脓毒症大鼠肺基质金属蛋白酶-2/9表达与血必净干预

Objective To detect the expression of MMP-2/9 and TIMP-1/2 in the lung of Vibrio vulnificus sepsis rats and observe the intervention of Xuebijing injection. Method One hundred and ten SD rats of clean grade were randomly(random number) divided into normal control group (group A, n = 10),Vibrio vulnificus sepsis group (group B, n = 50. Sepsis was reproduced in rats with subcutaneous injection in left lower limb with Vibrio vulnificus) and Xuebijin intervention group ( group C, n = 50. Rats were intraperitoneal(ip) with the dose of Xuebijing 4mL/kg at the time of 30 min later after infection). The rats in group B and C were sacrificed at 1 h, 6 h, 12 h, 24 h, 48 h after infection, the expression of MMP-2/9 and TIMP-1/2 were examined by PCR, Immunohistochemistry or ELISA methods, the lung permeability were measured by Coomassie Brilliant Blue method. Experimental data used single factor analysis of variance, and between groups by LSD method for pairwise comparison,P ＜0.05 statistically significant difference. Results The lung permeability increased both in group B and C compared with group A,and in group B were relatively higher. The lung MMP-2/9, TIMP-1/2mRNA expression in groups B and C compared with in group A was markedly higher, and reached the peak at 6 h(0. 344 ± 0. 108 ),6 h ( 1. 230 ± 0.377 ), 12 h (0.523 ±0.098),12 h(0.280±0.070) (P＜0.05) in group B while at 12 h(0.256 ±0.074),6 h(0.968±0.225) ,12 h(0.746 ±0. 316) ,12 h(0.356 ±0.035) (P ＜0. 05) in group C; the MMP-2/9mRNA expression in group C decreased(P＜0. 05) compared with the group B while the TIMP-1/2mRNA expression increased(P＜0. 05). The lung MMP-2/9, TIMP-1/2 protein expression in groups B and C compared with the group A(0.345±0.109) also increased, and the peak was at 12 h (0. 692 ± 0. 191 ), 12 h (0. 061 ±0.017) ,24 h(1384.42 ±91) ,24 h(41.04 ±3.60)in group B while at 24 h(0. 217 ±0.065) ,12 h(0. 045± 0. 013 ) ,24 h ( 1617.22 ± 103 ) ,24 h (47.66 ± 3.58 )in group C, the MMP-2/9 protein expression in group C was lower than in group B(P＜0.05), the TIMP-1/2 protein expression in group C was similar to in group B early while marked increased(P＜0.05)later. Conclusions MMP/TIMP imbalance was one of the mechanisms of the lung injury in the rats with Vibrio vulnificus sepsis, Xuebijing could restore the balance of MMP/TIMP ratio.     

高迁移率族蛋白B1在失血性休克致急性肺损伤中的作用

目的 探讨高迁移率族蛋白 B1(high mobility group box1,HNGB1)在失血性休克(hemarrhag-ic shock,HS)所致急性肺损伤(acute lung injury,ALI)小鼠体内的变化和意义.方法健康雄性BALB/c小鼠42只,随机分为对照组、HS致ALI组和抗HMGB1抗体干预组.心脏穿刺抽血复制小鼠HS致ALI模型,酶联免疫吸附(ELISA)法检测肺组织IL-1β,TNF-α,Westemblot检测肺组织HMGB1,比色法检测肺组织髓过氧化物酶(MPO)、伊文蓝(EBD),观察肺脏病理变化.统计学采用方差分析及LSD-t检验.结果 HS后2 h即可见肺血管通透性增高,肺脏出现弥漫件炎细胞侵润.肺绀织IL-1β和TNF-α在HS诱导的ALI早期(4 h)升高[IL-1β(333.83±31.18)vs.(284.83±30.49),P=0.014;TNF-α(805.00±114.67)vs.(584.17±181.17),P=0.023];肺组织HMCB1在ALI 16～48 h显著升高[HMGB1/β-actin(0.99±0.16)vs.(0.50±0.18),P=0.003].ALI组小鼠肺MPO及EBD水平显著增高,分别于ALI 4 h和24 h达峰值[MPO(38.33±3.88)vs.(8.00±0.89),P<0.01;EBD(20.05±2.79)vs.(4.63±0.43),P<0.01];抗HMGB1抗体干预组肺组织MPO及EBD峰值显著下降[MPO(25.67±1.63)vs.(38.33±3.88),P<0.01;EBD(5.68±0.53)vs.(20.05±2.79),P<0.01],抗体干预组小鼠肺组织病理切片见肺部弥漫性炎细胞浸润减轻,抗体干预24 h组尤为明显.结论 HNGB1是HS所致ALI的晚期炎症介质,HNGBl拮抗治疗能减轻小鼠HS所致ALI病理牛理改变.     

