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BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and down-regulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.
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Animals , Mice , Electroacupuncture , Microglia/physiology , G-Protein-Coupled Receptor Kinase 2/physiology , Pain Management , Pain/chemically induced , Inflammation/chemically induced , Inflammation/therapy , NeuronsABSTRACT
Rheumatoid arthritis (RA) is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes (FLS). The up-regulated cellular membrane expression of G protein coupled receptor kinase 2 (GRK2) of FLS plays a critical role in RA progression, the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor (EP4) signaling and FLS abnormal proliferation. Recently, although our group found that paeoniflorin-6'-
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OBJECTIVE Aryl hydrocarbon receptor (Ahr) is thought to be a crucial factor that regulates immune responses, which may be involved in the pathogenesis of autoimmune inflammation including rheumatoid arthritis (RA). The results of our group in recent years have shown that CP-25, a novel ester derivative of paeoniflorin, has a good effect on improving RA animal models. However, whether the anti-arthritis effect of CP-25 is related to Ahr remains unclear. METHODS CP-25 treatment ameliorated adjuvant-induced arthritis (AA), a mouse model of RA, by inhibiting Ahr-related activities in fibroblasts like synoviocytes (FLS). AA rats were treated with CP-25 or paroxetine from day 17 to 33 after immunization. RESULTS CP-25 alleviated arthritis symptoms and the pathological changes, decreased the expression of Ahr in the synovium and FLS of AA rats. Besides, treatment with CP-25 reduced the proliferation and migration of MH7A caused by Ahr activation. In addition, we also demonstrated that CP-25 down-regulated the co-expres?sion and co-localization of Ahr and G protein-coupled receptor kinase 2 (GRK2) in MH7A. CONCLUSION The data pre?sented here demonstrated that CP-25 suppressed FLS dysfunction in rats with AA, which were associated with reduced Ahr activation and the interaction between Ahr and GRK2.
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Objective@#To observe the effect of high-intensity interval training (HIIT) on G protein-coupled receptor kinase-2 (GRK2) levels in the hearts and adrenal glands of spontaneously hypertensive rats (SHRs) and explore how exercise might improve sympathetic over-excitation.@*Methods@#Twenty male SHRs were divided randomly into a sedentary control group and an HIIT group. Ten age-matched, male Wistar-Kyoto rats without hypertension served as the control group. The rats of the control and sedentary control groups were housed in cages at rest while those of the HIIT group underwent eight weeks of HIIT. Caudal artery pressure, cardiac structure and function, and heart rate variability (HRV) were determined using a non-invasive blood pressure tester, echocardiograms, and electrocardiograms. The plasma concentrations of epinephrine and norepinephrine, and the expression of GRK2 and β1-adrenoceptor (β1-AR) protein were measured in the rats′ hearts, and GRK2 and α2-adrenoceptor (α2-AR) protein were measured in their adrenal glands using high-pressure liquid chromatography and western blotting.@*Results@#Compared with the normotensive control group, animals in the sedentary control group showed elevated blood pressure, cardiac hypertrophy, reduced heart function, sympathetic over-excitation manifested by HRV, increased plasma epinephrine and norepinephrine concentrations, up-regulated GRK2 protein expression in the heart and adrenal gland, but down-regulated β1-AR in the heart and down-regulated α2-AR in the adrenal gland. Compared with the sedentary control group, the HIIT group did not show improved cardiac hypertrophy, but it did show reduced blood pressure, enhanced heart function, suppressed sympathetic over-excitation, as well as lowered plasma epinephrine and norepinephrine concentrations, on average. The expression of GRK2 in the heart and adrenal gland was significantly down-regulated, while that of β1-AR in the heart and of α2-AR in the adrenal gland were significantly up-regulated, on average.@*Conclusions@#HIIT can alleviate sympathetic over-excitation and enhance heart function despite spontaneous hypertension, at least in rats. The therapeutic mechanism may be related with the down-regulation of GRK2 expression in the heart and adrenal gland.
