Effect of chemically modified tetracycline on transforming growth factor-beta1 and caspase-3 activation in liver of septic rats

OBJECTIVES: We have previously demonstrated that hepatic matrixmetalloproteinase (MMP)-9 and gelatinase activity increased significantly after sepsis, and pretreatment with chemically modified tetracycline (CMT-3) inhibited these expressions and improved survivability. It has been established that MMP-9 release from hepatic nonparenchymal cells activates transforming growth factor (TGF)-beta1, which in turn catalyzes the conversion of procaspase-8 into active caspase-8. Caspase-8 activates caspase-3, which in turn degrades fibronectin and focal adhesion kinase and leads to disruption of hepatic architecture and integrity. We have been interested in investigating the role of posttreatment with CMT-3 on hepatic MMP-9, TGF-beta1, and caspase-3 activity following sepsis. DESIGN: Laboratory experiment. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: In this study, sepsis was induced in rats by cecal ligation and puncture (CLP), and 2 hrs later, half of the rats received CMT-3 (25 mg/kg), whereas the other half received vehicle by gavage. Twenty-four and 48 hrs after sepsis induction, blood and liver samples were collected. MEASUREMENTS AND MAIN RESULTS: Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were determined by enzymatic method, and the activation states of hepatic MMP-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, TGF-beta1, and caspase-3 were determined by Western immunoblotting. Plasma GOT, GPT, and hepatic MMP-9 activity increased 2.5-fold, and TFG-beta1 and caspase-3 activity increased 1.5- to 2-fold at 24 hrs and 48 hrs post-CLP; CMT-3 treatment blocked these increases. Furthermore, CMT-3 treatment also led to increased TIMP-1 level, an in vivo inhibitor of MMP-9. MMP-2 level was unaffected by CLP. The 24-hr and 48-hr mortality rates for CLP rats were 29% and 50%, whereas posttreatment with CMT-3 resulted in 0% mortality. CONCLUSIONS: Our results are consistent with an MMP-9-induced caspase-3 activation in response to CLP. CMT-3 posttreatment increased TIMP-1 level and thereby inhibited MMP-9, which in turn decreased TGF-beta1 and caspase-3 signaling pathways and improved survivability in septic rats.     

Role of chemically modified tetracycline on TNF-alpha and mitogen-activated protein kinases in sepsis

Chemically modified tetracyclines are orally active inhibitors of multiple proteases and cytokines. In this study, we focused on the regulation of tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinases (MAPKs) in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracycline-3 (CMT-3), which retains their antiinflammatory activity. Sepsis was induced in rats by cecal ligation and puncture (CLP). At 24 h and 1 h before CLP, treated rats received CMT-3 (25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. TNF-alpha was determined by ELISA, and MAPKs were determined by Western blot analysis. A significant activation of p38 MAPK was observed after 0.5 h and 1.5 h of sepsis that appeared to coincide with the increased circulating TNF-alpha level. The activation of p42/44 was increased after 24 h of sepsis, whereas that of SAPK/JNK was unaltered throughout the course of sepsis. CMT-3 pretreatment inhibited the TNF-alpha level as well as p38 MAPK activation seen after 0.5 and 1.5 h of CLP and also suppressed the activation of p42/44 after 24 h post-CLP. These results indicate increased activity of TNF-alpha and MAPK following sepsis and demonstrate the beneficial effect of CMT-3 in preventing the increase in TNF-alpha, p38 MAPK, p42/44 MAPK, and the progression of septic shock.     

