酮替芬对小鼠病毒性心肌炎的作用研究

目的研究肥大细胞膜稳定剂酮替芬对脑心肌炎病毒（EMCV）心肌炎的作用。方法4周龄DBA／2小鼠经腹腔接种EMCV制成病毒性心肌炎模型,评价灌胃给予酮替芬对小鼠病毒性心肌炎的作用。观察小鼠在接种EMCV后14d生存率的变化。小鼠心肌切片HE、Masson trichrome染色,观察心室肌坏死和炎症细胞浸润面积的变化。荧光定量RT—PCR检测小鼠心脏促炎细胞因子（IL-6、TNF—α）和肥大细胞蛋白酶类胰蛋白酶及糜蛋白酶（mMCP-4、mMCP-5、mMCP-6）mRNA表达。结果酮替芬治疗使小鼠的14d生存率明显改善（75％比25％,P〈0．01）;小鼠心脏／体重比值显著改善（P〈0．05）;小鼠左心室坏死和炎症细胞浸润面积明显减少（P〈0．05）;IL-6、TNF—α、mMCP－4、mMCP－5及mMCP－6mRNA的表达明显下调（P〈0．05）。结论酮替芬可改善EMCV心肌炎小鼠的心肌炎症和生存率,其作用机制可能与下词心肌促炎细胞因子和肥大细胞特异蛋白酶类胰蛋白酶和糜蛋白酶的表达有关。     

Calpastatin减轻病毒性心肌炎心脏炎症损伤的作用

目的探讨钙蛋白酶抑制蛋白(calpastatin)对柯萨奇B组病毒3型(coxsackievirus B3, CVB3)诱导的病毒性心肌炎小鼠心脏炎症损伤的作用。方法取全身过表达calpastatin的转基因小鼠(Tg-CAST)及野生型小鼠(WT)腹腔注射CVB3,感染病毒7 d后建立病毒性心肌炎模型。分为对照组、Tg-CAST组、WT病毒性心肌炎组、Tg-CAST病毒性心肌炎组。留取小鼠心肌、血清及脾脏组织标本,分别行组织病理学检查以及炎症损伤相关因子检测。结果①WT病毒性心肌炎小鼠心肌组织中炎症浸润面积、病毒包膜蛋白VP-I及血清cTnI水平均明显高于对照组(P0.001)。与WT病毒性心肌炎小鼠相比,Tg-CAST病毒性心肌炎小鼠心肌炎症浸润面积、VP-1及cTnl水平均明显降低(P0.05)。②WT病毒性心肌炎小鼠心肌、脾脏与血清中炎症因子HMGB1、TNF-α、IL-17水平及心肌β-NF-κB表达显著升高(P0.01),而Tg-CAST病毒性心肌炎小鼠相应指标均明显低于WT病毒性心肌炎小鼠(P0.05)。结论 calpastatin可能通过抑制NF-KB通路活化降低病毒性心肌炎小鼠HMGB1、TNF-α、IL-17水平,进而改善心脏炎症损伤。     

病毒性心肌炎肿瘤坏死因子-α白介素-2及白介素-1β的检测及其意义

目的探讨病毒性心肌炎患者血清肿瘤坏死因子-α(TNF-α)、白介素-2(IL-2)、白介素-1β(IL-1β)的变化及临床意义。方法采用酶联免疫吸附试验(ELISA)法测定2004年中山大学第三附属医院及第一附属医院心脏内科45例病毒性心肌炎患者血清TNF-α、IL-2、IL-1β,并与正常对照组35名进行比较。结果病毒性心肌炎患者急性期TNF-α、IL-2、IL-1β明显高于恢复期及正常对照组(P<0.01);病情越重,血清TNF-α、IL-2、IL-1β越高;并与磷酸肌酸激酶同工酶(CK-MB),乳酸脱氢酶同工酶(LDH1)呈明显的正相关;恢复期血清TNF-α、IL-2、IL-1β与正常对照组比较差异无显著性意义(P>0.05)。结论病毒性心肌炎患者存在细胞免疫功能紊乱,TNF-α、IL-2、IL-1β增高可能与其发生、发展有关,并可作为病情判断及疗效的观察指标。     

