Upregulation of TNF-α and IL-6 mRNA in mouse liver induced by bacille Calmette-Guerin plus lipopolysaccharide

AIM: To investigate the mechanism of immunological liver injury induced by bacille Calmette-Guerin (BCG) plus lipopolysaccharide (LPS). METHODS: Mice were injected via the tail vein with 125 mg/kg BCG, and 12 d later, the mice were injected intravenously with different doses of LPS (125, 250, or 375 microg/kg). Serum alanine aminotransferase (ALT) activity and liver pathological changes were examined. The expression of tumor necrosis factor (TNF)- alpha, interleukin (IL)-6, lipopolysaccharide binding protein (LBP) and CD14 mRNA, and NF-kappaB and IkappaB-alpha protein in mouse liver at different time points after BCG and LPS injection were measured using RT-PCR, immunohistochemistry and Western blotting analysis, respectively. RESULTS: The activity of serum ALT in mice treated with BCG and LPS was significantly increased. Different degrees of liver injury, such as inflammatory cell infiltration, spotty necrosis, piecemeal necrosis, even bridging necrosis, could be seen in liver sections from mice after BCG and LPS administration. Furthermore, the levels of TNF-alpha and IL-6 mRNA in mouse liver were significantly elevated after administration of BCG plus LPS (P<0.05). The levels of LBP and CD14 mRNA in mouse liver were markedly upregulated after treatment with BCG and LPS, and treatment with BCG alone led to an increase in CD14 mRNA in mouse liver. Finally, immunoreactivity for NF-kappaB p65 was predominantly detected in hepatocyte nuclei from mice treated with BCG plus LPS, compared with the normal group. Protein levels of IkappaB-alpha were strikingly decreased by LPS or BCG plus LPS treatment, compared with the normal group or BCG group. CONCLUSION: TNF-alpha and IL-6 mRNA were partially involved in early immunological liver injury induced by challenge with small doses of LPS after BCG priming. Upregulation of TNF-alpha and IL-6 mRNA might be related to increases in LBP and CD14 mRNA expression and activation of NF-kappaB. Furthermore, BCG priming in immunological liver injury may occur via upregulation of CD14 mRNA expression in mononuclear cell infiltration into the liver.     

来氟米特对免疫性肝损伤的影响

目的　研究来氟米特对小鼠免疫性肝损伤的影响。方法　利用BCG+LPS诱导免疫性肝损伤;分光光度法检测血中ALT ,AST和NO水平及肝匀浆MDA和GSHpx含量;ELISA法测定血中TNF-α含量;细胞增殖法测定IL-1,IL-2及ConA诱导的脾淋巴细胞增殖反应。结果　在BCG+LPS诱导的免疫性肝损伤中,来氟米特(4,12 ,36mg·kg^-1 )ig给药明显降低免疫性肝损伤小鼠增高的血浆ALT和AST活性及肝匀浆MDA含量,使降低的肝匀浆GSHpx水平增高。进一步研究发现来氟米特明显抑制免疫性肝损伤小鼠TNF-α的产生,降低增高的血清NO水平,抑制PM_Ф产生过高的IL-1,对低下的IL-2产生和ConA诱导的脾淋巴细胞增殖反应有进一步抑制作用。结论　来氟米特对免疫性肝损伤具有保护作用     

