ is presented. SPRC mediated S-sulfhydration of CaMKII was found to inhibit CaMKⅡ activity and to preserve cardiovascular homeostasis.
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OBJECTIVE To determine the functional role of hydrogen sulfide (H2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca2 +/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) using wild type and CSE knockout mouse models. METHODS Continuous subcutaneous injection isoprenaline (7.5 mg·kg-1 per day), once a day for 4 weeks to induce heart failure in male C57BL/6 (6-8 weeks old) mice and CSE-/- mice. 150 μmol·L-1 H2O2 was used to induce oxidative stress in H9c2 cells. Echocardiograph was used to detect cardiac parameters. H&E stain and Masson stain was to observation histopathological changes. Western blot was used to detect protein expression and activity. The siRNA was used to silence protein expression. HPLC was used to detect H2S level. Biotin assay was used to detect the level of S-sulfhydration protein. RESULTS Treatment with S-propyl-L-cysteine (SPRC) or sodium hydrosulfide (NaHS), modulators of blood H2S levels, attenuated the development of heart failure in animals, reduced lipid peroxidation, and preserved mitochondrial function. The inhibition CaMKⅡ phosphorylation by SPRC and NaHS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds. Interestingly, CaMKⅡ activity was found to be elevated in CSE-/- mice as compared to wild type animals and the phosphorylation status of CaMK Ⅱ appeared to relate to the severity of heart failure. Importantly, in wild type mice SPRC was found to promote S-sulfhydration of CaMKⅡ leading to reduced activity of this protein however, in CSE-/- mice S-sulfhydration was abolished following SPRC treatment. CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of CaMKⅡ is presented. SPRC mediated S-sulfhydration of CaMKⅡ was found to inhibit CaMKⅡ activity and to preserve cardiovascular homeostasis.
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AIM:To investigate the change of late sodium current (INaL) and the effect of Ca2+/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) inhibitor KN-93 on INaLin the cardiomyocytes after isoproterenol-induced heart fai-lure (HF) in rabbits. METHODS:The rabbit model of HF was induced by injecting isoproterenol (300 μg·kg-1· d-1) for 15 d. One month later, all rabbits received by echocardiography and HE staining to observe the morphological changes of myocardium for evaluating the HF model. The protein expression of NaV1.5, CaMKⅡδ and phosphorylated CaMKⅡδ was determined by Western blot. The ventricular myocytes were isolated from the rabbits of normal saline(NS) group and HF group by Langendorff perfusion, and the whole-cell patch-clamp technique was used to record INaL. RE-SULTS:Compared with NS group,the heart rate in HF group was increased (P<0.01), the ventricular cavity was en-larged (P<0.05),and the cardiac function was decreased(P<0.01). Compared with NS group,the cardiomyocytes in HF group arranged in disorder, vacuolar degeneration and myocardial interstitial edema were observed, and fibrous tissue increased. The protein levels of NaV1.5,CaMKⅡδ and phosphorylated CaMKⅡδ in HF group were higher than those in NS group(P<0.01). INaLin HF group significantly increased compared with NS group (P<0.01). After adding sea anemone toxin Ⅱ (ATXⅡ), the density of INaLin HF group and NS group was significantly increased, but that in HF group increased more obviously than that in NS group (P<0.01). After ATXⅡ had induced stable current, we added KN-93 into NS group and HF group,and we found that the ATXⅡ-increased INaLin NS group and HF group was signifi-cantly decreased(P<0.05).CONCLUSION:CaMKⅡinhibitor KN-93 inhibits the increase in INaLin HF rabbits,which may be related to the activity of CaMKⅡδ and the regulation of CaMKⅡ δ on INaL.
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Objective To elucidate the relationship between Ca2+/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)and autophagy during the development of cardiac hypertrophy. Methods Rat embryonic cardiac cell line H9c2 cells was treated by angiotensin Ⅱ to establish cardiomyocyte hypertrophy model in vitro and using antagonists and gene function gain and loss to analyze AMPK-LC3Ⅱ autophagy signaling pathway. Results The phosphorylation of CaMKⅡ and autophagy related signaling-AMPK and autophagy marker LC3Ⅱ were rapidly increased by angiotensinⅡtreatment at early stage. However ,the above changes were highly blocked by CaMKⅡinhibitor and HDAC4 inhibition. Conclusion CaMKⅡ is the center factor of regulating cardiac hypertrophy ,it mediates autophagy through directly regulating AMPK or indirectly regulating HDAC4 during the development of cardiac hypertrophy.
