温肾固疏方对PMOP大鼠骨密度及细胞因子表达的影响

通过观察温肾固疏方对去卵巢骨质疏松(PMOP)大鼠的骨密度及对白细胞介素-1β、肿瘤坏死因子-α、白细胞介素-6的血清含量和转化生长因子-β骨组织表达的影响,探讨其治疗骨质疏松的可能作用机制。将48只SPF级雌性SD大鼠分为6组:假手术组、模型组、雌激素组、温肾固疏方高、中、低剂量组。造模4周后开始给药,连续给药12周,酶联免疫法(ELISA)检测外周血清白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)含量;q RT-PCR法检测骨组织转化生长因子-β(TGF-β)m RNA含量。与假手术组相比较,去势模型组大鼠骨密度(BMD)、TGF-β mRNA显著降低(p0.05),而IL-1β、TNF-α、IL-6均显著升高(p0.01),符合绝经后骨质疏松症改变;干预治疗后,与模型组比较,温肾固疏方高、中剂量组椎骨BMD显著升高(p0.05),IL-1β、TNF-α、IL-6显著减少(p0.05),温肾固疏方高剂量组TGF-β mRNA显著增加(p0.05)。温肾固疏方治疗骨质疏松的作用机制可能与其下调IL-1β、TNF-α、IL-6等促炎细胞因子的表达,抑制骨吸收;同时上调TGF-β等抑炎细胞因子的表达,促进骨形成有关。     

复合致病因素诱导肝硬化大鼠TGF-α和TGF-β1的动态变化

目的:观察复合致病因素诱导肝硬化大鼠肝组织中转化生长因子-α(TGF-α)和转化生长因子-β1(TGF-β1)的动态变化。方法:采用复合致病因素法复制大鼠肝硬化模型:首次皮下注射CCl4原液(0.5 ml/100 g·w),之后每隔3天皮下注射40%CCl4油溶液(0.3 ml/100 g·w),同时辅以低胆碱、高胆固醇、高脂肪、高醇饮食。随机将大鼠分为肝硬化模型4周、6周和8周组,并分别设立同期正常对照组。检测各组大鼠血浆中谷丙转氨酶(ALT)、内毒素、肿瘤坏死因子-α(TNF-α)和同型半胱氨酸(Hcy)的水平;肝组织切片行HE染色,TGF-α和TGF-β1的免疫组化染色。结果:与相应的同期正常对照组比较,大鼠血浆中ALT、内毒素、TNF-α和Hcy水平在肝硬化模型4周、6周和8周组均逐渐显著升高(P0.05);肝组织中TGF-α的表达在肝硬化模型4周组明显增加(P0.05),而肝组织中TGF-β1的表达则随着肝硬化病程的进展持续显著增加(P0.05)。结论:在肝硬化形成过程中,TGF-α的表达先增加后被抑制,TGF-β1的表达持续增加,导致肝细胞再生先增强后被抑制,而肝纤维化程度不断加重,最终发生肝硬化。TGF-α和TGF-β1的这一特征性动态变化可能与内毒素血症、TNF-α水平增高以及高同型半胱氨酸血症有关。     

房颤不同时期结构重构的血清学变化

目的:探讨转化生长因子-β1(TGF-β1)、基质金属蛋白酶-9(MMP-9)、金属蛋白酶组织抑制因子-1(TIMP-1)、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、白介素-10(IL-10)与阵发性或持续性房颤结构重构的关系。方法:入选患者分为3组,其中持续性房颤组30例、阵发性房颤组45例,以及32例阵发性室上速(包括预激综合征)作为对照组,通过ELISA方法检测上述患者左房血清的TGF-β1、MMP-9、TIMP-1、TNF-α、IL-6、IL-10水平,超声测量左心房长径。分析上述细胞因子水平与阵发性房颤或持续性房颤的关系。结果:阵发性房颤组与对照组相比,左房内径增大、TGF-β1、TNF-α升高(P0.05),IL-10、TIMP-1降低(P0.05),MMP-9无统计学差异。持续性房颤组与对照组相比,TGF-β1无统计学差异(P0.05),MMP-9、TNF-α、IL-6均升高(P0.05),IL-10、TIMP-1降低(P0.05);阵发性房颤组TGF-β1高于持续性房颤组(P0.05),持续性房颤组左房内径及MMP-9高于阵发性房颤组(P0.05)。结论:阵发性房颤患者左房扩大,已出现结构重构,结构重构的血清学变化以TGF-β1升高为主;持续性房颤患者结构重构血清学变化以MMP-9、IL-6升高为主。     

