蛋白酶激活受体-1拮抗剂对兔心脏骤停后综合征中肾损伤的保护作用及机制

目的：探讨蛋白酶激活受体-1（PAR-1）拮抗剂对心脏骤停后综合征（PCAS）中肾损伤的影响及机制。方法：将日本大耳白兔随机分为假手术组（n ＝5）、PCAS 组（n＝10）、PAR-1拮抗剂组（n＝10）。采用窒息性心脏骤停法制备兔 PCAS 模型。PAR-1拮抗剂组于心肺复苏后10 min 给予静脉滴注 PAR-1拮抗剂 SCH79797（25μg／kg）,其余各组给予等量生理盐水静脉滴注。72 h 后取股静脉血检测血清肌酐和胱抑素 C 水平;取肾组织制备石蜡切片后行 HE 染色;采用 TUNEL 法测定肾脏细胞凋亡情况;应用Western blot 测定肾组织半胱天冬酶（casepase）-3、细胞外调节蛋白激酶（ERK）活化情况。结果：与假手术组相比,PCAS 组血清肌酐和胱抑素 C 水平明显上升,肾组织有大量炎性细胞浸润,凋亡细胞数显著增多,有催化活性的 caspase-3裂解片段（cleaved caspase-3）表达增高,磷酸化 ERK （p-ERK）表达减少（P ＜0．05）。与 PCAS 组相比, PAR-1拮抗剂组肾组织损伤减轻,血清肌酐和胱抑素 C 水平明显下降,凋亡细胞数减少, cleaved caspase-3表达降低,而 p-ERK 表达显著增高（P ＜0．05）。结论：PAR-1拮抗剂 SCH79797能抑制 PCAS 兔肾组织的炎症反应和细胞凋亡,可能与 ERK 信号通路激活有关。     

肥大细胞及蛋白酶激活受体-2在肠易激综合征中的作用

目的:探讨肥大细胞及蛋白酶激活受体-2(protease activated receptor-2,PAR-2)在肠易激综合征(irritable bowel syndrome,IBS)中的作用.方法:2012-01/2012-12自南京医科大学附属南京医院消化内科门诊随机选取腹泻型IBS患者60例及正常者30例,采用免疫组织化学检测各组肥大细胞、PAR-2蛋白的表达强度;采用Western blot检测各组Occludin的表达量.结果:免疫组织化学法结果显示IBS-D组与对照组相比,肥大细胞表达明显增多(5.20±2.78vs 1.40±0.55,P<0.05);PAR-2蛋白的表达明显增多(3.20±1.64 vs 1.20±0.45,P<0.05);Western blot结果示IBS-D组Occludin蛋白的表达明显低于正常组(2108.33±59.58 vs 3113.00±77.74,P<0.01).结论:本研究提示IBS-D患者肥大细胞过表达、激活后可刺激PAR-2活化,从而进一步损伤细胞间紧密连接.     

蛋白酶激活受体的研究进展

蛋白酶激活受体属于与G蛋白相偶联、有七个跨膜单位的受体家族,有四个成员:PAR-1,PAR-2,PAR-3和PAR-4.PAR-1是凝血酶受体,PAR-2是胰岛素、肥大细胞纤维蛋白溶酶、凝血因子Ⅶa和Ⅹa和其他未知蛋白水解酶的受体.PAR-2可诱导唾液腺和胰腺的分泌.PAR-1和PAR-2都对胃黏膜有保护作用,但机制不尽相同.在肠黏膜,PAR-2有前炎性和抗炎性双重作用,其和PAR-1,PAR-4均可调节胃肠道平滑肌.蛋白酶激活受体,尤其是PAR-1和PAR-2对调节胃肠道功能有非常重要的作用.     

