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Objective To evaluate the role of M3 receptors in penehyclidine hydrochloride ( PHC)-induced reduction of lipopolysaccharide ( LPS)-induced injury to human pulmonary microvascular endotheli-al cells ( PMVECs) . Methods Human PMVECs transfected with M3 shRNA were seeded in 6-well plates (2 ml∕hole) or in culture flasks (4 ml∕flask) at the density of 1×105 cells∕ml and divided into 5 groups ( n=5 each) using a random number table method: control group ( group C) , LPS group, PHC plus LPS group ( group P+LPS) , LPS plus M3 shRNA transfection group ( group LPS+shRNA) , and PHC plus LPS plus M3 shRNA transfection group ( group P+LPS+shRNA) . Group C received no mediation, and LPS was added at the final concentration of 0. 1 μg∕ml in the other groups. PHC 2 μg∕ml was added at 1 h before adding LPS in P+LPS and P+LPS+shRNA groups. The cells were transfected with plasmid containing 2. 5 nmol∕L M3 receptor shRNA in LPS+shRNA group and P+LPS+shRNA group. Contents of filamentous actin ( F-actin) in endothelial cells were measured by flow cytometry at 1 h after adding LPS. The expression of myosin light chain kinase ( MLCK) and VE-cadherin protein was examined by immunofluorescence. The ex-pression of nuclear factor kappa B ( NF-κB) p65 and IκB was detected by Western blot. Contents of tumor necrosis factor-alpha ( TNF-α) and interleukin-6 ( IL-6) were determined by enzyme-linked immunosorbent assay. The M3 receptor mRNA transcription was detected by real-time polymerase chain reaction at 10, 30 and 60 min after adding LPS. Results Compared with group C, F-actin content was significantly de-creased, the expression of VE-cadherin and IκB was down-regulated, the contents of TNF-αand IL-6 were increased, and the expression of MLCK and NF-κB p65 was up-regulated in LPS and P+LPS groups ( P<0. 05) . Compared with group C, the expression of M3 receptor mRNA was significantly up-regulated in group LPS ( P<0. 05) , and no significant change was found in group P+LPS ( P>0. 05) . Compared with group LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB was up-reg-ulated, the contents of TNF-αand IL-6 were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+LPS and group LPS+shRNA ( P<0. 05) . Compared with group P+LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB protein was up-regulated, TNF-α and IL-6 contents were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+LPS+shRNA ( P<0. 05) . Conclusion PHC re-duces LPS-induced injury to human PMVECs through interfering with M3 receptors and inhibiting NF-κB-mediated inflammatory responses.
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Objective To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells (PMVECs) caused by endotoxin and the relationship with mitogen-activated protein kinase (MAPK) signaling pathway.Methods Human PMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and randomly divided into 6 groups (n=5 each):control group (group C),M3 receptor shRNA transfection group (group shRNA),lipopolysaccharide (LPS) group,penehyclidine plus LPS group (group P+LPS),LPS plus M3 receptor shRNA transfection group (group LPS+shRNA) and PHC plus LPS plus M3 shRNA transfection group (group P+LPS+shRNA).The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA,LPS+shRNA and P+LPS+shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation,cells were incubated for 1 h,then LPS at the final concentration of 0.1 μg/ml was added,and cells were incubated for another l h in P+LPS and P+LPS+shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) was detected by Western blot,the expression of heat shock protein 27 (HSP27) using immunofluorescent staining,and the expression of M3receptor mRNA by real-time polymerase chain reaction.Results Compared with group C,M3 receptor mRNA expression was significantly down-regulated in group shRNA,and the permeability of cells was significantly increased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was up-regulated in group LPS (P＜0.05).The permeability of cells was significantly decreased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was down-regulated in P+ LPS,LPS+shRNA and P+LPS+shRNA groups as compared with group LPS,and in group P+LPS+shRNA as compared with group LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor.
