Amitriptyline inhibits NLRP3 inflammasome activation via the ASM/CE pathway in a cell model of NAFLD

Background:Nonalcoholic fatty liver disease(NAFLD)is a global health concern with the acid sphingomyelinase(ASM)/ceramide(CE)pathway and the NOD-like receptor family,pyrin domain-containing protein 3(NLRP3)inflammasome identified as pivotal players in lipid disorders and inflammation.This study explores the interaction mechanism between the ASM/CE pathway and NLRP3 in NAFLD cell models,aiming to understand the impact of amitriptyline(Ami),an ASM inhibitor,on lipid deposition and hepatocyte injury by regulating the ASM/CE-NLRP3 pathway.Methods:HepG2 and HL-7702 cells were exposed to free fatty acids(FFAs)to establish the NAFLD model.The cells were divided into 5 groups:control group,model group,Ami group,tumor necrosis factoralpha(TNF-α)group,and Ami+TNF-αgroup.Intracellular lipid droplets were visualized using Oil Red O staining,and Western blot analysis quantified ASM,NLRP3,and caspase 1 protein expression.Enzyme linked immunosorbent assay(ELISA)was measured CE and ASM levels,while qRT-PCR assessed mRNA expression.The apoptotic rate was evaluated by flow cytometry(FCM).Results:Following FFAs incubation,significant increases in ASM and CE levels were observed in HepG2 and HL-7702 cells,accompanied by elevated expression of NLRP3,and caspase 1,and IL-1β.TNF-αtreatment further amplified these indicators.Ami demonstrated a reduction in lipid deposition,suppressed ASM/CE pathway activation,downregulated NLRP3 and caspase 1 expression,and improved apoptosis.Additionally,MCC950,a selective inhibitor of the NLRP3,mitigated NLRP3,caspase 1,and IL-1βexpression,alleviating lipid deposition and apoptosis in the NAFLD cell model.Conclusion:The ASM/CE-NLRP3 pathway in NAFLD cells promotes hepatocyte steatosis,inflammation,and cell damage.Ami emerges as a promising therapeutic agent by inhibiting the ASM/CE-NLRP3 pathway,underscoring its potential as a key target for NAFLD treatment.

鉴别猪圆环病毒2型和3型双重TaqMan MGB探针FQ-PCR检测方法研究

建立一种快速、特异鉴别检测猪圆环病毒2型(PCV2)和猪圆环病毒3型(PCV3)的双重TaqMan MGB探针FQ-PCR方法,本研究以PCV2的Rep蛋白和PCV3的Cap蛋白基因作为靶基因,各设计1对特异性引物和1条TaqMan MGB探针,经优化各反应条件和进行敏感性、特异性、重复性和干扰性试验,建立鉴别检测PCV2/PCV3的双重FQ-PCR方法。结果显示:该方法可特异性扩增PCV2、PCV3核酸,与猪伪狂犬病病毒(PRV)等8种病原及阴性对照无交叉反应,特异性较强;对PCV2和PCV3阳性质粒标准品的最低检出限均可达10 copies/μL,敏感性较高;PCV2/PCV3批内/批间重复试验变异系数(CV)值均在3%以下,表明方法稳定性、重复性较好;干扰性试验表明在两种病毒阳性质粒起始模板相差较大时该方法不会影响对其中任一病毒核酸的检出和准确定量。对42份临床疑似PCV感染样品检测结果与PCV2、PCV3基因测序结果符合率100%。本研究建立的双重FQ-PCR方法具有敏感性高达10 copies/μL、特异性强、在同一反应体系中能同时快速鉴别检测PCV2、PCV3等优点,可用于临床PCV2/PCV3感染的快速鉴别检测。

Effect of amitriptyline on gastrointestinal function and brain-gut peptides: A double-blind trial

