清解化攻方调控NLRP3/TLR4/NF-κB信号通路对重症急性胰腺炎小鼠模型胰腺组织的保护作用

目的观察清解化攻方对重症急性胰腺炎(SAP)小鼠模型的治疗作用,探索清解化攻方抗炎症反应的作用机制。方法将36只C57BL/6J雄性小鼠随机分成空白组,模型组,清解化攻方低、中、高剂量组,西药组(乌司他丁),每组6只,除空白组小鼠,余各组小鼠采用逆行胰胆管注射5%牛黄胆酸钠建立SAP模型,清解化攻方低、中、高剂量组在造模后分别予以清解化攻方1、2、4 g/kg灌胃,西药组在造模后予以腹腔注射乌司他丁(5×10^(4) U/kg),共干预7 d。采用苏木素-伊红染色观察胰腺组织病理改变;酶联免疫吸附测定法(ELISA)检测小鼠α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α水平;RTqPCR检测胰腺组织NOD样受体蛋白3(NLRP3)、Toll样受体4(TLR4)、核因子-κB(NF-κB)mRNA表达水平;免疫组化检测胰腺组织NLRP3、TLR4、NF-κB的阳性表达率;Western Blot技术检测NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白的表达水平。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果与空白组相比,模型组小鼠胰腺组织结构弥漫性破坏、胰腺小叶间隔局灶性扩张、腺泡萎缩和大量炎症细胞浸润,α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α含量明显升高(P值均<0.05),NLRP3、TLR4、NF-κB mRNA表达水平及阳性表达率均明显上升(P值均<0.05),NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白表达均明显上调(P值均<0.05)。与模型组相比,清解化攻方各剂量组和西药组可见小鼠胰腺组织结构稍紧密、完整,胰腺腺泡细胞排列有序,伴少量炎症细胞浸润和胰腺小叶出血灶,α-淀粉酶、脂肪酶、IL-1β、IL-6、IL-8、IL-18和TNF-α含量明显下降(P值均<0.05),NLRP3、TLR4、NF-κB mRNA表达水平及阳性表达率均明显降低(P值均<0.05),NLRP3、TLR4、NF-κB、IL-1β、IL-6蛋白表达水平均明显减弱(P值均<0.05)。结论清解化攻方可能通过抑制NLRP3/TLR4/NF-κB信号通路相关蛋白的激活,减少炎症介质的释放,防止炎症级联反应增强,进而对SAP小鼠胰腺组织发挥保护作用。

4-硝基喹啉-1-氧化物诱导C57BL/6小鼠舌黏膜癌变的研究

目的用4-硝基喹啉-1-氧化物（4NQO）诱导C57BL/6小鼠舌黏膜癌变。方法将85只小鼠随机分为蒸馏水对照组（n=5）、1,2丙二醇对照组（n=5）、实验组（n=75）,分别给予蒸馏水、1,2丙二醇及4NQO溶液饮用。实验组小鼠再分为15笼,每2周处死一笼,对照组小鼠于28周处死。对小鼠进行称质量和大体观察及组织病理学检查。结果实验组死亡1只。实验组小鼠体质量在24周后体质量较对照组明显降低（P〈0.05）。实验动物的舌、口底、上颚、颊部共出现79处肉眼可见的病变,其中舌部病变70处（88.6%）。实验组动物在不同时间点黏膜病变不同,28周时均发生高分化鳞状细胞癌。对照组未见病变。结论舌癌动物模型被成功建立。实验12~16周及20~28周可作为研究舌黏膜癌变早期和中晚期阶段的时间点。

CTLA-4Ig治疗C57BL/6小鼠自身免疫性肝炎的实验研究

目的研究 CTL A- 4Ig在治疗 C5 7BL/ 6小鼠自身免疫性肝炎上的作用。方法通过用 C5 7BL/ 6近交系小鼠的肝特异性抗原与弗氏完全佐剂混合物免疫攻击小鼠 ,随后用 CTL A- 4Ig治疗 ,观察小鼠的临床经过、血生化、肝脏组织学改变。结果随着免疫次数的增多 ,治疗组临床经过、血生化、肝脏组织学改变逐步与正常对照组相似 ,而病理模型组与前两组有明显差异 :血生化可见天冬氨酸氨基转移酶、球蛋白显著升高 ;肝组织学检测可见炎细胞的浸润 ,肝细胞的肿胀、灶性坏死甚至广泛坏死 ;肝组织免疫荧光检测提示有大量免疫球蛋白沉着。结论 CTL A- 4Ig能有效地治疗 C5 7BL /

