Fermented Lentinus edodes extract containingα-glucan ameliorates concanavalin A-induced autoimmune hepatitis in mice

Autoimmune hepatitis(AIH)is a chronic inflammatory liver disease that threatens human health worldwide.The aim of this study was to detect the protective effect of a fermented Lentinus edodes extract containingα-glucan(FLA),in a concanavalin A(Con A)-induced AIH mouse model and to determine the underlying liver-protective mechanism.The results showed that compared with the model group,the level of proinflammatory cytokines in serum of FLA pretreated mice was significantly decreased,and the degree of inflammatory cell infiltration in liver,thymus and spleen was significantly reduced.Quantitative polymerase chain reaction,immunohistochemistry,and Western blotting showed that FLA pre-treatment inhibited the Con A-induced apoptosis of hepatocytes by down-regulating the expression of BAX and up-regulating the expression of BCL-2.Further research found that FLA may improve liver injury in mice by activating NRF2 signaling pathway and inhibiting TRAF6/NF-κB signaling pathway.Thus,FLA may improve liver injury in mice by shifting gut microbial composition to reduce the release of inflammatory cytokines in the serum and prevent the necrosis of hepatocytes.Up-regulation of NRF2 signaling pathway,down-regulation of TRAF6/NF-κB signaling pathway,and an increase in the relative abundance of Lactobacillus_johnsonii and Ligilactobacillus_murinus play a protective role in liver.

羟基红花黄色素A对实验性自身免疫性肝炎小鼠的保护作用

目的 利用刀豆蛋白A(concanavalin A,Con A)诱导的肝损伤小鼠模型,研究羟基红花黄色素A(hydroxylsafflor yellow A,HSYA)对实验性自身免疫性肝炎小鼠的保护作用。方法 36只BALB/c小鼠随机分为6组,Sham组、Con A组、HSYA低剂量组(5 mg/kg)、HSYA中剂量组(10 mg/kg)、HSYA高剂量组(20 mg/kg)、Sham+HSYA高剂量组(20 mg/kg),每组小鼠各6只。将各组小鼠称质量并记录,通过腹腔注射给药的方式对其进行3 d HSYA预处理,处死小鼠前12 h,尾静脉注射Con A,制作实验性自身免疫性肝炎小鼠模型,最终以二氧化碳吸入法处死小鼠并取材备用。使用全自动生化分析仪检测各组小鼠血浆AST和ALT水平,并进行组间比较。肝组织进行苏木精-伊红染色,镜下观察肝脏组织学变化。应用ELISA检测血浆中TNF-α、 IL-17、IL-10水平。结果 Con A组小鼠体内TNF-α、 IL-17、IL-10水平(F=21.19、F=13.12、F=3.209,P均<0.05)及ALT、AST水平(F=274.33、F=214.91,P均<0.05)高于Shan组,并伴有肝细胞大片坏死。HSYA中剂量和HSYA高剂量组小鼠体内炎症因子水平及氨基转移酶水平低于ConA组,同时伴肝细胞坏死区域缩小。结论 Con A成功诱导小鼠肝组织损伤,HSYA对实验性自身免疫性肝炎小鼠具有保护作用。

根皮素对免疫性肝炎小鼠的保护作用及机制

目的观察根皮素(Phloretin,PHL)对自身免疫性肝炎(autoimmune hepatitis,AIH)小鼠的保护作用及调控机制。方法SPF级Balb/C小鼠32只,随机分为对照(Control)组、刀豆蛋白A(concanavalin A,ConA)模型组、PHL低剂量组和PHL高剂量组。造模处理24 h后提取小鼠血清及肝脏组织,ELISA法检测血清转氨酶及炎症因子;HE染色观察肝脏组织病理学变化;TUNEL染色检测肝细胞凋亡;qRT-PCR检测转录表达水平;免疫组化和Western-blot检测肝组织炎症因子、自噬与凋亡蛋白及TRAF6-JNK通路信号蛋白表达水平。结果与正常对照组相比,AIH小鼠血清转氨酶及炎症因子表达显著升高(P<0.001),病理学见肝组织结构被广泛破坏,肝细胞大面积坏死及炎性细胞浸润。与ConA组相比,PHL组血清转氨酶及炎症因子水平下降(P<0.05),肝细胞结构完整,肝细胞坏死面积减少(P<0.05)。此外,与ConA组相比,PHL显著下调肝组织促凋亡蛋白及自噬蛋白的表达(P<0.05),下调TRAF6-JNK信号通路激活(P<0.05)。结论PHL可能通过减轻AIH小鼠肝细胞自噬和肝细胞凋亡,缓解AIH小鼠高炎症负荷,发挥对AIH小鼠的保护作用,可能是通过TRAF6-JNK信号通路发挥作用。

