M_(4) muscarinic receptors regulates dopamine/DARPP-32 signaling and glutamate transmis⁃sion to balance dopaminergic D1 function in mouse dorsal striatum

OBJECTIVE Abnormal striatal dopaminergic and glutamatergic neurotransmis⁃sion is central to the pathophysiology of schizo⁃phrenia.In this study,we investigated the roles of M4 receptor interplay with D1 signaling in stria⁃tal neurotransmission that affect glutamatergic transmission to control the etiology of neuropsy⁃chiatric disorders.METHODS To study dorsal striatum(DS)region-specific neuronal and behav⁃ioral responses modulated by M4 receptors,we used clustered regularly interspaced short palin⁃dromic repeats-associated protein 9 technology to generate mice lacking M4 in the dorsal stria⁃tum(DS-M4-KD).The M4 positive allosteric modu⁃lator,VU0467154,were used to study the phar⁃macologically profiles with M4 receptor stimula⁃tion in WT mice.Oxotremorine M(Oxo-M),a no subtype-selective muscarinic agonist,was used to show that mAchRs activation,in order to dissect the particular function of M4,in DS-M4-KD mice.Open filed test and forced swim test were used to assess the change of psychiatric-like behav⁃iors.Western blotting and immunohistochemistry were used to detect protein levels of phosphory⁃lation site of dopamine-and cAMP-regulated phosphoprotein of 32 ku(DARPP-32).Whole-cell patch-clamp recording was used to assess M4-mediated cholinergic inhibition of glutamater⁃gic synaptic input transmission.RESULTS West⁃ern blotting and immunohistochemistry assay showed VU0467154(5 mg·kg-1,ip)promoted phosphorylation of DARPP-32 at Thr75,and atten⁃uated D1-dependent phosphorylation of DARPP-32 at Thr34 within the mouse DS.Consistently,the Oxo-M(4μg,icv)also increased DARPP-32 phosphorylation at site Thr75 to reversed phos⁃phorylation at site Thr34 in WT mice,but not in DS-M4-KD mice.In parallel with altered DARPP-32 responses,VU0467154 or Oxo-M evoked a psychological stress response and reversed D1-induced hyperlocomotion in mice in open field test and force swim tests.However,Oxo-M sup⁃pression of D1-depengdeng behavioral respons⁃es was impaired in DS-M4-KD mice.Whole-cell patch recording showed that VU0467154 or Oxo-M mediated endogenous cholinergic inhibition of miniature excitatory postsynaptic currents through M4 receptors,which in turn suppressed D1-depen⁃dent glutamatergic synaptic transmission in the DS.CONCLUSION This study provides evidence for the role of M4 receptors in regulation of dopa⁃mine/DARPP-32 signaling and glutamate respons⁃es in the DS,and therefore modulation of psychi⁃atric behaviors associated with D1 signaling.This results indicate the mechanisms of treatments targeting M4 in psychiatric disorders.

METABOLIC KINETICS OF BRAIN MUSCARINIC CHOLINERGIC RECEPTORS IN NORMAL AND HYPOTHYROID MICE

A technique for studying in vivo the production rate and turnover rate constant of mouse brain M-receptors was established. A single injection of 25 mg / kg of Benzilylcholine Mustard to living mice resulted in 90 % irreversible block of brain M-receptors. The time course of the receptor density was then monitored by 3H-QNB binding assay and the production rate and turnover rate constant were calculated from the time course curve with a computer program. It was found that in normal mice the turnover rate constant was about 0.035 h-1 (half-life was about 20 h) and the production rate was 30-42 fmol / (h ·mg protein). Parallel experiments revealed a significant slow down of the turnover of brain M-receptors in hypothyroid mice (turnover rate constant was 0.0257±0.0012 h-1 in hypothyroid vs. 0.0356±0.0021 h-1 in normal) while the production rate was not changed significantly. The results suggest that thyroid hormones have a regulatory action on the turnover of brain M-receptors and the elevation of brain M-receptor density together with slow down of the turnover of brain M- receptors is probably one of the important mechanisms relevant to the brain dysfunction in hypothyroidism.