RS4型抗性淀粉对高脂饮食C57BL/6J小鼠肠绒毛形态及肠道菌群的影响

目的:探讨交联辛烯基琥珀酸酯化甘薯淀粉(CLOSA-SPS)、柠檬酸乙酰化甘薯淀粉(CAAC-SPS)及羟丙基交联甘薯淀粉(HPCL-SPS)3种RS4型抗性淀粉(RS)对高脂饮食C57BL/6J小鼠肠道微环境的影响作用。方法:72只雄性C57BL/6J小鼠随机分成基础对照组(CL,给予基础饲料)、高脂对照组(HF,给予高脂饲料)、甘薯原淀粉高脂对照组(HF-SPS,给予添加甘薯原淀粉的高脂饲料,150g/kg)及高脂饲料中添加CLOSA-SPS、CAAC-SPS、HPCL-SPS组(HF-CLOSA-SPS、HF-CAAC-SPS、HF-HPCL-SPS,各150g/kg),共6个实验组。连续饲喂12周后处死小鼠,测定其小肠各肠段绒毛高度、隐窝深度并通过PCR-DGGE技术探究这几种RS4型RS对高脂饮食小鼠肠道菌群的影响。结果:与CL组相比,HF组与HF-SPS组C57BL/6J小鼠小肠各肠段的绒毛高度减小,隐窝深度增大,绒腺比减小,且差异显著(P<0.05);而RS4型RS能不同程度使高脂饮食小鼠肠绒毛高度增大,隐窝深度减小,绒腺比增大,其中,HF-CAAC-SPS组及HF-HPCL-SPS组效果优于HF-CLOSA-SPS组,与HF组及HF-SPS组差异均显著(P<0.05)。高脂饮食会改变小鼠的肠道菌群,聚类分析表明HF组与CL组极不相似,前者多样性指数显著小于后者(P<0.05);RS4型RS组的肠道菌群更相似于CL组,且多样性指数均显著大于HF组及HF-SPS组(P<0.05)。结论:CAAC-SPS、HPCL-SPS及CLOSA-SPS这3种RS4型RS能不同程度改善高脂饮食C57BL/6J小鼠肠绒毛形态及肠道菌群,前两者效果优于第三者。

敲除SLC39A5基因对4-NQO诱发C57BL/6小鼠食管癌模型建立的影响

目的探索敲除SLC39A5基因对4-NQO诱导的C57BL/6小鼠食管癌模型的影响。方法选取10只C57BL/6野生型小鼠作为阴性对照组,饮用浓度为100μg/ml的丙二醇空白溶液;选取140只野生型小鼠与80只SLC39A5基因敲除小鼠,采用饮水法摄入诱癌剂,即浓度为100μg/ml的4-NQO(4-nitroquinoline 1-oxide),实验时间为28周,实验结束后处死三组小鼠。结果阴性对照组小鼠、野生型小鼠和SLC39A5基因敲除小鼠在实验结束时其存活率分别为100%、92.96%、91.25%;三组小鼠食管癌诱癌成功率分别为0、61.36%、28.77%,两实验组小鼠诱癌成功率差异有统计学意义(χ2=19.98,P<0.001)。结论成功建立了C57BL/6小鼠及SLC39A5基因敲除小鼠的食管癌模型,同时验证了SLC39A5基因在食管癌发生发展中的促进作用。

Vitamin D deficiency: Correlation to interleukin-17, interleukin-23 and PⅢNP in hepatitis C virus genotype 4

AIM: To assess vitamin D (Vit D) abnormalities in hepatitis C infected patients and their relationship with interleukin (IL)-17, IL-23 and N-terminal propeptide of type Ⅲ pro-collagen (PⅢNP) as immune response mediators. METHODS: The study was conducted on 50 Egyptian patients (36 male, 14 female) with hepatitis C virus (HCV) infection, who visited the Hepatology Outpatient Clinic in the Endemic Disease Hospital at Cairo University. Patients were compared with 25 ageand sexmatched healthy individuals. Inclusion criteria were based on a history of liver disease with HCV genotype 4 (HCV-4) infection (as new patients or under followup). Based on ultrasonography, patients were classified into four subgroups; 14 with bright hepatomegaly; 11 with perihepatic fibrosis; 11 with hepatic cirrhosis; and 14 with cirrhosis and hepatocellular carcinoma (HCC).Total Vit D (i.e., 25-OH-Vit D) and active Vit D [i.e., 1,25-(OH) 2 -Vit D] assays were carried out using commercial kits. IL-17, IL-23 and PⅢNP levels were assayed using enzyme linked immunosorbent assay kits, while HCV virus was measured by quantitative and qualitative polymerase chain reaction. RESULTS: Levels of Vit D and its active form were significantly lower in advanced liver disease (hepatic cirrhosis and/or carcinoma) patients, compared to those with bright hepatomegaly and perihepatic fibrosis. IL-17, IL-23 and PⅢNP levels were markedly increased in HCV patients and correlated with the progression of hepatic damage. The decrease in Vit D and active Vit D was concomitant with an increase in viral load, as well as levels of IL-17, IL-23 and PⅢNP among all subgroups of HCV-infected patients, compared to normal healthy controls. A significant negative correlation was evident between active Vit D and each of IL-17, IL-23 and PⅢNP (r = -0.679, -0.801 and -0.920 at P < 0.001, respectively). HCV-infected men and women showed no differences with respect to Vit D levels. The viral load was negatively correlated with Vit D and active Vit D (r = -0.084 and -0.846 at P < 0.001, respectively), and positively correlated with IL-17, IL-23 and PⅢNP (r = 0.951, 0.922 and 0.94 at P < 0.001, respectively). Whether the deficiency in Vit D was related to HCVinduced chronic liver disease or was a predisposing factor for a higher viral load remains to be elucidated. CONCLUSION: The negative correlations between Vit D and IL-17, IL-23 and PⅢNP highlight their involvement in the immune response in patients with HCV-4related liver diseases in Egypt.