蛋白酶激活受体1在癌旁组织中的高表达与肝癌早期患者术后预后不良相关

目的 研究蛋白酶激活受体1(PAR1)在肝细胞肝癌(HCC)及癌旁组织的表达水平,探讨其与HCC早期患者预后的相关性.方法 应用实时定量聚合酶链反应检测随机选择的41例经根治性切除的HCC早期患者癌组织及对应的癌旁组织中PAR1的mRNA表达水平,并分析其与肝细胞肝癌预后的相关性.另随机选择49例H C C早期患者组织石蜡切片做免疫组织化学染色,分析其与HCC临床病理特征及预后的相关性.计数资料比较采用卡方或Fisher精确概率法;计量资料比较用两组间比较的t检验或Wilcoxon符号秩检验;生存率的判定使用Kaplan-Meier方法,差异性使用Log-rank检验评估.结果 癌旁组织中,复发组的PAR1 mRNA表达水平明显高于非复发组(0.591±0.458比0.361±0.177,T=-2.379,P＜0.05).PAR1蛋白在癌旁组织的表达水平与肿瘤分化程度相关(P＜0.05).PAR1阳性组患者生存时间为(58.39±4.59)个月,1、3、5年累计生存率分别为90.3%、83.9%和61.8%; PAR1阴性组患者生存时间为(75.84±1.87)个月,1、3、5年累计生存率分别为100.0%、100.0%和94.1%.PAR1阳性组患者至复发时间为(51.97±4.30)个月,1、3、5年累计复发率分别为10.2%、25.2%和40.6%;阴性组患者至复发时间为(71.92±3.77)个月,1、3、5年累计复发率分别为0、5.6%和21.3%.癌旁组织PAR1蛋白阳性表达组1、3、5年总生存率明显低于阴性表达组(x2=5.297,P＜0.05),累计复发率明显高于阴性表达组(x2=4.682,P＜0.05).结论 PAR1在HCC早期患者癌旁组织中的表达与术后患者的预后密切相关,其可能参与了凝血酶介导的肝细胞肝癌的恶性侵袭行为,并可能成为判断预后的预测指标.

Abstract：

Objective To evaluate the relationship between PAR1 (Protease-Activated Receptor 1)expression and the clinicopathologic features and to investigate the prognostic value of PAR1 expression in hepatocellular carcinoma (HCC) in early stage after curative resection. Methods Real-time PCR was used to detect PAR1 expression in 41 pairs of tumors and matched peritumoral samples of HCC in early stage.Prognostic value of PAR1 mRNA expression was evaluated. Meanwhile, another 49 tissue paraffin slices of HCC were tested using immunohistochemistry (Envision) and the prognostic value of PAR1 expression and other clinicopathologic factors were evaluated. Results Peritumoral PAR1 mRNA expression was significantly increased in HCCs from the patients with tumor recurrence as compared with those without recurrence (P＜0.05). Peritumoral PAR1 protein expression was related to tumor differentiation (P＜0.05). KaplanMeier analysis showed that Peritumoral PAR1 protein expression was associated with the overall survival(OS) (P＜0.05) of HCC patients and the time to recurrence (TTR) (P＜0.05). The 1,3 and 5 -year overall survival time and the cumulative recurrence time in the high PAR1 protein expression group were significantly lower as compared to the low PAR1 expression group in the peritumoral liver tissue. Conclusion Peritumoral PAR1 expression is closely associated with the prognosis of early stage HCC patients after curable surgery. PAR1 may be involved in thrombin-mediated invasion process and may be used as a prognostic marker for HCC.     

肥大细胞及蛋白酶激活受体-2与肝纤维化

近年来研究表明肥大细胞与肝纤维化的发生发展有密切关系,但其具体作用途径不甚明确,目前推测肥大细胞可能通过蛋白酶激活受体-2途径在肝纤维化中发挥重要作用.现将肥大细胞、蛋白酶激活受体-2与肝纤维化的有关进展作一综述.     

蛋白酶激活受体-2激动剂对肝癌细胞增殖的影响

本研究通过体外培养人肝癌细胞株HepG2细胞,初步探讨了HepG2细胞表达蛋白酶激活受体(PAR)-2的水平,以及胰蛋白酶和人工合成的PAR-2激动剂SLIGKV对其增殖的影响.     