超声双重造影评价胃癌淋巴结转移的应用价值

Objective To investigate the clinical value of double contrast-enhanced ultrasonography (DCUS) in diagnosing lymph nodes metastasis of gastric carcinoma.Methods One hundred and sixteen patients with gastric cancer diagnosed by gastroscope and confirmed by pathology after operation were examined by DCUS preoperatively.The enhanced characteristic of gastric carcinoma tissues was assessed by autotracking contrast quantification(ACQ) software.The baseline intensity(BI), peak intensity(PI), arrival time(AT) and time to peak(TTP) of gastric cancer was measured automatically,and the enhanced intensity (EI) and wash-in time(WIT) of gastric cancer was calculated manually (EI=PI-BI; WIT=TTP-AT).All of the subjects were divided into two groups according to their lymph nodes status postoperatively:group N1,sixty-nine patients with lymph nodes metastasis; and group N0, forty-seven patients without lymph nodes metastasis.The DCUS quantitative analysis and pathological results of these two groups were compared each other.The Kappa's test was used for inter-rater reliability.Results BI of group N1 in the gastric carcinoma tissues was lower than that of group N0 significantly [(1.41 ± 1.56)dB vs (3.92 ± 2.82)dB, t = - 4.81, P = 0.000].EI of group N1 in the gastric carcinoma tissues was higher than that of group N0 significantly [(20.67±3.71)dB vs (14.12±3.75)dB, t=7.31, P=0.000].Moreover, there was a significant difference of WIT in the gastric carcinoma tissues between these two groups[(9.12±2.99)s vs (10.88±3.05)s, t =-2.43, P=0.018].The WIT in patients with lymph nodes metastasis was shorter than that without it. A cut-off value ＞17.05 dB of EI in gastric cancer tissues for assessing the lymph nodes metastasis had a sensitivity of 80.50% and specificity of 76.70% respectively obtained by the area under the ROC curve. The Kappa value of this method was 0.88.Conclusions EI of gastric cancer tissues can be considered as a new potential index to evaluate the lymph nodes metastasis of gastric cancer.     