AIM: To study the effects of low-dose amitriptyline (AMT) on gastrointestinal function and brain-gut peptides in healthy Chinese volunteers. METHODS: This was a double-blind, randomised, placebo-controlled, two-period cross-over trial. Twentyeight healthy volunteers were randomised and administered 1-wk treatments of AMT (12.5 mg tid) or placebo. Before and during the final two days of treatment, gastric emptying, proximal gastric accommodation and visceral sensitivity were measured by drinkingultrasonography test; the orocecal transit time (OCTT) was measured by lactulose hydrogen breath test, and fasting blood was collected. Plasma levels of ghrelin, motilin and neuropeptide Y (NPY) were measured by enzyme-linked immunosorbent assay kits.RESULTS: AMT slowed the OCTT (109.2 ± 29.68 min vs 96.61 ± 23.9 min, P = 0.004) but did not affect liquid gastric emptying and had no effect on proximal gastric accommodation. AMT resulted in decreases in the visual analogue scale (VAS) for difficulty in drinking 600 and 800 mL of water (3.57 ± 0.94 vs 2.98 ± 0.85, 5.57 ± 0.82 vs 4.57 ± 0.98, P < 0.01 for both), although it had no significant effect on the VAS for difficulty in drinking 200 mL and 400 mL of water. AMT significantly increased the plasma ghrelin level (442.87 ± 176.79 pg/mL vs 526.87 ± 158.44 pg/mL, P = 0.04) and the neuropeptide-Y level (890.15 ± 131.46 pg/mL vs 965.64 ± 165.63 pg/mL, P = 0.03), whereas it had no effect on the MTL level. CONCLUSION: Low-dose AMT could slow OCTT, make the stomach less sensitive and increase the plasma levels of ghrelin and NPY. Thus, we recommend the use of low-dose AMT for functional gastrointestinal disorders.

Effect of low-dose amitriptyline on globus pharyngeus and its side effects

AIM:To compare the efficacy and side effects of lowdose amitriptyline(AMT)with proton pump inhibitor treatment in patients with globus pharyngeus.METHODS:Thirty-four patients who fulfilled the RomeⅢcriteria for functional esophageal disorders were included in this study.Patients were randomly assigned to receive either 25 mg AMT before bedtime(AMT group)or 40 mg Pantoprazole once daily for 4 wk(conventional group).The main efficacy endpoint was assessed using the Glasgow Edinburgh Throat Scale(GETS).The secondary efficacy endpoints included the Medical Outcomes Study 36-item short form health survey[social functioning(SF)-36]and the Pittsburgh Sleep Quality Index.Treatment response was defined as a>50%reduction in GETS scores.All patients entering this study recorded side effects at days 1,8,15,22 and 29 using a visual analogue scale.RESULTS:Thirty patients completed the study.After 4 wk of treatment,the AMT group had a greater response than the conventional group(75%vs 35.7%,P=0.004).At day 3,the AMT group showed significantly more improvement than the Conventional group in GETS score(3.69±1.14 vs 5.64±1.28,P=0.000).After 4 wk of treatment,the AMT group showed significantly greater improvement in GETS score and sleep quality than the Conventional group(1.25±1.84 vs 3.79±2.33,4.19±2.07 vs 8.5±4.97;P<0.01 for both).Additionally,the AMT group was more likely than the Conventional group to experience improvement in the SF-36,including general health,vitality,social functioning and mental health(P=0.044,0.024,0.049 and 0.005).Dry mouth,sleepiness,dizziness and constipation were the most common side effects.CONCLUSION:Low-dose AMT is well tolerated and can significantly improve patient symptoms,sleep and quality of life.Thus,low-dose AMT may be an effective treatment for globus pharyngeus.

Sitagliptin,sitagliptin and metformin,or sitagliptin and amitriptyline attenuate streptozotocin-nicotinamide induced diabetic neuropathy in rats