刀豆凝集素快速检测技术的研究现状及发展趋势

刀豆凝集素主要存在于刀豆中,是一种能与多糖、糖蛋白等物质发生特异性结合的植物凝集素。刀豆凝集素具有抗营养和抗虫害作用,是植物自我保护的重要环节,也是造成食用豆类食物中毒和过敏反应的主要原因。因此刀豆凝集素的检测对豆类蔬菜的育种、食用安全性评估等工作具有重要意义。详细介绍了新型刀豆凝集素快速检测技术的研究进展,包括基于纳米材料的生物传感器、免疫传感技术、糖传感技术和核酸适配体传感技术等。基于纳米材料的生物传感器具有较高的精度但是缺乏生物特异性;免疫传感技术改进了传统免疫法的检测速度但仍存在信号重复性差的问题;糖传感技术大幅提升了血凝法的特异性和检测精度,但无法区分糖特异性相同的凝集素。基于核酸适配体的检测技术具有优秀的检测精度、特异性和可重复性,还可通过化学修饰获得非天然核酸适配体进一步加强特异性,是未来刀豆凝集素快速检测的发展趋势。

环状RNA参与自身免疫性肝炎小鼠肝损伤的相关性分析

背景:自身免疫性肝炎的发病机制尚未明确阐明,环状RNA(CircRNAs)是RNA领域的一个研究热点,参与了许多自身免疫性疾病的发病,但CircRNAs在自身免疫性肝炎中的作用尚不清楚。目的:探讨CircRNAs与刀豆蛋白A诱导自身免疫性肝炎小鼠肝损伤的关系。方法:对前期微阵列技术筛选出差异表达的CircRNAs谱进行生物信息学分析,包括基因本体论(GO)及京都基因与基因组百科全书(KEGG)富集分析,探讨上述差异表达基因潜在的生物学功能。采用随机数字表法将12只C57BL/6小鼠分为正常组和模型组,每组6只,模型组通过尾静脉注射刀豆蛋白A建立自身免疫性肝炎模型,造模12 h后处死小鼠,提取小鼠肝脏及外周血,利用qRT-PCR技术验证部分CircRNAs的表达水平,通过比色法检测小鼠血清中肝损伤指标谷丙转氨酶和谷草转氨酶的水平,通过微孔板法检测小鼠肝脏中氧化应激指标丙二醛和NO的水平,并将肝损伤指标和氧化应激水平的相关性进行分析。结果与结论:①GO分析结果显示,表达上调CircRNAs的靶基因参与的生物过程主要为“SNARE复合装配的调节”(P=0.004)等,分子功能主要为“金属离子结合”(P=0.00029)等,主要富集在“CORVET复合体”(P=0.075)等细胞组分;表达下调circRNAs的靶基因参与的生物过程主要为“胰液分泌的负调节”(P=0.00042)等,分子功能主要为“转录激活因子活性”(P=0.025)等,主要富集在“膜外组分”(P=0.006)等细胞组分;②KEGG富集分析结果显示,表达上调CircRNAs的靶基因主要富集于“碱基切除修复”(P=0.026)等信号通路;③与正常组比较,模型组小鼠血清谷丙转氨酶和谷草转氨酶及肝组织中丙二醛和NO的水平升高(P<0.01),mmu-circ-0001520和mmu-circ-0001577的表达升高(P<0.05);④Spearman相关分析显示,mmu-circ-0001520和mmu-circ-0001577的表达与谷丙转氨酶、谷草转氨酶、丙二醛、NO存在正相关关系;⑤结果显示,CircRNAs的差异表达与自身免疫性肝炎小鼠肝损伤过程具有相关性,其中mmu-circ-0001520和mmu-circ-0001577有望成为自身免疫性肝炎诊断的生物标志物及治疗靶点。