Inhibition of Matrix Metalloproteinase on Hepatic Transforming Growth Factor β1 and Caspase-3 Activation in Hemorrhage

Objectives: Hemorrhage initiates an inflammatory response that induces the systemic release of cytokines and sequestration of polymorphonuclear neutrophils. Sequestered polymorphonuclear neutrophils release proteases, including matrix metalloproteinases (MMPs) that degrade elements of the extracellular matrix, contributing to the morbidity and mortality seen from hemorrhage. Activation of MMPs may be associated with changes in transforming growth factor β1 (TGF‐β1) and caspase‐3 signaling pathways. In this study, the authors examined hemorrhage‐induced changes in the expression of rat hepatic MMP‐9, tissue inhibitor of metalloproteinase‐1 (TIMP‐l), TGF‐β1, and caspase‐3 activities in the presence and absence of the MMP inhibitor hydroxamate. Methods: Hemorrhagic shock was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure level of 40 mm Hg, maintained for 90 minutes by withdrawal and infusion of blood, followed by a resuscitation period of lactated Ringer's infusion. Rats received either hydroxamate (25 mg/kg) or vehicle by gavage before hemorrhage. Twenty‐four hours after resuscitation, plasma and liver samples were collected. Liver MMP‐9, TGF‐β1, and caspase‐3 levels were quantified by Western immunoblotting. Plasma glutamic oxaloacetic transaminase (GOT) and plasma glutamic pyruvic transaminase (GPT) were determined enzymatically. Results: Plasma GOT, plasma GPT, and liver MMP‐9, TGF‐β1, and caspase‐3 levels were all significantly elevated at 24 hours postresuscitation when compared with the control values. Hepatic TIMP‐1, an in vivo inhibitor of MMP‐9, was unaltered at 24 hours. Hydroxamate treatment reduced GOT, GPT, MMP‐9, TGF‐β1, and caspase‐3 levels at 24 hours. The mortality of hemorrhaged untreated rats was 29% after 24 hours, and pretreatment with hydroxamate reduced mortality to 0%. Conclusions: These results indicate the beneficial effects of MMP inhibitor in preventing an increase in GOT, GPT, MMP‐9, TGF‐β1, and caspase‐3 activity with the potential for improvement of hepatic injury due to hemorrhage.     

Inhibition of breast cancer cell extracellular matrix degradative activity by chemically modified tetracyclines

BACKGROUND: Inhibition of tumour cell proliferation, invasion and metastasis by chemically modified tetracyclines has been ascribed to inhibition of matrix metalloproteinase (MMP) activity. METHODS: Exposure of the human breast carcinoma cell line MDA-MB-231 or its MMP-9-overproducing transfected clone (E-10) to 6-demethyl, 6-deoxy, 4-de [dimethylamino]-tetracycline (CMT-3), a chemically modified non-antimicrobial tetracycline followed by analysis using gelatinase activity assay, zymography, degradation of radiolabelled extracellular matrix (ECM), Western blotting, TNF-alpha ELISA and cell viability assays. RESULTS: CMT-3 treatment results in diminution in extracellular MMP-9 protein levels as well as inhibition of gelatinase activity. This prevents cell-mediated ECM degradation without inducing general cytostasis or cytotoxicity. Culturing E-10 cells in 10 or 20 microM CMT-3 diminished secreted MMP-9 levels by 45% or 60%, respectively, but did not affect levels of most other secreted proteins, including tissue inhibitor of Metalloproteinases (TIMP-1). ECM degradation by E-10 cells or their conditioned medium was inhibited by approximately 20%-30% in the presence of 20 microM CMT-3, reflecting inhibition of MMP-9 activity in addition to diminution of released MMP-9 levels. TNF-alpha levels were also diminished in E-10 conditioned medium in the presence of CMT-3, but cell viability, measured by MTS reduction and cytosolic LDH retention, was unaffected. CONCLUSIONS: It is proposed that the reduction in ECM-degradative activity reflects diminished levels of expression as well as inhibition of enzymatic activity of MMPs released by cells in the presence of CMT-3. These multiple effects of CMT-3 may offer promise for use in suppressing tumour invasion, and if used in conjunction with other chemotherapy agents, may lead to more successful treatment of cancer.     