慢性血吸虫感染对脓毒症小鼠保护作用的研究

Objective To preliminarily study the protective effect of chronic schistosoma japonica (SJ)infestation against sepsis in mice and its mechanism. Methods BALB/c male mice were used, and the experiment was divided into three parts. Experiment 1: chronic SJ infestation model was reproduced by SJ cercaria inoculation through abdominal skin for 8 weeks. Twenty mice were randomly grouped into normal group (n=10) and SJ group (n=10). The levels of interleukins (IL-4, IL-10), tumor necrosis factor-α(TNF-α) and interferon-γ (IFN-γ) in serum were detected by enzyme linked immunosorbent assay (ELISA).Real-time polymerase chain reaction (PCR) was employed to detect the levels of IL-10 mRNA and TNF-αmRNA in abdominal macrophages. This experiment was meant to evaluate immune state in mice with chronic SJ infestation. Experiment 2: lipopolysaccharide (LPS) was intraperitoneally injected to reproduce sepsis model. Thirty mice were randomly grouped into LPS group (n=15) and SJ-LPS group (n=15). The levels of cytokines were determined by ELISA at 0, 24, 48 and 72 hours after LPS injection. This experiment was meant to detect the effect of chronic SJ infestation in mice during the septic process. Experiment 3 : two types of sepsis model were reproduced by cecal ligation and puncture (CLP) and LPS injection, respectively. The survival rate of mice with chronic SJ infestation in 72 hours in either type of sepsis was evaluated. Results Experiment 1, compared with normal group [IL-4 (56.32±8.66) ng/L, IL-10 (48.17±7.23) ng/L],chronic SJ infestation showed an increase in serum IL-4 [(151. 35 ± 12. 24) ng/L] and IL-10 [(133. 22 ±11. 09) ng/L, both P＜0. 05]. Chronic SJ infestation also resulted in an increase in IL-10 mRNA expression (SJ group 4. 46±1. 82, normal group 1. 52±0. 60) and inhibited TNF-α mRNA expression (SJ group 1. 61±0.93, normal group 2. 32±1.03) in abdominal macrophages (both P＜0. 05), indicating that macrophages could be differentiated into alternative activated macrophages. Experiments 2 and 3 showed that the levels of serum IL-4 and IL-10 were increased at 0 hour after LPS injection, and then gradually decreased in SJ-LPS group, but the levels were still higher than those in LPS group at 72 hours [IL-4 (ng/L): 92. 2±7. 6 vs.41.5±4. 5; IL-10 (ng/L): 92. 1±7. 8 vs. 35. 6±4. 0, both P＜0. 05]; the levels of TNF-α and IFN-γ were increased at 24 hours, and then decreased in SJ-LPS group, and the levels were lower than those in LPSgroup at 72 hours [TNF-α (ng/L): 82. 9±5. 6 vs. 91. 5±5. 2; IFN-γ (ng/L): 44.1±4. 8 vs. 52. 6±4. 0,both P＜0. 05]. Therefore, chronic SJ infestation could improve the survival rate of mice with sepsis induced by CLP or LPS (CLP: 80% vs. 20%, LPS: 70% vs. 30%, both P＜0.05). Conclusion Chronic SJ infestation could elevate anti-inflammatory factors in septic mice, thus ameliorating the survival rate, so it has protective effect on mice with sepsis.     

脓毒症大鼠肺高迁移率族蛋白B1的表达及意义

目的 观察创伤弧菌脓毒症大鼠肺组织HMGB1表达和肺损伤的动态变化,并探讨HMGB1在创伤弧菌脓毒症肺损伤中作用.方法 温州医学院生命科学院实验室,清洁级SD大鼠60只,随机分为正常组(A组,n=10)和创伤弧菌脓毒症组(B组,n=50),采用大鼠左下肢皮下注射创伤弧菌悬液(浓度为6×108cfu/mL,剂量为0.1 mL/100 g)制作大鼠创伤弧菌脓毒症模型),B组于染菌后1、6、12、24、48 h后活杀(各时间点n=10),采用逆转录聚合酶链式反应(RT-PCR)和蛋白免疫印迹(Western blot)分别检测大鼠肺组织HMGB1基因与蛋白的表达,检测肺含水分数和光镜观察肺组织病理变化,数据采用单因素方差分析,并用LSD法进行组间两两比较,P＜0.05为差异有统计学意义.结果 B组染菌后12 h(1.161±0.358,P=0.013)、24 h(1.679±0.235,P=0.000)及48 h(1.258±0.274,P=0.004)大鼠肺组织HMGB1 mRNA表达量较A组(0.652±0.177)明显增高(P＜0.05),并于24 h达到高峰;与A组(0.594±0.190)比较,B组HMGB1蛋白表达量于感染后6 h(1.408±0.567,P=0.026)(P＜0.05)逐渐增加,24 h达到高峰(2.415±1.064,P=0.000);与A组(0.699±0.054)比较,B组大鼠肺含水分数于感染后6 h(0.759±0.030,P=0.001)、12 h(0.767±0.023,P=0.000)、24 h(0.771±0.043,P=0.000)和48 h(0.789±0.137,P=0.000)明显增大(P＜0.05),呈逐渐递增趋势;感染后12 h,大鼠肺内血管明显充血,间质水肿并伴炎性浸润,且逐渐加重,到48 h肺泡腔塌陷明显,肺泡间隔分界不清.结论 大鼠创伤弧菌脓毒症可导致肺脏损伤,HMGB1的表达增加可能是创伤弧菌脓毒症大鼠肺组织损伤的机制之一.