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Objective To explore the effects of prenatal taurine supplementation on the expression of protein kinase A(PKA) and c-fos,Ca2+/calmodulin-dependent protein kinase Ⅱ (GaMK Ⅱ) in fetal rat brains with intrauterine growth restriction and its significance.Methods Fifteen pregnant Sprague-Dawley rats were randomly divided into 3 groups:the control group,the intrauterine growth restriction (IUGR) group and the IUGR with prenatal taurine supplementation group(the taurine group),with 5 fetal rats in each group.All the fifteen fetal rat brains were detected as following:the expression of PKA,c-fos,CaMK Ⅱ mRNA in fetal rat brains was detected by way of real-time polymerase chain reaction(real-time PCR),while the expression changes of PKA,CaMK Ⅱ,and c-fos protein in fetal rat brains were detected by using Western blot,and the number of PKA,CaMK Ⅱ,c-fos positive cells in fetal rat brains was detected by using immunohistochemistry.Results The control group,the IUGR group and the taurine group:Comparison of the expression of PKA,CaMK Ⅱ,c-fos mRNA among 3 groups were of significant differences(F =7.934,P =0.021 ; F =5.568,P =0.043 ;F =7.332,P =0.024).Comparison of the expression of PKA,CaMK Ⅱ,c-fos protein among the 3 groups were of significant differences(F =57.743,P =0.000 ; F =163.405,P =0.000 ; F =160.136,P =0.000).Comparison of the number of PKA,CaMK Ⅱ,c-fos positive cells among the 3 groups were of significant differences (F =42.903,P =0.000 ;F =329.123,P =0.000 ; F =43.674,P =0.000).Compared with the control group,the expression of mRNA,protein,positive cells of three indicators in IUGR group was less,and the difference was statistically significant (P ＜ 0.05).The expression of mRNA,protein,positive cells of three indicators in fetal rat brains of the taurine group were not different from the control group.The expression of mRNA,protein,positive cells of three indicators in fetal rat brains of the taurine group were significantly more than the IUGR group(P ＜ 0.05).Conclusions Prenatal taurine supplementation can improve PKA-CaMK Ⅱ,c-fos mRNA and protein levels in fetal rat brain tissue with IUGR,and increase its number of positive cells and may enhance the regenerative capacity of the central nervous system,so as to reduce the IUGR brain injury and promote its role in brain development.
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ObjectiveTo study the effects of intrathecal injection of CaMK Ⅱ inhibitor KN93 on the hyperalgesia induced by remifentanil in a incision pain model.MethodsSixty SD rats were divided randomly into 5 groups ( n =12):C group (control) ; (I) group ( incision pain ) ; R group ( incision pain + remifentanil ) ; DMSO group ( incision pain + remifentanil + DMSO) and KN93 group ( incision pain + remifentanil).In group R,DMSO and KN93,remifentanil (0.04 mg/kg,1 mg remifentanil was dissolved in 40 ml NS ) needed to be infused subcutaneously 30 min at the moment of surgery.Group DMSO and group KN93 were respectively intrathecal injected 20 μl DMSO( 10％ ) and 20 μl KN93 (50 μg/20 μl,dissolved in 10％ DMSO).Each rat received tests of the paw mechanical withdrawal threshold (PMWT) and the paw withdrawal thermal latency (PWTL) at the times of 24 h before and 2h,6h,24h,48h after surgery.ResultsCompared with the group C,the PWTL( ( 11.24 ± 0.69) s,(10.36 ±0.29)s,(11.29 ±1.12)s,(12.21 ±0.75)s) and PMWT( (25.5 ± 1.20)s,(24.92 ± 1.98)s,(25.47± 1.54 ) s,( 27.14 ± 1.04 ) s) of the group I were significantly decreased after surgery (P ＜ 0.05 ).Compared with the group Ⅰ,the PWTL( (8.48 ±0.72)s,(8.58 ±0.45)s,(8.46 ±0.92)s,(9.07±0.79) s and PMWT( (21.2± 2.42 ) s,( 19.58 ± 1.12 ) s,( 21.87 ± 1.56 ) s,( 22.26 ± 1.64 ) s ) of the group R were significantly decreased after surgery (P ＜ 0.05 ).Compared with the group R,the PWTL( ( 13.32 ± 0.73 ) s,( 11.79 ± 0.32) s,( 11.86 ±0.98)s,(12.76 ±0.82)s) and PMWT((29.75 ±1.38)s,(28.27± 1.16)s,(26.5 ± 1.02)s,(27.79 ± 1.22)s) of the group KN93 were significantly increased (P ＜ 0.05 ).ConclusionIntrathecal injection KN93 could relieve the hyperalgesia induced by remifentanil