老龄大鼠脑缺血/再灌注致脑微血管基底膜损伤的变化及与明胶酶系的关系

目的 研究老年脑缺血/再灌注(I/R)不同时期微血管基底膜损伤与明胶酶系的关系.方法 采用大脑中动脉阻塞(MCAO)线栓法制备大鼠局灶性脑I/R模型,将大鼠分为青年组和老龄组,各组又分为假手术组、制模后缺血(1)3 h和I/R 6 h、12 h、24 h、3 d、6 d时间点组.采用免疫组化和酶谱分析方法测定脑微血管结构、基底膜Ⅳ型胶原(Col Ⅳ)、层连蛋白(LN)和基质金属蛋白酶(MMPs)及其抑制剂(TIMPs)的变化.结果 与青年假手术组比较.老龄假手术组Col IV、LN升高和MMP-2、MMP-9表达增强.随I/R时间的延长,老龄与青年大鼠摹底膜成分Col IV和LN表达递减;MMP-2表达递增,MMP-9、TIMP-1表达呈先增强而后降低趋势.与青年模型组相同时间点比较,老龄模型组Col IV(I 3 h、1/R 6 h、I/R 12 h)、LN(1 3 h、I/R 6～24 h)、MMP-2(I 3 h,I/R 6 h～6 d)、MMP-9(I 3 h、1/R 6～24 h)表达水平增强,TIMP-1(I/R 24 h)表达水平降低,差异均有统计学意义(P＜0.05或P＜0.01).各组MMP-2与MMP-9的酶谱分析比较,量的变化与免疫表达规律基本一致.结论 随着增龄,大鼠腩微血管基底膜成分改变与MMPs、TIMP的变化有关.在脑I/R脑微血管基底膜损伤方面,老龄大鼠较青年严重,其损伤的特点与明胶酶系变化有关.     

Elevation of glutamic pyruvic transaminase and gamma-glutamyl transpeptidase in obesity

Study was made of glutamic pyruvic transaminase (GPT), glutamic oxalacetic transaminase (GOT), and gamma-glutamyl transpeptidase (gamma-GTP) in 729 obese subjects in various groups, namely, primary school children, high school children, university students, and outpatients. The incidences of abnormal GPT, GOT, and gamma-GTP in the obese subjects were frequently significantly higher than in the controls. It was most clearly shown in GPT. The incidences of abnormal GPT in the obese females were significantly lower than those in the obese males, but were significantly higher than the controls. Higher incidences of abnormality in the school children were ascribed to the higher degree of obesity in the children. The extent of increase in GPT was considerable. GPT was sometimes higher than 6 times the normal upper limit.     

Fluvastatin attenuates severe hemorrhagic shock-induced organ damage in rats

Objectives

Multiple organ dysfunction resulting from hemorrhagic shock (HS) and subsequent resuscitation was mediated by several inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10). The present study was designed to investigate the protective effects of fluvastatin on these mediators after HS in rats.

Methods

The experimental rats were randomly divided into three groups. The vehicle group received only vitamin K without HS, the HS-control group received vitamin K and HS, and the HS-experimental group received both vitamin K and fluvastatin (1 mg/kg) before HS. HS was produced by bleeding from a femoral arterial catheter to remove 60% of total blood volume (6 ml/100 g BW) over 30 min. The mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 12 h after the start of blood withdrawal. The biochemical parameters, including arterial blood gas, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and lactate were obtained at 30 min before induction of HS and at 0, 1, 3, 6, 9 and 12 h after HS. Equal volume of normal saline was given to replace blood volume loss. Cytokine levels including TNF-α and IL-10 in serum were measured at 1 h after HS. Kidney, liver, lung and small intestine were removed for pathology examination at 48 h after HS.

Results

HS significantly increased HR, blood GOT, GPT, BUN, Cre, LDH, CPK, lactate, TNF-α and IL-10 levels, and also induced metabolic acidosis and decreased MAP in rats. Pre-treatment with fluvastatin was found to improve survival rate, preserved MAP, decreased the markers of organ injury, suppressed the release of TNF-α and increased IL-10 after HS in rats.