蛋白酶激活受体在腹泻型肠易激综合征患者结肠黏膜组织中的表达及意义

目的 研究蛋白酶激活受体(PAR)家族在腹泻型肠易激综合征(D-IBS)患者结肠黏膜中的表达变化情况.方法 入选28例D-IBS患者及18名健康对照者,经结肠镜钳取乙状结肠黏膜组织,采用Western印迹法和实时荧光定量PCR法观察蛋白酶激活受体超家族的表达情况.应用SPSS 18.0软件进行统计分析,两组间差异采用Mann-Whitney U检验.结果 PAR1在D-IBS患者和对照组结肠黏膜中的表达差异无统计学意义(P=0.300).PAR2和PAR4在D-IBS患者的结肠黏膜中均表达增加,其灰度值分别为0.99±0.67和0.37±0.14,显著高于对照组的0.63±0.38和0.25±0.11(P值分别=0.038和0.013).实时荧光定量PCR显示PAR mRNA的表达与蛋白水平相一致.结论 PAR2、PAR4的表达在D-IBS患者结肠黏膜中明显增高,可能在D-IBS疾病过程中发挥某些作用.

Abstract：

Objective To investigate the expression of protease-activated receptors (PARs) in colon mucosal tissue of patients with diarrhea-predominant irritable bowel syndrome (D-IBS). Methods Colon mucosa tissues were obtained from 28 D-IBS patients and 18 normal controls by colonoscopy.The expression of PARs was detected using Western blotting and real-time PCR. Data were analysed by SPSS 18. 0 softwave, and comparisons between groups were done using Mann-Whitney U test.Results There was no difference in expression of PAR1 between D-IBS patients and normal controls (P=0. 300). The expressions of PAR2 and PAR4 were higher in D-IBS patients than those in normal controls (0.99±0.67 vs 0.63±0.38, P=0.038 and 0.37±0. 14 vs 0.25±0. 11, P=0.013,respectively). The results of real-time PCR were in accordance with those of Western blotting.Conclusion The increased expressions of PAR2 and PAR4 in colon mucosa of D-IBS patients indicate that these two PAR receptors may be involved in the process of D-IBS.     

蛋白酶激活受体-2激动剂对肝癌细胞增殖的影响

本研究通过体外培养人肝癌细胞株HepG2细胞,初步探讨了HepG2细胞表达蛋白酶激活受体(PAR)-2的水平,以及胰蛋白酶和人工合成的PAR-2激动剂SLIGKV对其增殖的影响.     

蛋白酶激活受体-2与消化系统疾病的研究进展

蛋白酶激活受体-2(protease activated receptor-2,PAR-2)属于G蛋白耦联受体,具有蛋白酶受体家族较特异的分子结构与激活方式,广泛分布于胃肠道,可被多种蛋白酶激活,产生多种生物学效应,包括影响多种消化腺的分泌,具有胃黏膜保护作用,影响胃肠道运动,参与胰腺炎的发生、发展,与消化道恶性肿瘤密切相关.PAR-2与消化系统疾病的关系正成为当前研究的一个热点.     