Abstract：

Objective To observe the dynamic changes of high mobility group protein B1 ( HMGB1 )expression in the lung of rats with Vibrio vulnificus sepsis so as to unravel the role of HMGB1 in lung injury.Methods Sixty rats of clean grade were randomly divided into normal control group ( A group, n = 10) and Vibrio vulnificus sepsis group (B group, n =50). Sepsis model was made in rats with subcutaneous injection of Vibrio vulnificus with concentration of 6 × 108 cfu/ml in dose of 0. 1 ml/100 g into left lower limb.The rats of group B were sacrificed 1 h, 6 h, 12 h, 24 h and 48 h after infection for taking lung tissues to detect the water content of lung and to observe the histopathological changes in lung under light microscope.The expression of HMGB1 mRNA and the level of HMGB1 protein in the lungs were detected by RT-PCR and Western blot, respectively. Data were analysed with ANOVA and LSD method for comparison between groups, and P ＜0.05 was considered statistically significant. Results Compared with the group A (0.652±0. 177), the expressions of HMGB1 mRNA in lung of rats of group B were significantly higher in 12 hours (1. 161 ±0.358, P=0.013), 24 hours (1.679 ±0.235, P =0.000) and 48 hours (1.258 ±0.274, P=0.004) and reached the peak in 24 h. Compared with group A (0.594 ±0. 190), the level of HMGB1 protein in rats of group B 6 h after infection ( 1. 408 ± 0. 567, P = 0. 026) was significantly increased (P＜0.05), and it reached peak in 24 h (2.415 ± 1.064, P =0.000) after infection. Compared with group A (0.699 ± 0.054), the lung water contents in rats of group B were significantly increased in 6 h (0.759±0.030, P=0.001), in 12 h (0.767 ±0.023, P =0.000), in 24 h (0.771 ±0.043, P=0.000) and in 48 h (0.789 ±0.137, P=0.000) after infection. Compared with group A, the pathological changes in the lung of rats in group B showed clearly marked pulmonary vascular congestion, interstitial edema and inflammatory cell infiltration, and those changes became more and more serious until alveolar sacs entirely collapsed and the boundaries of the alveolar septa could not be clearly identified in 48 h. Conclusions Vibrio vulnificus sepsis leads to the lung injury of infected rats, and the increase in the expression of HMGB1 mRNA in lung might be one of the mechanisms of lung injury in rats with Vibrio vulnificus sepsis.     