Lactobacilli inhibit interleukin-8 production induced by Helicobacter pylori lipopolysaccharide-activated Toll-like receptor 4

AIM： To investigate the effect of Lactobacillus bulgaricus （LBG） on the Toll-like receptor 4 （TLR4） pathway and interleukin-8 （IL-8） production in SGC-7901 cells treated with Helicobacter pyloriSydney strain 1 lipopolysaccharide （HpyloriSS1-LPS）. METHODS： SGC-7901 cells were treated with HpyloriSS1-LPS in the presence or absence of pretreatment for 1 h with viable LBG or supernatant recovered from LBG culture MRS broth （LBG-s）. Cellular lysates were prepared for Western blot with anti-TLR4, anti-transforming growth factor β-activated kinase 1 （TAK1）, anti-phospho-TAK1, anti-nuclear factor κB （NF-κB）, anti-p38 mitogen-activated protein kinase （p38MAPK）, and anti-phospho-p38MAPK antibodies. The amount of IL-8 in cell culture medium was measured by ELISA. RESULTS： H pyloriSS1-LPS up-regulated the expression of TLR4, stimulated the phosphorylation of TAKI, subsequently enhanced the activation of NF- κB and the phosphorylation of p38MAPK in a time- dependent manner, leading to augmentation of IL-8 production in SGC-7901 cells. Viable LBG or LBG-s pretreatment attenuated the expression of TLR4, inhibited the phosphorylation of TAK1 and p38MAPK, prevented the activation of NF-κB, and consequently blocked IL-8 production.CONCLUSION： H py/oriSS1-LPS induces IL-8 production through activating TLR4 signaling in SGC-7901 cells and viable LBG or LBG-s prevents H pyloriSS1-LPS-mediated IL-8 production via inhibition of the TLR4 pathway.

Association between Promoter Polymorphisms of Interleukin-4 Gene and Allergic Rhinitis Risk: a Meta-analysis

Summary： The relationship of interleukin-4 （IL-4） C-33T and C-590T （C-589T） gene polymorphisms with allergic rhinitis was analyzed. Data about the case control studies of IL-4 gene promoter polymorphisms [C-33T and C-590T （C-589T）] and their association with allergic diseases and correlation between serum IL-4 levels and allergic rhinitis were retrieved. The Stata 12.0 statistical soitvcare was applied to analyze the correlation between IL-4 gene polymorphisms and allergic rhinitis. The meta-analysis result of TT/CC genotype of -590 （-589） polymorphism showed a significant association with allergic diseases [OR=1.93, 95% CI （1.61 2.31）, P=0.00]. Meta-analysis of the TT＋TC versus CC genotype of IL-4 C-33/T polymorphism revealed significant associations with allergic diseases [OR=3.23, 95% CI （1.13-9.25）, P=0.03]. Meanwhile, there was a significant correlation between serum IL-4 levels and allergic rhinitis [OR=2.52, 95% CI-（1.80-3.23）, P=0.00]. IL-4 gene -590 TT genotype may increase the risk of allergic rhinitis and the T allele mutation of -33 might be correlated with aller- gic rhinitis.

The Relationship between Polymorphisms of Interleukin-4 Gene and Silicosis

Objective To explore the relationship between polymorphisms of interleukin-4 （IL-4） gene （-33, ＋45, intron3, ＋429, ＋448） and the susceptibility of silicosis. Methods A case-control study was carried out. 101 silicosis patients were selected as cases. As strictly matching, 121 of non silicosis workers were selected as the controls. The polymophisms of IL-4 （five locus） were detected by the method of polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） techniques. Results The GA genotype in the IL-4＋429 locus and the CC genotype in the IL-4＋448 locus were found. The frequencies ofAA, GG and AG of IL-4＋45 locus in the cases were 55.4%, 10.9%, and 33.7% and in the controls were 62.0%, 12.6%, and 26.4%. The differences between cases and controls were not significant. The frequencies of B1B1, B2B2, and B1B2 of intron3 VNTR locus in the cases were 73.3%, 1.0%, and 25.7% and in the controls were 68.6%, 1.7%, and 29.8%. The differences were not significant. The frequencies of TT, CC, and CT in -33 locus in the cases were 55.4%, 11.9%, and 32.7% and in the controls were 69.4%, 4.1%, and 26.4%. The differences were significant （P=0.034）. Conclusion The relationship between genetic polymorphism of IL-4-33 site and silicosis has been found and -33TT is a protective genotype for silicosis.