Conclusion

Pre-treatment with fluvastatin can suppress the release of serum TNF-α and can also increase serum IL-10 level to protect HS-induced multi-organ damage in rats.     

Peptidoglycan of S. aureus causes increased levels of matrix metalloproteinases in the rat

Matrix metalloproteinases (MMPs) have been suggested to contribute to the organ injury in septic patients. We recently demonstrated that peptidoglycan (PepG) of S. aureus causes organ injury in the rat. A possible role for MMPs in the septic response to PepG is unknown. In the present study, we have examined whether the release of MMP-9 (gelatinase B) and MMP-2 (gelatinase A) is induced by PepG in the anesthetized rat. Male Wistar rats were injected intravenously with PepG (10 mg/kg), LPS (1 mg/kg), or a combination of LPS and PepG (1 mg/kg of each). After 1 or 3 h, liver, lung, and plasma were harvested. MMP-9 and MMP-2 levels were analyzed in organ homogenates and in plasma samples by zymography. MMP-9 levels were significantly increased in the lung within 1 h after injection of PepG, LPS, or combined treatment, compared with sham animals (P < or = 0.05). In the liver and plasma, MMP-9 was clearly increased by PepG or LPS at both 1 and 3 h compared with sham animals (P < or = 0.05). Considerable basal amounts of MMP-2 protein were seen in the liver and in plasma. In the lung, MMP-2 levels were elevated by combined LPS/PepG at 1 h and by LPS at 3 h (P < or = 0.05). In contrast, MMP-2 activity in the liver was significantly reduced by bacterial products. In the plasma, no major alterations of MMP-2 levels were observed. Our data show that PepG of S. aureus causes a rapid elevation of MMP-9 protein in the liver, lung, and blood of the rat. Based on these and previous data, we hypothesize that the release of MMP-9 in lung, liver, and blood is part of the septic host response to systemic PepG.     

Alcohol, liver function tests, and high density lipoprotein cholesterol in university students

Among non-obese students, those consuming daily 44 g or more of alcohol showed significantly higher incidences of abnormality in glutamic oxalacetic transaminase (GOT) and gamma-glutamyl transpeptidase (gamma-GTP). When daily alcoholic intake of 44 g or more was combined with obesity, highly significant increase in incidences of abnormality in GOT, glutamic pyruvic transaminase (GPT), and gamma-GTP was observed. In normal weight students, even lower range of alcoholic consumption was associated with significant increase in high density lipoprotein (HDL) cholesterol. As compared with normal weight groups, obesity groups showed significantly lower HDL cholesterol, and leanness group significantly higher HDL cholesterol.     

Changes in serum enzyme activity after transcatheter arterial embolization for hepatic neoplasm

We assayed serum levels of certain enzymes and tumor markers in patients after transcatheter arterial embolization (TAE) to evaluate the effectiveness of this treatment. Twenty patients had hepatocellular carcinoma and two patients had metastases to the liver from colon cancer. Assays were first done immediately after TAE and were continued for the next 12 days. Glutamic oxaloacetic transminase (GOT; EC 2.6.1.1, -aspartate:2-oxoglutarate aminotransferase), glutamic pyruvic transaminase (GPT; EC 2.6.1.2, -alanine:2-oxoglutarate aminotransferase), and lactate dehydrogenase (EC 1.1.1.27; (S)-lactate:NAD⁺ oxidoreductase) peaked 24 to 48 h after TAE and returned to the base lines in 7 to 10 days. Mitochondrial GOT (mGOT) and glutamate dehydrogenase (GLDH; EC 1.4.1.2, -glutamate:NAD⁺ oxidoreductase) also peaked at the same time after TAE. -Fetoprotein peaked 2 h after TAE and decreased to half of the baseline on day 7. Carcinoembryonic antigen peaked at 24 h and fell at 48 h only in the patients with colon cancer. The total amount of cytosolic GOT, GPT, mGOT, and GLDH released was correlated to the volume of the necrotic mass estimated by computed tomography scans. The correlation coefficients for mGOT and GLDH were r = 0.919 and r = 0.939 (both p < 0.001), respectively. Assays of mGOT and GLDH may be useful to estimate the volume of the necrotic mass of a hepatoma or metastatic carcinoma in the liver.     