M3 Muscarinic Acetylcholine Receptor Antagonist Darifenacin Protects against Pulmonary Fibrosis through ERK/NF-κB/miR-21 Pathway

Idiopathic pulmonary fibrosis is an untreatable lethal lung disease, which is related to the aberrant proliferation of fibroblasts. M3 muscarinic acetylcholine receptor (M3-mAChR) activation exerts proliferative effect on various kinds of cells. However, whether M3-mAChR inhibition has a protective effect on pulmonary fibrosis remains unexplored. A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin. Darifenacin was used to block M3-mAChR. Histological changes were observed using Masson’s Trichrome and hematoxylin and eosin (HE) staining. Hydroxyproline was measured by Hydroxyproline detection kit. Transforming growth factor β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, pulmonary fibroblasts were isolated from lungs of neonatal rat. After treatment, the cell viability, Hydroxyproline level was measured by MTT and Hydroxyproline detection kit respectively. The expression level of extracellular signal-regulated kinase (ERK), nuclear factor kappa-B (N-NF-κB), and microRNA-21 (miR-21) was detected by western blot or quantitative real-time PCR (qRT-PCR). Darifenacin relieved the fibrotic effects provoked by bleomycin. The expression level of hydroxyproline, TGF-β1 and TNF-α level was all downregulated after darifenacin treatment. In lung fibroblasts, darifenacin decreased cell viability and hydroxyproline level induced by bleomycin. Besides, phosphorylation-ERK and nuclear N-NF-κB protein level was downregulated, as well as miR-21 level. M3-mAChR antagonist darifenacin attenuates bleomycin-induced pulmonary fibrosis in rats, which may relate to the ERK/NF-κB/miRNA-21 signaling pathway.

Involvement of M3 Cholinergic Receptor Signal Transduction Pathway in Regulation of the Expression of Chemokine MOB-1, MCP-1 Genes in Pancreatic Acinar Cells

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10 -3 mol/L, 10 -4 mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10 -3 mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10 -3 mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10 -5 mol/L atropine) or NF-κB inhibitor (10 -2 mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1genes in pancreatic acinar cells in vitro through the activation of NF-κB.

A Decreased Expression and Functionality of Muscarinic Cholinergic Receptor in Acute Chagas Myocarditis

Background: Chagas disease is still a public health problem because of the remaining high prevalence, the expansion of the disease into developed countries and the re-emergences by oral transmission outbreaks. Chagas cardiomyopathy evolves as a consequence of an autonomic un-balance where the parasympathetic tone is undermined. Objective: To determine the functionality and expression of muscarinic cholinergic receptors in acute Chagas disease. Methodology: 62 male, 3-week-old Sprague Dawley rats were assayed;32 were infected with Trypanosoma cruzi trypo-mastigotes and 30 were healthy controls. Electrocardiographic studies were conducted in the absence or presence of direct muscarinic (oxotremorine and McN-A-343) or indirect agonists (phenylephrine) or antagonist (pirenzepine). Muscarinic M1 and M2 receptor expression was determined by radioligand [3H]-QNB binding assay and immunoblot. Results: Chagasic acute myocarditis was sustained by electrocardiographic signs and histopathological findings. Bradycardia induced by oxotremorine was significantly higher in healthy rats (HR) and the differences were enhanced by CsCl. In the absence of the agonist, CsCl induced a greater bradycardia in chagasic rats (ChR). In HR McN-A-343 induced tachycardia, however it induces bradycardia in the presence of a acetylcholinesterase inhibitor (neostigmine);no effects were observed in ChR. Pirenzepine induced a higher tachycardia in HR. Phenylephrine in the presence of pirenzepine induced a similar bradycardia in both groups, but recovery was faster in ChR. Muscarinic M1 and M2 receptor density was higher in HR. Conclusion: Muscarinic receptor expression and functionality are decreased in the acute Chagas disease that could impact the evolution and prognosis of the disease.