Production of interleukin-1β related to mammalian target of rapamycin/Toll-like receptor 4 signaling pathway during Aspergillus fumigatus infection of the mouse cornea

AIM：To elucidate the effect of rapamycin on regulating the production of interleukin（IL）-1β in Aspergillus fumigatus（A.fumigatus）-induced keratitis and to verify whether the expression of IL-1β in A.fumigatus keratitis is associated with the mammalian target of rapamycin（mT OR）/Toll-like receptor 4（TLR4） signaling pathway.METHODS：Fungal keratitis mouse models of susceptible C57 BL/6 mice were established using A.fumigatus.The mice were subsequently treated with rapamycin.The protein levels of p-mT OR,TLR4,and IL-1β in normal and infected corneal tissue were measured by Western blot.The TLR4 and IL-1β m RNA levels were determined by real-time polymerase chain reaction（PCR）.RESULTS：In C57 BL/6 mice,rapamycin treatment decreased the clinical scores and production of the pro-inflammatory cytokine,IL-1β.The expression of TLR4,stimulated by A.fumigatus,was reduced as well when the mT OR signaling pathway was suppressed by rapamycin.CONCLUSION：Rapamycin is beneficial for the outcome of fungal keratitis and has an inhibitory effect expression of the inflammatory cytokine IL-1β.The inhibitory effect on IL-1β expression can be associated with the mT OR/TLR4 signaling pathway in A.fumigatus infection in mice.

Interleukin-4 promotes microglial polarization toward a neuroprotective phenotype after retinal ischemia/reperfusion injury

Glaucoma results from irreversible loss of retinal ganglion cells(RGCs)through an unclear mechanism.Microglial polarization and neuroinflammation play an important role in retinal degeneration.Our study aimed to explore the function of microglial polarization during glaucoma progression and identify a strategy to alleviate retinal neuroinflammation.Retinal ischemia/reperfusion injury was induced in C57BL/6 mice.In a separate cohort of animals,interleukin(IL)-4(50 ng/mL,2μL per injection)or vehicle was intravitreally injected after retinal ischemia/reperfusion injury.RGC loss was assessed by counting cells that were positive for the RGC marker RNA binding protein,mRNA processing factor in retinal flat mounts.The expression of classically activated(M1)and alternatively activated(M2)microglial markers were assessed by quantitative reverse transcription-polymerase chain reaction,immunofluorescence,and western blotting.The results showed that progressive RGC loss was accompanied by a continuous decrease in M2 microglia during the late phase of the 28-day period after retinal ischemia/reperfusion injury.IL-4 was undetectable in the retina at all time points,and intravitreal IL-4 administration markedly improved M2 microglial marker expression and ameliorated RGC loss in the late phase post-retinal ischemia/reperfusion injury.In summary,we observed that IL-4 treatment maintained a high number of M2 microglia after RIR and promoted RGC survival.

隔河岩升船机BL4梁预应力张拉的应力应变特性

为降低隔河岩升船机BL4梁拉应力,对BL4梁采用了预应力锚索措施。为了解预应力锚索张拉时的应力分布,在BL4梁悬臂的混凝土表面粘贴应变片,进行现场监测。监测成果表明:随锚索预应力增大,梁悬臂的压应力也逐步增大,最大压应力为-5.54MPa,未出现拉应力,表明张拉效果良好。

Constituents of the anti-asthma herbal formula ASHMI^(TM) synergistically inhibit IL-4 and IL-5 secretion by murine Th2 memory cells,and eotaxin by human lung fibroblasts in vitro