Matrix metalloproteinases: potential therapeutic target in spinal cord injury.

Mediators of extracellular matrix proteins degradation, the matrix metalloproteinases (MMPs), involved in inflammation as well as facilitation of process outgrowth of oligodendrocytes are interesting targets for neural repair. Recent data reported their activation after seizures, cerebral ischemia and spinal cord injury. The present study was designed to localize at cellular level the gelatinase activity by in situ zymography in a rat spinal cord contusion model. The kinetic of gelatinase activation was monitored by in situ zymography on 20 microm cryostat sections. The fluorescein-quenched DQ gelatin digestion yielded cleaved fluorescent peptides enabling the detection of gelatinase activity at cellular level. Twenty four hours and 48 h after injury, a strong gelatinase activity was detected at the lesion site in and around vascular structures and infiltrated cells. A preincubation with either MMP-2 or MMP-9 antibodies significantly decreases the gelatinase activity pattern, suggesting the involvement of at least both MMPs. Our results are consistent with a role for MMPs in the blood spinal barrier disruption, the leukocytes infiltration, the disruption of the extracellular matrix and the clearance of debris.     

Chemically modified tetracycline (COL-3) improves survival if given 12 but not 24 hours after cecal ligation and puncture

Sepsis can result in excessive and maladaptive inflammation that is responsible for more than 215,00 deaths per year in the United State alone. Current strategies for reducing the morbidity and mortality associated with sepsis rely on treatment of the syndrome rather than prophylaxis. We have been investigating a modified tetracycline, COL-3, which can be given prophylactically to patients at high risk for developing sepsis. Our group has shown that COL-3 is very effect at preventing the sequelae of sepsis if given before or immediately after injury in both rat and porcine sepsis models. In this study, we wanted to determine the "treatment window" for COL-3 after injury at which it remains protective. Sepsis was induced by cecal ligation and puncture (CLP). Rats were anesthetized and placed into five groups: CLP (n = 20) = CLP without COL-3, sham (n = 5) = surgery without CLP or COL-3, COL3@6h (n = 10) = COL-3 given by gavage 6 h after CLP, COL3@12h (n = 10) = COL-3 given by gavage 12 h after CLP, and COL3@24h (n = 20) = COL-3 given by gavage 24 h after CLP. COL-3 that was given at 6 and 12 h after CLP significantly improved survival as compared with the CLP and the CLP@24h groups. Improved survival was associated with a significant improvement in lung pathology assessed morphologically. These data suggest that COL-3 can be given up to 12 h after trauma and remain effective.     

Blood levels of glutamate oxaloacetate transaminase are more strongly associated with good outcome in acute ischaemic stroke than glutamate pyruvate transaminase levels

Ischaemic stroke is associated with an excessive release of glutamate in brain. GOT (glutamate-oxaloacetate transaminase) and GPT (glutamate-pyruvate transaminase) are two enzymes that are able to metabolize blood glutamate facilitating the lowering of extracellular levels of brain glutamate. Our aim was to study the association between blood levels of both enzymes and stroke outcome in patients with acute ischaemic stroke. We prospectively studied 365 patients with first ischaemic stroke<12 h. Glutamate, GOT and GPT levels were determined in blood samples obtained at admission. We considered functional outcome at 3 months [good outcome: mRS (modified Rankin Scale)≤2; poor outcome mRS >2], END (early neurological deterioration) in the first 72 h [increment ≥4 points in NIHSS (National Institutes of Health Stroke Scale)] and infarct volume [CT (computed tomography) at 36-72 h] as end points. We have found an inverse correlation between GOT and GPT levels and blood glutamate levels. Patients with poor outcome showed lower levels of GOT (11.9±8.2 compared with 22.7±10.2 m-units/ml, P<0.0001) and GPT (19.5±14.3 compared with 24.7±20.3 m-units/ml; P=0.004). A negative correlation has been found between GOT (Pearson coefficient=-0.477, P<0.0001) and GPT (Pearson coefficient=-0.116; P=0.027) levels and infarct volume. Patients with END showed higher levels of blood glutamate (381.7±97.9 compared with 237.6±114.0 μmol/l, P<0.0001) and lower levels of GOT (10.8±6.7 compared with 18.1±10.8 m-units/ml; P<0.0001). This clinical study shows an association between high blood GOT and GPT levels and good outcome in ischaemic stroke patients, this association being stronger for GOT than GPT levels.     