Expression Imbalance of Cholinergic M₂and M₃Receptors Contributes to the Motility Reduction of the Small Intestine in Spleen Qi Deficiency

Objective: To study roles of cholinergic M2 and M3 receptors in the motility reduction of small intestine (SI) in spleen qi deficiency. Methods: 16 male SD rats were randomly divided in the control group and spleen qi deficiency group (model group)—8 rats each group;spleen qi deficiency model of the improper diet and overfatigue was established;the SI propelling rate (SIPR) was used to evaluate the SI motility;ELISA was used to measure concentrations of acetylcholine (ACh), cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in the SI tissue;immohistochemistry was employed to detect expressions of cholinergic M2 and M3 receptors. Results: Compared with those in the control group, SIPR was reduced;expression of M2 receptors was increased;and expression of M3 receptors and concentrations of cAMP and PKA were decreased, significantly, in the model group. Conclusions: Expression imbalance of cholinergic M2 and M3 receptors might contribute to the motility reduction of the SI in spleen qi deficiency.

Expression and Significance of ECP, 25-(OH)D3 and M2 receptors in Children with Acute Attack of Asthma

Objective:To study the expression and significance of ECP, 25-(OH)D3 and M2 receptors in children with acute attack of asthma.Methods: Seventy children with bronchial asthma who first visited our hospital from September 2016 to September 2018 were divided into chronic persistence group, remission group and acute attack group. Thirty healthy children who underwent physical examination in our hospital were selected and divided into control group. The levels of ECP, 25-(OH) D3 and M2 receptors were analyzed by ELISA, and Pearson correlation analysis was performed.Results: Compared with the control group, the levels of ECP and M2 receptors in chronic persistence group, remission group and acute attack group increased, while the levels of 25-(OH)D3 decreased, with statistical difference (P<0.05). The levels of ECP and M2 receptors in acute attack group were higher than those in chronic persistence group, and the levels of 25-(OH)D3 were lower than those in chronic persistence group (P<0.05). The levels of ECP and M2 receptors in acute attack group were higher than those in remission group, and the levels of 25-(OH)D3 were lower than those in remission group (P<0.05). Compared with mild children, the levels of ECP and M2 receptors increased and 25-(OH)D3 decreased in moderate and severe children (P<0.05). Compared with moderate children, the levels of ECP and M2 receptors increased and 25-(OH)D3 decreased in severe children (P<0.05).There was a negative correlation between ECP and 25-(OH)D3 (r=-0.380, P=0.038);a negative correlation between 25-(OH)D3 and M2 receptor (r=-0.448,P=0.013);and a positive correlation between ECP and M2 receptor (r=0.450,P=0.013).Conclusions:The expression of ECP and M2 receptors increased during the acute attack of bronchial asthma in children, while the expression of 25-hydroxyvitamin D3 decreased during the acute attack of bronchial asthma in children. The correlation among ECP, 25-(OH) D3 and M2 receptors is significant in the clinical diagnosis of acute attack of bronchial asthma in children.

Muscarinic acetylcholine receptor M1 mediates prostate cancer cell migration and invasion through hedgehog signaling

The autonomic nervous system contributes to prostate cancer proliferation and metastasis. However, the exact molecular mechanism remains unclear. In this study, muscarinic acetylcholine receptor M1 （CHRM1） expression was measured via immunohistochemical analysis in human prostate cancer tissue array slides. PC-3, LNCaP, and A549 cells were treated with pirenzepine or carbachol, and the cell migration and invasion abilities were evaluated. Western blotting and quantitative real-time PCR were performed to measure GLI family zinc finger 1 （GLI1）, patched 1 （PTCH1）, and sonic hedgehog （SHH） expression levels. High expression of CHRM1 was found in early-stage human prostate cancer tissues. In addition, the selective CHRM1 antagonist pirenzepine inhibited PC-3, LNCaP, and A549 cell migration and invasion, but the agonist carbachol promoted the migration and invasion of these three cell lines. Muscarinic signaling can be relayed by hedgehog signaling. These data show that CHRM1 is involved in the regulation of prostate cancer migration and invasion through the hed^eho~ si^nalin~ ~athwav.

