Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma

Summary. All-trans retinoic acid (ATRA) inhibits human myeloma cell growth in vitro, presumably through the down-regulation of interleukin 6 receptors (IL-6R). Based on these and other studies, we initiated a phase II clinical trial using ATRA in patients with advanced refractory multiple myeloma (MM). We report that three out of six treated patients developed severe hypercalcaemia following administration of ATRA, which was accompanied by a significant rise in serum IL-6 levels. Normal calcium levels were restored after the discontinuation of the drug and the administration of standard anti-hypercalcaemic care. We suspect that down-regulation of IL-6R resulted in increased serum IL-6 levels, leading to advanced bone resorption and hypercalcaemia. We conclude that the use of ATRA in patients with advanced MM is not warranted.     

Down-regulation of bcl-2 in AML blasts by all-trans retinoic acid and its relationship to CD34 antigen expression

High levels of expression of the bcl-2 oncoprotein in acute myeloblastic leukaemia (AML) cells have been associated with low complete remission rates and poor survival. The sensitivity of AML blasts to drugs such as Ara-C can be increased by the down-regulation of bcl-2 expression by antisense oligonucleotides. All-trans retinoic acid (ATRA) has been reported to increase the sensitivity of AML cell lines to Ara-C and to induce differentiation in the HL60 promyelocytic cell line, with both effects being accompanied by a decrease in bcl-2 expression. Using flow cytometry and a monoclonal antibody to bcl-2, we have investigated the effects of ATRA (1 μM ) on bcl-2 expression in the blast cells of 25 AML patients and the K562 cell line after incubation for 72 or 24 h, respectively. Using Kolmogorov-Smirnov statistical analysis where a D value of > 0.12 was statistically significant, we found that in 8/25 AML samples and the K562 cells there was a significant decrease in bcl-2 protein expression after incubation with ATRA (D value range 0.14–0.44). The mean peak fluorescence (MPF) values for the bcl-2 levels of the ATRA responders (n = 8) was reduced to 35.5 ± 6.9 following incubation with ATRA compared to 47.6 ± 8.2 (mean ± SEM) for control samples incubated in the absence of ATRA (P = 0.014). There was no significant difference between the baseline bcl-2 molecules of equivalent soluble fluorochrome (MESF) levels in the ATRA responders (48.9 ± 5.7, n = 8) and the non-responders (41.3 ± 3.9, n = 17) (mean ± SEM) (P = 0.28). The down- regulation of bcl-2 expression by ATRA was particularly associated with CD34-negative AML and of the eight AML patients’ cells that responded to ATRA by down-regulating bcl-2, seven were CD34 negative (P<0.05). Our data suggest that the addition of ATRA to combination chemotherapy would increase the chemosensitivity of some patients with AML, particularly CD34-negative AML, due to down-regulation of bcl-2 expression.     

All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

Preventive effect of cyclosporin A on experimentally induced acute liver injury in rats

Abstract: We examined the effect of cyclosporin A (CsA) on the pathogenesis of acute experimental liver injury in rats induced by injection of heat-killed Propionibacterium acnes (P. acnes) and subsequent injection of lipopolysaccharide (LPS). Pretreatment with CsA significantly reduced serum alanine aminotransferase (ALT), serum tumor necrosis factor-α (TNF-α) production, without changing the TNF-α mRNA level in the liver, and plasma interferon-γ (IFN-γ), following LPS injection in this model. Twenty-four-hour mortality was also markedly improved, from 100% in the P. acnes plus LPS group to 0% in the CsA-pretreated group. Although direct addition of CsA to isolated hepatic macrophages from P. acnes-pretreated rats did not prevent the production of TNF-α and active oxygen species, isolated hepatic macrophages from P. acnes plus CsA-pretreated rats significantly reduced their production in response to the addition of LPS. These results suggest that CsA protects against P. acnes plus LPS-induced acute liver injury, not by direct inhibition of hepatic macrophage activation, but by indirect prevention of hepatic macrophage activation, presumably related to the reduction in plasma IFN-γ levels.     

Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells

The production of interleukin-8 (CINC: cytokine-induced neutrophil chemo-attractant) from different cell populations in the rat liver was studied and cells related to the initiation of CINC production in lipopolysaccharide (LPS)-injected endotoxaemic rats were characterized. Sinusoidal endothelial cells (16.4 ± 10.6 ng/mL) produced significantly higher amounts of CINC in 24 h primary cultures compared with hepatocytes (0.9 ± 0.9 ng/mL; P < 0.05) and Kupffer cells (6.5 ± 5.1 ng/mL; P < 0.05). Lipopolysaccharide, tumour necrosis factor-α (TNF-α), and interleukin-1α (IL-1α) stimulated different liver cell populations to produce CINC; LPS mainly stimulated Kupffer cells, TNF-α stimulated hepatocytes and IL-1α stimulated all three types of cells. Intraperitoneal injection of LPS (4 mg/kg) caused CINC accumulation in non-parenchymal cells of the rat liver within 1 h of injection, as shown by immunohistochemical staining. In contrast, CINC-positive hepatocytes were not seen until 3 h after injection of LPS. Ethanol was not a direct inducer of CINC production by rat hepatocytes in vitro. These findings strongly suggest that non-parenchymal liver cells, including sinusoidal endothelial cells, are the main source of CINC. Our data also suggest that during endotoxaemia, CINC production is initiated by non-parenchymal cells and this is followed by production from hepatocytes.     

Protective effects of regulatory amino acids on ischaemia-reperfusion injury in the isolated perfused rat liver

Objective. Some amino acids (AAs) display potent regulatory activities on cell metabolism, including via anti-oxidative defences. The aim of this study was to evaluate the protective effect of these AAs on warm ischaemia-reperfusion (I/R) injury in the isolated perfused rat liver.

Material and methods. Livers from fasted male Sprague-Dawley rats were isolated and perfused without (control group) or with (AP group) a mixture of regulatory AAs (glutamine, histidine, leucine, methionine, proline, phenylalanine, tryptophan and alanine). After 45 min of perfusion, warm ischaemia was induced for 45 min by clamping the portal vein catheter; thereafter, reperfusion was performed for 30 min.

Results. TNF-α production was significantly lower in the AP group during reperfusion (Control: 39±7 versus AP: 16±2 pg min^?1 g^?1, p<0.05), and lactate dehydrogenase (LDH) release decreased significantly during the last 15 min of reperfusion (Control: 0.13±0.03 versus AP: 0.04±0.02 IU min^?1 g^?1, p<0.05), despite similar levels of oxidative stress. The addition of regulatory AAs was not associated with variations in portal flow, bile flow, hepatic glucose or urea metabolism. However, significant changes in intrahepatic glutamine (Control: 1.4±0.2 versus AP: 2.6±0.5 µmol g^?1, p<0.05) together with higher glutamate release in the AP group (Control: 10.2±5.4 versus AP: 42.6±10.9 nmol min^?1 g^?1, p<0.05) indicated modifications in nitrogen metabolism.

Conclusions. Taken together, the lower TNF-α production, suggesting decreased inflammatory response, the decrease in LDH release in the AP group, demonstrating a better preservation of liver viability, and the increase in hepatic glutamine indicate that AAs play an important role in the liver's response to I/R.     

