Upregulated FAAH expression in PVN contributes to sympathoexcitation in rat with chronic heart failure

AIM: To investigate the expression of fatty-acid amide hydrolase (FAAH) in paraventricular nucleus (PVN) and its contribution to renal sympathetic nerve activity in rats with chronic heart failure (CHF). METHODS: A rat model of CHF was established by ligation of the left coronary artery to induce acute myocardial infarction. Eight weeks after ischemia, the rat model of CHF was identified by echocardiogram and histopathological observation. The plasma level of norepinephrine (NE) was detected by ELISA. The protein expression levels of FAAH in the PVN were determined by Western blot. The N-arachidonoylethanolamide (AEA) generation in PVN was analyzed by high-performance liquid chromatography. After microinjection of AEA, PF3845 (an FAAH inhibitor) or rAAV2-FAAH shRNA virus in PVN, the sympathetic drive indexes were recorded in different experiment groups. RESULTS: Compared with the rats in sham group, the cardiac function and AEA concentration in PVN were significantly reduced, while the plasma NE level and FAAH expression in PVN were obviously increased in the CHF rats (P<0.05). After microinjecion of PF3845, AEA or rAAV2-FAAH shRNA virus in PVN, the sympathetic drive indexes were decreased significantly and the cardiac function were improved in the CHF rats. CONCLUSION: Upregulated FAAH expression in PVN may result in sympathoexcitation in the rat with CHF.     

Role of central PGE2 on sympathetic excitation in chronic heart failure

AIM: To observe the effect of central prostaglandin E₂ (PGE₂) on sympathetic activation in chronic heart failure (CHF) and to explore the underlying mechanism. METHODS: Male SD rats were subjected to coronary artery ligation to induce heart failure (HF), and the intracerebroventricular infusion was performed by osmotic pump continuously. The rats in sham group and HF group were given artificial cerebrospinal fluid (0.25 μL/h). The rats in HF plus treatment group was given celecoxib (CLB; 20 mg/h). After 4 weeks, the levels of PGE₂ in cerebrospinal fluid (CSF), the sympathetic nerve excitability and cardiac function were measured, and the changes of corticotropin-hormone releasing hormone (CRH)-containing neurons activation and neurotransmitter contents in the hypothalamic paraventricular nucleus (PVN) were also determined. RESULTS: Compared with the sham-operated rats, the HF rats had raised level of PGE₂ in CSF, up-regulated renal sympathetic nerve activity and plasma norepinephrine, increased left ventricular end diastolic pressure, lung-to-body weight and right ventricular-to-body weight ratios, and decreased maximal increase and decreased rate of left ventricular pressure (P<0.05). In addition, the number of CRH positive neurons in PVN and the level of plasma adrenocorticotropic hormone were higher in HF rats than those in sham-operated rats (P<0.05). After administration of CLB into the lateral ventricle of HF rats, the contents of PGE₂ in CSF were significantly reduced, the number of activation CRH neurons in PVN was decreased, the excitability of sympathetic nerves was down-regulated and cardiac function was improved (P<0.05). Compared with the sham-operated rats, the content of glutamic acid in PVN of HF rats was increased, the content of γ-aminobutyric acid and the number of glutamate decarboxylase 67-positive neurons were decreased (P<0.05). After the CLB was given, the above indexes were reversed (P<0.05). CONCLUSION: These findings indicate that in CHF, the increased central PGE₂ may activate CRH-containing PVN neurons and contribute to the augmented sympathetic drive possibly by modulating the neurotransmitters within the PVN.     

Effect of Ca2+/calmodulin-dependent protein kinaseⅡinhibitor KN-93 on late sodium current in rabbits model of heart failure

AIM: To investigate the change of late sodium current (I_NaL) and the effect of Ca²⁺/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) inhibitor KN-93 on I_NaL in 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 Na_V1.5, 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 I_NaL. RESULTS: Compared with NS group, the heart rate in HF group was increased (P<0.01), the ventricular cavity was enlarged (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 Na_V1.5, CaMKⅡδ and phosphorylated CaMKⅡδ in HF group were higher than those in NS group (P<0.01). I_NaL in HF group significantly increased compared with NS group (P<0.01). After adding sea anemone toxin Ⅱ (ATXⅡ), the density of I_NaL in 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 I_NaL in NS group and HF group was significantly decreased (P<0.05).CONCLUSION: CaMKⅡ inhibitor KN-93 inhibits the increase in I_NaL in HF rabbits, which may be related to the activity of CaMKⅡδ and the regulation of CaMKⅡ δ on I_NaL.     