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Type 2 diabetes mellitus (T2DM) is a common cause of erectile dysfunction (ED). It has been demonstrated that G protein-coupled receptor kinase 2 (GRK2) overexpression contributes to diabetic endothelial dysfunction and oxidative stress, which also underlies ED in T2DM. We hypothesized that GRK2 overexpressed and attenuated endothelial function of the cavernosal tissue in a rat model of T2DM. T2DM rats were established by feeding with a high-fat diet (HFD) for 2 weeks and then administering two intraperitoneal (IP) injections of a low dose of streptozotocin (STZ), followed by continuous feeding with a HFD for 6 weeks. GRK2 was inhibited by IP injection of paroxetine, a selective GRK2 inhibitor, after STZ injection. Insulin challenge tests, intracavernous pressure (ICP), GRK2 expression, the protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit gp91phox, nitric oxide (NO), reactive oxygen species (ROS) production, and apoptosis in cavernosal tissue were examined. Less response to insulin injection was observed in T2DM rats 2 weeks after HFD. Markedly increased GRK2 expression, along with impaired Akt/eNOS pathway, reduced NO production, increased gp91phox expression and ROS generation, increased apoptosis and impaired erectile function were found in T2DM rats. Inhibition of GRK2 with paroxetine ameliorated Akt/eNOS signaling, restored NO production, downregulated NADPH oxidase, subsequently inhibited ROS generation and apoptosis, and ultimately preserved erectile function. These results indicated that GRK2 upregulation may be an important mechanism underlying T2DM ED, and GRK2 inhibition may be a potential therapeutic strategy for T2DM ED.
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Objective@#To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and G protein-coupled receptor kinase 2 (GRK2) in the development of persistent postoperative pain in rats.@*Methods@#Pathogen-free healthy male Sprague-Dawley rats, weighing 200-250 g, aged 2 months, were used in this study.Sixty rats in which intrathecal catheters were successfully implanted were divided into 6 groups (n=10 each) using a random number table method: sham operation group (group S), sham operation plus dimethyl sulfoxide (DMSO) group (group D), sham operation plus GRK2 degradation inhibitor MDL28170 group (group M), persistent postoperative pain group (group PPP), persistent postoperative pain plus DMSO group (group PPP+ D) and persistent postoperative pain plus MDL28170 group (group PPP+ M). Persistent postoperative pain was evoked by skin/muscle incision and retraction (SMIR). Immediately after operation and at 1, 2 and 3 days after operation, normal saline 20 μl was intrathecally injected once a day in S and PPP groups, 5% DMSO 10 μl was intrathecally injected once a day in D and PPP+ D groups, and MDL28170 10 μl (50 μg) was intrathecally injected once a day in M and PPP+ M groups.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before operation and 3, 7, 14 and 21 days after operation (T1-4). Four rats in each group were selected after behavioral testing at T3 and sacrificed, and the L4-6 segments of the spinal cord were removed for determination of the expression of phosphorylated p38MAPK (p-p38MAPK) by Western blot.@*Results@#There was no significant difference in MWT at each time point or expression of p-p38MAPK among group S, group D and group M (P>0.05). Compared with group S, the MWT was significantly decreased, and the expression of p-p38MAPK was up-regulated in PPP, PPP+ D and PPP+ M groups (P<0.05). Compared with group PPP, the MWT was significantly increased, and the expression of p-p38MAPK was down-regulated in group PPP+ M (P<0.05), and no significant change was found in the MWT or expression of p-p38MAPK in group PPP+ D (P>0.05).@*Conclusion@#Down-regulated expression of spinal GRK2 can promote the activation of p38MAPK in the spinal cord and is involved in the development of persistent postoperative pain in rats.
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Objective To evaluate the relationship between p38 mitogen-activated protein kinase (p38MAPK) and G protein-coupled receptor kinase 2 (GRK2) in the development of persistent postoperative pain in rats.Methods Pathogen-free healthy male Sprague-Dawley rats,weighing 200-250 g,aged 2 months,were used in this study.Sixty rats in which intrathecal catheters were successfully implanted were divided into 6 groups (n =10 each) using a random number table method:sham operation group (group S),sham operation plus dimethyl sulfoxide (DMSO) group (group D),sham operation plus GRK2 degradation inhibitor MDL28170 group (group M),persistent postoperative pain group (group PPP),persistent postoperative pain plus DMSO group (group PPP+D) and persistent postoperative pain plus MDL28170 group (group PPP+M).Persistent postoperative pain was evoked by skin/muscle incision and retraction (SMIR).Immediately after operation and at 1,2 and 3 days after operation,normal saline 20 μl was intrathecally injected once a day in S and PPP groups,5％ DMSO 10 μl was intrathecally injected once a day in D and PPP+D groups,and MDL28170 10 μl (50 μg) was intrathecally injected once a day in M and PPP+M groups.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before operation and 3,7,14 and 21 days after operation (T1-4).Four rats in each group were selected after behavioral testing at T3 and sacrificed,and the L4-6 segments of the spinal cord were removed for determination of the expression of phosphorylated p38MAPK (p-p38MAPK) by Western blot.Results There was no significant difference in MWT at each time point or expression of p-p38MAPK among group S,group D and group M (P＞0.05).Compared with group S,the MWT was significantly decreased,and the expression of p-p38MAPK was up-regulated in PPP,PPP+D and PPP +M groups (P＜ 0.05).Compared with group PPP,the MWT was significantly increased,and the expression of p-p38MAPK was down-regulated in group PPP+M (P＜0.05),and no significant change was found in the MWT or expression of p-p38MAPK in group PPP+D (P＞0.05).Conclusion Down-regulated expression of spinal GRK2 can promote the activation of p38MAPK in the spinal cord and is involved in the development of persistent postoperative pain in rats.