视黄酸相关孤核受体α对脓毒症小鼠心肌损伤的影响

目的 本研究旨在探索视黄酸相关孤核受体α（RORα）对脓毒症炎症反应及其所致的心脏功能损害影响。 方法 通过腹腔注射脂多糖（LPS）建立小鼠脓毒症模型，通过LPS刺激巨噬细胞，建立脓毒症细胞模型，采用细胞转染和小鼠尾静脉注射过表达质粒，以在细胞和组织中过表达RORα，进而探究RORα在脓毒症炎症反应和脓毒症所致心肌损伤中的作用。采用实时定量PCR试验、蛋白免疫印记试验检测RORα、NF-κB p65和炎症因子的变化，采用酶联免疫试验、病理切片等检测脓毒症小鼠心肌损伤的变化。 结果 RORα在LPS刺激巨噬细胞中显著降低（P<0.05）；过表达RORα能够显著抑制LPS刺激巨噬细胞中炎症因子白介素(IL)-1β和肿瘤坏死因子 (TNF)-α的表达（P<0.05）、NF-κB p65的核移位水平（P<0.05）、脓毒症小鼠血清中炎症因子IL-1β和TNF-α的释放、降低脓毒症小鼠肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）的含量以及改善脓毒症所致的心肌病理损害。 结论 RORα能够通过抑制NF-κB p65的核移位进而负性调控LPS刺激巨噬细胞中的炎症反应，从而缓解脓毒症小鼠的炎症反应及心肌损害。     

In vitro down-regulation of bcl-2 expression by all-trans retinoic acid in AML blasts

 Using flow cytometry, we have investigated the effects of 0.5 μM all-trans–retinoic acid (ATRA) on bcl-2 expression in the blast cells of 25 acute myeloblastic leukemia (AML) patients and the HL-60 cell line after incubation for 6 days. We observed a significant decrease of bcl-2 expression after treatment with ATRA in 12 of 25 AML samples and the HL-60 cells. The mean fluorescence intensity (MFI) ratio for the bcl-2 levels of the ATRA responders (n=12) was reduced to 7.9±4.8 following incubation with ATRA compared with 10.9±6.5 (mean±SD) for control samples incubated without ATRA (p=0.011). There was no significant difference between the baseline bcl-2 MFI ratio in the ATRA responders (11.14±7, n=12) and the non responders (14.18±11.3, n=13;p=0.432). The down-regulation of bcl-2 expression by ATRA was not significantly associated with CD34-negative or -positive AML. There was no correlation between AML subtypes and regulation of bcl-2 expression by ATRA. Complete remission and overall survival were not significantly improved in bcl-2 down-regulated cases. Our data confirm that ATRA can down-regulate the bcl-2 expression in AML blasts. Because many chemotherapeutic agents also operate through the activation of programmed cell death and bcl-2 levels are positively associated with resistance to apoptosis, ATRA can be used in combination chemotherapy to increase the chemosensitivity of some patients with AML.     

Overexpression of granulocyte‐macrophage colony‐stimulating factor in mouse liver enhances the susceptibility of lipopolysaccharide leading to massive apoptosis of hepatocytes

Abstract: Background/Aims: We examined whether antigen‐nonspecific accumulation of dendritic cells (DCs) and macrophages in the liver by the overexpression of granulocyte macrophage‐colony stimulating factor (GM‐CSF) could prime severe liver injury after LPS injection. Methods: We injected a recombinant adenovirus encoding GM‐CSF intravenously (AdGM), and LPS was administered 7 days later. Liver histology, serum alanine aminotransferase (ALT) levels and apoptosis of hepatocytes were examined. Results: Liver histology of the AdGM‐primed mice showed marked infiltrates of mononuclear cells (DCs and macrophages) without granuloma formation on day 7. Expression of toll‐like receptor‐4 on intrahepatic mononuclear cells isolated from AdGM‐primed mice was up‐regulated. After LPS injection, serum ALT levels in AdGM‐primed mice reached about 6000 IU/l at 12 h, and all those mice died within 24 h. Hemorrhagic liver injury with massive apoptosis of hepatocytes was histologically recognized. When AdGM and LPS were injected in FasL‐deficient C57BL/6J‐gld/gld mice, serum ALT levels were not elevated by the pretreatment with a neutralizing anti‐TNF‐α antibody. Conclusions: Our present study provides a new model of severe liver injury, in which antigen‐nonspecific accumulation of DCs and macrophages in the liver by overexpressing GM‐CSF enhances the susceptibility to LPS, leading to hemorrhagic liver injury with massive hepatocyte apoptosis after LPS injection.     