大承气汤对急性坏死性胰腺炎大鼠胰腺水通道蛋白1的影响

目的 观察急性坏死性胰腺炎( ANP)大鼠胰腺组织水通道蛋白1(AQP1)的表达以及大承气汤对其的影响.方法 160只雄性SD大鼠按随机数字法分为对照组、ANP组、地塞米松组、乙酰唑胺组及大承气汤组,每组32只.采用胆胰管逆行注射5％牛磺胆酸钠方法制备ANP模型.地塞米松组于造模后即刻静脉给予地塞米松4 mg/kg体重;乙酰唑胺组于造模前2h用含乙酰唑胺的生理盐水1ml灌胃;大承气汤组于造模前48、24、2h分别用大承气汤2ml/次灌胃;对照组仅开腹触摸胰腺数次后关腹.制模后3、6、12、18 h分批处死8只大鼠.记录腹水量;检测血清淀粉酶;胰腺组织病理检查及电镜观察;伊文思兰(EB)血管外渗法检测毛细血管通透性;实时PCR和蛋白质印迹法检测AQP1 mRNA和蛋白表达.结果 ANP组血清淀粉酶水平显著升高,胰腺损伤明显;地塞米松组和大承气汤组淀粉酶水平较ANP组降低,胰腺损伤减轻;乙酰唑胺组淀粉酶水平高于ANP组,胰腺病理损伤较ANP组加重.造模后6h,对照组、ANP组、地塞米松组、乙酰唑胺组、大承气汤组胰腺组织EB含量分别为(13.44±2.56)、( 126.35±14.80)、(86.31±14.46)、(108.99±15.07)、(78.29±16.85) mg/L;AQP1 mRNA表达量为(170.07±22.48)％、(83.93±8.98)％、(117.09±10.70)％、(69.00±8.98)％、(112.82±11.79)％;AQP1蛋白表达量为0.23±0.06、0.10±0.02、0.32±0.03、0.13±0.02、0.45±0.04.ANP组的EB量显著高于对照组,而AQP1mRNA及蛋白的表达显著低于对照组(P值均＜0.05);地塞米松组及大承气汤组EB含量显著低于ANP组,而AQP1 mRNA及蛋白的表达显著高于ANP组(P值均＜0.05).结论 AQP1在ANP大鼠胰腺组织毛细血管渗漏的发生中起重要作用.大承气汤通过调控AQP1的表达可减轻ANP大鼠的胰腺损伤.     

Alterations in serotonin,transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction

AIM:To determine the molecular mechanisms of Shugan decoction(SGD) in the regulation of colonic motility and visceral hyperalgesia(VHL) in irritable bowel syndrome(IBS).METHODS:The chemical compounds contained in SGD were measured by high-performance liquid chromatography.A rat model of IBS was induced by chronic water avoidance stress(WAS).The number of fecal pellets was counted after WAS and the pain pressure threshold was measured by colorectal distension.Morphological changes in colonic mucosa were detected by hematoxylin-eosin staining.The contents of tumor necrosis factor(TNF)-αin colonic tissue and calcitonin-gene-related peptide(CGRP)in serum were measured by ELISA.The protein expression of serotonin[5-hydroxytryptamide(5-HT)],serotonin transporter(SERT),chromogranin A(Cg A)and CGRP incolon tissue was measured by immunohistochemistry.RESULTS:SGD inhibited colonic motility dysfunction and VHL in rats with IBS.Blockers of transient receptor potential(TRP)vanilloid 1(TRPV1)(Ruthenium Red)and TRP ankyrin-1(TRPA1)(HC-030031)and activator of protease-activated receptor(PAR)4 increased the pain pressure threshold,whereas activators of PAR2and TRPV4 decreased the pain pressure threshold in rats with IBS.The effect of SGD on pain pressure threshold in these rats was abolished by activators of TRPV1(capsaicin),TRPV4(RN1747),TRPA1(Polygodial)and PAR2(AC55541).In addition,CGRP levels in serum and colonic tissue were both increased in these rats.TNF-αlevel in colonic tissue was also significantly upregulated.However,the levels of 5-HT,SERT and Cg A in colonic tissue were decreased.All these pathological changes in rats with IBS were attenuated by SGD.CONCLUSION:SGD alleviated VHL and attenuated colon motility in IBS,partly by regulating TRPV1,TRPV4,TRPA1,PAR2,5-HT,Cg A and SERT,and reducing CGRP and TNF-αlevel.     

In Vivo and protease-activated receptor-1-mediated platelet activation in patients presenting for cardiac catheterization