辛伐他汀预处理对脓毒症大鼠血清血管性血友病因子的影响

目的 评价辛伐他汀预处理对脓毒症大鼠血管内皮细胞功能的影响.方法 清洁级雌性Wistar大鼠96只,体质量200～250 g,4月龄.采用随机数字表法,将大鼠随机分为3组:假手术组32只、脓毒症组32只和辛伐他汀组32只.脓毒症组和辛伐他汀组采用盲肠结扎穿孔法制备脓毒症模型.辛伐他汀组制模前胃内灌注辛伐他汀20 mg·kg-1 ·d-1,每日l次,连续2周;假手术组和脓毒症组胃内灌注等容量生理盐水.分别于术后3、6、24和48小时取8只大鼠,采集颈动脉血样,测定白细胞计数;采用酶联免疫吸附测定(ELISA)法检测血清血管性血友病因子(vWF)水平;记录术后3小时内、＞3～6小时、＞6～24小时和＞24～48小时各时段大鼠的死亡情况.结果 与假手术组比较,脓毒症组和辛伐他汀组术后各时点白细胞计数和血清vWF浓度升高,术后6小时达峰值,白细胞计数分别为(7.45±0.69)×109/L,(12.79±0.51)×109/L,(10.36±0.19)×109/L; vWF水平分别为(0.543±0.001)μg/L,(1.198±0.035)μg/L,(1.018±0.005)μg/L( P＜0.05);与脓毒症组比较,辛伐他汀组术后各时点白细胞计数和血清vWF水平降低,术后24～48小时内病死率降低(脓毒症组75.0％,辛伐他汀组50.0％)(P＜0.05).结论 辛伐他汀可保护脓毒症大鼠的内皮细胞功能,从而降低48小时内的病死率.     

Enhanced systemic matrix metalloproteinase response in Helicobacter pylori gastritis

Background. Helicobacter pylori causes chronic gastritis, peptic ulcer disease, and is the most important risk factor for non-cardia gastric cancer, and has been shown to upregulate matrix metalloproteinases (MMPs) in infected gastric mucosa. MMPs are proteolytic enzymes regulated by tissue inhibitors of metalloproteinases (TIMPs).

Aims. We set up this study to find out whether H. pylori gastritis induces systemic MMP response.

Methods. Serum samples were collected from patients undergoing gastroscopy; 26 patients had H. pylori gastritis and 18 were H. pylori-negative controls with normal gastric mucosa. Serum MMP levels were analysed by enzyme-linked immunosorbent assay.

Results. Significantly elevated serum levels of collagenase-2 (MMP-8), gelatinase B (MMP-9), neutrophil elastase (NE), and myeloperoxidase (MPO), and reduced serum levels of gelatinase A (MMP-2) and TIMP-1 were demonstrated in patients with H. pylori gastritis as compared to H. pylori-negative controls. No significant differences were shown in serum matrilysin-1 (MMP-7) levels.

Conclusions. For the first time, we show enhanced MMP-8 response in H. pylori infection together with other neutrophil degranulation products (MMP-9, MPO, NE). Elevated circulating neutrophil degranulation product levels in serum of H. pylori-positive patients reflect accelerated proteolysis and oxidative stress, and may contribute to extraintestinal sequelae, such as cardiovascular diseases.     