Predictive Value of Preoperative Serum Cholinesterase Concentration in Patients with Liver Dysfunction Undergoing Cardiac Surgery

Abstract Objective There are an increasing number of patients with severe liver dysfunction subjected to open heart surgery. This retrospective study was designed to assess operative results and clarify the degree of liver injury in patients with liver dysfunction undergoing open heart surgery. In addition, determinants influencing their prognosis were assessed. Methods In a 9-year period from 1988 to 1996, we operated on 31 patients with posthepatitis liver dysfunction and 16 with chronic passive congestion of the liver. This group was 2.3% and 1.6% of the 1368 patients undergoing cardiac surgery in the same period. We compared several perioperative factors between survivors and nonsurvivors to determine risk factors affecting mortality. Results In the group with posthepatitis liver dysfunction, the postoperative course of 5 patients among 31 (16.1%) was poor. Serum cholinesterase concentration was lower only in the nonsurvivor group (nonsurvivor vs survivor: 1979 ± 949 vs 3515 ± 1424 lU/l, p < 0.05). All patients with cholinesterase < 2000 IU/L died. The duration of CPB (212 ± 53 vs 150 ± 54 minutes, p < 0.03) and ACC time (151 ± 38 vs 96 2 40 minutes, p < 0.02) was longer in the nonsurvivor group. In the group with chronic passive congestion, the postoperative course of 5 of 16 (31.3%) patients with valvular disease was poor. Serum cholinesterase concentration was lower only in the nonsurvivor group (nonsurvivor vs survivors: 2006 ± 435 vs 3483 ± 1442 IU/L, p < 0.021, and all patients with cholinesterase < 2000 IU/L died. Postoperative bleeding was greater in the nonsurvivor group (3327 ± 2106 vs 1428 ± 643 mL, p < 0.05). Multivariate logistic regression analysis including the described pre- and intraoperative factors identified only serum cholinesterase concentration (F = 9.18) as significant. Conclusions A low value of preoperative serum cholinesterase (< 2,000 IU/L) is thought to be the predictor of prognosis after open heart surgery in patients with severe posthepatitis and congestive liver dysfunction. operative factors (cardiopulmonary time in posthepatitis liver dysfunction and postoperative bleeding in the congestive liver dysfunction) also influenced the prognosis.     

Inhibition of concanavalin A-induced acute T cell dependent hepatic damage in mice by hypothyroidism

Abstract: Aims/Background: Concanavalin A (Con A) activates T lymphocytes and causes acute T-cell-mediated hepatic injury in mice. Decreased thyroid hormonal production is associated with a variety of immunological manifestations, including inactivation of macrophages with reduced TNF production and reduced soluble IL-2 receptors in the serum. We have recently shown that hypothyroidism prevents the development of cirrhosis and also minimizes hepatic damage in rats with fulminant hepatic failure. In the present study we examined the effects of hypothyroidism on a mouse model of Con A induced T cell-mediated acute hepatitis. Methods: Hypothyroidism was induced both medically (MMI, PTU) and surgically. Eight groups of 10 mice each were studied: euthyroid controls (2 groups: water, Con A) and hypothyroid (6 groups: MMI, PTU, Surgical, MMI-Con A, PTU-Con A, Surgical-Con A). Results: Hepatic inflammation was significantly decreased in each of the Con A treated hypothyroid groups of mice. The serum transaminases, TNF-α and IL-6 levels were significantly elevated in the Con A treated group while near normal levels were found in the hypothyroid Con A treated groups (mean±SE AST: 1499±18 vs 78±10 IU/1, p<0.001; TNF: 2500±250 vs 135± 15 pg/ml. p<0.001, IL-6: 12,200±300 vs 1260±140 pg/ml, p<0.001, respectively). Conclusions: Hypothyroidism, independent of the mode of induction, can effectively inhibit the development of acute T cell-mediated liver damage in mice. These results suggest that some decrease in thyroid function might have a role in the prevention of immune mediated liver diseases.     