Notch signaling mediated by TGF-β/Smad pathway in concanavalin A-induced liver fibrosis in rats

AIM To explore the exact interaction between Notch and transforming growth factor(TGF)-β signaling in liver fibrosis. METHODS We established a rat model of liver fibrosis induced by concanavalin A. Peripheral blood mononuclear cells(PBMCs) were isolated from the modeled rats, and cultured with γ-secretase inhibitor DAPT and TGF-β inhibitor for 24 h. The m RNA levels of Notch and TGF-β signaling were detected by quantitative real-time polymerase chain reaction. Expression of Notch and TGF-β proteins was analyzed by western blotting.RESULTS Compared to control rats, Notch and TGF-β signaling was activated in PBMCs of model rats. Administration of DAPT and TGF-β inhibitor suppressed Notch and TGF-β signal transducer in PBMCs of model rats. DAPT reduced m RNA and protein expression of TGF-β signaling, such as TGF-β1 and Smad3. TGF-β inhibitor also downregulated Notch1, Hes1 and Hes5, and m RNA and protein expression of the Notch signaling pathway.CONCLUSION Notch and TGF-β signaling play a role in liver fibrosis. TGF-β signaling upregulates Notch signaling, which promotes TGF-β signaling.

Immune mechanisms of Concanavalin A model of autoimmune hepatitis

As a chronic inflammatory disease of the liver,the pathogenic mechanisms of autoimmune hepatitis (AIH) have not yet been elucidated,with prognosis and diagnosis remaining unsatisfied.Currently the only viable treatments of AIH are immunosuppressant application and liver transplantation.It is considered that lack of good animal AIH models is the main reason for the shortage of a simple and efficient cure.The Concanavalin A (Con A) model is a typical and well established model for investigating T-cell and macrophage dependent liver injury in mice,which closely mimics the pathogenesis mechanisms and pathological changes of patients,and is regarded as the best experimental model for AIH research so far.In this paper we eluci-dated the pathogenic mechanisms of AIH and the evolution of relative animal models.We go on to further focus on Con A-induced liver injury from the point of immunological mechanisms and the change of cytokine levels.Finally,we manifested the clinical significance of the AIH animal models and the challenges they would meet during their future development.

Interleukin-22 contributes to liver regeneration in micewith concanavalin A-induced hepatitis after hepatectomy

AIM: To investigate the therapeutic effects and mechanisms of interleukin(IL)-22 in liver regeneration in mice with concanavalin A(Con A)-induced liver injury following 70% hepatectomy.METHODS: Mice were injected intravenously with Con A at 10 μg/g body weight 4 d before 70% hepatectomy to create a hepatitis model, and recombinant IL-22 was injected at 0.125 μg/g body weight 30 min prior to 70% hepatectomy to create a therapy model. Control animals received an intravenous injection of an identical volume of normal saline.RESULTS: IL-22 treatment prior to 70% hepatectomy performed under general anesthesia resulted in reductions in the biochemical and histological evidence of liver injury, earlier proliferating cell nuclear antigen expression and accelerated recovery of liver mass. IL-22 pretreatment also significantly induced signal transducer and activator of transcription factor 3(STAT3) activation and increased the expression of a variety of mitogenic proteins, such as Cyclin D1. Furthermore, alpha fetal protein m RNA expression was significantly elevated after IL-22 treatment.CONCLUSION: In this study, we demonstrated that IL-22 is a survival factor for hepatocytes and prevents and repairs liver injury by enhancing pro-growth pathways via STAT3 activation. Treatment with IL-22 protein may represent a novel therapeutic strategy for preventing liver injury in patients with liver disease who have undergone hepatectomy.