病毒性心肌炎中肿瘤坏死因子-α与心室重构的关系

目的:探讨肿瘤坏死因子(TNF)-α与CVB3m重复感染导致的病毒性心肌炎的心室重构的关系。方法:连续4次注射CVB3m病毒液感染小鼠,分别于末次感染后5、10、306、0 d处死动物,Western Blot法测定心肌中基质金属蛋白酶(MMP)和组织型MMP抑制剂(TIMP)的蛋白含量;ELISA法检查血清中TNF-α水平;氯胺T氧化法检测心肌中胶原蛋白的含量。结果:心肌中的MMP-1和TIMP-1蛋白水平和血清中TNF-α变化趋势一致,即先升高,然后下降,但TIMP-1的变化稍延迟。心肌中的胶原蛋白水平则与MMP-1和TIMP-1蛋白含量比值呈负相关(r=-0.9903,P<0.01)。结论:CVB3m重复感染可导致血清中TNF-α水平持续升高,增加的TNF-α与MMP-1和TIMP-1的高水平表达有关,TNF-α与病毒重复感染导致的心室重构关系密切。     

老年慢性心力衰竭血清标志物TNF-α和MMP-9的检测及对预后判断的价值

目的:观察老年慢性心力衰竭(CHF)血清标志物肿瘤坏死因子-α(TNF-α)和基质金属蛋白酶(MMP-9)的表达及对预后判断的价值。方法:将我院收治的97例老年CHF患者作为观察组,选择同期体检的30例健康者作为对照组,观察两组患者TNF-α、MMP-9及白细胞介素(IL)-6等血清表达水平,同时观察心力衰竭(心衰)严重程度及患者预后情况,并分析其相关性。结果:观察组TNF-α、MMP-9及IL-6等血清表达水平显著高于对照组(P0.05);观察组患者心功能等级与TNF-α、MMP-9及IL-6等血清表达水平呈显著正相关(P0.05),与预后改善程度也呈显著正相关(P0.05)。结论:CHF血清标志物TNF-α和MMP-9的表达水平与心功能等级及预后显著相关,对于判断病情及预后有重要的临床价值。     

卡维地洛治疗对病毒性心肌炎小鼠心肌组织中细胞因子影响的分析

目的探讨卡维地洛治疗对病毒性心肌炎小鼠心肌组织中细胞因子的影响。方法雄性4周龄BALB/c小鼠75只,随机分为对照组、心肌炎组、心肌炎+卡维地洛治疗组,每组各25只。将存活小鼠取血后处死并留取心脏标本。光镜观察各组小鼠心肌组织病理改变情况,采用高效液相色谱-电化学法检测血浆去甲肾上腺素含量,荧光定量实验检查心肌组织中白介素-6(IL-6)m RNA表达,Western blot检测心肌组织中IL-6蛋白表达水平。结果在治疗后的第7 d与第14 d,心肌炎组小鼠的心脏重量(HW)/身体重量(BW)比值、血浆去甲肾上腺素浓度明显高于对照组,有明显统计学差异(P0.05),卡维地洛组小鼠的HW/BW比值、血浆去甲肾上腺素浓度明显低于心肌炎组,有明显统计学差异(P0.05);对照组小鼠未见异常病理变化,而在治疗后的第7 d与第14 d,卡维地洛组小鼠的心肌炎症浸润、心肌细胞变性坏死较心肌炎组小鼠明显减轻,其组织评分也低于心肌炎组,有显著统计学差异(P0.05);在治疗后的第7 d心肌炎组小鼠的心肌组织IL-6 m RNA和蛋白的表达明显高于对照组小鼠,有明显统计学差异(P0.05),卡维地洛组的小鼠的心肌组织IL-6 m RNA和蛋白的表达较心肌炎组明显降低,有明显统计学差异(P0.05),在治疗后的14 d,三组小鼠的心肌组织IL-6 m RNA和蛋白的表达无明显统计学差异(P0.05)。结论卡维地洛可以抑制病毒性心肌炎小鼠IL-6的表达,保护心肌组织,具有较好的疗效。     