Effect of angiotensin II on primary cardiac fibroblast matrix metalloproteinase activities

Left ventricular dysfunction is associated with reperfusion injury occurring during open-heart surgery. There is an increased secretion of angiotensin II (Ang II) and increased matrix metalloproteinases (MMPs) activities associated with open-heart surgery that may affect the cardiac extracellular matrix (ECM). The goal of this study was to determine the effects of Ang II and selective angiotensin II receptor (AT1-R and AT2-R) blockers on the enzymatic activities of MMPs in primary adult murine cardiac fibroblasts (CF). Our hypothesis is that Aug II, with and without a selective receptor blocker, differentially affects CF MMPs activities. The CF were treated with Ang II (10(-6) M) and doses of AT1-R and AT2-R blockers (losartan and PD123319, respectively) at doses of 10(-7) to 10(-5) M for 48 hours. The Ang II-stimulated CF reduced collagenase activities by only 24% (p = 0.004); however, the MMP-2 and MMP-9 gelatinase activities were reduced by 42% and 39%, respectively (p = 0.022). The losartan dose dependently increased MMP-2 (p = 0.02) and MMP-9 (ns). PD123319 at 10(-5) M significantly reduced MMP-2 and MMP-9 activities compared with the Ang II group (p = 0.014 and p = 0.02, respectively). The doses of PD123319 at 10(-6) and 10(-7) M increased the MMP-2 and MMP-9 enzymatic activities significantly above the Ang II only group. Thus, Ang II and AT1-R and AT2-R differentially affect the collagenase and gelatinase MMPs activities released by cardiac fibroblasts.     

Protective effects of atorvastatin in rat models of acute pulmonary embolism: involvement of matrix metalloproteinase-9

OBJECTIVE: Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of acute pulmonary embolism (APE)-induced pulmonary hypertension. Here, we evaluate the effects of atorvastatin pretreatment on APE-induced pulmonary hypertension, 24-hr mortality rate, and changes in plasma and lung MMP-2 and MMP-9 activities. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats. INTERVENTIONS: Rats received atorvastatin (30 mg/kg/day orally) or tap water for 2 wks. In study 1, we examined whether atorvastatin affected APE-induced pulmonary hypertension by using a rat isolated lung perfusion model of APE. In study 2, we examined whether atorvastatin affects the survival rate after APE, which was induced by rapid intravenous injection of 14 mg/kg of a suspension of microspheres (or saline) into the tail vein. MEASUREMENTS AND MAIN RESULTS: Plasma nitrite/nitrate concentrations were measured by chemiluminescence. Pretreatment with atorvastatin was associated with 49% higher nitrite/nitrate levels compared with controls (p < .05). In study 1, whereas APE increased mean pulmonary artery pressure (MPAP) by 13.0 +/- 1.6 mm Hg in perfused lungs isolated from rats pretreated with water, pretreatment with atorvastatin attenuated by 27% the increases in MPAP after APE. In study 2, pretreatment with atorvastatin was associated with a significant increase in 24-hr survival rate after APE, which was 48% in embolized rats pretreated with water and 64% in rats pretreated with atorvastatin (p < .05). Gelatin zymography of lung and plasma MMP-2 and MMP-9 was performed. Lungs and plasma from embolized rats showed higher levels of both pro- and activated forms of MMP-9 compared with those from nonembolized animals (all p < .05). However, pretreatment with atorvastatin attenuated by 32% the increases in lung-activated MMP-9 levels after APE (p < .05). CONCLUSIONS: These results suggest that pretreatment with atorvastatin attenuates APE-induced pulmonary hypertension and increases 24-hr survival rate by mechanisms that result in attenuated increases in lung activated MMP-9 after APE.     