OBJECTIVE： Anti-asthma herbal medicine intervention （ASHMITM）, a combination of three tradi- tional Chinese medicinal herbs developed in our laboratory, has demonstrated efficacy in both mouse models of allergic asthma, and a double-blind placebo-controlled clinical trial in patients with asthma. This study was designed to determine if the anti-inflammatory effects of individual herbal constituents of ASHMITM exhibited synergy. METHODS： Effects of ASHMI and its components aqueous extracts of Lingzhi （Ganoderma lucidum）, Kushen （Sophora flavescens） and Gancao （Glycyrrhiza uralensis）, on Th2 cytokine secretion by murine memory Th2 cells （D10.G4.1） and eotaxin-1 secretion by human lung fibroblast （HLF-1） cells were determined by measuring levels in culture supernatants by enzyme- linked immunosorbent assay. Potential synergistic effects were determined by computing interaction indices from concentration-effect curve parameters. RESULTS： Individual Lingzhi, Kushen and Gancao extracts and ASHMI （the combination of individual extracts） inhibited production of interleukin （IL）-4 and IL-5 by murine memory Th2 cells and eotaxin-1 production by HLF-1 cells. The mean 25%-inhibitory-concentration （IC2s） values （mg/mL） forASHMI, Lingzhi, Kushen and Gancao for IL-4 production were 30.9, 79.4, 123, and 64.6, respectively; for IL-5 production were 30.2, 263, 123.2 and 100, respectively; for eotaxin-1 were 13.2, 16.2, 30.2, and 25.1, respectively. The IC50values （mg/mL） for ASHMI, Lingzhi, Kushen and Gancao for IL-4 production were 158.5, 239.9, 446.7, and 281.8, respectively; for eotaxin-1 were 38.1, 33.1, 100, and 158.5, respectively. The interaction indices of ASHMI constituents at IC25 were 0.35 for IL-4, 0.21 for IL-5 and 0.59 for eotaxin-l. The interaction indices at IC^0 values were 0.50 for IL-4 and 0.62 for eotaxin-1 inhibition. Inhibition of IL-5 did not reach IC^0 values. All interaction indices were below 1 which indicated synergy. CONCLUSION： By comparing the interaction index values, we find that constituents in ASHMITM synergistically inhibited eotaxin-1 production as well as Th2 cytokine production.

Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4^(-/-) mouse model

AIM: To study the interleukin-1(IL-1) pathway as a therapeutic target for liver fibrosis in vitro and in vivo using the ATP-binding cassette transporter b4^(-/-)(Abcb4^(-/-)) mouse model.METHODS: Female and male Abcb4^(-/-) mice from 6 to 13 mo of age were analysed for the degree of cholestasis(liver serum tests), extent of liver fibrosis(hydroxyproline content and Sirius red staining) and tissue-specific activation of signalling pathways such as the IL-1 pathway [quantitative polymerase chain reaction(q PCR)]. For in vivo experiments, murine hepatic stellate cells(HSCs) were isolated via pronasecollagenase perfusion followed by density gradient centrifugation using female mice. Murine HSCs were stimulated with up to 1 ng/m L IL-1β with or without 2.5 μg/m L Anakinra, an IL-1 receptor antagonist, respectively. The proliferation of murine HSCs was assessed via the Brd U assay. The toxicity of Anakinra was evaluated via the fluorescein diacetate hydrolysis(FDH) assay. In vivo 8-wk-old Abcb4^(-/-) mice with an already fully established hepatic phenotype were treated with Anakinra(1 mg/kg body-weight daily intraperitoneally) or vehicle and liver injury and liver fibrosis were evaluated via serum tests, q PCR, hydroxyproline content and Sirius red staining. RESULTS: Liver fibrosis was less pronounced in males than in female Abcb4^(-/-) animals as defined by a lower hydroxyproline content(274 ± 64 μg/g vs 436 ± 80 μg/g liver, respectively; n = 13-15; P < 0.001; MannWhitney U-test) and lower m RNA expression of the profibrogenic tissue inhibitor of metalloproteinase-1(TIMP)(1 ± 0.41 vs 0.66 ± 0.33 fold, respectively; n = 13-15; P < 0.05; Mann-Whitney U-test). Reduced liver fibrosis was associated with significantly lower levels of F4/80 m RNA expression(1 ± 0.28 vs 0.71 ± 0.41 fold, respectively; n = 12-15; P < 0.05; Mann-Whitney U-test) and significantly lower IL-1β m RNA expression levels(1 ± 0.38 vs 0.44 ± 0.26 fold, respectively; n = 13-15; P < 0.001; Mann-Whitney U-test). No gender differences in the serum liver parameters [bilirubin; alanine aminotransferase(ALT); aspartate aminotransferase and alkaline phosphatase(AP)] were found. In vitro, the administration of IL-1β resulted in a significant increase in HSC proliferation [0.94 ± 0.72 arbitrary units(A.U.) in untreated controls, 1.12 ± 0.80 A.U. at an IL-1β concentration of 0.1 ng/m L and 1.18 ± 0.73 A.U. at an IL-1β concentration of 1 ng/m L in samples from n = 6 donor animals; P < 0.001; analyses of variance(ANOVA)]. Proliferation was reduced significantly by the addition of 2.5 μg/m L Anakinra(0.81 ± 0.60 A.U. in untreated controls, 0.92 ± 0.68 A.U. at an IL-1β concentration of 0.1 ng/m L, and 0.91 ± 0.69 A.U. at an IL-1β concentration of 1 ng/m L; in samples from n = 6 donor animals; P < 0.001; ANOVA) suggesting an anti-proliferative effect of this clinically approved IL-1 receptor antagonist. The FDH assay showed this dose to be non-toxic in HSCs. In vivo, Anakinra had no effect on the hepatic hydroxyprolinecontent, liver serum tests(ALT and AP) and profibrotic(collagen 1α1, collagen 1α2, transforming growth factor-β, and TIMP-1) and anti-fibrotic [matrix metalloproteinase 2(MMP2), MMP9 and MMP13 ] gene expression after 4 wk of treatment. Furthermore, the hepatic IL-1β and F4/80 m RNA expression levels were unaffected by Anakinra treatment.CONCLUSION: IL-1β expression is associated with the degree of liver fibrosis in Abcb4^(-/-) mice and promotes HSC proliferation. IL-1 antagonism shows antifibrotic effects in vitro but not in Abcb4^(-/-) mice.