Over-expression of SK2 channel in PVN reduces renal sympathetic nerve activity in chronic heart failure rats

AIM：To investigate the effect of over-expression of small-conductance calcium-activated potassium channel subtype 2 (SK2) in the paraventricular nucleus (PVN) of hypothalamus on heart rate(HR), mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) of the rats with chronic heart failure (CHF). METHODS：Adenovirus vector encoding SK2 (pDC316-mCMV-EGFP-rKCNN2) was constructed.The CHF model of the male Sprague Dawley (SD) rats was established by the ligation of anterior descending coronary artery. Echocardiogram was used at the 6th week after the operation to identify the CHF model. Adenovirus vector encoding SK2 (pDC316-mCMV-EGFP-rKCNN2) or control adenovirus vector (pDC316-mCMV-EGFP) were transfected into the PVN in vivo. The cardiac function was monitored by echocardiogram. The expression of SK2 at mRNA and protein levels was determined by RT-PCR, Western blotting and immunofluorescence. The HR, MAP and RSNA were measured by PowerLab 8/30 in anesthetized rats with bilateral PVN microinjection of SK channel blocker apamin. RESULTS：Treatment with pDC316-mCMV-EGFP-rKCNN2 significantly decreased the renal sympathetic nerve activity in the rat with CHF. Injection of pDC316-mCMV-EGFP did not change the renal sympathetic nerve activity in the rats in sham group. CONCLUSION：The expression and function of SK channels in PVN in the heart failure rats were decreased, suggesting a reduced negative regulation of sympathetic activity in central neural system. Increased expression of SK2 in the PVN leads to a reduction in sympathetic outflow under the condition of CHF, which may provide a new target for the treatment of heart failure.     

Microinjection of AT1 and AT2 receptor antagonists into PVN affects renal sympathetic nerve activity in chronic heart failure rats

AIM: To examine the renal sympathoexcitation affected by microinjection of angiotensin Ⅱ type 1 (AT₁) receptor antagonist L-158809 and angiotensin Ⅱ type 2 (AT₂) receptor antagonist PD123319 into paraventricular nucleus (PVN) in heart failure rats.METHODS: Left anterior descending coronary artery ligation was used to induce rat heart failure (HF) . Four weeks after operation, the left ventricular end-diastolic pressure (LVEDP), the ratios of heart weight/body weight and lung weight/body weight, and the ratio of infarct area of the left ventricle were observed. Under anesthesia, SD rats were fixed into the brain stereo controller to locate PVN for microinjection and the artificial cerebrospinal fluid (ACSF) was used for control. The left kidney was exposed by retroperitoneal approach and the renal sympathetic nerve was separated under surgical microscope. The heart rate, blood pressure and the activity of renal sympathetic nerve discharge (RSNA) were recorded by POWERLAB 8/30 system. RESULTS: Microinjection of AT₁ receptor antagonist into PVN induced a decrease in RSNA in both HF rats and sham rats. The RSNA responses to L-158809 in the HF rats were significantly greater (P<0.05) than those in the sham rats. However, microinjection of AT₂ receptor antagonist and ACSF into PVN induced no change of RSNA in both HF and sham rats. CONCLUSION: There are some differences of sympathetic nerve outputs between using AT₁ receptor antagonist and AT₂ receptor antagonist on PVN, indicating the up-regulation of AT₁ receptors in PVN during HF. The central renin-angiotensin-aldosterone system(RAAS) may be affected by AT₁ receptor, not by AT₂ receptor.     