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Objective To investigate the change of spinal G protein-coupled receptor kinase 2 (GRK2) expression in persistent postoperative pain evoked by skin/muscle incision and retraction (SMIR) in rats.Methods 40 male Sprague Dawley (SD) rats were divided into 2 groups (n =20) using a random number table:sham operation group and skin/muscle incision and retraction group (SMIR group).A rat model of peristent postoperativepain evoked by SMIR was made according to the method described by Flatters.Pain behavior was assessed by paw mechanical withdrawal threshold (MWT) to yon Frey filament stimulationintensity at 1 d before operation (T0) and 3 d (T1),7 d (T2),14 d (T3) and 21 d (T4) after operation.4 rats in each group were sacrificed at T0-4,the L4-L6 segments of the spinal cord were obtained for determination of GRK2 expression in the spinal cord by Western blot.Results Compared with T0,the MWT was significantly decreased and the expression of spinal GRK2 was down-regulated at T1-T4 in SMIR group (P ＜ 0.05).Compared with sham operation group,the MWT and the expression of spinal GRK2 was decreased at T1-T4 in SMIR group (P ＜ 0.05).Conclusions The down-regulation of expression of spinal GRK2 may be involved in the development and maintenance of persistent postoperative pain in rats evoked by SMIR.
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G protein-coupled receptor kinase 2 (GRK2), as a key Ser/Thr protein kinase, belong to the member of the G protein-coupled receptor kinase (GRK) family. The C-terminus of GRK2 including a plekstrin homology domain and the N-terminus of GRK2 including the RGS homology domain with binding sites for several proteins and lipids such as G protein-coupled receptors (GPCRs), G protein, phospholipase C, phosphatidylinositol 4,5-bisphosphate, extracellular signal-regulated kinase, protein kinase A and Gβγ, which can regulate the activity of GRK2. GRK2 can regulate GPCR desensitization and internalization by phosphorylating the GPCR, promoting the affinity of binding to arrestins, and uncoupling the receptors from G proteins, which play an important role in maintaining the balance between the receptors and signal transduction. Previous studies have indicated that cardiac GRK2 overexpression can promote the phosphorylation of β-adrenergic receptor (βAR) leading to βAR desen?sitization and internalization, which play a pivotal role in inducing heart failure (HF)-related dysfunction and myocyte death. GRK2, as a regulator of cell function, is overexpression in hypertension. Overex?pression GRK2 can inhibit Akt/eNOS signaling pathway and decreased the production and activation of eNOS leading to endothelial dysfunction. Collagen-induced arthritis induces the upregulation of GRK2 expression in fibroblast- like synoviocytes. In this review, we mainly discussed the evidence for the association between GRK2 overexpression and various diseases, which suggests that GRK2 may be an effective drug target for preventing and treating heart failure, hypertension and inflammatory disease.