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Objective To investigate the role of Ca2+/calmodulin-dependent protein kinase Ⅱ on pain behavior in a mouse model of bone cancer pain. Methods 40 male C3H/HeN mice were divided randomly into 5 groups:sham group (S group, n=8) ,control group (C group, n=8) and KN93 treat group (T1, n=8;T2, n=8;T3, n = 8 ). Group C and T were induced mouse models of bone cancer pain by intra-left-femur inoculations of osteolytic NCTC2472 cells while group S were injected only α-MEM. On the 14 d after inoculations,group S and C received intrathecal injection of 20% DMSO 5 μl . While group T1, T2, T3 received intrathecal injection of KN93 15nmol,30nmol,60nmol which dissolved in 5 μl 20% DMSO respectively. Mice received pain behavior tests including quantification of spontaneous flinches, paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) before and at 0.5 h,2 h,4 h,8 h after administration. Results Treatment with KN93(15 nmol) have no effect on bone cancer pain,while treatment with KN93(30 nmol,60 nmol) can dose-dependently reverse quantification of spontaneous flinches, mechanical allodynia and thermal hyperalgesia which were induced by bone cancer pain, At 0. 5h after administration, the quantification of spontaneous flinches of the two groups ( (7.25 + 1.49 ), (4. 12 + 1.36 ) ) were decreased when compared with control group ( 11.62 + 1.92 ),PWMT((1.28 +0.14)g;(1.75 +0.46)g),PWTL((14.64 +2.12) s; (16.85 + 1.61)s)were increased when compared with control group ( (0.47 + 0. 16) g, ( 11.32 + 1.68 ) s) (P ＜ 0.05 ＝. The effect lasts for at least 4 h and disappears at 8 h. Conclusion CaMK Ⅱ may play an important role in the mechanism of bone cancer pain. Intrathecal KN93 injection can effectively attenuated bone cancer pain.
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Objective To investigate the alteration of phosphorylated GluR1Ser831 and behavioural effects in a rat model of levodopa-induced motor complications after Ca2 +/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) inhibitor KN-93 treatment. Methods The hemi-parkinsonian rat model was produced by injecting stereotaxically 6-OHDA to right medial forebrain bundle. Then, rats were intraperitoneally treated with levodopa ( 50 mg/kg with benserazide 12.5 mg/kg,twice daily) for 22 days. On day 23 ,rats received KN-93 before levodopa administration. Rotational duration was estimated. After sacrificed, subcellualr distribution of GluR1 and phosphorylated GluR1Ser831 were observed by western blot. Results The study showed that CaMKⅡ inhibitor KN-93 prolonged rotational duration. Moreover, KN-93 could regulate subcellular distribution of GluR1 and reduce hyperphosphorylation of GluR1 Ser831, which was closely associated with levodopa-induced motor complications. The expression of membrane GluR1 and phosphorylated GluR1Ser831 was (83.4 ±4.2)% and (47.2 ±5.2)% ,respectively. Conclusions These results indicated that activation of CaMKⅡ contributed to development of motor complications, through a mechanism that involved an increase in phosphorylated GluR1 Ser831. Pharmaceuticals which act to inhibit CaMKⅡ may be useful in the treatment of the motor complications in parkinsonian patients.