Downregulated transient outward potassium channel protein Kv4.2 and Kv4.3 expression in PVN contributes to sympathoexcitation in rats with chronic heart failure

AIM: To investigate the transient outward potassium channel protein expression in paraventricular nucleus(PVN) and its contribution to renal sympathetic nerve activity(RSNA) in rats with chronic heart failure(CHF).METHODS: A rat model of CHF was prepared by acute myocardial infarction that was induced by ligation of the left anterior descending coronary artery. Four weeks after heart failure, echocardiogram was applied to identify the CHF model and plasma norepinephrine(NE), serum NH₂-terminal pro-brain natriuretic peptide(NT-proBNP) were detected by ELISA. The expression of ransient outward potassium channel proteins Kv4.2 and Kv4.3 at mRNA and protein levels was determined by real-time PCR and Western blot. The mean arterial pressure(MAP), heart rate(HR) and RSNA were measured in anesthetized rats with PVN microinjection of potassium channel blockers 4-AP. RESULTS: In CHF group, the rat cardiac function and Kv4.2 and Kv4.3 expression in PVN were obviously lower while plasma NE and serum NT-proBNP were obviously higher than those in sham group. Microinjection of 4-AP into PVN induced an increase in MAP, HR and RSNA in both sham and CHF rats, while the CHF rats exhibited smaller responses to 4-AP than sham-operated rats.CONCLUSION: Downregulation of Kv4.2 and Kv4.3 expression in the PVN may be a potential mechanism for sympathoexciation in the rats with chronic heart failure.     

CaMKII aggravates hypoxia/reoxygenation injury in H9c2 cells by activating apoptosis

AIM: To investigate the effect of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: H9c2 cells were randomized into 4 groups:control group, KN-93 (an inhibitor of CaMKⅡ; 1 μmol/L) treatment group, H/R group and H/R+KN-93 (1 μmol/L) treatment group. The cells in KN-93 group and KN-93+H/R group were pretreated with KN-93 for 2 h before the other treatment was performed. The viability of H9c2 cells in each group was measured by CCK-8 assay. Lactate dehydrogenase (LDH) activity in the culture medium was detected. The protein levels of phosphorylated CaMKⅡ (p-CaMKⅡ), phosphorylated phospholamban (p-PLN) and cleaved caspase-3 were determined by Western blot. The apoptosis was analyzed by TUNEL staining and the flow cytometry. RESULTS: No significant difference of all indexes tested between control group and KN-93 group was observed. H/R treatment significantly reduced the cell viability, and increased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). KN-93 (1 μmol/L) significantly increased the cell viability, and decreased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). CONCLUSION: CaMKⅡ aggravates hypoxia/reoxygenation injury in the H9c2 cells by activating apoptosis.     

Activation of corticotrophin releasing hormone-containing neurons in hypothalamic paraventricular nucleus contributes to sympathoexcitation in rats with congestive heart failure

AIM:To observe the expression of corticotropin releasing hormone (CRH) within the paraventricular nucleus of hypothalamus (PVN) and to explore the relationship between the activated CRH-containing neurons and sympathetic activity in rats with heart failure (HF).METHODS:Healthy male Sprague-Dawley (SD) rats were subjected to coronary artery ligation to induce HF,and chronic intracerebroventricular (ICV) infusion was performed by osmotic pump for 4 weeks.The rats in sham group and HF group were given vehicle (VEH;artificial cerebrospinal fluid 0.25 μL/h).The rats in HF plus treatment group were treated with CRH competitive inhibitor αh-CRH (15 mg/h).Meanwhile,the Lewis rats and Fischer 344 rats for control study also underwent coronary ligation to induce HF or sham surgery.After 4 weeks,left ventricular end-diastolic pressure (LVEDP) and maximum positive/negative change in pressure over time (±dp/dt_max) were determined.The right ventricular-to-body weight (RV/BW) and lung-to-body weight (lung/BW) ratios were calculated.The renal sympathetic nerve activity (RSNA) was recorded and the plasma norepinephrine (NE) level was measured.The expression of CRH in the PVN combined with the plasma adrenocorticotrophic hormone (ACTH) levels were measured.RESULTS:Compared with the sham-SD rats,the HF-SD rats had a greater number of CRH positive neurons in the PVN (accordingly the plasma ACTH levels were increased),accompanied by decreased±dp/dt_max and increased RSNA,plasma NE,LVEDP,lung/BW and RV/BW.However,ICV treatment with αh-CRH attenuated these changes in the HF-SD rats (P<0.05).Compared with the sham-Fisher 344 rats,the HF-Fisher 344 rats also had a greater number of CRH positive neurons in the PVN (accordingly the plasma ACTH levels were increased).In addition,they had significantly increased RSNA and plasma NE level,higher LVEDP,RV/BW and lung/BW,and lower±dp/dt_max(P<0.05).Compared with the SHAM-Lewis rats,the HF-Lewis rats had not significantly changed in the above parameters.CONCLUSION:In CHF,the CRH-containing neurons in PVN are activated,thus aggravating cardiac function by increasing sympathoexcitation.     