Diabetic neuropathies are a family of nerve disorders caused by diabetes. Symptoms of the disease include nerve palsy, mononeuropathy, mononeuropathy multiplex, diabetic amyotrophy, painful polyneuropathy, autonomic neu- ropathy, and thoracoabdominal neuropathy. In this study, type 2 diabetes in rats was induced with nicotinamide- streptozotocin. Drug treatment was initiated on the d 15, with the combination regimen of metformin, pioglitazone and glimipiride or metformin and sitagliptin or sitagliptin, amitriptyline and sitagliptin and led to significantly im- proved glycemic control, increased grip strength and paw jumping response on d 21, 28 and 35 （P 〈 0.001）. Signif- icant increases in blood protein levels and decreases in urinary protein levels were observed in the animals treated with the different regimens on d 21, 28 and 35 （P 〈 0.001）. Combined treatment of streptozotocin and nicotinamide caused marked degeneration of nerve cells, while administration of metformin and sitagliptin showed tissue regen- eration and no body weight gain. In conclusion, treatment with sitagliptin and sitagliptin combined with metformin or amitriptyline results in no body weight gain, but causes an increase in grip strength and pain sensitivity, exhibits neural protection, and reverses the alteration of biochemical parameters in rats with streptozotocin-nicotinamide induced type 2 diabetes.

Low-dose amitriptyline combined with proton pump inhibitor for functional chest pain

AIM:To investigate the efficacy of amitriptyline with proton pump inhibitor(PPI)for the treatment of functional chest pain(FCP).METHODS:This was a randomized,open-label trial investigating the addition of low dose amitriptyline(10 mg at bedtime)to a conventional dose of rabeprazole(20 mg/d)(group A,n = 20)vs a double-dose of rabeprazole(20 mg twice daily)(group B,n = 20)for patients with FCP whose symptoms were refractory to PPI.The primary efficacy endpoints were assessed by global symptom score assessment and the total number of individuals with > 50% improvement in their symptom score.RESULTS:The between-group difference in global symptom scores was statistically significant during the last week of treatment(overall mean difference;3.75 ± 0.31 vs 4.35 ± 0.29,the between-group difference;P < 0.001).Furthermore,70.6% of patients in group A had their symptoms improve by > 50%,whereas only 26.3% of patients in group B had a similar treatment response(70.6% vs 26.3%,P = 0.008).Specifically,patients in group A had a significantly greater improvement in the domains of body pain and general health perception than did patients in group B(52.37 ± 17.00 vs 41.32 ± 12.34,P = 0.031 and 47.95 ± 18.58 vs 31.84 ± 16.84,P = 0.01,respectively).CONCLUSION:Adding amitriptyline to a PPI was more effective than a double-dose of PPI in patients with FCP refractory to a conventional dose of PPI.

Electrocardiograms changes in children with functional gastrointestinal disorders on low dose amitriptyline

AIM: To study the effects of low dose amitriptyline on cardiac conduction in children.

THE EFFECTS OF ELECTRO-ACUPUNCTURE VS. AMITRIPTYLINE ON PLATELET 3H-PAROXETINE BINDING SITES IN DEPRESSED PATIENTS

In this study platelet 3H-paroxetine binding site was studied in 16 depressed pa-tients and 16 healthy volunteers. We found that the mean Bmax of 3H-paroxetine binding on theplatelets of depressed patients was significantly lower than that of normal controls. After treated withamitriptyline or electro-acupuncture for 6 weeks, the density of paroxetine binding sites increased to-wards normal in well responded patients. But no significant difference was found between electro-acupuncture and amitriptyline as compared in their effects on 3H-paroxetine binding sites.

New Approach for the Determination of Tricyclic Antidepressant Amitriptyline Using <i>β</i>-Cyclodextrin-PEG System via Spectrophotomerty

A new and simple procedure for the spectrophotometric determination of the tricyclic antidepressant drug amitriptyline is proposed. The method is based on enhancement of sensitivity of the [AMIYTP]+ β-cyclodextrin and PEG molecules involved in formation of molecules inclusion complex, in presence of polyethylene glycol (PEG) medium. The mole ratio of [AMIYTP]+ β-cyclodextrin and PEG molecules in inclusion complex were determined by the curve fitting method. The value of molar absorptivity of {[AMIYTP: (β CD)] PEG} complex in term of the drug lies in rage of (2.20 - 2.23) × 104 L·mole–1·cm–1 at absorption maximum 242 nm. The slope, intercept and correlation co-efficient were found to be 14.21, 0.0046, and 0.998, respectively. The effect of analytical variables on the determination of the drug and composition of the ion associated complex are discussed in the paper. This method is applicable in the determina-tion of amitriptyline in the pharmaceutical preparations.