基于实时体温监测技术探讨酵母菌、脂多糖、2,4-二硝基酚对小鼠致热作用及呼吸功能的影响

目的基于实时体温监测技术比较酵母菌、脂多糖(LPS)、2,4-二硝基酚对小鼠致热作用及呼吸功能的影响。方法SPF级C57BL/6J雌性小鼠12只,进行动物核心体温监测胶囊腹腔埋入,将小鼠按体重、体温随机分为空白对照组、酵母菌组、LPS组、2,4-二硝基酚组。小鼠分别注射相应发热介质后,每天记录动物的体重,动物核心体温监测胶囊的记录频率为:每次15 min。3 d后对小鼠进行无创呼吸监测,分别测定特殊气道阻力(sRaw)、潮气量(TV)、呼吸频率(F)和气道传导率(sGaw)。实验结束后对小鼠进行解剖,取其胸腺、脾、肺进行称重并计算脏器指数。结果实验过程中,空白对照组小鼠体重持续上升,酵母菌组、2,4-二硝基酚组小鼠体重持续下降,LPS组小鼠48 h内体重升高,48 h后体重下降。酵母菌组脾指数对比空白对照组极显著升高(P<0.01),酵母菌组、2,4-二硝基酚组的胸腺指数对比空白对照组显著降低(P<0.05)。酵母菌组小鼠体温在注射后3 h开始升高,在48 h内持续处于高温水平,在24~48 h期间体温达到最大值,与空白对照组比,差异具有极显著性(P<0.01);48~72 h期间,酵母菌组小鼠体温开始逐渐下降,与空白对照组之间差异无显著性(P>0.05)。LPS组小鼠体温在注射后4 h开始升高,高温状态维持至8 h后开始降低,但仍高于空白对照组。无创呼吸监测发现,酵母菌组、LPS组、2,4-二硝基酚组sRaw均出现升高,TV、F、sGaw均出现降低。结论从发热时长比较,酵母菌作为发热介质引起发热时间最久,其次为LPS、2,4-二硝基酚;以发热趋势比较,酵母菌表现出先降后升的状态,于注射10 h达到峰值状态,LPS表现为持续升温,于注射4 h达到峰值状态,2,4-二硝基酚表现为先降后升,于注射8 h达到峰值状态;从呼吸功能比较,小鼠注射发热介质后,均出现气道阻力增强、抑制呼吸频率、降低潮气量、气道传导减弱情况,其中对比3种发热介质对小鼠呼吸影响,酵母菌对小鼠呼吸频率、潮气量、气道阻力、气道传导作用较强。因此,酵母菌无论是发热时长、发热高峰以及对机体呼吸功能抑制作用都更显著。

The Akt/glycogen synthase kinase-3β pathway participates in the neuroprotective effect of interleukin-4 against cerebral ischemia/reperfusion injury

Interleukin-4(IL-4) has a protective effect against cerebral ischemia/reperfusion injury. Animal experiments have shown that IL-4 improves the short-and long-term prognosis of neurological function. The Akt(also called protein kinase B, PKB)/glycogen synthase kinase-3β(Akt/GSK-3β) signaling pathway is involved in oxidative stress, the inflammatory response, apoptosis, and autophagy. However, it is not yet clear whether the Akt/GSK-3β pathway participates in the neuroprotective effect of IL-4 against cerebral ischemia/reperfusion injury. In the present study, we established a cerebral ischemia/reperfusion mouse model by middle cerebral artery occlusion for 60 minutes followed by a 24-hour reperfusion. An IL-4/anti-IL-4 complex(10 μg) was intraperitoneally administered 30 minutes before surgery. We found that administration of IL-4 significantly alleviated the neurological deficits, oxidative stress, cell apoptosis, and autophagy and reduced infarct volume of the mice with cerebral ischemia/reperfusion injury 24 hours after reperfusion. Simultaneously, IL-4 activated Akt/GSK-3β signaling pathway. However, an Akt inhibitor LY294002, which was injected at 15 nmol/kg via the tail vein, attenuated the protective effects of IL-4. These findings indicate that IL-4 has a protective effect on cerebral ischemia/reperfusion injury by mitigating oxidative stress, reducing apoptosis, and inhibiting excessive autophagy, and that this mechanism may be related to activation of the Akt/GSK-3β pathway. This animal study was approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University, China(approval No. WDRY2017-K037) on March 9, 2017.