Kupffer cells contribute to concanavalin A-induced hepatic injury through a Th1 but not Th17 type response-dependent pathway in mice

BACKGROUND:Increasing evidence suggests that a close interaction of Kupffer cells with T cells plays a central role in concanavalin A-induced hepatic injury in mice,but the underlying mechanisms remain obscure.The present study aimed to determine the relative roles of Th1 and Th17 type responses in concanavalin A-induced hepatic injury in mice, and to investigate whether or not Kupffer cells contribute to hepatic injury via a Th1 or Th17 type response-dependent pathway. METHODS:Immune-mediated hepatic injury was induced in C57BL/6 mice by intravenous injection of concanavalin A. Kupffer cells were inactivated by pretreatment with gadolinium chloride 24 hours before the concanavalin A injection.The interferon-gamma(IFN-γ)and interleukin-17(IL-17)pathways were blocked by specific neutralizing antibodies.Hepatic injury was assessed using serum transferase activity and pathological analysis.Expression of inflammatory cytokines within the liver was detected by real-time polymerase chain reaction and immunohistochemistry. RESULTS:Neutralization of IFN-γsignificantly attenuated concanavalin A-induced hepatic injury.However,neutralization of IL-17 failed to suppress the injury.Inactivation of Kupffer cells by gadolinium chloride pretreatment protected against concanavalin A-induced injury and significantly reduced hepatic cytokine levels including TNF-α,IL-6 and IFN-γbut not IL-17.CONCLUSION:Our findings suggest that Kupffer cells contribute to concanavalin A-induced hepatic injury via a Th1 type response-dependent pathway and production of inflammatory cytokines including TNF-α,IL-6 and IFN-γ.

Stem cells from human exfoliated deciduous teeth ameliorate concanavalin A-induced autoimmune hepatitis by protecting hepatocytes from apoptosis

BACKGROUND Autoimmune hepatitis is a serious autoimmune liver disease that threatens human health worldwide,which emphasizes the urgent need to identify novel treatments.Stem cells from human exfoliated deciduous teeth(SHED),which are easy to obtain in a non-invasive manner,show pronounced proliferative and immunomodulatory capacities.AIM To investigate the protective effects of SHED on concanavalin A(ConA)-induced hepatitis in mice,and to elucidate the associated regulatory mechanisms.METHODS We used a ConA-induced acute hepatitis mouse model and an in vitro co-culture system to study the protective effects of SHED on ConA-induced autoimmune hepatitis,as well as the associated underlying mechanisms.RESULTS SHED infusion could prevent aberrant histopathological liver architecture caused by ConA-induced infiltration of CD3+,CD4+,tumor necrosis-alpha+,and interferon-gamma+inflammatory cells.Alanine aminotransferase and aspartate aminotransferase were significantly elevated in hepatitis mice.SHED infusion could therefore block ConA-induced alanine aminotransferase and aspartate aminotransferase elevations.Mechanistically,ConA upregulated tumor necrosisalpha and interferon-gamma expression,which was activated by the nuclear factor-kappa B pathway to induce hepatocyte apoptosis,resulting in acute liver injury.SHED administration protected hepatocytes from ConA-induced apoptosis.CONCLUSION SHED alleviates ConA-induced acute liver injury via inhibition of hepatocyte apoptosis mediated by the nuclear factor-kappa B pathway.Our findings could provide a potential treatment strategy for hepatitis.

A comparative study of changing patterns of concanavalin A-binding proteins in early stage of cholesterol gallstone

IM To elucidate the importance and the changing patterns of biliary concanavalin Abinding proteins (CPs) in the early stage of cholesterol gallstone.METHODS CPs concentrations and nucleation activities were measured by lectin affinity chromatography in biles of patients with cholesterol gallstone, pigment gallstone, gallbladder cholesterosis and nonbiliary diseases.RESULTS The concentrations of CPs were much higher in patients with cholesterol gallstone (039g/L±011g/L, n=36, P<001) or gallbladder cholesterosis (040g/L±009g/L, n=9, P<001) than in those with pigment gallstone (026g/L±012g/L, n=7) and/or nonbiliary diseases (027g/L±009g/L, n=10). Pronucleating activities were much stronger in patients with cholesterol gallstones (nucleation time ratio: 057±021, n=5, P<001 vs pigment gallstone and/or nonbiliary diseases) and gallbladder cholesterosis (nucleation time ratio: 044±023, n=5, P<001 vs pigment gallstone or nonbilliary diseases). The binding percentages of CPs to model biliary vesicles were also higher in patients with cholesterol gallstones (n=6) than those with pigment gallstones (n=6) (24%±09% vs 09%±05%, P<001).CONCLUSION Hypersecretion of CPs, especially those in vesicular phase may be the important changes in the early stage of cholesterol gallstone.