Serum IgA against type 3 muscarinic acetylcholine receptor is a novel marker in diagnosis of Sjoegren＇s syndrome

Background Antibodies against type 3 muscarinic acetylcholine receptor （M3R） are involved in the pathogenesis of Sj6gren＇s syndrome （SS）, but the clinical value of them in SS patients has been controversial. The aims of this study were to： （1） establish an improved enzyme-linked immunosorbent assay （ELISA） to detect IgA antibodies against M3R; （2） evaluate the value of IgA antibodies against the second extracellular loop of M3R205-220 （c2M3RP） in diagnosis of SS. Methods To increase the ELISA sensitivity, c2M3RP was coupled to bovine serum albumin （BSA） by the glutaraldehyde method and a 96-well microplate was treated by ultraviolet rays before coated. Concentrations of anti-c2M3RP, anti-SSA, and anti-SSB were measured in the sera of 240 individuals： 91 patients with primary SS and 149 controls （16 secondary SS, 27 systemic lupus erythematosus, 40 rheumatoid arthritis and 66 healthy controls）. Diagnostic properties of anti-c2M3RP were determined by receiver-operating characteristic curve analysis. Results The prevalence of serum IgA anti-c2M3RP antibodies in patients with pSS （46%, 42/91） was significantly higher than that in patients with systemic lupus erythematosus （19%, 5/27）, in rheumatoid arthritis （15%, 6/40） and in healthy controls （5%, 3/66）. However, there was no significant difference between the two $S groups （P=-0.727）. The diagnostic performance of IgA anti-M3RP antibodies was similar to anti-SSA assay, but had 22% higher sensitivity than anti-SSB. By analyzing of IgA anti-c2M3RP antibodies, combination of anti-SSA and anti-SSB resulted in increased sensitivity, whereas their specificity was not significantly changed. Conclusions The improved anti-c2M3RP ELISA is a novel, sensitive, and specific serological test for the diagnosis of SS. The combined application of anti-c2M3RP, anti-SSA and anti-SSB tests can improve the laboratory diagnosis of SS. The IgA anti-c2M3RP antibodies may serve as a novel diagnostic marker for SS.

益气养阴祛瘀方对干燥综合征NOD小鼠颌下腺AQP5、M3R表达的影响

目的研究益气养阴祛瘀方(黄芪、生地黄、玄参、丹参、益母草等)对干燥综合征NOD小鼠颌下腺水通道蛋白5(AQP5)以及毒蕈碱样胆碱能受体3(M3R)表达的影响。方法将30只NOD小鼠随机分为5组:模型组、西药组(硫酸羟氯喹60 mg·kg^(-1))及益气养阴祛瘀方低、中、高剂量组(11.147、22.295、44.590 g·kg^(-1)),每组6只。每日灌胃给药1次,连续给药8周。给药后第0、4、8周连续测量小鼠每日饮水量;给药结束后,分离小鼠双侧颌下腺并称定其质量,计算小鼠颌下腺指数;采用HE染色法进行颌下腺组织病理学观察,计算灶性指数评分;RT-qPCR法检测小鼠颌下腺组织AQP5、M3R mRNA表达水平;Western Blot法检测小鼠颌下腺组织AQP5、M3R蛋白表达水平;免疫组化法检测颌下腺组织AQP5蛋白表达水平;ELISA法检测血清干扰素γ(IFN-γ)、白细胞介素10(IL-10)含量。结果给药前,与模型组比较,益气养阴祛瘀方低、中、高剂量组及西药组NOD小鼠的日饮水量无明显变化(P>0.05);给药第4、8周,与模型组比较,益气养阴祛瘀方低、中、高剂量组及西药组NOD小鼠的日饮水量均显著减少(P<0.01)。模型组小鼠颌下腺淋巴细胞浸润明显并形成较多浸润灶;与模型组比较,益气养阴祛瘀方低、中、高剂量组及西药组NOD小鼠的颌下腺淋巴细胞浸润情况得到明显改善,浸润灶数量明显减少,灶性指数评分显著降低(P<0.01),颌下腺组织AQP5、M3R蛋白表达水平明显升高(P<0.05,P<0.01),血清IL-10水平明显升高(P<0.05,P<0.01);益气养阴祛瘀方高剂量组和西药组NOD小鼠的颌下腺指数明显升高(P<0.05);益气养阴祛瘀方中、高剂量组和西药组NOD小鼠颌下腺组织AQP5、M3R mRNA表达明显上调(P<0.05,P<0.01),血清IFN-γ含量显著降低(P<0.01)。结论益气养阴祛瘀方对NOD小鼠唾液腺损伤具有改善作用,可能通过上调AQP5、M3R表达,抑制颌下腺淋巴细胞浸润,改善唾液分泌功能,从而减缓干燥综合征进程。