MMP-9在心肌缺血再灌注中的作用和机制研究

目的探讨血清基质金属蛋白酶-9(MMP-9)在心肌缺血再灌注中的表达和作用机制。方法建立大鼠急性心肌缺血再灌注模型,构建MMP-9-siRNA慢病毒,并分为对照组、缺血再灌注(I/R)组、I/R+control-siRNA组、I/R+MMP-9-siRNA组。观察并记录术后28 d各组大鼠生存情况,Western Blot检测在大鼠I/R术后1 d,7 d,14 d,28 d心肌中MMP-9的表达;M型超声评价各组大鼠心功能改变;Real time PCR法检测干扰素-γ(IFN-γ)、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的变化。结果I/R+MMP-9-siRNA组大鼠生存率达80%,生存率显著提高,与I/R组比较,具有统计学意义(P0.05)。I/R组和I/R+control-siRNA组左室收缩末期内径(LVESD)、左室舒张末期内径(LVEDD)以及左心室质量指数(LVMI)增加(P0.05),对I/R组大鼠进行MMP-9-siRNA干预后,可见3个指标减少,与I/R组比较,差异具有统计学意义(P0.05)。I/R术后,MMP-9蛋白表达明显增加,与术后1 d比较,差异具有统计学意义(P0.05)。当缺血再灌注时IFN-γ、IL-6、TNF-α急剧增加(P0.05),MMP-9-siRNA干预后,可见IFN-γ、IL-6、TNF-α不同程度的减低,与I/R组大鼠比较,差异具有统计学意义(P0.05)。结论以MMP-9-siRNA作为靶标可减轻缺血再灌注损伤和心室重构,改善心室功能。     

白藜芦醇对脑组织缺血再灌注大鼠肿瘤坏死因子-α、白介素-6、基质金属蛋白酶9及白介素-1β水平影响

目的探讨白藜芦醇(Res)对脑组织缺血再灌注损伤的影响,以及其对炎症因子表达水平的影响。方法选取健康成年SPF级SD大鼠30只,体重200～220 g,按随机数表法分为三组:假手术组(Sham组)、脑组织缺血再灌注组(MI/R组)、脑组织缺血再灌注+白藜芦醇组(MI/R+Res组)。采用改良线栓法建立大鼠大脑中动脉栓塞模型,建模前连续7 d予MI/R+Res组腹腔注射Res 30 mg/kg,余两组腹腔注射等剂量的0.9%氯化钠溶液。采用酶联免疫吸附法(ELISA)检测三组血清中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、基质金属蛋白酶9(MMP-9)及白介素-1β(IL-1β)的水平;提取缺血脑组织,通过逆转录-聚合酶链式反应(RT-PCR)测TNF-α、IL-6、MMP-9 mRNA的水平。结果与Sham组相比,MI/R组及MI/R+Res组大鼠血清炎症因子TNF-α、IL-6、MMP-9及IL-1β的水平明显增加(P 0.05);与MI/R组相比,MI/R+Res组TNF-α、IL-6、MMP-9及IL-1β的水平显著减少(P 0.05);与Sham组相比,MI/R组及MI/R+Res组缺血脑组织TNF-α、IL-6、MMP-9 mRNA的表达明显增加(P 0.05);与MI/R组相比,MI/R+Res组TNF-α、IL-6、MMP-9 mRNA的表达显著降低(P 0.05)。结论 Res可以降低脑组织缺血再灌注大鼠炎症因子的表达水平,减轻炎症反应。     

COPD稳定期患者血清IL-6、TNF-α、MMP-9水平与BODE指数的相关性研究

目的 探讨COPD稳定期患者血清IL-6、肿瘤坏死因子α(TNF-a)、基质金属蛋白酶9(MMP-9)与BODE指数(B为体质量指数、O为气流阻塞、D为呼吸困难、E为运动能力)的关系.方法 45例符合纳入标准的COPD稳定期患者作为观察组,25例体检健康者作为对照组,测定2组血清IL-6、TNF-α、MMP-9水平,评价观察组患者BODE指数.结果 与对照组相比,COPD稳定期组患者血清IL-6、TNF-α、MMP-9水平均显著高于对照组(t值分别为3.341、3.922、2.345,P值均＜0.05);COPD稳定期患者血清IL-6、TNF-α、MMP-9水平与BODE指数呈显著正相关(r值分别为0.808、0.567、0.568,P值均＜0.05).结论 COPD稳定期患者血清IL-6、TNF-α、MMP-9仍维持较高水平,且与患者BODE指数相关,可作为预测COPD患者病情的有效指标.     