Immunomodulatory effect of schisandrae oil in mouse model of autoimmune hepatitis induced by concanavalin A

Objective:To study the immunomodulatory effect of schisandra oil (SCO) in mouse model of autoimmune hepatitis induced by concanavalin A (ConA). Methods: C57BL/6 mice were divided into control group, model group and SCO group. Mice in SCO group were given SCO at 5 mg/kg by intragastric administration every day for 7 days, followed by intravenous injection of ConA at 10 mg/kg. 10 hours after ConA injection, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured by the kits, the expression of inflammatory cytokines like interferon-γ(IFN-γ), interleukin-4 (IL-4), interleukin-17 (IL-17), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6 (IL-6) in liver was detected by real-time quantitative PCR, and the T cell activation and IFN-γ expression in spleen and MLN were examined by flow cytometry. Results: Compared with control group, each indicator in model group were significantly higher. In SCO preventive treatment group, the levels of serum ALT, AST and LDH were significantly reduced (all P < 0.001), the expression levels of inflammatory cytokines in liver were downregulated, the T cell activation in spleen and MLN was inhibited (P = 0.006 and P = 0.008), the percentages of IFN-γ+ CD8+ and IFN-γ+ CD4+ T cells were decreased, and the frequencies of Th2 and Th17 cells in spleen and MLN were also decreased at the same time. Conclusion: SCO has a protective effect on immune liver injury by inhibiting the activation of T cells and reducing the expression of inflammatory cytokines, which reflects that SCO plays a role in the immunomodulation of autoimmune hepatitis, indicating that SCO is of great significance for the maintenance of autoimmune homeostasis.

Concanavalin A-induced changes in lysosomal morphology of macrophages under confocal microscope

Changes in lysosomal morphology of cultured mouse peritoneal macrophages after stimu1ation by Concanavalin A (Con A) were observed with a laser scanning confocal microscope (LSCM). A series of images were obtained including phase-contrast images, optical sectioning images, 3-dimensional reconstruction images. The changes of lysosomal fluorescence intensity and pH were measured. It was found that macrophage lysosomes were Cllstributed mainly at the periphery of the cells in resting conditions, the lysosomal area containing fluorescence probe became markedly enlarged after stimulation by Con A for 30 min, and the fluorescence intensity in the medium increased about 15 min after suggesting that Con A could induce outflow of the fluorescence probe within the macrophage lysosomes. The lysosomal pH rose from 4. 6 to 5. 7 in 7 min after Con A was added, and maintained at that level hereafter.

Effects of interleukin-18 and Anti-interleukin-18-mAb on Experimental immunological Liver Fibrosis induced by Repeatedly Administered Concanavalin A and its Mechanism

Objective To explore the prevention of IL-18 or anti-IL-18-m Ab to the immune liver fibrosis model induced by repeated injection of concanavalin A in BALB/c mice and its mechanism.Methods Total of 120 BALB/c mice were divided into four groups, control group mice(Ga) were injected weekly with normal saline, concanavalin A group was divided into Gb, Gc, Gd. All mice were injected with concanavalin A(15 mg/kg) once a week. Moreover, Gc, Gd mice were injected weekly with IL-18(7.5 mg/kg) and anti-IL-18-m Ab(10 mg/kg) 2 hours before treatment with concanavalin A, respectively. Twenty-four hours after concanavalin A challenge at 1, 5, 12 and 20 weeks, 3 mice were killed by vena orbitalis, repectively. The sera were storaged at 4℃ for detecting of up TNF-α and IFN-γ by ELISA. The liver of mice in different groups were excised and fixed in 10% formalin for HE staining and Masson staining or frozen in liquid nitrogen for immunohistochemical staining for α-SMA. After extracting of total RNA from liver tissue, MMP-2 and TIMP-1A messenger RNA were amplified by reverse transcription polymerase chain reaction(PCR). Products were electrophoresed on agrose gel containing ethidium bromide and visualized under ultraviolet light. Densitometric RT-PCR data were standardized with β-actin signals. Results After experiment, the number of dead mice of Ga, Gb, Gc and Gd were 0, 6, 15 and 3, respectively. There were significant difference on each group(P < 0.05). At the fifth week of experiment, hepatocellular necrosis in IL-18 administered group mice had become widespread throughout the lobule. Evidence of liver fibrosis was observed during this period. However, at the twelfth week of experimemt, bridging fibrosis and large fibrosis strip in the parenchyma with hepatocellular necrosis was detectable in Gb, but at twentieth week, only the small fibrosis strip had been found in anti-IL-18-mA b administered group mice by HE staining and Masson staining. The serum levels of TNF-α and IFN-γ in IL-18 administered group were higher than that in concanavalin A group and anti-IL-18 administered groups(P < 0.05). Moreover, immunohistochemical staining for α-SMA indicated that the semi-quantu scores in IL-18 administered group were more than concanavalin A group and anti-IL-18-mA b administered groups(P < 0.05). MMP-2-mR NA, TIMP-1- mR NA expression levels increased signifigantly compared with concanavalin A group and anti-IL-18-mA b administered group(P < 0.05).Conclusions The immune liver fibrosis model induced by repeated injection of concanavalin A in BALB/c mice could be worsened by IL-18 administration and block by anti-IL-18 mA b administraion.