Changes in the expression of endothelin system in rats with chronic heart failing

AIM: To study the changes of endothelin system during chronic heart failure (CHF) and imply the relationship between endothelin system and the course of CHF by observing the mRNA expression of endothelin receptors (ET_AR and ET_BR) and PreproET₁ in early stage and later stage of CHF caused by left coronary artery ligation. METHODS: The mRNA expression of ET_A, ET_B receptors and PreproET₁ were detected by RT-PCR technique. The plasma concentrations of ET₁ and ANP were determined by RIA method. RESULTS: The plasma concentrations of ET₁ and ANP, and the mRNA expression of ET receptors and PreproET₁ in the lefe ventricle increased significantly in early stage (myocardial infarction 10 d). While the plasma concentrations of ET₁ and ANP in later stage (myocardial infarction 70 d) were higher than those in the early stage. However, the mRNA expression of ET_AR, ET_BR and PreproET₁ decreased significantly. The mRNA expression of ET_AR in myocardial infarction (MI) 70 d rats had no difference with those in sham-operated rats and the mRNA expression of ET_BR and PreproET₁ in MI 70 d rats was lower significantly than those in MI 10 d rats, but significantly higher than those in sham-operated rats. CONCLUSION: The changes of ET receptors and PreproET₁ mRNA expression are involved in the cardiac function modulation during the different stages in chronic heart failure.     

Inhibitors of CaMKⅡ suppress the expression of TNF-α, TGF-β1 and collagen I,III in cardiac fibroblasts treated with angiotensin II or electrical field stimulation

AIM: To observe the role of calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) in the proliferation, released cytokines and expression of collagen Ⅰ and Ⅲ in rat cardiac fibroblasts induced by angiotensin Ⅱ (AngⅡ) or electrical field stimulation (EFS).METHODS: The cultured cardiac fibroblasts were isolated from the neonatal rats of 1-3 days and used in the 3rd passage. The cells were divided into 10 groups: control group, 0.1 μmol/L AngⅡ group, 0.1 μmol/L AngⅡ+0.5 μmol/L KN92 group, 0.1 μmol/L AngⅡ+0.5 μmol/L KN93 group, 0.1 μmol/L AngⅡ+0.5 μmol/L AIP group; 10V 1.0 Hz EFS group, 10 V 1.0 Hz EFS+0.5 μmol/L KN92 group, 10 V 1.0 Hz EFS+0.5 μmol/L KN93 group, 10 V 1.0 Hz EFS+0.5 μmol/L AIP group, 10 V 1.0 Hz EFS+0.1 μmol/L AngⅡ group.MTT was used to detect the proliferation of cardiac fibroblasts. The release of cytokines was measured by ELISA. The mRNA expression of TNF-α, TGF-β₁ and collagen Ⅰ, Ⅲ was determined by RT-PCR.RESULTS: CaMKⅡ inhibitors (0.5 μmol/L KN93 or 0.5 μmol/L AIP) prevented the proliferation and the increase in the expression of TGF-β₁ and TNF-α in cardiac fibroblasts induced by AngⅡ (0.1 μmol/L) or EFS (10 V 1.0 Hz). CaMKⅡ inhibitors (0.5 μmol/L AIP or 1.0 μmol/L AIP) also prevented the increase in mRNA expression of collagen Ⅰ and Ⅲ induced by 0.1 μmol/L AngⅡ. CONCLUSION: Inhibition of CaMKⅡ prevents the proliferation of cardiac fibroblasts induced by AngⅡ or EFS. The possible mechanism of CaMKⅡ inhibitors may be involved in preventing the mRNA expression and release of cytokines (TGF-β₁ and TNF-α), and regulating collagen I and III expression.     