Outcome of pregnancy in women treated with all-trans retinoic acid; A case report and review of literature

Abstract

All-trans-retinoic acid (ATRA) has been proved to be an effective treatment for acute promyelocytic leukemia (APL), inducing remission in more than 90% of cases. Treatment of APL in pregnancy is controversial as the use of ATRA has been questioned due to the teratogenic effect of retinoids. We report a case of pregnancy in a woman exposed to ATRA during the first trimester. The baby was born healthy, without any anomalies. Review of all reported cases of the use of ATRA in pregnancy revealed no serious adverse outcomes or congenital anomalies although only very few cases had exposure in the first trimester.     

Chronic Alcohol Administration Stimulates Nitric Oxide Formation in the Rat Liver With or Without Pretreatment by Lipopolysaccharide

This study examines the effect of chronic alcohol consumption on nitric oxide release from the liver of rats with or without lipopolysaccharide (LPS) (Escherichia coli) treatment. Reactive nitrogen intermediates (RNIs) in plasma were monitored with an NO_x Analyzer, and nitric oxide (NO) production was measured as nitrite or nitrite + nitrate accumulation in perfusates of the perfused liver, and in supematants of the freshly isolated hepatic cells after incubation for 3 hr in Hank's balanced salt solution buffer containing 1 Mm l -arginine. RNI concentration in plasma of control rats was 32.0 ± 3.4 μm (mean ×se ). Livers from diet-fed control rats produced RNIs at the barely detectable rate of 7.8 ± 1.5 nmol/hr × g wet liver. Six hr after administration of LPS (1 mg/kg, iv), plasma RNI levels in diet-fed control rats increased to 426.9 ± 29.4 μm , and RNI release from the perfused liver was also markedly elevated to 97.7 ± 7.7 nmol/hr × wet g liver, indicating hepatic NO release as a potentially important source for the increased RNI in plasma. The presence of N^G-monomethyl-l -arginine (0.5–1 mm ) or the absence of l -arginine in the perfusate inhibited LPS-induced stimulation of RNI release. EGTA (1 mm ) had little effect, indicating that the increased RNI release was likely to be due to inducible NO synthase activity. The release of RNIs by freshly isolated Kupffer cells increased 13-fold, and this small cell mass contributed almost half of the hepatic RNI production under these conditions. Plasma ALT concentration was elevated after LPS administration, indicating incipient liver damage. Chronic alcohol administration resulted in increased RNI levels in plasma (44.6 ± 4.2) that were accompanied by increased spontaneous release in the perfused liver (16.4 ± 1.5 nmol/hr wet g). Enhanced activity of Kupffer cells was responsible for this increase. After administration of LPS, the increase in plasma RNIs (565.72 ± 49.6 μm ) was slightly higher in chronic alcoholic rats than in control animals. This was also accompanied by a somewhat higher RNI release from the perfused liver (125.40 ± 12.2 nmol/hr × wet g) in the same group. Hepatocytes were responsible for the post-LPS increase in alcoholic rats. Plasma ALT concentration was higher in alcoholic than control, diet-fed rats after LPS, indicating more liver damage in that group. The exact role of the elevated hepatic RNI release in affecting hepatic function in chronic alcoholic animals remains to be clarified.     

Preventive Effect of FK 506 (Tacrolimus Hydrate) on Experimentally Induced Acute Liver Injury in Rats