白芍总苷对卡介苗加脂多糖引起的小鼠免疫性肝损伤的保护作用

目的　研究白芍总苷对小鼠免疫性肝损伤的保护作用。方法　在建立BCG +LPS诱导免疫性肝损伤小鼠模型的基础上 ,分光光度法检测血清中ALT、AST、NO水平和肝匀浆MDA、GSH Px、SOD含量 ;放免法检测TNF α的生物学活性 ;细胞增殖法测定脾淋巴增殖反应。结果　白芍总苷 (6 0、12 0、2 4 0mg·kg-1)ig给药可明显降低免疫性肝损伤小鼠增高的血清ALT、AST活性 ,同时能减少肝匀浆MDA含量 ,使降低的肝匀浆GSH Px、SOD活性升高 ,进一步研究发现白芍总苷可明显抑制免疫性肝损伤小鼠血清NO和TNF α的产生。白芍总苷还可抑制小鼠腹腔巨噬细胞TNF α的产生 ,对ConA诱导的脾淋巴增殖反应具有恢复作用 ,而对LPS诱导的脾淋巴增殖反应无明显影响。结论　白芍总苷对免疫性肝损伤具有保护作用。     

二硫代氨基甲酸吡咯烷对小鼠免疫性肝损伤的抑制作用

目的探讨二硫代氨基甲酸吡咯烷(PDTC)对免疫性肝损伤的抑制作用及其机制。方法设正常对照、脂多糖(LPS)、卡介苗(BCG)、BCG+LPS、PDTC和BCG+PDTC+LPS组。除正常对照、LPS和PDTC组外,其余各组小鼠经尾静脉注射BCG(每只2.5mg)。10d后,LPS和BCG+LPS组分别给予LPS(0.2mg·kg-1,ip),PDTC和BCG+PDTC+LPS组在给予LPS前24和2h分别给予PDTC(100mg·kg-1,ip),对照组给予等体积生理盐水。每组15只小鼠用于观察LPS处理后72h的死亡率;每组6只小鼠于LPS处理后1.5h处死,取肝脏,用RT-PCR检测肝脏组织肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)mRNA表达水平,用凝胶电泳迁移率分析法测定肝脏核因子κB(NF-κB)结合活性;每组12只小鼠于LPS处理后6h取血,处死,留取肝脏,测定血清谷丙转氨酶(GPT)活性、一氧化氮(NO)水平和肝组织还原型谷胱甘肽(GSH)含量,制备肝组织切片进行HE染色,观察组织病理变化。结果与正常对照组比较,BCG和LPS组小鼠肝脏炎症细胞明显增加,血清GPT活性升高,肝脏GSH含量显著下降,肝脏TNF-α与IL-1β mRNA表达增强,各组均未见小鼠死亡;PDTC组除血清GPT活性升高外,上述其他指标均未发生明显改变。与BCG和LPS组比较,BCG+LPS组血清GPT活性进一步升高,并伴有大面积肝脏坏死和大量炎症细胞浸润,肝脏NF-κB结合活性显著升高,TNF-α和IL-1β表达进一步增强,GSH水平下降,血清NO水平增加,小鼠死亡率40%。与BCG+LPS组比较,PDTC预处理明显抑制BCG+LPS引起的肝脏NF-κB活性、TNF-α及IL-1β mRNA表达增强,升高肝脏GSH含量,降低血清GPT活性和NO水平,减轻BCG+LPS引起的肝脏炎症和坏死,未见小鼠死亡。结论PDTC可抑制BCG+LPS引起的小鼠免疫性肝损伤,其机制可能与其抗炎和抗氧化作用有关。     

N-乙酰半胱氨酸对小鼠免疫性肝损伤的影响

目的研究N-乙酰半胱氨酸(NAC)对卡介苗(BCG)与细菌脂多糖(LPS)引起小鼠免疫性肝损伤的影响。方法建立BCG/LPS引起的小鼠免疫性肝损伤模型。采用两种处理方式给予NAC:方式A,于LPS处理前4h和15min分别经腹腔注射给予NAC(预处理);方式B,于LPS处理后0h和4h分别经腹腔注射NAC(后处理)。LPS处理后8h剖杀动物,取血和肝脏,并检测血清丙氨酸氨基转移酶(ALT)活性与一氧化氮(NO)水平、肝脏组织谷胱甘肽(GSH)与丙二醛(MDA)含量以及肿瘤坏死因子α(TNF-α) mRNA表达水平。结果与模型组比较,NAC预处理组小鼠血清ALT活性下降,肝脏TNF-α mRNA表达明显减少,而体内NO生成和肝脏脂质过氧化水平无改变;NAC后处理组与模型组相比,小鼠死亡率升高,血清NO生成增加,肝脏GSH含量进一步下降,而小鼠血清ALT活性未见明显改变。结论NAC对小鼠免疫性肝损伤有双重效应,NAC预处理对抗BCG/LPS引起的小鼠免疫性肝脏损伤,NAC后处理加重BCG/LPS引起的氧化应激并升高动物死亡率。     