急性前壁心肌梗死伴心力衰竭患者急诊经皮冠状动脉介入治疗围手术期应用重组人B型钠尿肽的心肾保护效应

Objective To evaluate the protective effect of recombinant human B-type natriuretic peptide (rhBNP) on cardiac and renal functions in heart failure (HF) patients as a result of acute anterior myocardial infarction (AAMI) in peri-operative period of primary percutaneous coronary intervention (pPCI).Methods One hundred and twenty-six patients with AAMI-HF were enrolled into this study.All patients undertaken pPCI were randomly assigned to the rhBNP group (n=62) or the control group(n=64).rhBNP or nitroglycerin was intravenously administered on the basis of conventional treatment from first day of admission to 24 hours after pPCI in both groups.Heart rate (HR), systolic blood pressure (SBP), B-type natriuretic peptide (BNP), estimated lomerular filtration rate (eGFR) and heart function were observed.All patients were followed up for 30 days for the observation of main adverse cardiac events (MACE).Results The HR was significantly decreased compared with that at admission in rhBNP group, but such condition was not found in the control group.The SBP was reduced obviously in both groups.The plasma level of BNP, left ventricular ejection fraction (LVEF) and left ventricular end-diastolic dimension (LVEDD) were improved significantly at different time points compared with those before administration in both groups.The improvement of above parameters in rhBNP group was more significant than that in the control group[BNP (ng/L) 30 hours after pPCI: 303.5±128.4 vs.354.0± 133.6, 14 days after pPCI:157.8±78.6 vs.201.1±91.7; LVEF 1dayafter pPCI: 0.420±0.052 vs.0.378±0.055, 14 days after pPCI:0.444±0.050 vs.0.393±0.055, 30 days after pPCI: 0.469±0.053 vs.0.413±0.052; LVEDD (mm) 1 day after pPCI: 53.5±4.4 vs.57.6±4.4, 14 days after pPCI: 49.6±5.1 vs.53.4±4.6, 30 days after pPCI: 46.5±4.4 vs.50.2±4.8, P＜0.05 or P＜0.01].The eGFR was reduced obviously 1 day after pPCI than that at admission in both groups, and eGFR recovered to baseline 3 days after pPCI.The level of eGFR was significantly increased 7 days and 14 days after pPCI than that at admission, but there was no difference between rhBNP group and control group.The incidence of contrast-induced nephropathy showed a lowering tendency in the rhBNP group than that in the control group[19.4% (12/62) vs.29.7% (19/64),P=0.178].The incidence of ventricular arrhythmias was obviously lowered 7 days after pPCI in the rhBNP group than that in the control group[48.4% (30/62) vs.75.0% (48/64), P＜0.01].The rate of MACE was lower in rhBNP group than that in control group in 30 days[12.9% (8/62) vs.26.6% (17/64), P＜0.05].Conclusion Administration of rhBNP can effectively improve the heart function in AAMI-HF patients undergoing pPCI, and it lowered the incidence of MACE in 30 days, without influence on renal function, and it can reduce the incidence of contrast-induced nephropathy.     

Effect of ulinastatin on expressions of serum cardiac troponin Ⅰ, myocardial tumor necrosis factor-α and endothelin-1 in septic rats

Objective To investigate the myocardial protective effects of different dosage of ulinastatin (UTI) and the possible mechanism in septic rats.Methods Forty male Sprague-Dawley (SD) rats were randomly divided into five groups: control group, sham group, model group, UTI in low dose or high dose group.Cecal ligation and puncture (CLP) was adopted to reproduce animal model of sepsis.Left ventricular myocardium was harvested and blood samples were collected at 24 hours after successful establishment of animal model.Serum cardiac troponin Ⅰ (cTnI), the contents of myocardial tumor necrosis factor-α (TNF-α)and endothelin-1 (ET-1) were measured, and myocardial pathological changes were observed.Results In the model group, the level of serum cTnI, and the expressions of myocardial TNF-α and ET-1 were much higher than those in control group [cTnI (μg/L): 7.58 ± 0.53 vs.1.05 ± 0.21, TNF-α (pg/g): 945.6 ±72.0 vs.238.2±35.2, ET-1 (pg/g): 776.8±123.9 vs.170.1±28.3, all P＜0.01].There were no differences in the levels of serum cTnI, myocardial TNF-α and ET-1 between low dose UTI group and the model group [cTnI (μg/L): 7.21± 0.51 vs.7.58 ± 0.53, TNF-α(pg/g):910.5 ± 96.6 vs.945.6 ± 72.0,ET-1 (pg/g): 714.0±66.7 vs.776.8±123.9, all P＞0.05].However, serum cTnI, myocardial TNF-α and ET-1 were lower significantly in high dose UTI group than in model group [cTnI (μg/L): 4.30±0.84 vs.7.58±0.53, TNF-α(pg/g): 430.5±75.6 vs.945.6±72.0, ET-1 (pg/g): 377.1±39.0 vs.776.8±123.9, all P＜0.01].Conclusion High dose (but not low dose) UTI may protect myocardium from the damage resulted from sepsis in a rat model, probably by lowering expressions of TNF-α and ET-1.     