The aim of the study was to investigate the effect of the immunosuppressant FK 506 (tacrolimus hydrate) on acute liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS). Acute liver injury was induced in male Wistar rats by injecting the animals with P. acnes (10 mg/rat), and administering LPS (10 g/rat) seven days later. One group was given FK 506 (1 mg/kg) 24 and 2 hr before administration of LPS, and the other group was given the same dose of saline. The 24-hr survival rate, serum alanine aminotransferase (ALT) concentration, and tumor necrosis factor (TNF) - mRNA and protein concentrations in the liver and spleen were then compared. Hepatic macrophages were also isolated from rats seven days after P. acnes injection, LPS, and FK 506 or saline were added to the culture supernatant, and TNF- production was studied. The 24-hr survival rate was 100% in the FK 506-treated group, in contrast with 16.6% in the saline group. Four hours after LPS injection, the serum ALT concentration was 755 ± 401 in the saline group versus 119 ± 42 units/ml (P < 0.01) in the FK 506-treated group. The serum TNF- concentration was lower in the FK 506-treated group (1419 ± 957 pg/ml) than in the saline group (9205 ± 2215) (P < 0.01). The mRNA and protein concentrations in the liver and spleen in the two groups did not differ significantly 1 hr after LPS injection but were significantly lower in the FK 506-treated group after 4 hr. FK 506 did not directly inhibit TNF- production by isolated cultured hepatic macrophages. FK 506 is unable to inhibit initial TNF- production by hepatic macrophages (or probably that by splenic macrophages either) stimulated by injection of LPS in P. acnes + LPS-induced acute liver injury. However, the immunosuppressant does limit hepatic damage by inhibiting subsequent aggravation of inflammation by the cytokine network.     

Growth hormone treatment affects plasma LH pulsatile release in women with secondary amenorrhoea

OBJECTIVE Since growth hormone (GH) is administered as a co-gonadotrophic factor in ovulation induction, this study aimed to assess the action of GH on the episodic pulsatile release of LH and FSH in amenorrhoeic patients. PATIENTS AND DESIGN Nineteen patients affected by hypothalamic amenorrhoea were enrolled for this study: group A, 9 patients with normal gonadotrophins; group B, 10 patients with low gonadotrophins. Both groups were studied during GH infusion (0015 IU/min for 4 hours) and after 7 days of GH administration (0 1 IU/kg/day). Patients underwent a 4-hour pulsatility study, with blood sampling every 10 minutes. A standard GnRH test (10 μ g i.v. bolus) was performed immediately after the pulsatility evaluation. MEASUREMENTS LH and FSH were assayed with an IFMA method; oestradiol and IGF-I were assayed by RIA and IRMA, respectively. PULSE DETECTION Time series were analysed with Detect program. RESULTS All patients showed similar LH and FSH pulsatile characteristics both under baseline conditions and during GH infusion. After 7 days of GH administration, episodic FSH release showed no change in either group. On the contrary, LH pulse frequency (mean ± SE) significantly increased in group A (4 0±0 2 peaks/4h, P&kt;0 05), while putse amplitude (baseline, 3.9± 0 6 IU/I; after 7 days, 2.9±0.3 IU/I, P<0 05), and integrated LH plasma concentrations (baseline, 7.6 ±1–1 IU/I; after 7 days, 5±0 8 IU/I, P<0 05) were significantly decreased. No significant changes were observed for LH pulse frequency, amplitude or integrated LH plasma concentrations in hypogonado-trophinaemic patients (group B). Plasma oestradiol levels were significantly increased only in group A (baseline, 154 18±23 8 pmoI/I; after 7 days, 380 3±110 1 pmoI/I, P < 005), while IGF-I levels were significantly increased in both groups after 7 days of GH administration (P<0 05). No significant differences were observed in the gonadotropin responses to GnRH test before and after GH administration. CONCLUSIONS The present study showed that the administration of GH in amenorrhoeic patients determines the significant changes in episodic LH release in those subjects with normal LH plasma levels and suggests that the action of GH may be dependent upon the ovarian-pituitary feedback action.     