小鼠脾T淋巴细胞增殖实验条件优化

目的探讨刀豆球蛋白A刺激小鼠脾T淋巴细胞增殖的最佳浓度与最佳作用时间。方法以小鼠脾T淋巴细胞与刀豆球蛋白A共同培养后,采用CCK-8法检测淋巴细胞的增殖情况。结果不同浓度的刀豆球蛋白A对小鼠脾T淋巴细胞的刺激效果不同,在作用后48和72 h检测,10~25μg/mL浓度范围内增殖最为明显。结论刀豆球蛋白A对小鼠脾T淋巴细胞的增殖具有双重作用,推荐浓度为10~25μg/mL,检测时间为48~72 h。

基于MAPK信号转导通路探究异甘草酸镁对刀豆蛋白A诱导小鼠急性肝损伤的保护机制

目的 探讨异甘草酸镁(magnesium isoglycyrrhizinate,MgIG)在刀豆蛋白A(concanavalin A,Con A)诱导的小鼠急性肝损伤中的作用及机制。方法 按照简单随机分组法将20只无特定病原体(specific pathogen free,SPF)级Balb/c小鼠分为正常对照组(4只)、MgIG组(4只)、Con A组(6只)、Con A+MgIG干预组(6只)。小鼠Con A(25 mg/kg)尾静脉注射12 h,构建急性肝损伤模型,干预组提前1 h给予MgIG(30 mg/kg)腹腔注射。检测丙氨酸氨基转移酶(alanine aminotransferase,ALT)和天门冬氨酸转氨酶(aspartate aminotransferase,AST)水平、白细胞介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子(tumor necrosis factor α,TNF-α)、干扰素诱生蛋白10(interferon-inducibleprotein-10,IP-10)水平,检测IL-6、IL-1β、TNF-α、IP-10的mRNA相对表达量。体外实验中小鼠腹腔单核巨噬细胞用脂多糖(lipopolysaccharide,LPS,200 ng/ml)分别处理30 min、1 h、2 h和4 h,干预组小鼠腹腔单核巨噬细胞用MgIG(25μg/ml)预处理1h,检测IL-6、IL-β、TNF-α、IP-10炎症因子及磷酸化-p38丝裂原活化蛋白激酶(phospho-p38 mitogen activited protein kinase, p-p38)、磷酸化-cJun氨基末端激酶(phospho-c-JunN-terminalkinases,p-JNK)、磷酸化-细胞外调节蛋白激酶(phospho-extracellular regulated protein kinases,p-Erk)蛋白表达。结果 与Con A组相比,Con A+MgIG干预病理切片炎性细胞浸润明显减少,血清炎症因子[IL-6:(10695.71±4861.94)pg/ml vs (27650.88±5701.79)pg/ml;IL-1β:(13.37±8.18)pg/ml vs (56.55±9.29)pg/ml;IP-10:(3298.43±534.95)pg/ml vs (7413.38±1497.78)pg/ml;TNF-α(63.27±13.97)pg/ml vs (97.06±21.26)pg/ml]及mRNA相对表达量(IL-6:5.23±1.63 vs 16.06±4.55;IL-1β:0.88±0.45 vs 5.44±0.94;IP-10:126.24±29.54vs 454.40±114.81;TNF-α:9.55±2.75 vs 16.46±3.98)均显著降低(P均<0.05)。体外实验表明,与LPS诱导的模型组相比,MgIG干预组p-p38、p-Jnk、p-Erk蛋白水平明显降低,同时炎症因子mRNA相对表达量(IL-6:3627.91±1491.16 vs 6630.40±1149.59;IL-1β:259.92±49.47 vs 658.06±95.06;IP-10:4088.38±790.20 vs 7762.08±1007.42;TNF-α:117.09±15.29 vs 194.56±25.14)也显著降低(P均<0.05)。结论MgIG通过MAPK信号转导通路降低炎症反应显著改善Con A诱导的小鼠急性肝损伤,为MgIG在改善肝损伤的功能提供了理论支持。