Interleukin-28 and hepatitis C virus genotype-4:Treatment-induced clearance and liver fibrosis

AIM:To investigate the association between interleukin-28B(IL28B) genotype and response to treatment and hepatic fibrosis in patients with hepatitis C virus(HCV) genotype 4.METHODS:Two hundred and one HCV-genotype 4 patients were included.All patients were treated with Peginterferon alph2a/Ribavirin for 48 wk.End of treatment response(ETR) was defined as loss of detectable serum HCV RNA at the end of treatment.Sustained viral response(SVR) was defined as loss of detectable serum HCV RNA at the end of 24 wk follow up.Genotyping of IL28B rs12979860 was performed using the TaqMan assay.We used logistic regression to estimate the adjusted odds ratio(aOR) and 95%CI.RESULTS:The study included 201 HCV-genotype 4 patients.The majority of patients were men(89.6%),with a median age of 47 years,inter-quartile range(40-51).Approximately 62.5% of patients had ETR,and 49.6% had SVR.Individuals who achieved SVR were more likely to be younger(χ 2 = 4.91,P = 0.027),and less likely to have fibrosis(χ 2 = 15.54,P < 0.0001),or inflammation(χ 2 = 7.58,P = 0.006).The genotype distribution of rs12979860 was 36.2%,49.0% and 14.8% for genotypes CC,CT,and TT,respectively.In these participants,rs12979860 genotype distribution did not differ by gender(P = 0.466),pretreatment viral load(P = 0.600),inflammation(P = 0.435),or fibrosis(P = 0.291).The frequencies of IL28B rs12979860 genotypes were TT(14.8%),CT(49.0%),and CC(36.2%).Compared to rs12979860 genotype TT,aORs(95%CI) for ETR and SVR were:CC genotype,[17.55(5.34-57.69) and 5.92(2.09-16.76),respectively];CT genotype,[5.15(1.80-14.78) and 2.48(0.94-6.52),respectively].In the current study,the patients who did not achieve ETR or SVR had a lower prevalence of rs12979860 CC(17.4% and 23.3%,respectively) than individuals who had ETR or SVR(47.9% and 47.2%,respectively).Individuals with rs12979860 CC genotype had approximately 6 times the odds of SVR compared to individuals with TT genotype(aOR = 5.92;95%CI:2.09-16.76).Similarly,patients with CT genotype had SVR more often than patients with TT genotype(aOR = 2.48;95%CI:0.94-6.52).Carrying at least one copy of the C allele(genotypes CT and CC) had almost 8 times the probability of ETR compared to those with genotype rs12979860 TT(aOR = 7.87;95%CI:2.84-21.82),and approximately 3 times the odds of SVR compared to those with genotype rs12979860 TT(aOR = 3.46;95%CI:1.37-8.74).In addition,data were consistent with a significant gene-dose relationship(aOR = 4.05/allele;95%CI:2.27-7.22).The association between rs12979860 genotype and SVR was similar among those who achieved and those who did not achieve SVR.CONCLUSION:In HCV-genotype 4 patients,rs12979860 is a sensitive predictor of viral clearance,independent of viral load,age,gender or fibrosis,with no similar relation to severity of fibrosis.

四神丸加减方对溃疡性结肠炎模型小鼠结肠组织白细胞介素-6、白细胞介素-10及TLR4/NF-κB信号通路的影响

目的:探讨四神丸加减方治疗溃疡性结肠炎的疗效机制。方法:选择30只雌性C57BL/6小鼠为实验对象,将小鼠随机分为正常组、模型组、阳性对照组、四神丸加减方高剂量组(简称高剂量组)和四神丸加减方低剂量组(简称低剂量组)各6只。在适应性喂养1周后,构建葡聚糖硫酸钠诱导的溃疡性结肠炎小鼠模型,并灌胃给药,实验期间每天观察小鼠精神状态并称重。实验结束后,比较5组小鼠疾病活动指数(DAI),结肠组织病理学指标、白细胞介素-6(IL-6)和白细胞介素-10(IL-10)表达水平、Toll样受体4(TLR4)、p65分子量、磷酸化P65(p-p65)、β-actin蛋白表达水平。结果:与正常组比较,模型组小鼠DAI评分、粪便黏稠度评分、便血评分、IL-6水平、结肠组织中TLR4、p65及p-p65表达水平均显著升高(P<0.05),结肠长度、IL-10水平均显著降低(P<0.05)。与模型组比较,阳性对照组和高剂量组小鼠DAI评分均显著下降(P<0.05),而低剂量组小鼠DAI评分无明显变化(P>0.05);高剂量组小鼠结肠长度明显增加(P<0.05),阳性对照组、低剂量组小鼠结肠长度增加不明显(P>0.05);阳性对照组及高、低剂量组小鼠粪便黏稠度评分均明显降低(P<0.05);阳性对照组及高、低剂量组小鼠便血评分无明显变化(P>0.05);高剂量组IL-6水平显著降低(P<0.05),阳性对照组及低剂量组IL-6水平下降不明显(P>0.05);高、低剂量组IL-10水平显著升高(P<0.05),阳性对照组IL-10水平升高不明显(P>0.05);阳性对照组、高剂量组、低剂量组小鼠结肠组织中TLR4、p65和p-p65的表达水平均明显降低(P<0.05)。病理变化上,模型组小鼠结肠组织中杯状细胞明显减少,隐窝结构损伤严重,出现明显的炎症浸润;与模型组比较,阳性对照组、高剂量组小鼠结肠组织上述病理变化改善明显,而低剂量组上述病理病化略有改善。结论:四神丸加减方可在一定程度上改善溃疡性结肠炎小鼠的临床症状及结肠组织病理变化,其作用机制可能与TLR4/NF-κB信号通路有关。