TGF-β与转录因子在肝纤维化中的相互作用

肝纤维化的形成是由于肝脏持续性损伤以及细胞外基质合成和降解失衡所引起的。转化生长因子-β(Transforming growth factor-β,TGF-β)是肝纤维化形成中的关键细胞因子,在肝纤维化发生、发展过程中起着至关重要的作用。一些与TGF-β相关的转录因子如AP1、STAT3及Foxo3a等也参与肝纤维化的调控过程。现就在肝纤维化中TGF-β与转录因子AP1、STAT3和Foxo3a的相互作用作一综述。     

肝脏发育相关信号通路

肝脏发育从肝芽的出现开始,到肝祖细胞的形成,接着肝祖细胞的增殖、分化和迁移,直至最后器官的形成,经历了复杂的细胞信号调控过程。本文综述了肝脏发育过程中常见的信号调控作用,包括成纤维生长因子(fibroblast growth factor,FGF)、骨形态发生蛋白(bone morphogenetic protein,BMP)、β-转化生长因子(transforming growth factor-β,TGF-β)、肝细胞生长因子(hepatocyte growth factor,HGF)和Wnt等信号通路,并重点讨论了在胚胎阶段调控肝脏发育的信号途径以及肝细胞和胆管细胞发育成熟过程中的信号因子作用,最后对肝脏再生相关的信号调控进行了简要介绍。     

蜜环菌多糖对小鼠血清IL-2和TGF-β1水平的影响

目的:观察蜜环茵多糖对小鼠血清中IL-2和TGF-β1水平的干预作用。方法:实验组小鼠腹腔注射(i.p)不同浓度的蛮环菌多糖,阴性对照组给予生理盐水,阳性对照组给予香菇多糖,连续给药14天。给药结束后,用形态学方法测定各组小鼠在植物血凝素(PHA)诱导下淋巴细胞转化率,用ELISA法测定各组小鼠血清中白介素2(IL-2)、转化生长因子β1(TGF-β1)水平。结果:蜜环菌多糖具有提高小鼠淋巴细胞转化率,增加血清中IL-2水平,并同时下调血清中TGF-β1水平的作用。结论:蜜环菌多糖可通过调节小鼠免疫细胞及免疫分子的作用,增强小鼠的免疫功能。     

小鼠肝再生过程中ROS及线粒体代谢变化规律的研究

目的:肝脏是维持人体发挥功能的重要器官,同时肝脏再生能力十分强大。本文通过部分肝切除术后小鼠肝再生模型,观察肝再生过程中氧化应激及线粒体代谢变化规律,以期为将来的调控肝再生提供新的干预靶点。方法:选择雄性健康体重均匀的Balb/c小鼠,采用经典70%肝切除模型,随机分为假手术对照组(Sham组)以及70%肝切除组(70%PH组)。肝切除术后6 h、1d、2 d、3 d、5 d、7 d不同时间点取肝组织,制备冰冻切片检测活性氧(ROS)水平,Western blot分别检测细胞增殖相关蛋白PCNA、Cyclin D1;氧化应激相关蛋白SOD1、SOD2、CAT、GPX1;以及线粒体代谢相关蛋白PGC-1α、Nrf1、TFAM、Drp1、Fis1、Mfn1、Mfn2、OPA1的表达并分析其变化规律。结果:70%肝切除术后小鼠肝脏增长迅速,细胞增殖关键蛋白PCNA和Cyclin D1表达显著增加;在此过程中细胞ROS水平呈现先升高后降低的变化,细胞主要抗氧化酶SOD1、SOD2、CAT、Gpx1与ROS相一致出现先升高后降低的变化。线粒体生物合成调控因子PGC-1α、Nrf1、TFAM呈现先降低后升高的趋势,而线粒体分裂蛋白Drp1和Fis1呈现先降低后显著升高的趋势,线粒体融合相关蛋白Mfn1、Mfn2和OPA1总体为先降低后恢复至正常水平。结论:在小鼠70%肝切除再生过程中,存在着明显的氧化应激,线粒体生物合成增加,线粒体分裂/融合平衡偏向分裂,并且这些变化呈现具有一定的时间变化规律,这些变化及规律很可能作为将来调控肝再生的重要的潜在干预靶点。     