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Objective To investigate the adjustment and value of lymphocyte G protein-coupled receptor kinase-2 (GRK2) protein levels in patients with acute coronary syndrome(ACS). Methods Forty-two patients with stable angina pectoris (SAP), 44 patients with unstable angina pectoris (UAP), 43 patients with acute myocardial infarction (AMI), and 46 patients with normal coronary angiography (NCA) in hospital were enrolled in this study. Lymphocyte GRK2 protein levels were analyzed by Western blot in 24 h after admitted to hospital. Heart rate variability (HRV) analysis was performed based on 24 h Holter electrocardiogram (ECG) monitoring. Cardiac functions were measured using ultrasonic cardiogram. The results were compared and the relationship between GRK2 protein levels and HRV, cardiac functions index was analyzed. Results The level of lymphocyte GRK2 in AMI group, UAP group, SAP group and NCA group was (209.8 ± 63.9)%, (165.6 ± 60.2)%, (131.7 ± 51.8)% and (125.3 ± 50.6)%. The levels of lymphocyte GRK2 in AMI group and UAP group was significantly higher than that in SAP group and NCA group .Moreover, the level of GRK2 was the highest in AMI group, and there were significant differences (P<0.01). The level of lymphocyte GRK2 had negative correlation with high frequency(HF), low frequency(LF), LF/HF, standard deviation NN interval (SDNN), standard deviation of the average normal RR interval for 5-minute segments (SDNNI) and left ventricular ejection fraction (LVEF) (r =-0.52,-0.47,-0.53,-0.56,-0.49,-0.51, P < 0.01). Conclusions The rise of lymphocyte GRK2 protein levels is significantly associated with increased sympathetic nerve excitability and deterioration of cardiac function.
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Objective To investigate the G protein-coupled receptor kinase 2 (GRK 2) level in peripheral blood lymphocytes with cardiac func-tion in elderly patients with acute myocardial infarction. Methods This study enrolled 40 patients with acute ST-segment elevation myo-cardial infarction (STEMI) and 40 patients with unstable angina. All patients were 65 years or older. Cardiac function was evaluated by echocardiography, and the GRK 2 level in peripheral blood lymphocytes was measured. Patients with STEMI were followed up for 2 years. Results The GRK 2 level in peripheral blood lymphocytes was significantly higher in patients with STEMI than in patients with unstable angina, and was negatively correlated with left ventricular ejection fraction, cardiac output, stroke volume, and left ventricular fractional shortening. The GRK 2 level was significantly elevated in some patients with acute STEMI and poor cardiac function. Conclusions In-creased GRK 2 level in patients with acute STEMI may contribute to poor myocardial systolic function and myocardial remodeling. Meas-urement of the GRK 2 level in peripheral blood lymphocytes may assist in the evaluation of cardiac function and myocardial remodeling in elderly patients with acute STEMI.
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Objective To establish mild cognitive dysfunction (MCI) models in elderly rats,and to investigate the pathophysiological features.Methods Totally 40 SD rats (14 to 18-month-old) were randomly divided into 2 groups:the model group (n=20) and the sham operation group (n=20).Bilateral carotid artery stenosis was prepared in the model group while bilateral carotid artery was seperated with no bilateral narrowing in the sham operation group.30 days after the operation,Morris water maze test was performed,pathomorphological and electron microscopic observations of the cerebral tissue were examined and the expression of G protein-coupled receptor kinase 2(GRK2) in hippocampus tissue w detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blottin.Results The mortality in model group was only 10％.Pathological morphology and ultrastructure showed that hippocampal tissue structure was almost normal in sham operated group,but in model group group,hippocampal CA1 pyramidal cells were in ischemic demyelination,arranged loose,and part of the cells showed nucleus pyknosis,deeply stained; there was no obvious infarct in white matter,part of the white matter fiher hecame thinner and disorder,nucleolus became smaller and steped aside,cytoplasmic electron density increased,lipofuscin appeared occasionally.Rough endoplasmic reticulum and Golgi were expanded,cytosolic free ribosomes increased,part of mitochondria became swelled,vacuolated.Morris water maze test results showed that the average escape latency in model group was longer than in sham group (P＜0.05).In spatial probe test,the average time of crossing the first original platform in model rats was significantly longer than the sham operated group [(36.80±7.68) s vs.(20.87±6.16)s,P＜0.05].The average number of crossing the original platform in 60 seconds in model group was significantly less than in sham group(1.43±0.51 vs.3.10±1.45,P＜0.05).The expressiones of GRK2 mRNA and protein in the hippocampus were significantly increased in model group rats than in sham group (P＜0.05).Conclusions The model of severe CCA stenosis in elderly rats can be applied for MCI animal models with good stability and repeatability.Compared with sham group,the cells morphology and ultrastructure in model group appeare more obvious pathological changes and mild impairments in cognitive function.GRK2 may play an important role in the development of MCI.