Pathways of platelet activation that are not targeted by current antithrombotic therapy may be crucial for the development of ischemic events in patients undergoing coronary angiography. We therefore investigated whether in vivo and thrombin receptor activating peptide (TRAP)-stimulated platelet activation and monocyte-platelet aggregate (MPA) levels can serve as independent risk markers for adverse outcomes in aspirin-treated patients presenting for cardiac catheterization. In vivo and TRAP-stimulated platelet surface P-selectin, activated glycoprotein IIb/IIIa (GPIIb/IIIa) and MPA levels were determined in 682 consecutive patients undergoing cardiac catheterization and in 47 healthy controls. Two-year follow-up data were obtained from 562 patients. In vivo platelet surface P-selectin, activated GPIIb/IIIa and MPA levels were significantly higher in patients with angiographically-proven coronary artery disease than in healthy controls (all p≤0.02). Patients with an acute coronary syndrome (ACS; n=125) had significantly higher levels of in vivo MPA than patients without ACS (n=437; p=0.01). In the overall study population (n=562) the surface expression of P-selectin and activated GPIIb/IIIa, and the levels of MPA in vivo and in response to TRAP were similar in patients without and with subsequent ischemic events (all p>0.05). Similar results were obtained when only patients with angiographically-proven coronary artery disease (n=459), stent implantation (n=205) or ACS (n=125) were analyzed. Receiver-operating characteristic curve analyses did not reveal cut-off values for P-selectin, activated GPIIb/IIIa, and MPA levels for the prediction of ischemic events. In conclusion, in vivo and TRAP-stimulated platelet activation and MPA levels did not predict adverse ischemic outcomes in aspirin-treated patients presenting for cardiac catheterization.     

Unbalanced expression of protease-activated receptors-1 and -2 in the colon of diarrhea-predominant irritable bowel syndrome patients

Objective  The aim of this study is to determine whether a changed expression ratio of PAR-1 and PAR-2 in the colon is associated with diarrhea-predominant IBS patients. Methods  PAR-1, -2, thrombin, mast cell tryptase, tryptophan hydroxylase (TPH), and chromgranin A (ChrA) in colonic biopsy samples from 10 diarrhea-predominant IBS patients and 13 healthy control subjects were semiquantified with immunofluorescence and image analysis. Serotonin concentrations in biopsy samples were evaluated by capillary electrophoresis. Results  Significantly lower expression of PAR-1 and higher expression of mast cell tryptase was detected in the colons of patients, with statistically unchanged expression of PAR-2. Thrombin-, TPH-, and ChrA-positive cells were markedly increased in IBS patients, but no significant difference in serotonin concentration existed in the colons between two groups. The ratio of PAR-1/PAR-2 expression was significantly decreased in patients (0.33 ± 0.19 versus 0.66 ± 0.22, P = 0.001) and negatively correlated to ChrA-positive cells. Conclusions  Changed expression ratio of PAR-1 to PAR-2 in the colon is connected with diarrhea-predominant IBS patients. Methods to restore an appropriate balance of PAR-1 and PAR-2 activation in the colon may offer a promising future therapeutic strategy for IBS patients.     

半夏泻心汤合丹参饮治疗胃心综合征疗效评价

[目的]观察半夏泻心汤合丹参饮治疗胃心综合征,并评价其循证医学治疗性疗效.[方法]经预实验获得94例样本量,按患者就诊序号,随机分为A组和B组各47例.A组给予半夏泻心汤合丹参饮,B组给予奥美拉唑肠溶片、颠茄片、丹参片,2组观察周期均为15d,观察2组综合疗效,疗效积分,并评价其比值比(OR)、相对危险度(RR)、相对危险度降低率(RRR)、绝对危险度降低率(ARR)、需要治疗的病例数(NNT)及其95％的可信区间(CI).[结果]A组、B组的综合疗效分别为100.0％、82.9％,A组治疗后的症状、体征及心电图的积分较B组明显改善(P＜0.05及0.01);A组的OR=10.0,OR95％CI=3.4～15.6;RR=0.43,RR95％CI=0.09～0.72;RRR=57.0％,RRR95％CI=28.0％～91.0％;ARR=23.4％,ARR95％CI=5.2％～32.0％;NNT=4,NNT95％ CI=3～5(例).2组在观察期间均未出现不良事件,治疗后检查肝、肾功能均无异常.[结论]半夏泻心汤合丹参饮治疗胃心综合征疗效好;循证医学治疗性疗效评价其优于奥美拉唑肠溶片、颠茄片、丹参片联合治疗.     