草莓白化相关病毒中国分离物全基因组分析

草莓白化相关病毒(strawberrypallidosis-associatedvirus,SPa V)属于长线形病毒科(Closteroviridae)毛形病毒属(Crinivirus),可引起草莓病害,2017年在中国首次报道。采用高通量测序、RACE和RT-PCR技术获得了SPa V中国分离物(FJ)的基因组全长。该病毒含有两条正单链基因组RNA1和RNA2。RNA1全长8 048 nt,5′和3′非编码区序列分别为264和197 nt,含有3个开放阅读框(ORF),分别编码ORF 1a/1b融合蛋白和p9蛋白。RNA2全长7 977 nt,5′和3′非编码区序列分别为248和186 nt,含有8个开放阅读框(ORF),分别编码HSP70h、CPh、CP、CPm、p7、p6、p9和p28等8个蛋白。RNA1和RNA2与美国M1分离物分别具有98.5%和99.0%的核苷酸一致性;系统发育分析结果表明,SPa V中国分离物(FJ)单独处在一个分支。对SPa V来源的小RNA的分析表明,来源于SPa V的小RAN长度以21和22 nt为主。     

Effects of Dryk1A,ASF and alternative splicing of CaMKⅡδ on myocardial hypertrophy in renovascular hypertensive rats

AIM: To investigate the role of dual-specificity tyrosine phosporylation-regulated kinase 1A (Dyrk1A)-alternative splicing factor (ASF)-calcium/calmodulin-dependent protein kinase Ⅱδ (CaMK Ⅱδ) pathway in the progression of myocardial hypertrophy in renovascular hypertensive rats. METHODS: The renovascular hypertension was induced by two-kidney one-clip (2K1C) method. The changes of blood pressure and myocardial hypertrophy were measured. The techniques of RT-PCR and Western blotting were used to detect CaMKⅡδ alternative splicing and the protein expression of Dyrk1A and ASF, respectively. RESULTS: Eight weeks after operation, systolic blood pressure (SBP) and diastolic blood pressure (DBP) in 2K1C rats increased (P<0.05). The increases in left ventricular weight (LVW), the ratio of LVW to body weight (BW) and the area of myocardial cells indicated that the hypertensive rats developed significant cardiac hypertrophy. The protein expression of Dyrk1A and mRNA expression of CaMKⅡδA and δB were significantly increased, while the protein expression of ASF and mRNA expression of CaMKⅡδC were decreased compared with sham-operated control rats (P<0.05). Treatment with Dryk1A inhibitor epigallocatechin gallate (EGCG) or harmine effectively attenuated cardiac hypertrophy and reversed the changes in the protein expression of Dyrk1A, ASF and alternative splicing of CaMKⅡδ (all P<0.05). CONCLUSION: Dyrk1A-ASF-CaMKⅡδ pathway plays a role in the development of myocardial hypertrophy in renovascular hypertensive rats.     

Effects of arginine vasopressin on subunit phosphorylation of GABAA receptors in rat preoptic area

AIM: To investigate the effects of arginine vasopressin (AVP) on the expression and phosphorylation of γ-aminobutyric acid (GABA) subtype A receptor (GABA_A receptor) subunits in the preoptic area (POA) of rats. METHODS: The rats were divided into AVP group (n=10), AVP+V1a receptor blocker group (n=10), V1a receptor blocker group (n=10) and control group (n=10). After intraperitoneal injection of AVP or V1a receptor antagonist for 0.5 h, the changes of the expression and phosphorylation of POA GABA_A receptor subunits (α, β and γ2) were analyzed by RT-qPCR and Western blot. RESULTS: Compared with control group, no significant change of GABA_A receptor subunit expression in the rats injected with AVP or V1a receptor antagonist was observed. AVP up-regulated the phosphorylation level of POA GABA_A receptor γ2 subunit (P<0.05), and significantly increased the expression and phosphorylation of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ). CONCLUSION: Exogenous AVP has no effect on expression of POA GABA_A receptor subunits (α, β and γ2) and is involved in modulation of GABAergic synaptic transmission in the POA by activation of PKC and CaMKⅡ and phosphorylation of γ2 subunit via V1a receptor.     