丹参多糖对小鼠免疫性肝损伤的保护作用

目的探讨丹参多糖对卡介苗（BCG）和脂多糖（LPS）诱导的小鼠免疫性肝损伤的保护作用。方法尾静脉注射BCG和LPS诱导小鼠免疫性肝损伤,对比小鼠脏器系数,检测血清谷草转氨酶（AST）、谷丙转氨酶（ALT）、一氧化氮（NO）的水平以及比较肝组织匀浆中肿瘤坏死因子α（TNF-α）、白介素1β（IL-1β）的含量。结果丹参多糖可显著改善免疫性肝损伤小鼠的胸腺、肝脏、脾脏系数,降低血清中ALT、AST、NO的活性以及肝组织匀浆中TNF-α、IL-1β的含量。结论丹参多糖对免疫性肝损伤具有显著的保护作用。     

七味净肝灵对卡介苗联合脂多糖诱导的小鼠免疫性肝损伤的保护作用及其机制

目的：研究七味净肝灵（牛大力、黑老虎、地耳草、叶下珠、虫牙药、白花蛇舌草、甘草）对卡介苗（BCG）+脂多糖（LPS）诱导的免疫性肝损伤小鼠的保护作用及其作用机制。方法：建立BCG+ LPS致小鼠免疫性肝损伤模型,取血,收集肝脏,脾脏和胰腺。生化法测定血清中ALT, AST, MDA, SOD的活性或含量,ELISA法检测肝组织中IL-4,IL-6和NO含量,Western-blot分析肝组织中TNF-α和NF-κB蛋白的表达情况;HE染色,显微镜观察肝组织病理改变。结果：七味净肝灵各剂量组可降低免疫性肝损伤小鼠血清或肝组织中ALT、AST、MDA、IL-4,IL-6和NO的活性或含量,抑制肝组织中TNF-α和NF-κB蛋白的表达,升高SOD活性;病例检查显示其各剂量组均能显著改善肝组织坏死损伤。结论：七味净肝灵对BCG+ LPS诱导的小鼠免疫性肝损伤有明显的保护作用,其机制可能通过抗脂质过氧化、抑制肝内的炎症反应及细胞毒性作用来介导保肝作用。     

Treatment with Y-40138, a multiple cytokine production modulator, inhibits lipopolysaccharide- or tumour necrosis factor-alpha-induced production of pro-inflammatory cytokines and augments interleukin-10

N-[1-(4-[4-(pyrimidin-2-yl)piperazin-1-yl]methyl phenyl)cyclopropyl] acetamide . HCl (Y-40138) suppresses liver injury in concanavalin A- and D-galactosamine/lipopolysaccharide (LPS)-induced mouse hepatitis models. However, the mechanism of action of Y-40138 has not been fully investigated. In this study, we examined the effect of Y-40138 on cytokine production in mice. Cytokine production was induced by intraperitoneal injection of LPS (0.5 mg kg(-1)) or intravenous injection of recombinant mouse tumour necrosis factor (TNF)-alpha (10 mug mouse(-1)) in BALB/c mice. TNF-alpha and interleukin (IL)-10 reached maximum levels 1.5 h after the LPS injection. IL-12 and interferon-sigma (IFN-sigma) reached maximum levels 3 to 9 h after the injection. When Y-40138 was orally administered 30 min prior to the injection, it inhibited TNF-alpha, IL-12 and IFN-sigma production and augmented IL-10 production. Y-40138 also inhibited IL-12 production and augmented IL-10 production in TNF-alpha-stimulated mice. In IL-10 knockout mice, Y-40138 inhibited TNF-alpha and IL-12 production 1.5 h after the LPS injection but not after 3 h or later, unlike in wild mice. In addition, TNF-alpha production was inhibited by Y-40138 at concentrations that could not augment IL-10 production. These data suggest that Y-40138 modulates pro-inflammatory cytokine production by both IL-10-dependent and -independent mechanisms.     