多糖佐剂FQ_2对日本血吸虫rBCG-Sj26GST疫苗增效作用及其机理的初步探讨

为探讨多糖佐剂FQ2 对血吸虫疫苗保护性免疫力的增效作用及其机理 ,采用 1 0 8CFU日本血吸虫重组BCG -Sj2 6GST疫苗分别与 5 %、1 0 %、2 0 %浓度的佐剂FQ2 混匀皮下接种BALB/c小鼠 ,同时设对照组。接种后第 8W以日本血吸虫尾蚴攻击感染 ,感染后 6W剖杀小鼠 ,计算减虫率 ,测定血清中特异抗体水平和胸腺T淋巴细胞及其亚群。结果实验组获得了 33 49% - 4 0 50 %的减虫率 ,与对照组相比 ,胸腺细胞总数、胸腺活性细胞率、CD8+ 细胞率、CD4 + 细胞率均显著升高 ,尤其是疫苗 +1 0 %佐剂 ,疫苗 +2 0 %佐剂组差别有非常显著意义 (P <0 0 1 )。而各实验组IgG抗体水平无统计学差别。提示多糖佐剂FQ2 对日本血吸虫rBCG -Sj2 6GST疫苗有一定的增效作用 。

FQ-PCR检测BALF中结核杆菌-DNA对肺结核诊断的应用研究

目的:对FQ-PCR检测BALF中结核杆菌-DNA对肺结核诊断的应用进行研究。方法:随机选取2013年12月-2014年11月期间本院收治的肺结核患者110例作为本次研究的对象,所有患者实施纤支镜检查、活检、刷检,BALF中结核杆菌-DNA行FQ-PCR检测,对检查结果进行分析研究。结果:在本次研究中,确诊为肺结核的患者有40.00%(44/110);FQ-PC检测的敏感性明显高于常规涂片镜检,差异有统计学意义(P<0.05)。结论:在肺结核的诊断中,FQ-PCR检测的敏感度高,操作简单方便,检测速度较快,是BALF中结核杆菌-DNA检查的重要方法,在肺结核的诊断中有重要的应用价值。

草鱼呼肠孤病毒HZ08株FQ-PCR检测方法的建立及应用

草鱼出血病是危害中国淡水养殖最为重要的病害之一,其病原为草鱼呼肠孤病毒(grass carp hemorrhagevirus,GCRV),其中,HZ08株是当前引起草鱼出血病的主要流行毒株。为建立一种快速、灵敏、特异的草鱼呼肠孤病毒主要流行株检测方法,本研究利用草鱼呼肠孤病毒HZ08株S7基因保守区,设计了一对能特异性扩增154 bp片段的引物和TaqMan探针,用含有S7基因全长的重组质粒PAVX1-S7作为标准品,构建质粒拷贝数与CT值的标准曲线,并对该方法的特异性、可重复性、敏感度进行评价。结果显示:标准曲线在6.0×1010～6.0拷贝数之间有很好的线性关系(r=0.999);实时荧光定量PCR最少可检测到6个阳性质粒,有较高的敏感性;试验内及试验间变异系数分别为0.82%与0.41%～0.52%,重复性强;对水生动物其他病毒均无扩增反应,具有很好的特异性。应用该方法对采集的32份草鱼出血病样品进行检测,其中28份为阳性,而以常规RT-PCR检测同样的样品,仅23份为阳性。本研究建立的草鱼呼肠孤病毒流行株实时荧光定量PCR检测方法在特异性、灵敏度、重复性方面具有较好的测试结果,在GCRV的快速检测和病毒初步定量中应用前景乐观。