两种不同种属小鼠急性免疫性肝损伤模型的比较

目的探讨刀豆蛋白A(ConA)引起两种不同种属小鼠急性免疫性肝损伤的剂量差异。方法雄性BALB/C小鼠和雄性C57BL/6小鼠分别为36只和40只。BALB/C小鼠分为对照组和5个(5、10、15、20和25 mg/kg ConA)实验组,C57BL/6小鼠分为对照组和5个(5、7.5、10、12.5和15 mg/kg ConA)实验组,实验组尾静脉注射设定剂量ConA。禁食不禁水,16 h后摘眼球采血,并取肝、脾和胸腺组织称重计算脏器指数,病理学检测肝组织,比色法测定血清中天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)活性,ELISA检测血清中肿瘤坏死因子α(TNF-α)含量,并结合各组动物死亡率,以确定最佳ConA剂量。结果在BALB/C小鼠中,25 mg/kg剂量ConA死亡2只;AST、ALT活性和TNF-α含量与ConA呈剂量依赖性升高(P<0.05);肝脏指数与ConA的剂量呈现正相关(P<0.05);脾脏指数在5 mg/kg时高于10和15 mg/kg,表现出先升高再降低再升高的趋势。在C57BL/6小鼠中,ConA剂量在10、12.5和15 mg/kg时分别死亡1只、1只和2只;AST、ALT活性和TNF-α含量与ConA也呈剂量依赖性升高(P<0.05);肝脏指数与ConA的剂量呈正相关(P<0.001);脾脏指数在7.5 mg/kg时高于其余剂量组,总体表现为先升高后降低的趋势。在两个品系的小鼠中,胸腺指数均随着剂量的升高而降低。两种小鼠肝脏病变程度与ConA剂量呈正相关,从低剂量时的炎性细胞浸润发展为高剂量时的肝细胞坏死,从灶状坏死发展为片状坏死。结论C57BL/6小鼠较BALB/C小鼠对ConA敏感性更高,BALB/C小鼠复制动物模型的合适剂量为20 mg/kg,而C57BL/6小鼠复制动物模型的合适剂量为10 mg/kg。

4种方法测定小米直链淀粉含量的比较

选取全国小米主产省份的24份商品小米样品,分别用改进的GB/T 15683—2008《大米直链淀粉含量的测定》法、NY/T 2639—2014《稻米直链淀粉的测定分光光度法》、双波长法及伴刀豆球蛋白A(concanavalin A,ConA)法测定其直链淀粉的含量,确定不同方法测定小米直链淀粉含量的差异,以期建立小米直链淀粉含量测定的标准方法。结果表明,4种方法测定小米直链淀粉含量的结果相比较,GB/T 15683—2008法≈双波长法>NY/T 2639—2014法>Con A法;且NY/T 2639—2014法与GB/T 15683—2008法测定结果的相关系数最高,达到0.752(P=0)。NY/T 2639—2014测定结果的1.41倍与GB/T 15683—2008测定结果相近,可以较好实现小米直链淀粉含量的简易预测。总之,改进的GB/T 15683—2008法、双波长法及NY/T 2639—2014(×1.41)法均能准确反映小米中直链淀粉的含量,且极大地缩短了样品的测试时间。

Ratiometric fluorescence probe for accurate detection of Concanavalin A by coupling fluorescent microsphere with boric acid functionalized carbon dots