胸腺肽对免疫性肝损伤小鼠外周血T细胞亚群的影响

目的观察胸腺肽对免疫性肝损伤小鼠外周血T细胞亚群的调节作用。方法采用卡介苗联合脂多糖法建立免疫性肝损伤的小鼠模型,观察胸腺肽对免疫性肝损伤合并败血症小鼠外周血T细胞亚群的调节作用。结果免疫性肝损伤合并败血症小鼠的CD3+、CD4+和CD8+的值均明显低于正常小鼠,导致免疫功能低下,而胸腺肽对免疫性肝损伤合并败血症小鼠可有效地提高CD3+、CD4+和CD4+/CD8+的值,从而提高机体的免疫功能,增强机体的抵抗力。结论胸腺肽可通过调整CD4+和CD8+之间的平衡,有效地提高机体的免疫功能。     

丝毛飞廉总黄酮对小鼠免疫性肝损伤的保护作用

目的研究丝毛飞廉总黄酮对卡介苗(BCG)+脂多糖(LPS)致小鼠急性免疫性肝损伤的保护作用。方法小鼠尾静脉注射BCG+LPS造模后,ig给予丝毛飞廉总黄酮,测定小鼠脏器指数、血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)的含量及小鼠肝组织中超氧化物歧化酶(SOD)的活性及丙二醛(MDA)的含量。结果模型组脏器指数及ALT、AST与空白组均有显著性差异,表明模型建立成功;给药组与造模组比较,小鼠脏器指数、AST、ALT的含量、SOD的活性及MDA的含量均有显著性差异。结论丝毛飞廉总黄酮对BCG+LPS致小鼠免疫性肝损伤具有保护作用。     

Melatonin-selenium nanoparticles protects liver against immunological injury induced by bacillus Calmette-Guerin and lipopolysaccharide

AIM: Melatonin-selenium nanoparticle (MT-Se), a novel complex, was synthesized by preparing selenium nanoparticles in a melatonin medium. The present investigation was designed to determine the protective effects of MT-Se against immunological liver injury in mice induced by bacillus Calmette-Guerin (BCG)/lipopolysaccharide (LPS). METHODS: The model of immunological liver injury in mice was prepared. The levels of alanine aminotransferase, aspartate amino-transferase, nitric oxide (NO) in serum, malondialdehyde content, superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) activities in a liver homogenate were assayed by spectrophotometry. The content of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) were determined by ELISA. The splenocyte proliferation was assayed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye reduction. Meanwhile, a hepatic pathological examination was observed. Results: In the BCG/LPS-induced hepatic injury model, MT-Se administered at doses of 5, 10, or 20 mg/kg to the BCG/LPS-treated mice for 10 d significantly reduced the increase in serum aminotransferase, reduced the severe extent of hepatic cell damage and the immigration of inflammatory cells. It also attenuated the increase in the content of thiobarbituric acid-reactive substances and enhanced the decrease in activities of SOD and GSH-px. In contrast, the treatment with MT-Se suppressed the increase in NO level in both the serum and liver tissue. Furthermore, MT-Se significantly lowered an increase in TNF-alpha and IL-1beta levels in the liver and inhibited the production of TNF- alpha and IL-1beta by peritoneal macrophages. A downregulation effect of MT-Se on splenocyte proliferation was also observed. CONCLUSION: MT-Se showed a hepatic protective action on immunological liver injury in mice.     