葛根素对Lewis肺癌荷瘤小鼠的作用及机制

目的探讨葛根素对Lewis肺癌荷瘤小鼠的作用机制。方法建立Lewis肺癌荷瘤小鼠模型,分为模型组、环磷酰胺组、低、中、高剂量葛根素组,计算原发瘤抑制率及肺转移灶数;通过对脾脏指数、胸腺指数及TNF-α、IL-2含量的测定,评价其对免疫功能的影响;免疫印迹法检测原发瘤组织基质金属蛋白酶(MMP)-2,9的表达。结果同模型组比较,葛根素可以显著抑制原发瘤的生长及肺转移灶数(P<0.05或P<0.01);葛根素可以增加荷瘤小鼠的脾脏指数、胸腺指数及TNF-α、IL-2含量,与模型组相比具有统计学差异(P<0.05或P<0.01);葛根素各剂量组MMP-2、MMP-9蛋白表达水平与模型组相比均显著降低(P<0.05或P<0.01)。结论葛根素能明显抑制小鼠Lewis肺癌原发瘤的生长和转移,可能与其增强免疫功能、抑制肿瘤组织MMP-2、MMP-9蛋白表达有关。     

Role of substance P in viral myocarditis in mice

Substance P (SP) is widely expressed in the central nervous system and in peripheral tissues such as myocardial nerves. We examined SP in viral myocarditis in mice induced by encephalomyocarditis virus (EMCV). Localization of SP in the hearts was examined immunohistochemically, and concentrations of SP in hearts and sera were measured by enzyme immunoassay. Substance P levels and density of SP-containing cells in murine hearts on day 6 after EMCV inoculation were decreased compared with those in normal controls. There was a negative correlation between SP levels in the hearts and ratio of heart weight to body weight of the mice at 6 days. Circulating SP levels were decreased in mice on day 6 after EMCV inoculation, and further decreased on day 14. Substance P in hearts and sera is decreased in viral myocarditis in mice, suggesting that SP may play a role in the pathogenesis of viral myocarditis, and that interaction of the neuropeptide nervous system and mast-cell immune system is important in the pathogenesis of viral myocarditis.     

High doses of digitalis increase the myocardial production of proinflammatory cytokines and worsen myocardial injury in viral myocarditis: a possible mechanism of digitalis toxicity

Results of recent studies suggest that proinflammatory cytokines cause myocardial contractile dysfunction, and that the drugs used to treat heart failure modulate the production of cytokines. This study was designed to examine the effects of digoxin in a murine model of heart failure induced by viral myocarditis. Four-week-old inbred DBA/2 mice were inoculated intraperitoneally with encephalomyocarditis virus (EMCV). Digoxin was given orally in doses of 0.1, 1 or 10 mg/kg daily from the day of virus inoculation. Interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha production in the heart were measured on day 5 after EMCV inoculation by enzyme-linked immunosorbent assay. The 14-day mortality tended to be increased in mice treated with 1 mg/kg, and was significantly increased in the group treated with 10 mg/kg per day. Myocardial necrosis and cellular infiltration on day 6 were significantly more severe in the high-dose digoxin group than in the control group. In the animals treated with 1 mg/kg digoxin, IL-1beta was significantly higher than in the control group. Intracardiac TNF-alpha levels were increased in a dose-dependent manner. These results suggest that digoxin worsens viral myocarditis, and that its use in high doses should be avoided in patients suffering from heart failure due to viral myocarditis.     