Effect of zacopride on angiotensin Ⅱ-induced myocardial fibrosis

AIM:To investigate the effect of inwardly rectifying potassium channel (I_K1) agonist zacopride (Zac) on angiotensin Ⅱ (Ang Ⅱ)-induced viability and apoptosis of cardiac fibroblasts (CFb) and to explore the underlying anti-fibrosis mechanism.METHODS:The ventricular fibroblasts from neonatal SD rats were isolated and cultured by tissue digestion and differential adherence methods. The model of rat cardiac fibroblast activation induced by angiotensin Ⅱ was established. The CFb were randomly divided into control group, Ang Ⅱ model group, Ang Ⅱ+Zac group, Ang Ⅱ+Zac+BaCl₂ group, AngⅡ+Zac+chloroquine group and Ang Ⅱ+captopril group. CCK-8 assay was used to detect the effect of Zac on the viability of CFb. The amount of collagen I and collagen Ⅲ secreted by CFb was determined by ELISA. The apoptosis of the CFb was analyzed by flow cytometry. The protein expression of Kir2.1 was determined by Western blot. RESULTS:Compared with the control group, the viability and collagen synthesis of the CFb were significantly increased, along with decreased Kir2.1 expression (P<0.05). Compared with the Ang Ⅱ model group, Zac treatment inhibited the viability and collagen synthesis of the CFb, induced apoptosis and up-regulated Kir2.1 expression (P<0.05). I_K1 blockers BaCl₂ and chloroquine reversed the effect of Zac. CONCLUSION:By enhancing I_K1 (Kir2.1) expression, Zac attenuates Ang Ⅱ-induced ventricular fibrosis, in response to the inhibition of cell viability and induction of apoptosis.     

Expression profiles of CaMKⅡδ at different stages of osteoclast differentiation

AIM: To study the expression profiles and the role of Ca²⁺/calmodulin-dependent kinase Ⅱ delta (CaMKⅡδ) during osteoclast differentiation.METHODS: Mouse RAW264.7 cells were induced by receptor activator of nuclear factor κB ligand (RANKL) at 50 μg/L for osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) staining and bone resorption lacunae examination were performed to verify osteoclast formation. The expression of CaMKⅡδ at mRNA and protein levels was also determined by immunofluorescent cytochemistry, RT-qPCR and Western blot at days 0, 1, 3 and 5.RESULTS: TRAP positive multinuclear cells with bone resorption function were formed after 5 d of induction. The mRNA levels of CaMKⅡδ detected by RT-qPCR were 1.028±0.041, 2.478±0.087, 10.524±1.284 and 42.914±2.667 at days 0, 1, 3 and 5, respectively, while the protein levels of CaMKⅡδ detected by Western blot were 0.762, 0.963, 1.802 and 3.136, respectively. The changes of protein level were also verified by immunofluorescence cytochemistry, in which the fluorescence intensity increased in a time-dependent manner (P<0.05).CONCLUSION: The expression of CaMKⅡδ increases with the differentiation of osteoclasts. CaMKⅡδ may play a key role in the osteoclastogenesis.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.     

Role and regulation of calcineurin in angiotensin Ⅱ-stimulated cardiac myocyte hypertrophy of rats

AIM: To study the role and regulation of calcineurin(CaN) in angiotensin II(AngⅡ)-stimulated cardiacmyocyte hypertrophy of rats. METHODS: Using AngⅡ to induce the cultured cardiac myocyte hypertrophy of rats, and investigating the effect of CaN inhibitor on [³H]-leucine incorporation of AngⅡ-stimulated cardiomyocytes and the regulation of various factors on CaN activity in cardiomyocytes.RESULTS: AngⅡ can stimulate the CaN activity in cultured neonatal rat cardiomyocytes in a dose- and time-dependent manner. In cardiac myocytes incubated with 10, 100, 1000 nmol·L^-1 of AngⅡ for 12h, the CaN activities increased respectively by 13%,57%(P＜0.05) and 228%(P＜0.01) compared with that in non-stimulated cardiomyocytes. The CaN activities in AngⅡ-stimulated cardiomyocytes were significantly inhibited by losartan(50 μmol·L^-1), H₇(50 μmol·L^-1)and Fura-2/AM(4 μmol·L^-1),while no effect was observed with PD98059(50 μmol·L^-1).The [³H]-leucine incorporation in AngⅡ-stimulated cardiomyocytes increased by 46%(P＜0.01) compared with that in control group, which was dramatically inhibited by cyclosporin A(0.5~5μg/mL). CONCLUSIONS: Calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, may play an important role in AngⅡ-induced cardiac myocyte hypertrophy. The activation of CaN may dependent on the sustained increases of [Ca²⁺]i and be regulated by some protein kinases (such as PKC,etc.).