呼吸机相关肺损伤兔细胞间黏附分子-1和白细胞介素-10的表达及激活蛋白-1活性的变化

目的 研究呼吸机相关肺损伤(VILI)兔细胞间黏附分子-1(ICAM-1)、白细胞介素(IL)-10的表达及激活蛋白-1(AP-1)DNA结合活性的变化,并探讨其在VILI炎症反应中的意义.方法 应用40 ml/kg的大潮气量(VT)机械通气建立兔VILI模型.将40只新西兰兔按随机数字表法分为非机械通气对照组、常规VT组、大VT 1h组、大VT 2 h组及大VT 4 h组.用酶联免疫吸附法(ELISA)检测肺组织匀浆可溶性细胞间黏附分子-1(sICAM-1)和IL-10含量,逆转录多聚酶链反应(RT-PCR)检测mRNA表达.凝胶电泳迁移率分析法(EMSA)测定AP-1的活性,同时测定PaO2、髓过氧化物酶(MPO)、肺湿重/干重比值(W/D).结果 (1)肺组织匀浆中sICAM-1含量:大VT2 h组[(23±5)ng/L]及大VT 4 h组[(35±5)ng/L]均高于常规VT组[(16±4)ng/L],均P＜0.05;大VT4 h组高于大VT 1 h组[(19±4)ng/L]及大VT 2 h组(均P＜0.01).肺组织匀浆IL-10含量:大VT2 h组[(24±4)ng/L]和大VT4 h组[(26±5)ng/L]均高于常规VT组[(15±2)ng/L,均P＜0.05],大VT4 h组高于大VT 1 h组[(19±4)ng/L,P＜0.05].ICAM-1 mRNA含量:大VT 2 h组(1.18±0.19)及大VT4 h组(1.29±0.19)均高于常规VT组(0.84±0.13,均P＜0.05).大VT4 h组高于大VT1 h组(0.96±0.24,P＜0.05).IL-10 mRNA含量:大VT 4 h组(1.13±0.17)高于常规VT组(0.84±0.20)和大VT 1 h组(0.86±0.12,均P＜0.05).(2)AP-1的DNA结合活性:大VT 2 h组(34±8)和大VT4 h组(38±9)均高于常规VT组(23±9,均P＜0.01),大VT4 h组高于大VT 1 h组(25±9,P＜0.01).(3)病理学检查显示大VT机械通气后随时间延长肺损伤逐渐加重,大VT机械通气2 h可见MPO升高,4 h可见PaO2下降及W/D升高.结论 ICAM-1、IL-10参与了VILI的炎症反应过程,二者升高可能与其mRNA的表达增高有关.核转录因子AP-1可能参与了上述炎性介质基因的转录调节.     

大黄附子汤加红景天治疗胆心综合征临床疗效评价

[目的]观察并评价大黄附子汤加红景天治疗胆心综合征临床疗效.[方法]采用随机数与就诊顺序相结合方法,将符合人选标准的88例患者随机分为观察组（A组）44例和对照组（B组）44例.观察组给予大黄附子汤加红景天;对照组给予舒胆通和参麦注射液,严重者给予氧氟沙星.观察2组综合疗效、疗效积分,并评价其比值比（OR）、相对危险度（RR）、相对危险度降低率（RRR）、绝对危险度降低率（ARR）、需要治疗的病例数（NNT）及其95％的可信区间（CI）.[结果]与B组比较,A组在总有效率（100.0％ v.s.94.4％,P＜0.01）,症状缓解时间、症状、体征及心电图改善明显,差异有统计学意义（P＜0.05或P＜0.01）,尤其胸前憋痛症状的改善更加明显（P＜0.01）;在复发率方面明显低于B组,差异具有统计学意义（30.67％ v.s.63.33％,P＜0.05）.大黄附子汤加红景天治疗胆心综合征的OR=2.78,OR95％CI=2.44～3.16;RR=0.59,RR95％CI=0.38～0.93;RRR=41.0％,RRR95％CI=7.0％～62.0％;ARR=27.0％,ARR95％CI=7.4％～47.0％;NNT=4,NNT95％ CI=2～14例.2组在观察期间均未出现不良事件,治疗后检查肝、肾功能均无异常.[结论]大黄附子汤加红景天治疗胆心综合征,疗效好,且稳定;无论近期,还是远期疗效均优于西药对照组.