α7nAChR调控STAT3磷酸化在星形胶质细胞机械性损伤后炎症反应中的作用

目的:通过建立星形胶质细胞机械性损伤模型,研究烟碱型乙酰胆碱受体α7亚单位(α7nAChR)在创伤性脑损伤后星形胶质细胞炎症反应中的作用及调控机制。方法:建立星形胶质细胞机械性损伤模型,通过ELISA检测炎症因子IL-1β、TNF-α、IL-10和TGF-β的表达;利用α7n ACh R抑制剂α-BGT和激动剂PHA-543613处理星形胶质细胞,检测相关炎症因子表达,并通过Western blot检测信号传导及转录活化因子3(STAT3)和磷酸化STAT3(p-STAT3)的表达;利用α-BGT和STAT3抑制剂Stattic处理星形胶质细胞,检测相关炎症因子表达。结果:①星形胶质细胞机械性损伤后,促炎因子IL-1β、TNF-α表达增加,抗炎因子IL-10、TGF-β表达降低(P0.05)。②利用α-BGT抑制α7nAChR可增加损伤后IL-1β、TNF-α的表达,减少IL-10、TGF-β的表达(P0.05);而利用PHA-543613激活α7nAChR功能,则发挥相反作用(P0.05)。③α-BGT可促进STAT3磷酸化,而PHA-543613抑制STAT3磷酸化(P0.05)。④STAT3抑制剂Stattic可减少IL-1β和TNF-α的表达,增加IL-10和TGF-β的表达,并部分阻断α-BGT对IL-1β、TNF-α、IL-10及TGF-β表达的影响(P0.05)。结论:机械性损伤后,激活α7nAChR可减轻星形胶质细胞炎症反应,而抑制STAT3磷酸化是其重要的下游机制。     

儿童过敏性紫癜血清IL-21、TGF-β1、TNF-α、IgA1的变化与紫癜性肾炎发生的相关性研究

探讨儿童过敏性紫癜(henoch-schonlein purpura,HSP)患儿血清中白细胞介素-21(Interleukin-21,IL-21)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、免疫球蛋白A1(Immunoglobulin A1,Ig A1)和转化生长因子-β1(Transforming growth factor-β1,TGF-β1)的含量变化,并研究这些因子与HSP发展为紫癜性肾炎(Henoch-SchOnlein purpura nephritis,HSPN)的相关性。选取我院2015年1月~2016年1月确诊的急性期过敏性紫癜患儿共72例作为本研究的实验对象。根据HSP患儿病症是否累积到肾脏,再分为普通HSP组(39例)和HSPN组(33例),另对照组为30例同期健康体检儿童。通过酶联免疫吸附(ELISA法)对检测血清中白细胞介素-21、转化生长因子-β1以及肿瘤坏死因子-α含量,采用全自动化生化分析仪检测血清免疫球蛋白A1的含量,同时研究此类因子与紫癜性肾炎是否存在一定的相关性。3组结果对照,HSPN组和HSP组转化生长因子-β1、肿瘤坏死因子-α和免疫球蛋白A1水平均高于对照组,差异具有显著意义(p0.05);HSPN组IL-21、TGF-β1、TNF-α和Ig A1水平均高于HSP组,差异具有显著意义p0.05)。HSP患儿血浆TGF-β1水平与TNF-α呈正相关(r=0.478,p0.05)。IL-21、TGF-β1、TNF-α和Ig A1可能参与HSP和HSPN的发病过程,HSP患儿TNF-α水平的升高可能和TGF-β1水平改变有关,了解这些因子在HSP发病和发展过程的作用可以更好地指导临床对过敏性紫癜肾脏合并症的发生率和预后判断。     