黄芩苷对脓毒症大鼠早期肾功能损伤的保护作用

目的：通过观察黄芩苷对脓毒症大鼠血清中性粒细胞明胶酶相关载脂蛋白（NGAL）浓度的影响,探讨黄芩苷对脓毒症大鼠肾功能损伤的早期保护作用。方法：采用盲肠结扎穿孔术（CLP）制备SD大鼠脓毒症模型。实验大鼠随机分成假手术组、模型组、黄芩苷干预组（每纽各24只）,每组大鼠再随机分为0h、3h、6h、24h4个亚组（每组各6只）。黄芩苷组在造模后立即经腹腔给予黄芩苷120mg／kg共2mL液体,假手术组和模型组则给予等量％0．9氯化钠液腹腔注射。每组大鼠在4个时间点（CLP术后0h、3h、6h、24h）采集血标本和肾组织标本。HE染色观察肾组织病理改变;检测血清NGAL和肌酐浓度和用免疫组织化学法测定肾组织肿瘤坏死因子“（TNF-α）水平。结果：与假手术组相比,模型组大鼠血清NGAL和肌酐浓度显著升高（P〈0．05）,术后3h开始升高,术后24h继续升高;肾组织TNF-α水平也显著升高（P〈0．05）,术后3h开始升高,术后24h仍升高;术后24h肾组织HE染色显示肾组织内大量炎症细胞浸润。与模型组相比,黄芩苷干预组术后3h、6h和24h血清NGAL和肌酐浓度以及肾组织TNF-α水平均显著降低（P〈0．05）,肾脏组织的炎症病理改变也明显减轻。结论：黄芩苷对脓毒症大鼠早期肾功能损伤有保护作用。     

Suppression of autophagy in rat liver at late stage of polymicrobial sepsis

Sepsis develops as a result of the host response to infection, and its mortality rate in ICU remains high. Severe inflammation usually causes overproductions of proinflammatory cytokines, i.e., TNF-α and reactive oxygen species, which lead to mitochondrial damage and energetic depletion. Autophagy is a survival mechanism for eukaryote to recycle intracellular nutrients and maintain energy homeostasis. We hypothesize that autophagy plays a beneficial role in the pathogenesis of organ failure during sepsis. A rat model of cecal ligation and puncture (CLP) that simulate peritonitis-induced sepsis was used, and indicators for liver dysfunction, serum glutamic oxaloacetic, serum glutamic pyruvic, alkaline phosphatase, and bilirubin were measured. Levels of LC3-II and LC3 aggregation were quantified by Western blot analysis and by immunohistochemistry, respectively. The tissue localization of autophagy was identified by immunohistochemistry and transmission electron microscopy. Our results showed that (a) increase in LC3-II level in liver tissue occurs at 3 h, peaks at 6 h, and then surprisingly declines quickly until 18 h after CLP (CLP18h); (b) significant hepatic dysfunction was observed at CLP18h; (c) ratio of LC3 aggregation is significantly higher in hepatocytes than in Kupffer cells, and (d) loss of Atg7, an essential gene for autophagosome formation, exaggerates the TNF-α-induced cell death, depletion of ATP, and decrease of albumin production in hepatocytes. These results indicate that autophagy occurs transiently in hepatocytes at early stage, and the decline in autophagy at late stage may contribute to the functional failure in liver during polymicrobial sepsis.     

脓毒症大鼠肠道肠三叶因子mRNA表达的变化

目的：探讨脓毒症大鼠肠道肠三叶因子（trefoil factor family,TFF3）mRNA表达的变化。方法：32只SD大鼠随机分为对照组（n=8）和脓毒症组（n=24）。采用盲肠结扎穿孔术（cecal ligation and puncture,CLP）制作大鼠脓毒症模型。于模型建立后3h,6h及12h（n=8）取回肠黏膜,以RT-PCR法检测TFF3 mRNA的表达。结果：脓毒症模型建立3h始TFF3 mRNA表达即显著下降（P〈0.01）,随着时间延长表达进一步下降。结论：脓毒症大鼠肠道TFF3 mRNA表达明显下降,可能是脓毒症肠屏障功能障碍的机制之一并延缓肠道黏膜修复。