毒蕈碱型胆碱能受体M3调控肝癌细胞侵袭转移的研究

目的探讨毒蕈碱型胆碱能受体M3(muscarinic cholinergic receptors 3,ChRM3)活化对人肝癌细胞侵袭、转移能力的影响及其分子机制。方法 Western blot检测两种人肝癌细胞株Hep G2和SMMC-7721中ChRM3受体的表达;采用氯贝胆碱(Beth)及达菲那新(UK88525)处理肝癌Hep G2和SMMC-7721细胞,分为空白对照组、Beth组、Beth+UK88525组、UK88525组,经处理48 h后,Transwell实验检测两种肝癌细胞经不同分组处理后侵袭、迁移能力的变化,Western blot检测各组细胞中上皮-间质转化(epithelial-to-mesenchymal transition,EMT)标志蛋白的表达及下游信号通路PI3K/Akt的变化;采用shRNA干扰质粒经Lipofectamine 2000TM转染肝癌细胞Hep G2以沉默ChRM3,分为空白对照组、Beth组、Beth+sh ChRM3组、sh ChRM3组,分别检测细胞的侵袭、转移能力及EMT、下游PI3K/Akt的改变。结果 Hep G2和SMMC-7721两种肝癌细胞中均有ChRM3受体的表达;Transwell实验显示:Beth单独处理组对肝癌细胞的迁移和侵袭有明显的促进作用,而UK88525能够抑制Beth对肝癌细胞迁移侵袭能力的促进作用(P<0.05);Western blot结果显示:Beth单独处理组能够上调EMT标志蛋白N-Cadherin和Vimentin,下调E-Cadherin的表达,UK88525处理后能够明显抑制Beth促进Hep G2细胞EMT的过程(解除Beth对Hep G2细胞EMT的促进过程),同时也能够抑制PI3K/Akt的活化(P<0.05);干扰质粒沉默ChRM3受体同样能够抑制Beth促肝癌细胞迁移、侵袭的作用,并且抑制Beth诱导的EMT和下游PI3K/Akt的活化(P<0.05)。结论 ChRM3活化能够通过PI3K/Akt信号通路促进肝癌细胞的侵袭和转移。

小细胞肺癌中M3R的表达及意义

目的观察毒蕈碱胆碱受体3(muscarinic cholinergic receptor 3,M3R)在小细胞肺癌(small cell lung cancer,SCLC)中的表达,探讨M3R表达与SCLC分期及临床病理特征的相关性。方法采用免疫组化SP两步法检测60例SCLC(局限期和广泛期各30例)癌组织中M3R的表达,与正常肺组织对比观察M3R表达的差异;回顾性分析所有SCLC病例的病史、体征、支气管镜、胸部CT、颅脑CT、骨扫描、腹部CT等检查,电话随访患者的生存时间。结果 M3R在SCLC中的表达明显高于正常肺组织;M3R在局限期和广泛期SCLC中的表达差异无统计学意义;M3R表达与患者年龄、性别、淋巴结转移无关,与是否吸烟有关;Kaplan-Meier生存曲线显示:M3R阴性者的生存期较阳性者长,差异有统计学意义。结论 SCLC癌组织中M3R的表达水平明显高于正常肺组织,M3R表达与SCLC预后有相关性。

人血单个核细胞上M2、M3受体与冠脉痉挛的相关性研究

目的探讨人外周血单个核细胞上M2、M3受体表达的变化与冠状动脉痉挛的相关性。方法选择因静息性胸痛住院,且冠状动脉造影未见血管狭窄的患者29例,经乙酰胆碱激发试验分为冠状动脉痉挛组(痉挛组,20例)和非痉挛组(对照组,9例),应用ELISA分别检测并比较两组外周血单个核细胞上M2、M3受体的表达量。结果冠状动脉痉挛患者M3受体表达量显著低于对照组(P<0.01)。M2受体表达量两组无显著性差异(P>0.05)。在排除有组间差别的血中低密度脂蛋白胆固醇的影响后,两组间M3受体表达量的差别依然存在。结论M3受体与冠状动脉痉挛的发生相关,M2受体与冠状动脉痉挛的发生无相关,M3受体表达量的下降可能导致内皮功能紊乱,从而引起冠状动脉痉挛。