FQ-PCR方法检测女性CA患者HPV_(6、11)和HPV_(16、18)的实验研究

目的:探讨女性尖锐湿疣(CA)患者HPV6、11型和HPV16、18型的感染情况。方法:采用荧光定量聚合酶链反应(FQ-PCR)方法检测216例女性CA患者疣体组织或分泌物中的HPV6、11型和HPV16、18型。结果:HPV6、11型阳性率为94.91%(205/216),HPV16、18型阳性率为16.20%(35/216),HPV6、11型和HPV16、18型同时阳性的感染率为12.50%(27/216)。定量测定结果显示,HPV6、11型患者病毒载量最高1.0×108 copies/ml,最低2.8×103copies/ml;HPV16、18型患者病毒载量最高1.0×108 copies/ml,最低2.57×104 copies/ml。结论:FQ-PCR技术具有灵敏、简捷、安全、特异性强的特点,避免了单纯PCR扩增产生的假阳性。女性尖锐湿疣患者应用此方法检测HPV阳性率高,可以快速区分高危型及低危型的HPV感染,有助于对尖锐湿疣的复发和癌变作出可能性预测。

中、英大学生15FQ^+英文版人格问卷测量比较

目的: 探讨中、英大学生人格特征的差异性.方法:采用英国Psytech国际有限公司修订的 15FQ+英文版人格测量问卷,对 159名中国大陆大学生和 157名英国大学生进行测量,将二者结果进行比较.结果: 不同英语水平的中国大学生(CET4、CET6),在 15FQ+的 16项因子中除fI因子外,均无显著性差异,中、英大学生在 15FQ+的 12项因子上存在显著差异: 英国学生显示出高外向性、低焦虑性、思维开放、独立性强等特点,而中国学生则显示出高内向性、高紧张度、思维保守、自制力强等特点.结论: 15FQ+英文版具有很强的通用性,可以在中国进一步推广使用.

番茄内生放线菌Fq24发酵条件的优化

【目的】确定番茄内生放线菌Fq24的最佳发酵液培养基配方和最佳发酵条件。【方法】通过测定Fq24菌株发酵液对灰葡萄孢的抑菌作用,研究了Fq24菌株在不同发酵培养基、培养时间、接种量、装液量、起始pH、培养温度、碳源、氮源条件下的生长情况及其代谢物活性,以确定其最佳发酵条件。【结果】Fq24发酵液培养基的最适配方为:葡萄糖20 g,黄豆饼粉20 g,玉米面30 g,淀粉10 g,牛肉膏1 g,KNO32 g,NaCl 2 g,MgSO40.3 g,CaCO36g,KH2PO40.2 g,H2O 1 000 mL;筛选出的最佳发酵条件为:培养时间7 d,接种量10%,500 mL容量瓶中装液量100mL,起始pH值6.5,培养温度28℃,碳、氮源分别为葡萄糖和KNO3。【结论】在最佳发酵液培养基和培养条件下,Fq24菌株发酵液对灰葡萄孢的抑菌率可达到92.1%。

应用FQ-PCR方法快速产前诊断Down综合征

目的为探讨FQ-PCR方法快速、准确的应用于产前诊断Down综合征。方法分别提取46例外周血(包括16例确诊为Down综合征患儿和30例正常对照)和40例唐氏筛查高风险羊水细胞基因组DNA,FQ-PCR方法扩增基因D21S11、S100β(位于21号染色体)及基因UFD1L(位于22号染色体)。结果正常人羊水基因定量R1(D21S11/UFD1L)=1.095±0.213,R2(S100β/UFD1L)=1.087±0.146,Down综合征R1、R2分别为1.597±0.156、1.604±0.235,经t检验两组差异都有统计学意义。结论该方法无须细胞培养、操作简单,是一种快速准确产前诊断Down综合征的良好方法。