Preventive effects of sevoflurane treatment on lung inflammation in rats

ObjectiveTo observe the effects of sevoflurane treatment on lung inflammation in rats with lipopoIysaccharide-induced acute lung injury (ALI).MethodsThe rat model of ALI was established by intratracheal instillation of lipopolysaccharide (LPS). 45 infantile SD rats [body weight (272±15) g] were randomly divided into 3 groups (n=15): control group, LPS group, sevoflurane group. NS (1 mL/kg) was instillated in rats' airways of control group; LPS (5 mg/kg) was instillated in rats' airways of LPS group. Sevoflurane group rats received sevoflurane (2.4%) inhalation for a hour after LPS was instillated in rats' airways. Six hours after NS or LPS instillation, all rats were exsanguinated. Lung tissues were examined by HE staining. Expressions of TNF-α and ICAM1 mRNA were detected by semiquantitative RT-PCR techniques. The protein level of TNF-α and ICAM1 were assessed by western blot techniques.ResultsIn LPS group the permeability of lung tissues increased, organizational structure severely damaged and the alveolar wall tumed thick, with interstitial edema and Europhiles infiltrated increasingly. The LPS group had higher mRNA expressions of TNF-α and ICAM1 than control group and sevoflurane group (P<0.05), and LPS group had higher protein level of TNF-α and ICAM1 than control group and sevoflurane group (P<0.05).ConclusionsSevoflurane treatment can attenuate lung inflammation in rats with lipopolysaccharide-induced acute lung injury.     

Suppression of Rat Lower Extremity Postoperative Reperfusion Injury with Edaravone

In this study we evaluated whether the free radical scavenger (edaravone) could suppress rat lower extremity postoperative reperfusion injury by evaluating dose response and skeletal muscle viability. Fifteen Lewis male rats (450–570 gm) were divided into three groups by the dosage of edaravone (3.0 and 9.0 mg/kg, n = 5 each). Both common femoral arteries were clamped for 5 h and then declamped. At 5 h after reperfusion, serum creatine phosphokinase (CPK) levels were examined. The muscles of lower extremity were harvested, in order to count the numbers of viable rat muscle cells under 400 × magnifications. After 3.0 mg/kg of edaravone was given preoperatively, serum CPK levels were lower (797 ± 173 IU/ml) than the control group (1,438 ± 280 IU/ml). The mean number of rat muscle cells in the 3.0 mg/kg edaravone group was significantly greater (951 ± 168 cells/mm², p = 0.002) than both the control group (258 ± 31 cells/mm²) and 9.0 mg/kg edaravone group (390 ± 82 cells/mm²). This study suggests that the preoperative administration of “3.0 mg/kg of edaravone” could suppress postoperative reperfusion injury in a rat model. This study is partially supported by Medical Research Fund of Hyogo Medical Association 2001.     

Immunohistochemical study of tumor necrosis factor-alpha in acute liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

The effect of intravenous injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) on the distribution of tumor necrosis factor-α (TNF-α) in different organs have not previously been investigated. Immunohistochemistry and histological examination were employed in evaluating the distribution of TNF-α in the liver, spleen, lungs and bone marrow in rats injected intravenously with P. acnes followed by LPS 7 days later. Granulomas containing ED1-positive macrophages were observed in the liver 7 days after P. acnes injection. Subsequent LPS injection resulted in proliferation of ED1-positive macrophages in the sinusoids and coagulation necrosis of hepatocytes after 6 h. TNF-α was detected in ED2-positive macrophages (Kupffer cells) 1 day after P. acnes injection and in macrophages constituting the granulomas 7 days later, but prior to LPS injection. TNF-α was also detected in ED1-positive macrophages in the spleen, predominantly in the marginal zone. When granulomas were formed 7 days after P. acnes injection, TNF-α was observed in macrophages of the granulomas. TNF-α was also detected in macrophages of the granulomas found in the lung 1 day after P. acnes injection. No macrophages expressing TNF-α were found in the granulomas of bone marrow. The highest expression was in the liver at any time interval and in macrophages constituting granulomas. Our results suggest that the high expression of TNF-α in the liver results in selective hepatic necrosis. The expression of TNF-α in macrophages of the liver after P. acnes injection and the subsequent development of hepatic necrosis after LPS injection suggest that P. acnes acts as an inducer of TNF-α production in macrophages while LPS acts as a trigger for the release of TNF-α from macrophages.