Increased severity of viral myocarditis in mice lacking lymphocyte maturation

The role of lymphocytes in the pathogenesis of viral myocarditis is controversial. To better understand how lymphocyte maturation controls a virus-induced myocarditic process, a murine model of viral myocarditis was utilized. Encephalomyocarditis virus (EMCV) was inoculated intraperitoneally into three kinds of mice; virus-susceptible C57BL/6, virus-resistant 129/SV and recombination activity gene (RAG)-2 knockout 129/SV mice. The RAG2 participate in the maturity of T and B lymphocytes. Survival rate, heart weight (HW), HW to body weight (BW) ratio, viral genome, cardiac inflammation and myocardial necrosis were evaluated after EMCV (500 plaque forming unit/mouse) inoculation. On post-inoculation day 10, the survival rate of C57BL/6, 129/SV and RAG2 knockout mice were 42, 90 and 0%, respectively. Myocardial viral titer was significantly (P<0.05) higher in C57BL/6 and RAG2 knockout mice than in 129/SV mice. In situ hybridization demonstrated the EMCV genome in the myocardium of RAG2 knockout and C57BL/6 mice, but not in 129/SV mice. At day 8, HW and HW/BW ratios were elevated (P<0.05) in RAG2 knockout mice as well as C57BL/6 mice compared with 129/SV mice. Myocardial necroses were more severe in RAG2 knockout mice than in wild-type 129/SV mice. In conclusion, matured lymphocytes protect the development of viral myocarditis which includes viral replication and myocardial apoptosis.     

Inhibitory effect of oxymatrine on serum hepatitis B virus DNA in HBV transgenic mice

AIM: To study the inhibitory effect of oxymatrine on serum hepatitis B virus (HBV) DNA in HBV transgenic mice. METHODS: HBV transgenic mice model was established by microinjection, and identified by HBV DNA integration and replication. Transgenic mice with replicating HBV were divided into 3 groups, and injected with normal saline (group A, n=9), 50 mg/kg (group B, n=8) and 100 mg/kg (group C, n=9) oxymatrine intraperitoneally once a day for 30 d, respectively. Quantitation of serum HBV DNA in HBV transgenic mice was performed by competitive polymerase chain reaction (PCR) in combination with DNA hybridization quantitative detection technique before and after treatment. RESULTS: Compared with pre-treatment, the serum HBV DNA in group A (F=1.04, P=0.9612) and group B (F=1.13, P=0.8739) had no changes after treatment. However, in group C serum HBV DNA was significantly decreased (F=13.97, P=0.0012). The serum HBV DNA after treatment was lower in group C than in groups B and A (F=8.65, P=0.0068; F=12.35, P=0.0018; respectively). The serum HBV DNA after treatment was lower in group B than in group A, but there was no statistical significance (F=1.43, P=0.652). CONCLUSION: Oxymatrine has inhibitory effects on serum HBV DNA in HBV transgenic mice.     

Injection of mice with antibody to interferon renders peritoneal macrophages permissive for vesicular stomatitis virus and encephalomyocarditis virus.

Vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) multiply in only a small percentage of peritoneal macrophages freshly explanted from 4- to 6-week-old male or female DBA/2, BALB/c, C3H, C57BL/6, or Swiss mice. However, when these mice were injected intraperitoneally with potent sheep (or goat) anti-mouse interferon alpha/beta globulin 4 days prior to harvesting peritoneal macrophages, the viruses multiplied to high titers and most of the cells were infected, as determined by total virus yield (VSV and EMCV), percentage of VSV antigen-positive cells (immunofluorescence), and determination of VSV infectious centers. This effect was not observed when mice were inoculated with other sheep hyperimmune or normal serum globulins. Anti-interferon globulin appeared to act in vivo because incubation of this globulin with peritoneal macrophages during the period of cell attachment or during the 18 hr after virus absorption did not render these cells permissive for VSV. Injection of mice with anti-interferon globulin did not affect the binding and uptake of labeled VSV by peritoneal macrophages. Although the underlying mechanism of this phenomenon is unknown, the results suggest that there may be low levels of endogenous interferon that contribute to host defense by maintaining some cells in an antiviral state.     

Synergistic infection with murine cytomegalovirus and Pseudomonas aeruginosa in mice.