栀子苷通过TGF-β1/Smad信号通路抑制肝纤维化和肝星状细胞活化

本研究旨在观察栀子苷对肝纤维化和肝星状细胞活化的影响,并探讨其可能机制。人肝星状细胞LX-2用5 ng/mL转化生长因子-β1 (transforming growth factor-β1, TGF-β1)处理,并用不同浓度(0, 1, 2.5, 5, 10, 20, 40, 60, 80, 100μmol/L)的栀子苷联合培养,MTT法进行细胞活力测定,LX-2分别经5 ng/mL TGF-β1、TGF-β1+栀子苷(20μmol/L)处理后,用qPCR和Western blot分别检测I型胶原蛋白(collagen I)、纤连蛋白(fibronectin)、α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、p-Smad2和p-Smad3基因和蛋白的表达。BALB/c小鼠用CCl₄ (25%, 1 mL/kg)诱导肝纤维化,设立对照组、CCl₄组和CCl₄+栀子苷组(40 mg/kg),4周后检测肝功能及血清肝纤维化指标,用HE和Masson染色进行组织学观察,免疫组织化学分析组织α-SMA...     

Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor beta and interleukin 6.

In a previous study, we revealed that tumor necrosis factor (TNF) was secreted in mouse liver at an early phase of liver regeneration after partial hepatectomy. Here, we investigated direct actions of TNF on the in vitro DNA synthesis of adult mouse hepatocytes in primary culture. TNF enhanced both 3H-TdR uptake and the number of 3H-TdR-labeled nuclei of hepatocytes. Their time courses were similar to those by epidermal growth factor (EGF) with about a 15 h lag period and a peak period of 24-48 h. This action of TNF was abrogated by DNA polymerase alpha inhibitor, aphidicolin and blocked specifically by anti-TNF antibody. The actions of rmTNF and rhTNF were not distinguishable; ED50 was about 7.5U/ml (5ng/ml) and 30U/ml (20ng/ml) for maximal response (about 2-fold or more of control). Other inflammatory monokines showed differential effects on in vitro DNA synthesis of hepatocyte. Neither type of interleukin 1 affected hepatocyte DNA synthesis in the range examined (up to 50 ng/ml). IL-6 markedly inhibited the hepatocyte DNA synthesis stimulated by TNF and EGF. The action of TNF was completely suppressed by transforming growth factor beta, which is known as a potent inhibitor of hepatocyte growth. Interferon gamma also blocked this TNF action when added simultaneously. These results indicate that the activation of tissue macrophages and local secretion of TNF in liver after partial hepatectomy is of physiological importance in liver regeneration, in part by a direct stimulation of hepatocyte DNA synthesis. Cytokines induced by TNF may also participate in the later termination of liver regeneration.     

Rapid activation of protein kinase B/Akt has a key role in antiapoptotic signaling during liver regeneration

Liver regeneration is controlled by multiple signaling pathways induced by a variety of growth factors, hormones, and cytokines. Here we report that protein kinase B (PKB)/Akt, part of a key cell survival signaling pathway, is markedly activated after partial hepatectomy (PHX). The antiapoptotic protein Bad, a downstream target of PKB/Akt, is also phosphorylated. This cascade can be activated by various factors in primary hepatocytes, with the strongest activation by insulin and the alpha1-adrenergic agonist phenylephrine (PE), followed by IL-6, epidermal growth factor (EGF), and hepatocyte growth factor (HGF). Pretreatment of cells with the specific PI3 kinase inhibitor LY294002 abolished insulin- or PE-activation of PKB/Akt, suggesting that activation of PKB/Akt is mediated by a PI3 kinase-dependent mechanism. In vivo administration of PE, insulin, IL-6, HGF, or EGF to mice markedly stimulated PKB/Akt in the liver, with the strongest stimulation induced by insulin and PE. Moreover, HGF and insulin were able to attenuate transforming growth factor beta-induced apoptosis in hepatic cells, and these effects were antagonized by LY294002. Taken together, these findings suggest that rapid activation of PKB/Akt is a key antiapoptotic signaling pathway involved in liver regeneration.     

Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration

The mechanisms that regulate the transition between the initial priming phase and DNA replication in liver regeneration are poorly understood. To study this transition, we compared events occurring after standard two-thirds partial hepatectomy, which elicits full regeneration, with response to a reduced hepatectomy, one-third partial hepatectomy (1/3PH), which leads to little DNA replication. Although the initial response to partial hepatectomy at the priming phase appeared to be similar between the two procedures, cell cycle progression was significantly blunted in 1/3PH mice. Among the main defects observed in 1/3PH mice were an almost complete deficiency in retinoblastoma phosphorylation and the lack of increase in kinase activity associated with cyclin E. We report that, in two-thirds partial hepatectomy mice, the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) preceded the start of DNA replication and was not detectable in 1/3PH animals. Injection of HB-EGF into 1/3PH mice resulted in a >15-fold increase in DNA replication. Moreover, we show that hepatocyte DNA replication was delayed in HB-EGF knock-out mice. In summary, we show that HB-EGF is a key factor for hepatocyte progression through G(1)/S transition during liver regeneration.     

Temporal expression profiles indicate a primary function for microRNA during the peak of DNA replication after rat partial hepatectomy

The liver has the unique capacity to regenerate after surgical resection. However, the regulation of liver regeneration is not completely understood. Recent reports indicate an essential role for small noncoding microRNAs (miRNAs) in the regulation of hepatic development, carcinogenesis, and early regeneration. We hypothesized that miRNAs are critically involved in all phases of liver regeneration after partial hepatectomy. We performed miRNA microarray analyses after 70% partial hepatectomy in rats under isoflurane anesthesia at different time points (0 h to 5 days) and after sham laparotomy. Putative targets of differentially expressed miRNAs were determined using a bioinformatic approach. Two-dimensional (2D)-PAGE proteomic analyses and protein identification were performed on specimens at 0 and 24 h after resection. The temporal dynamics of liver regeneration were characterized by 5-bromo- 2-deoxyuridine, proliferating cell nuclear antigen, IL-6, and hepatocyte growth factor. We demonstrate that miRNA expression patterns changed during liver regeneration and that these changes were most evident during the peak of DNA replication at 24 h after resection. Expression of 13 miRNAs was significantly reduced 12-48 h after resection (>25% change), out of which downreguation was confirmed in isolated hepatocytes for 6 miRNAs at 24 h, whereas three miRNAs were significantly upregulated. Proteomic analysis revealed 65 upregulated proteins; among them, 23 represent putative targets of the differentially expressed miRNAs. We provide a temporal miRNA expression and proteomic dataset of the regenerating rat liver, which indicates a primary function for miRNA during the peak of DNA replication. These data will assist further functional studies on the role of miRNAs during liver regeneration.     

PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy

Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85alpha regulatory and a p110alpha catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In this study, we used the potent PI3K inhibitor wortmannin and small interfering RNA (siRNA) targeting the p85alpha and p110alpha subunits to determine whether total or selective PI3K inhibition would abrogate the proliferative response of the liver after partial hepatectomy in mice. Hepatic resection is associated with an induction in PI3K activity; total PI3K blockade with wortmannin and selective inhibition of p85alpha or p110alpha with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest time points. Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85alpha or p110alpha, as reflected by a paucity of F4/80-positive cells. Additionally, PI3K inhibition led to an aberrant architecture in the regenerating hepatocytes characterized by vacuolization, lipid deposition, and glycogen accumulation; these changes were not noted in the sham livers. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, most likely by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.     

Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice

We previously demonstrated that adiponectin has anti-fibrogenic and anti-inflammatory effects in the liver of mouse models of various liver diseases. However, its role in liver regeneration remains unclear. The aim of this study was to determine the role of adiponectin in liver regeneration. We assessed liver regeneration after partial hepatectomy in wild-type (WT) and adiponectin knockout (KO) mice. We analyzed DNA replication and various signaling pathways involved in cell proliferation and metabolism. Adiponectin KO mice exhibited delayed DNA replication and increased lipid accumulation in the regenerating liver. The expression levels of peroxisome proliferator-activated receptor (PPAR) α and carnitine palmitoyltransferase-1 (CPT-1), a key enzyme in mitochondrial fatty acid oxidation, were decreased in adiponectin KO mice, suggesting possible contribution of altered fat metabolism to these phenomena. Collectively, the present results highlight a new role for adiponectin in the process of liver regeneration.