C5aR和P38-MAPK在脓毒性休克大鼠心肌损伤中的作用

Objective To investigate effects of complement C5a receptor and P38-MAPK on myocardial injury brought about by septic shock in rats. Method The early septic shock models were established by the method of cecal ligature and incision (CLI). A total of 30 Sprague-Dawley rats were randomly( random number) divided into normal control group ( n = 6 ) and model group ( n = 24 ) and the model group was further 12 hours later divided into 12 h subgroup (n = 12) and 24 h subgroup (n = 12). The arterial blood samples were collected 12 hours later for detecting the levels of lactate dehydrogenase (LDH) and creatine kinase (CK), and then the rats were sacrificed and the myocardial tissues were taken to assay the expressions of C5a receptor and P38-MAPK by using immunohistochemistry after HE staining. And the above procedure as did in 12 h subgroup was done 24 hours later. Results Compared with the control group, the levels of LDH and CK in rats of Model group were significantly higher (P ＜ 0. 05). There were significant differences in LDH and CK between 24 h subgroup and 12 h subgroup [(2 568.9 ± 280) vs. (2 201.2 ± 149)] and [(5 029.7±458) vs. (2 629.4±140)] ,P＜0. 05, P＜0.05. The analysis of C5aR and P38-MAPK gray values showed that there were significant differences between the model group and normal control group [(702.77 ±122) vs. (388.36±113)], P＜0. 05 and [(646.40±181) vs. (307.32 ±61)] ,P＜0.05,and those differences also found between the 24 h subgroup and 12 h subgroup. There was a significant positive correlation between C5aR and P38-MAPK (P＜0.05 ), and also the P38-MAPK had significant positive relationships with LDH(P＜0.05) and CK (P＜0.05). Conclusions The C5aR strongly potentiates the P38-MAPK to induce myocardial injury by septic shock.     

The role of erlotinib in the acute lung injury and the expression of surfactant protein A北大核心CSCD

Objective: To investigate the role of erlotinib in the expression of surfactant protein A (SP-A) in LPS-induced acute lung injury (ALI) of mice model. Methods: C57BL/6 mice were randomly (random number) divided into control group (n=6), ER group (n=6), LPS group (n=6), and ER+LPS group (n=6). In the LPS group, 2 mg/kg LPS was instilled into trachea of mice to induce lung injury. In control group, normal saline was instilled into trachea of mice instead. In the ER+LPS group and ER group, 100 mg/kg of erlotinib was instilled into stomach of mice, and one hour later. 2 mg/kg LPS was instilled into trachea of mice in ER+PLS group to induce lung injury. Twenty-four hours later, bronchoalveolar lavage fluid (BALF) and lung tissue of mice in four groups were collected. HE staining were used for evaluating pathological changes of lung injury. Lung wet/dry weight ratio, protein concentrations and total cell numbers in the BALF were measured to determine the degree of pulmonary edema. Immunohistochemical staining and Western Blot were used for testing the protein expression of SP-A, Data of multiple groups were analyzed by one way variance (ANOVA) and inter-group comparisons were made by the least significant difference (LSD) tests. Results: There was no significant difference in lung injury score (LIS) between control group (0.056±0.008) and ER (0.064±0.037) group, The LIS in LPS group (0.846±0.047) was higher than that in control group, however the LIS in ER+LPS group (0.279±0.020) was significant lower than that in LPS group (P < 0.05). Lung wet/dry weight, SP-A concentration and total cell numbers in the bronchoalveolar lavage fluid revealed that the degree of pulmonary edema in LPS group was higher than that in control group, and this pulmonary edema was reversed by erlotinib treatment. Immunohistochemical staining and Western blot showed that the expression of SP-A in LPS group was decreased compared with control group, but it was recovered after erlotinib treatment (P < 0.05). Conclusions: Erlotinib could protect the LPS-induced ALI, and it may be related to the regulation of SP-A. © 2018 Chinese Medical Association. All rights reserved.