外周血PLA2R抗体、25(OH)D_(3)、ADAMTS13与原发性膜性肾病患者病情、肾功能损伤的关系

目的探讨原发性膜性肾病患者外周血M型磷脂酶A2受体(PLA2R)抗体、25羟维生素D_(3)[25(OH)D_(3)]、血管性血友病因子裂解酶13(ADAMTS13)与其病情程度及肾功能损伤的关系。方法选取2021年1月至2023年1月该院收治的122例原发性膜性肾病患者作为研究对象,所有患者均经肾穿刺活检病理检查确诊,根据病理分期结果分为1期组(55例)、2期组(24例)、3期组(43例)。另选同期于本院体检的40例健康者作为健康组。所有患者均随访6个月,并评价患者肾功能情况,患者24 h尿蛋白量减少≥50%、水肿症状减轻或消失,纳入良好组,其余纳入不良组。比较1期组、2期组、3期组、健康组研究指标[PLA2R抗体、25(OH)D_(3)、ADAMTS13活性]、肾功能指标(24 h尿蛋白、ALB)水平。采用Pearson相关分析PLA2R抗体、25(OH)D_(3)水平、ADAMTS13活性与24 h尿蛋白、ALB水平的关系。比较良好组与不良组的PLA2R抗体、25(OH)D_(3)水平、ADAMTS13活性。绘制受试者工作特征(ROC)曲线分析PLA2R抗体、25(OH)D_(3)、ADAMTS13单独及3项指标联合检测对原发性膜性肾病患者肾功能损伤的预测价值。结果PLA2R抗体、24 h尿蛋白水平为3期组>2期组>1期组>健康组,且任意两组间比较,差异均有统计学意义(P<0.05)。25(OH)D_(3)水平、ADAMTS13活性、ALB水平比较,3期组<2期组<1期组<健康组,差异均有统计学意义(P<0.05)。Pearson相关分析结果显示,PLA2R抗体水平与24 h尿蛋白水平呈正相关(r=0.620,P<0.05);25(OH)D_(3)水平、ADAMTS13活性与24 h尿蛋白水平均呈负相关(r=-0.625、-0.607,P<0.05);PLA2R抗体水平与ALB水平呈负相关(r=-0.591,P<0.05);25(OH)D_(3)水平、ADAMTS13活性与ALB水平均呈正相关(r=0.742、0.899,P<0.05)。良好组患者63例,不良组患者59例。良好组PLA2R抗体水平低于不良组,而25(OH)D_(3)水平、ADAMTS13活性均高于不良组,差异均有统计学意义(P<0.05)。ROC曲线分析结果显示,PLA2R抗体、25(OH)D_(3)、ADAMTS13单独检测预测原发性膜性肾病患者肾功能损伤的曲线下面积分别为0.831、0.836、0.713,均低于3项指标联合检测预测原发性膜性肾病患者肾功能损伤的0.860(P<0.05)。结论原发性膜性肾病患者外周血PLA2R抗体水平会随着患者疾病恶化而逐渐升高,而25(OH)D_(3)水平、ADAMTS13活性会随病情加重而逐渐降低,且3项指标联合检测对原发性膜性肾病患者肾功能损伤有较高预测价值。