Lipopolysaccharide-induced down-regulation of organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is independent of tumor necrosis factor-alpha, Interleukin-1beta, interleukin-6, or inducible nitric oxide synthase.

Organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is expressed almost exclusively in liver, where it mediates uptake of a variety of compounds, including bile acids, as well as other endo- and xenobiotics, across hepatic sinusoidal membranes in a Na+-independent manner. Lipopolysaccharide (LPS) has been shown to decrease Oatp4 mRNA levels in a dose- and time-dependent manner in Toll-like receptor 4 (TLR4)-normal (C3H/OuJ) mice, but not in TLR4-mutant (C3H/HeJ) mice. Moreover, after LPS administration, serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) are markedly lower in TLR4-mutant mice than in TLR4-normal mice. Thus, TLR4 is considered an upstream mediator of LPS-induced decrease in mouse Oatp4 mRNA. LPS is thought to alter liver gene expression through LPS-induced cytokines or nitric oxide (NO). TNF receptor p55 (TNFRp55) and type I IL-1 receptor (IL-1RI) mediate the biological functions of TNF-alpha and IL-1beta, respectively. Therefore, to determine whether endogenous cytokines or NO are mediators of LPS-induced down-regulation of Oatp4, Oatp4 mRNA levels were determined in mice deficient in the TNFRp55, IL-1RI, IL-6, or inducible nitric oxide synthase (iNOS) after LPS administration. Mice homozygous for a targeted deletion of genes for TNFRp55, IL-1RI, IL-6, or iNOS exhibited similar decreases in Oatp4 mRNA levels as wild-type mice after LPS administration. Moreover, in mouse hepatoma cells, treatment with TNF-alpha, IL-1beta, or IL-6 individually or in combination did not suppress activity of mouse Oatp4 promoter (-4.8 kb to +30). Therefore, LPS-induced down-regulation of Oatp4 appears to be independent of TNF-alpha, IL-1beta, IL-6, or iNOS.     

褪黑素对免疫性肝损伤小鼠自由基和细胞因子的影响

目的　研究褪黑素对小鼠免疫性肝损伤的保护作用。方法　在诱导BCG +LPS免疫性肝损伤模型的基础上 ,用分光光度法检测血浆中ALT、AST、NO水平和肝匀浆MDA、SOD含量 ;L92 9细胞株法检测血浆中TNF α的生物学活性 ;胸腺细胞增殖法测定血浆中IL 1活性。结果　在BCG+LPS诱导的免疫性肝损伤中 ,褪黑素 (0 2 5 ,1,4mg·kg-1)ig预防给药均明显降低免疫性肝损伤小鼠增高的血浆ALT、AST活性 ,且以 1mg·kg-1的剂量作用最明显 ;并能减少肝匀浆MDA含量 ,使降低的肝匀浆SOD活性升高 ,抑制免疫性肝损伤小鼠血浆NO、TNF α和IL 1的升高。结论　褪黑素对小鼠免疫性肝损伤具保护作用     

二草清肝汤对免疫性肝损伤大鼠细胞因子表达的影响

目的：观察中药二草清肝汤对大鼠免疫性肝损伤的保护作用并探讨其药理学机制。方法：采用卡介苗（BCG）＋脂多糖（LPS）建立大鼠免疫性肝损伤模型。肝组织病理切片观察肝组织损伤情况;采用ELISA法检测血清IL-10及IL-12水平。结果：大鼠免疫性肝损伤时,血清IL-10及IL-12水平显著升高;中药组及西药对照组（环磷酰胺）IL-10及IL-12水平明显低于模型组（P〈0．01）,中药组和西药组肝组织病理改变较模型组明显减轻,中药组一般情况明显好于西药组。结论：二草清肝汤能降低免疫性肝损伤大鼠的血清IL-10及IL-12水平,对实验性免疫肝损伤有明显保护作用。     