A previous report from this laboratory demonstrated that mice infected intraperitoneally with a 0--20% lethal inoculum of murine cytomegalovirus (CMV) exhibited markedly enhanced mortality rates (90%--100%) within 48 hr after an intravenous injection of a 0--20% lethal inoculum of Pseudomonas aeruginosa. The current study demonstrated that mice infected with murine CMV alone had high titers of virus in multiple organs over a 20-day period, whereas mice injected with P. aeruginosa alone had a self-limited infection confined to the kidney. In the combined murine CMV-P. aeruginosa infection, titers of virus in tissues were changed very little. In contrast, P. aeruginosa was recovered in high concentrations from multiple organs, a finding which demonstrated a progressive systemic infection closely resembling that produced by a 100% lethal inoculum of P. aeruginosa alone in normal mice. An increased mortality rate due to P. aeruginosa in murine CMV-infected mice was dependent on inoculation of live bacteria and could not be explained by enhanced susceptibility to endotoxin. These results indicate that murine CMF markedly enhanced the suceptibility of mice to infection with P. aeruginosa and suggest that the virus altered mechanisms of host resistance important in recovery from bacterial infections.     

Role of NF-κB and cytokine in experimental cancer cachexia

AIM: To assess the putative involvement of NF-κB and proinflammatory cytokines in the pathogenesis of cancer cachexia and the therapeutic efficacy of indomethacin (IND)on cachexia.METHODS: Thirty young male BABL/c mice were divided randomly into five groups: (a) control, (b) tumor-bearing murine were inoculated subcutaneously to induce cachexia.Saline and IND were given intraperitoneally daily for 7 days from the onset of cachexia to sacrifice. Food intake and body composition were documented, serum levels of TNFα and IL-6 and activity of NF-κB in the spleen were investigated in all animals.RESULTS: Weight loss was observed in all tumor-bearing mice. By day 16, body weights of non-tumor mice were about 72 % of healthy controls (P＜0.01), and the weight of gastrocnemius was decreased by 28.7 % (P＜0.01). No difference was found between groups in food intake (P＞0.05).Gastrocnemius weight was increased markedly (P＜0.01)body weights were not significantly elevated. Tumor-bearing caused a 2-3 fold increase in serum levels of both TNF-αand IL-6 (P＜0.01). The concentration of TNF-α (P＜0.05)and IL-6 (P＜0.01) in tumor-bearing mice was reduced after of IL-6 was slightly elevated following treatment of IND 2.0tumor-bearing mice in comparison with controls in electrophoretic mobility shift assay (ENSA). NF-κB activity a higher NF-κB activity was observed in mice treated with CONCLUSION: Colon 26 adenocarcinoma cells can induce severe cancer cachexia experimentally, and the mechanism may be partially due to the enhanced TNF-αand IL-6 in tumor-bearing animals, which is controlled by NF-κB. Low dose of indomethacin alleviates the cachexia,decreases the activation of NF-κB and the serum levels of TNF-α and IL-6, and prevents body weight loss and muscle atrophy, while no further effect is gained by a higher dosage.     

Murine glutathione S-transferase A1-1 in sickle transgenic mice

Patients with sickle cell anemia exhibit mild to moderate renal and liver damage. Glutathione S-transferase A1-1 is produced during kidney and liver damage. We hypothesized that cellular damage in sickle transgenic mice would lead to increased serum and urine murine glutathione S-transferase A1-1 levels. Levels of murine glutathione S-transferase A1-1 in the serum and urine of S+S-Antilles, NY1DD, and control mice were measured by ELISA, which revealed that the serum of S+S-Antilles mice, relative to controls, had elevated levels of murine glutathione S-transferase A1-1 (P = 0.005) as did NY1DD mice (P = 0.02, baseline vs. 2-day hypoxia). Serum liver enzymes, such as aspartate amino transferase and alanine amino transferase, as well as lactate dehydrogenase were increased in S+S-Antilles mice relative to controls (P = 0.000006, P = 0.0003, and P = 0.029, respectively). Urine murine glutathione S-transferase A1-1 of S+S-Antilles mice, as well as NY1DD mice under hypoxic stress, was not significantly different from controls. Murine glutathione S-transferase class-mu was measured by ELISA in the urine of sickle transgenic mice and control mice to define the location of tubular damage at the proximal convoluted tubule; murine Glutathione S-transferase class-mu was below the limit of detection. These findings suggest that elevated levels of murine glutathione S-transferase A1-1 in the serum reflect release during liver damage and that proximal tubular damage does not lead to appreciable urinary murine glutathione S-transferase A1-1.