Accurate and sensitive strategies for Concanavalin A(Con A)sensing are conducive to the better cognition of various important biological and physiological processes.Here,by designing dextran-functionalized fluorescent microspheres(DxFMs)and boric acid-modified carbon dots(BCDs)as recognition unit and built-in signal reference respectively,a ratiometric fluorescent detection platform was proposed for Con A detection with high reliability.In this protocol,the BCDs/DxFMs precipitation was formed due to the covalent interactions between cis-diol of DxFMs and boronic acid groups of BCDs,thus only fluorescence of BCDs could be detected in the supernatant.When Con A was presented,it could bind to DxFMs through its carbohydrate recognition ability and suppress the subsequent assembly between DxFMs and BCDs,leading to the simultaneous capture of DxFMs and BCDs fluorescence in the supernatant.Since the BCDs content was superfluous,their fluorescence intensities were basically constant in all cases.Based on the unchanged BCDs fluorescence signal and target-dependent DxFMs fluorescence signal in supernatant,the ratiometric detection of Con A was realized.Under optimized conditions,this ratiometric fluorescent platform displayed a linear detection range from 0.125μg/mL to 12.5μg/mL with a detection limit of 0.089μg/mL.Moreover,satisfied analytical outcomes for Con A detection in serum samples were obtained,manifesting huge application potential of this ratiometric fluorescent platform in clinical diagnosis.

雄芍汤对刀豆蛋白A所致肝纤维化大鼠TGF-β1/p38MAPK信号通路的影响

目的:探讨雄芍汤抗刀豆蛋白A(ConA)所致大鼠肝纤维化的作用及其对TGF-β1/p38MAPK信号通路的影响。方法:50只Wistar雄性大鼠随机取10只作为正常组。40只大鼠尾静脉注射ConA-PBS溶液法复制大鼠肝纤维化模型。将38只造模成功大鼠随机分为模型组10只、雄芍汤组10只、雄芍汤加味组8只、扶正化瘀胶囊组10只。HE染色和Masson染色观察大鼠肝组织病理改变,检测大鼠肝功能指标(AST、ALT)、肝纤维化四项指标(HA、LN、PCⅢ、IV-C)、肝组织羟脯氨酸(HYP)含量。ELISA法检测大鼠血清α-平滑肌激动蛋白(α-SMA)、炎症因子(TNF-α、IL-1、IL-10)水平,Western blotting法检测TGF-β1、p38MAPK蛋白相对表达量,荧光定量PCR检测大鼠肝组织p38MAPK mRNA相对表达量,免疫组化法检测大鼠肝组织TGF-β1、NF-κBp65蛋白表达。结果:模型组大鼠肝组织可见炎症细胞浸润、肝细胞结构异常、纤维增生并异常沉积,各治疗组均能显著改善大鼠肝纤维化程度,减轻肝脏炎症,减少胶原纤维增生。与正常组比较,模型组大鼠血清AST、ALT、肝纤维化四项、α-SMA、TNF-α、IL-1水平明显升高(P<0.01),肝组织HYP含量、TGF-β1、p38MAPK、NF-κBp65蛋白相对表达量及p38MAPK mRNA相对表达量均明显升高(P<0.01);与模型组比较,各治疗组大鼠血清AST、ALT、肝纤维化四项、α-SMA、TNF-α、IL-1水平明显降低(P<0.01),肝组织HYP含量、TGF-β1、p38MAPK、NF-κBp65蛋白相对表达量及p38MAPK mRNA相对表达量均明显降低(P<0.01);与扶正化瘀胶囊组比较,雄芍汤组及雄芍汤加味组大鼠肝组织TGF-β1蛋白表达明显降低(P<0.05);除TGF-β1指标外,各治疗组上述其他指标之间差异无统计学意义(P>0.05);各组大鼠血清IL-10水平比较,差异无统计学意义(P>0.05)。结论:雄芍汤具有抗ConA所致大鼠肝纤维化、改善肝功能的作用,其抗肝纤维化的机制可能与抑制HSCs活化、减轻炎症反应、抑制TGF-β1/p38MAPK信号通路有关。雄芍汤和雄芍汤加味抑制肝组织TGF-β1蛋白表达作用优于扶正化瘀胶囊,而雄芍汤、雄芍汤加味和扶正化瘀胶囊抗肝纤维化作用及机制差异不明显。