M3R激动剂通过激活PI3K/AKT信号途径促进肺癌细胞A549的上皮间质转化

目的:探讨毒蕈碱胆碱受体3(muscarinic receptor 3,M3R)激动剂卡巴胆碱促进人肺癌A549细胞上皮间质转化的可能信号通路。方法:用400μmol/L卡巴胆碱刺激人肺癌A549细胞,在倒置相差显微镜下观察细胞形态的变化,应用划痕愈合实验和Transwell实验观察细胞迁移和侵袭能力;应用q PCR技术检测上皮间质转化相关蛋白波形蛋白(vimentin)和E钙黏蛋白(E-cadherin)m RNA水平的变化;应用Western blot技术检测p-AKT、vimentin和E-cadherin蛋白水平的变化。结果:卡巴胆碱刺激人肺癌A549细胞后,细胞形态发生明显改变,由不规则多边形逐渐向梭形转化、细胞间紧密结合逐渐变得松散,细胞迁移和侵袭能力增强;vimentin的m RNA和蛋白表达量明显增加,E-cadherin的m RNA和蛋白水平降低,磷酸化的AKT蛋白水平增加,且这些变化均可被M3R特异性抑制剂4-DAMP所抑制(P<0.05)。结论:卡巴胆碱可通过激活PI3K/AKT信号途径促进人肺癌A549细胞发生上皮间质转化。

酸甘化阴法对NOD小鼠唾液腺中AQP5、M3R及外周血干燥综合征特异性抗体的影响

目的探讨酸甘化阴法对NOD小鼠唾液腺中水分子通道蛋白-5(AQP5)、毒蕈碱受体3(M3R)及外周血干燥综合征特异性抗体的影响。方法将8周龄NOD小鼠随机分为5组,每组15只。适应性饲养1周后,芍药甘草汤低、中、高剂量组分别给予相应浓度芍药甘草汤19.5 g/kg灌胃,同时给予生理盐水腹腔注射,1次/d;毛果芸香碱组给予毛果芸香碱注射液1.4 mg/kg腹腔注射,1次/d;生理盐水组给予生理盐水灌胃,1次/d。各组均连续干预6周后取颌下腺,HE染色观察病理形态,real-time PCR法检测腺体内AQP5和M3R mRNA表达情况;取血清,采用ELISA法检测血清中抗SSA、SSB、M3R、α-Fodrin抗体含量。结果生理盐水组和毛果芸香碱组NOD小鼠颌下腺淋巴细胞浸润明显,芍药甘草汤各剂量组小鼠淋巴细胞浸润灶较少。芍药甘草汤各组AQP5及M3R mRNA相对表达量和血清中抗M3R抗体含量均明显高于生理盐水组(P均<0.05),血清中抗SSA、SSB、α-Fodrin抗体含量均明显低于生理盐水组(P均<0.05);芍药甘草汤中剂量组AQP5及M3RmRNA相对表达量均明显高于芍药甘草汤低剂量组(P均<0.05),且AQP5 mRNA相对表达量明显高于芍药甘草汤高剂量组(P均<0.05)。芍药甘草汤中、高剂量组血清中抗SSA、SSB、α-Fodrin抗体含量均明显低于芍药甘草汤低剂量组(P均<0.05),且芍药甘草汤高剂量组均明显低于芍药甘草汤中剂量组(P均<0.05);芍药甘草汤中、高剂量组血清中抗M3R抗体含量均明显高于芍药甘草汤低剂量组(P均<0.05),且芍药甘草汤高剂量组明显高于芍药甘草汤中剂量组(P<0.05)。结论酸甘化阴法可上调NOD小鼠唾液腺中AQP5和M3R的表达,降低血清中抗SSA、SSB、α-Fodrin抗体含量,从而改善腺体分泌能力,缓解口干症状。

M3R拮抗剂对人小细胞肺癌增殖、凋亡及粘附的影响

背景与目的研究表明毒蕈碱胆碱受体3(muscarinic receptor 3,M3R)在多种肿瘤的发生、发展中发挥重要的作用。本研究旨在探讨M3R在人小细胞肺癌(small cell lung cancer,SCLC)细胞株SBC3的表达,M3R拮抗剂对细胞增殖、凋亡及粘附的影响。方法体外培养SBC3细胞,RT-PCR和Western blot检测M3R的表达。MTT法及流式细胞法检测M3R拮抗剂(4-diphenylacetoxy-N-methylpiperidine methiodide,4-DAMP)对细胞增殖和凋亡的影响。流式细胞法检测细胞整合素的表达及碘化乙酰胆碱(acetylcholine iodide,Ach)和4-DAMP对整合素表达的影响。纤维结合蛋白(Fn)包被的96孔板用以研究Ach、4-DAMP及整合素抗体对细胞粘附的作用。结果 SBC3细胞表达M3R,4-DAMP浓度依懒性抑制细胞增殖。与对照组比较,10-4 M 4-DAMP能够明显地增加SBC3细胞凋亡。SBC3细胞表达αvβ1和α5β1整合素,10-4 M Ach刺激细胞粘附(P<0.01)的作用几乎被10-5M 4-DAMP、5μg/m L抗-β1抗体或抗-αv和α5抗体完全阻断(P<0.01),但Ach及4-DAMP不影响αv、α5和β1的表达水平。结论 SBC3细胞表达M3R,M3R拮抗剂能抑制细胞的增殖并促进凋亡。其抑制粘附的作用是通过抑制细胞含β1的整合素(αvβ1和α5β1)的功能实现的。