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Introdução: A HD está presente em aproximadamente 50% dos pacientes com OIV devido HPB e 30% dos casos não apresentarão melhora após o tratamento cirúrgico. Até o momento, nenhuma característica clínica pode predizer acuradamente quais pacientes serão beneficiados. Neste estudo nós analisamos o papel de seis marcadores moleculares na resolução da HD após a RTUP. Método: Um estudo prospectivo e controlado analisou 43 pacientes com OIV devido HPB, submetidos a RTUP de 2011 a 2012. O grupo controle foi composto por espécimes de músculo vesical de 10 pacientes com menos de 60 anos, submetidos a prostatectomia radical devido câncer de próstata, apresentando IPSS menor que 8 e volume prostático menor que 30 gramas. Todos os pacientes realizaram estudo urodinâmico no pré-operatório e com 6 meses de pós-operatório. Nós analisamos a presença, o período de início (primeira vs segunda metade do enchimento vesical) e a amplitude (< 40 vs > 40 cmH2O) das CVIs, assim como sua resolução após 6 meses de tratamento cirúrgico. Uma biópsia de músculo vesical foi efetuada no final da RTUP para análise do perfil de expressão gênica do NGF, NGFr, VEGF, CD-105, CHRM2 e CHRM3. Para este propósito foi utilizado a técnica de qRT-PCR. Além disso, correlacionamos variáveis clínicas pré-operatórias com a evolução da HD no pós-operatório. Resultados: A idade média dos pacientes foi 63 anos (50 a 75). A HD estava presente em 21 (48,8%) pacientes. De acordo com aferições pré-operatórias, a média de expressão gênica do NGF foi 3,3 vezes maior nos pacientes que iniciaram CVI precocemente quando comparados àqueles que iniciaram as contrações na fase final de enchimento vesical (p=0,047). A presença e a amplitude das CVIs não apresentaram correlações estatísticas com os genes estudados. Em relação a resolução da HD, a média de expressão de CHRM2 foi 2 vezes maior entre os pacientes que evoluíram com melhora da HD (p=0,072). Após 6 meses da RTUP, 77,8% dos pacientes que possuíam...
Objective: Non-inhibited contractions (NIC) are present in about 50% of patients with bladder outlet obstruction (BOO) due to benign prostatic hyperplasia (BPH) and 30% of cases persist after surgery. To date, no clinical characteristic can predict accurately which patients are going to improve. We analyzed the role of six detrusor molecular markers in the resolution of NIC after transurethral resection of the prostate (TURP). Methods: We performed a prospective and controlled analysis of 43 patients with BOO due to BPH who underwent TURP from 2011 to 2012. The control group comprised 10 bladder specimens from patients younger than 60 years who underwent radical prostatectomy with an IPSS < 8 and prostate volume < 30 grams. All patients underwent urodynamic analysis pre and post operatively after 6 months. We analyzed the presence, time to occurrence (first vs second half of the filling phase) and grade (<40 vs >40 cmH2O) of NIC as well as its resolution after 6 months of surgery. A biopsy of the bladder muscle was performed at the end of TURP for analysis of nerve growth factor receptor (NGFr), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), endoglin (CD105), muscarinic cholinergic receptor 2 (CHRM2) and muscarinic cholinergic receptor 3 (CHRM3) genes expression. For this purpose, we used the quantitative real time polymerase chain reaction method (qRT-PCR). Results: Mean patient age was 63 years (50 to 75). NIC were present in 21 (48.8%) patients. According to pre-operative measures, NGF gene expression was 3.3 times greater in patients who presented early NIC as compared to those who presented late contractions (p=0.047). The presence or grade of NIC failed to present statistical correlations with the genes. With regard to the outcome, CHRM2 expression was 2.0 times greater among patients who presented resolution of NIC (p=0.072). After 6 months of TURP, 77,8% of patients with DO resolution had increased expression...
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Humans , Male , Middle Aged , Endothelial Growth Factors , Gene Expression , Nerve Growth Factor , Polymerase Chain Reaction , Prostatic Hyperplasia , Transurethral Resection of Prostate , Urinary Bladder Neck Obstruction , Urinary Bladder, Overactive , UrodynamicsABSTRACT
Objective To evaluate the role of M3 muscarinic acetylcholine receptor (M3 mAChR) in the release of glycinergic neurotransmitter by using oxotremorine-M (Oxo-M: a nonselective mAChR agonist) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP: a highly selective M3mAChR antagonist). Methods Twenty male 3-4 weeks old SD rats weighing 160-180 g after successful intrathecal catheterization were randomized into 2 groups (n = 10 each): normal saline group (group NS) and pertussis toxin (group PTX).Pertussis toxin 1.5 μg/10 μl was injected IT in group PTX, while in group NS normal saline 10 μl was injected IT instead. The animals were killed at day 7 after injection. The spinal cords were removed and sliced and placed in artificial CSF. Glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were measured in spinal lamina Ⅱneurons using whole-cell voltage-clamp technique. Five minutes after sealing, Oxo-M (final concentration 3 μ mol/L) was added. Oxo-M was then completely washed out 3 min later and 4-DAMP (final concentration 25 nmol/L) was added after 5 min of stabilization. In the presence of 4-DAMP, Oxo-M (final concentration 3 μmol/L) was added again 3 min later. sIPSCs were recorded before addition of Oxo-M (T1), 3 min after addition of Oxo-M (T2), 3 min after addition of 4-DAMP (T3), 3 min after the second addition of Oxo-M (T4). Results Compared with the baseline value at T1 , Oxo-M significantly increased the frequency of glycinergic sIPSCs at T2without changing the amplitude at T2-4 in both groups. The frequency of sIPSCs was significantly lower at T4 than at T2 in both groups (P ＜ 0.05 or 0.01). There was no significant difference in both frequency and amplitude of glycinergic sIPSCs between the two groups. Conclusion M3 mAChR plays a predominant role in the release of glycinergic transmitter in the spinal lamina Ⅱ neurons in rats.