Interleukin-13 promotes cellular senescence through inducing mitochondrial dysfunction in IgG4-related sialadenitis

Immunoglobulin G4-related sialadenitis(IgG4-RS)is an immune-mediated fibro-inflammatory disease and the pathogenesis is still not fully understood.The aim of this study was to explore the role and mechanism of interleukin-13(IL-13)in the cellular senescence during the progress of IgG4-RS.We found that the expression of IL-13 and IL-13 receptorα1(IL-13Rα1)as well as the number of senescent cells were significantly higher in the submandibular glands(SMGs)of IgG4-RS patients.IL-13 directly induced senescence as shown by the elevated activity of senescence-associatedβ-galactosidase(SA-β-gal),the decreased cell proliferation,and the upregulation of senescence markers(p53 and p16)and senescence-associated secretory phenotype(SASP)factors(IL-1βand IL-6)in SMG-C6 cells.Mechanistically,IL-13 increased the level of phosphorylated signal transducer and activator of transcription 6(p-STAT6)and mitochondrial-reactive oxygen species(mt ROS),while decreased the mitochondrial membrane potential,ATP level,and the expression and activity of superoxide dismutase 2(SOD2).Notably,the IL-13-induced cellular senescence and mitochondrial dysfunction could be inhibited by pretreatment with either STAT6 inhibitor AS1517499 or mitochondria-targeted ROS scavenger Mito TEMPO.Moreover,IL-13 increased the interaction between p-STAT6 and c AMP-response element binding protein(CREB)-binding protein(CBP)and decreased the transcriptional activity of CREB on SOD2.Taken together,our findings revealed a critical role of IL-13 in the induction of salivary gland epithelial cell senescence through the elevated mitochondrial oxidative stress in a STAT6–CREB–SOD2-dependent pathway in IgG4-RS.

Interleukin-4 affects microglial autophagic flux

Interleukin-4 plays an important protective role in Alzheimer’s disease by regulating microglial phenotype,phagocytosis of amyloid-β,and secretion of anti-inflammatory and neurotrophic cytokines.Recently,increasing evidence has suggested that autophagy regulates innate immunity by affecting M1/M2 polarization of microglia/macrophages.However,the role of interleukin-4 in microglial autophagy is unknown.In view of this,BV2 microglia were treated with 0,10,20 or 50 ng/mL interleukin-4 for 24,48,or 72 hours.Subsequently,light chain 3-II and p62 protein expression levels were detected by western blot assay.BV2 microglia were incubated with interleukin-4(20 ng/mL,experimental group),3-methyladenine(500μM,autophagy inhibitor,negative control group),rapamycin(100 nM,autophagy inductor,positive control group),3-methyladenine+interleukin-4(rescue group),or without treatment for 24 hours,and then exposed to amyloid-β(1μM,model group)or vehicle control(control)for 24 hours.LC3-II and p62 protein expression levels were again detected by western blot assay.In addition,expression levels of multiple markers of M1 and M2 phenotype were assessed by real-time fluorescence quantitative polymerase chain reaction,while intracellular and supernatant amyloid-βprotein levels were measured by enzyme-linked immunosorbent assay.Our results showed that interleukin-4 induced microglial autophagic flux,most significantly at 20 ng/mL for 48 hours.Interleukin-4 pretreated microglia inhibited blockade of amyloid-β-induced autophagic flux,and promoted amyloid-βuptake and degradation partly through autophagic flux,but inhibited switching of amyloid-β-induced M1 phenotype independent on autophagic flux.These results indicate that interleukin-4 pretreated microglia increases uptake and degradation of amyloid-βin a process partly mediated by autophagy,which may play a protective role against Alzheimer’s disease.