M3受体在慢性鼻-鼻窦炎合并鼻息肉患者鼻腔黏膜中的过度表达

目的:探讨M 3受体在人类鼻腔慢性炎症中的表达情况。方法:选择慢性鼻-鼻窦炎合并鼻息肉(CRS/NP)的患者30例作为研究对象。于鼻内镜手术过程中严格无菌操作,取鼻息肉组织及水肿黏膜用免疫组织化学和反转录PCR(RT-PCR)分别从蛋白质和基因水平确定M 3受体表达部位及密度。患者术后3个月换药时取恢复期黏膜,同法测定上述指标,最后进行统计学分析。结果:30例患者鼻腔或鼻窦黏膜的黏液腺和毛细血管上均有M 3受体亚型的过度表达,功能性鼻内镜手术后30例患者鼻腔黏膜M 3受体表达下降。结论:研究揭示在鼻腔慢性炎症过程中有M 3受体的过度表达,随着病情痊愈,M受体表达下调。

抗M3R抗体在干燥综合征发病机制及治疗中的研究进展

干燥综合征(SS)是一种主要累及外分泌腺的自身免疫性疾病。抗毒蕈碱型3型乙酰胆碱受体(M3R)抗体因潜在的致病作用,可作为SS的诊断标志物及潜在的治疗靶标。近年来,通过不同的检测手段显示抗M3R抗体在SS中具有较高的阳性率,不仅可造成SS的外分泌障碍,还可干扰自主神经、胃肠道、血液系统等功能并引起全身性表现。M3R激动剂改善SS口干症的疗效显著,针对M3R反应性T细胞靶点的治疗也是当前的研究热点,其可能靶向治疗唾液腺炎症,但需要进行更多的靶点研究,以深入挖掘抗M3R抗体在SS发病机制中的作用及治疗中的意义。

Maraviroc promotes recovery from traumatic brain injury in mice by suppression of neuroinflammation and activation of neurotoxic reactive astrocytes

Neuroinflammation and the NACHT,LRR,and PYD domains-containing protein 3 inflammasome play crucial roles in secondary tissue damage following an initial insult in patients with traumatic brain injury(TBI).Maraviroc,a C-C chemokine receptor type 5 antagonist,has been viewed as a new therapeutic strategy for many neuroinflammatory diseases.We studied the effect of maraviroc on TBI-induced neuroinflammation.A moderate-TBI mouse model was subjected to a controlled cortical impact device.Maraviroc or vehicle was injected intraperitoneally 1 hour after TBI and then once per day for 3 consecutive days.Western blot,immunohistochemistry,and TUNEL(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)analyses were performed to evaluate the molecular mechanisms of maraviroc at 3 days post-TBI.Our results suggest that maraviroc administration reduced NACHT,LRR,and PYD domains-containing protein 3 inflammasome activation,modulated microglial polarization from M1 to M2,decreased neutrophil and macrophage infiltration,and inhibited the release of inflammatory factors after TBI.Moreover,maraviroc treatment decreased the activation of neurotoxic reactive astrocytes,which,in turn,exacerbated neuronal cell death.Additionally,we confirmed the neuroprotective effect of maraviroc using the modified neurological severity score,rotarod test,Morris water maze test,and lesion volume measurements.In summary,our findings indicate that maraviroc might be a desirable pharmacotherapeutic strategy for TBI,and C-C chemokine receptor type 5 might be a promising pharmacotherapeutic target to improve recovery after TBI.