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Objective To establish the immunization model with synthetic M3-muscarinic receptor peptides in rats and investigate the role of autoantibodies against M3-muscarinic receptor in the pathogenesis of chronic obstructive pulmonary disease. Methods The control and immunization models were established. The rate of positive of autoantibodies against M3-muscarinic receptor and the pulmonary function were detected. The blood gas analysis was also detected on the next day after finishing immunization. The tissues of lung were observed by light microscope. Results In immunization group, the rate of positive of autoantibodies against M3-muscarinic receptor were 100%, the geometric means of autoantibody were 1:152. There was a statistical difference between the immunization group and control group (x2 =6. 68, P ＜0. 01). The inspiratory resistance (Ri) was [ ( 1. 77 ±0. 22) cm H2O/ml. sec] and [ ( 1.39±0. 21 )cmH2O/ml. sec] in immunization and control group, respectively. It was increased while the lung compliance (C1) and the percentage of forced expiratory volume in first 0. 3 second to forced vital capacity ( FEV0. 3/FVC% ) were decreased in immunization group. There was a statistical difference compared with control group ( t = 3. 11,2. 82,3.23, allP ＜ 0. 01 ). In immunization group, the PaCO2 was higher and the pH as well as the PaO2 were lower than that in control group. The values of blood gas analysis showed a statistical difference between the immunization group and control group ( t =3.86,3. 47 ,allP ＜0. 01 ). Lung tissueswere severely destroyed in immunization group. There were many inflammation cells and hyperplasia glands,alveolar septum was very thick, and part of pulmonary alveoli even expanded to form bullae lung. Positive correction were found between autoantibodies against M3-muscarinic receptor and pulmonary function as well as blood gas analysis in immunization group. Conclusion The autoantibodies against M3-muscarinic receptor maybe play an important role in the pathogenesis of chronic obstructive pulmonary disease.
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Objective To study the relationship between M3 receptor and prostastic tumor by analyzing the expressions of M3 and vascular endothelial growth factor(VEGF)in adult human normal and neoplastic prostatic gland tissue. Methods The specimens included 36 normal prostates(fresh),36 benign prostatic hyperplasia(BPH)tissue(fresh),and 36 cancer tissue(8 fresh).RT-PCR was used to detect M3 receptor,VEGPs genetic expression.At protein level,VEGF,Ms receptor,CD34 were detected by western-blot and immunohistochemical method. Results VEGF and M3 receptor's genetic expressions were higher in prostate cancer tissue(O.8354±0.1897,0.7824±0.2047)than in BPH tissue(0.6735±0.1603,0.6021±0.1637),while the expressions of these genes were lowest in normal prostate tissue(0.5425±0.1629,0.3436±0.1581)(P<0.001).There was a positive correlation between M3 and VEGFs gene expression(r=0.4999,P<O.001).The protein expression of Ms receptor and VEGF was similar to their genetic expression pattern,the expressions in prostate cancer tissue(0.4777±0.1638,0.5718±0.2245)were higher than those in BPH tissue(0.3655±0.1474,0.4342±0.1538)and in normal prostate tissue(0.2659±0.1076,0.3380±0.1527)(P<0.05).Conclusions M3 receptor might be related with development of prostatic tumor.It could be used as a new target 0f diagnosis and treatment for prostastic tumor.
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Our previous studies document the expression of adrenoceptors and purinoceptors in the rat prostate neuroendocrine cells (RPNECs). However, a direct investigation of the receptors for acetylcholine (ACh) is still lacking in the prostate neuroendocrine cells. RPNECs were freshly isolated from the ventral lobes of rat prostate by using collagenase. Effects of ACh and various muscarinic antagonists on the intracellular Ca2+ concentration ([Ca2+]c ) were investigated by using the fura-2 spectrofluorimetry. Single-cell RT-PCR analysis was applied to identify the transcripts for the muscarinic receptor subtypes. ACh (5 micrometer) induced a sharp transient increase in the [Ca2+]c of RPNECs, which was independent of the extracellular Ca2+. In the same RPNECs, high KCl (60 mM), phenylephrine (5micrometer), UTP (P2Y1/2 agonist, 50, micrometer), and alpha, beta-meATP (P2X1/3 agonist, 0.5micrometer) also increased the [Ca2+]c. The ACh-induced [Ca2+]c change (delta[Ca2+]c ) was blocked by atropine or by para-fluorohexahydrosiladifenidol (M3 antagonist, 0.3micrometer), but not by telenzepine (M1 antagonist, 1 micrometer) and himbacine (M2 and M4 antagonist, 1 mircoM). The single-cell RT-PCR demonstrated the selective expression of mRNAs for M3 in RPNECs. In summary, RPNECs express M3 muscarinic receptors that are linked to the release of Ca2+ from intracellular stores. The Ca2+ signals of RPNECs might mediate the parasympathetic regulation of prostate gland.