Experimental study on the efficacy of Fuganling granula on protecting against immunological hepatic injury

To study the efficacy of Fuganling granula (FGL, 复肝灵颗粒) in treating mouse immunological hepatic injury that was caused by Bacille Calmette Guerin (BCG) and lipopolysaccharide (LPS), a total of 60 mice were adopted, among which, 50 mice were given intraperitoneal injection with BCG and LPS to establish an immunological liver injury model and then were randomly divided into 5 groups (10 mice/group): 4 groups received treatment of FGL orally at the doses of 100 mg/kg (high-dosage), 50 mg/kg (middle-dosage), 25 mg/kg (low-dosage) and bifendate orally at the dose of 80 mg/kg, respectively. One group was treated with distilled water orally. The remaining 10 mice were given distilled water intraperitoneally as the normal control group. The indices of thymus, liver and spleen, and the activities of the alanine aminotransferase (ALT), aspartate aminotransferase (AST) in the serum were detected. Compared to the normal rat, the model group’s thymus index decreased significantly. The liver index and spleen index increased significantly. The activities of serum ALT and AST increased significantly (all P<0.01). Compared to the model control group, the group treated with FGL in high-dosage, middledosage or low-dosage can decrease the activities of ALT and AST and the group treated with FGL in high-dosage and middle-dosage can increase the thymus index significantly (P<0.01). This experiment established the immunological liver injury model successfully and found that FGL has a remarkably protective effect on this kind of immunological hepatic injury.     

白芍总苷在免疫性肝损伤大鼠体内的药代动力学研究

目的比较白芍总苷(TGP)在免疫性肝损伤大鼠和正常大鼠体内药动学参数的异同,为临床制定给药方案提供参考。方法采用猪血清腹腔注射法建立大鼠免疫性肝损伤模型,以HPLC法测定模型大鼠和正常大鼠灌胃给予3个剂量白芍总苷后15、30、60、90、120、150、180、240、360、480、720min血浆中芍药苷和芍药内酯苷浓度,根据药时曲线计算药动学参数,采用SPSS 11.5软件分析各组各剂量间药动学参数异同。结果与正常组相比,模型组大鼠体内白芍总苷的Cmax、AUC0-t和AUC0-∞明显增大,Tmax明显提前,T12明显延长;各剂量间白芍总苷Tmax和T21没有差异,剂量与Cmax、AUC0-t和AUC0-∞有一定的相关性。结论肝损伤大鼠对TGP的吸收速度较正常大鼠快,吸收量较大,消除较慢,提示临床要针对不同的机体状态,设计合理安全的剂量,以免给药量过大引起蓄积和毒性反应。     

Effect of amodiaquine, a histamine N-methyltransferase inhibitor, on, Propionibacterium acnes and lipopolysaccharide-induced hepatitis in mice

We examined whether treatment with amodiaquine, a potent inhibitor of histamine N-methyltransferase protects mice from Propionibacterium acnes (P. acnes)-primed and lipopolysaccharide (LPS)-induced hepatitis. The subcutaneous injection of amodiaquine (2 and 5 mg/kg) significantly increased the histamine levels in the liver in comparison to saline treated mice. Pretreatment with amodiaquine also improved the survival rate of the hepatitis mice, and this improvement was partially associated with the decrease in serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Amodiaquine partially suppressed increases of tumor necrosis factor (TNF)-alpha in the serum and TNF-alpha mRNA expression in the liver, whereas the expression of interleukin (IL)-18, interferon (IFN)-gamma and IL-12 in the liver was not changed by amodiaquine treatment. In conclusion, the present findings suggested that the elevation of endogenous histamine by amodiaquine may thus play a protective role through the regulation of TNF-alpha production in endotoxin-induced hepatic injury mice.