氮素胁迫下强、弱化感水稻萜类代谢途径中关键酶基因差异表达的FQ-PCR分析

运用实时荧光定量PCR技术研究低氮条件下化感与非化感水稻异戊二烯代谢途径中关键酶基因的表达差异。结果表明,与正常氮条件下相比,低氮条件下化感和非化感水稻与萜类物质合成相关的12个关键酶基因表达发生了不同程度的变化,其中,化感水稻PI312777有6个基因表达下调,6个基因则上调;而非化感水稻Lemont有7个基因表达下调,5个基因表达上调。在供氮条件从高向低改变的过程中,两水稻中有11个基因的表达行为(上调或下调)相同,从分子水平上,证实了它们萜类代谢相关基因在响应供氮环境变化的行为生态上是相同或相似的。低氮引起两水稻催化生成与化感物质相关的单萜、倍半萜、二萜、三萜类物质的合成酶基因表达量均显著下降,从而认为营养胁迫引起水稻化感抑草作用增强与萜类物质代谢变化无关。还讨论了营养胁迫引起萜类物质代谢变化与内源激素合成和生长速率减缓的关系,认为水稻甲羟戊酸代谢途径支路中3-羟基-3-甲基戊二单酰CoA合酶、3-羟基-3-甲基戊二单酰CoA还原酶和甲羟戊酸激酶相关基因表达量不同程度的上调,维持了生长所必需的激素水平。

应用荧光定量聚合酶链反应(FQ-PCR)检测孕妇巨细胞病毒感染

目的探讨荧光定量聚合酶链反应对孕妇巨细胞病毒（CMV）感染的临床诊断价值。方法用FQ—PCR检测1016例孕妇尿的CMV—DNA，同时用ELISA检测血中CMV IgG和CMV IgM。对20例感染孕妇的胎儿进行了产前诊断。结果FQ—PCR检测出36例CMV—DNA阳性，阳性率为3．5％（36／1016）；阳性样本CMV—DNA在3．9×10^5～6．8×10^8拷贝／ml之间，平均为4．23×10^5拷贝／ml；ELISA检测CMV—IgG阳性率72．83％（740／1016）；CMV—IgM阳性率3．15％（32／1016），CMV—IgG阴性率24．02％（244／1016）。20例产前诊断者有4例发生了宫内传播。结论FQ—PCR对CMV感染的诊断有重要的作用。

草鱼呼肠孤病毒JX-0901株FQ-PCR检测方法的建立及其在定量分析中的应用

根据GCRV JX-0901株S7基因保守区设计特异性引物和TaqMan探针,建立了针对GCRV JX-0901株病毒的TaqMan实时荧光定量PCR(FQ-PCR)检测方法,并利用FQ-PCR方法对该毒株在不同鱼类细胞中的增殖情况和不同温度条件下的稳定性进行了定量分析。结果显示,建立的FQ-PCR方法的Ct值与标准品在1.0×101～1.0×108拷贝/μL浓度范围呈良好的线性关系,R2高达0.999;该方法有较好的敏感性和特异性,最低模板检出量为10个拷贝,只能特异地检测出GCRV JX-0901,不能检出其他鱼类常见病毒。该毒株能在10种常见鱼类细胞中增殖,其中在L8824细胞中的增殖量最大,含量达到4.1933×107拷贝/μL;此外,该毒株对温度敏感性较弱,病毒在48、56℃条件下放置96 h后才失去对CIK细胞感染性,而在4、15、28、37℃条件下放置32d,-20℃条件下储存2年仍具有较高的含量和较强的感染性。

肠炎沙门氏菌种特异性FQ-PCR快速检测方法的建立

根据肠炎沙门氏菌（SE）种特异性DNA序列，设计合成了一对能特异性扩增130bp片段的引物和TaqMan探针，首次建立了SE的种特异性荧光定量聚合酶链式反应（FQ-PCR）诊断方法。该法在SEDNA含量为9×10^8-9X104拷贝有较好的线性关系；检测SEDNA模板的灵敏度为4拷贝／μL，检测SE细菌数的灵敏度为6CFU／mL；特异性试验表明只对SE基因组呈阳性反应。分别应用该技术和传统细菌分离法检测60份来自人工感染鸭、小白鼠和肉兔的心、肝、脑和直肠粪便，结果显示两种方法对心、肝、直肠粪便的阳性检测结果符合率为100％，而FQ-PCR对脑的阳性检测率极显著（P〈0．01）高于细菌分离。研究结果表明，该方法快速、灵敏、特异、重复性好且能实时定量检测，可用于SE分离鉴定、SE感染疑似病例检测、SE体内动态分布规律研究及其分子流行病学调查。