Functional genomic studies of the effect of selected phytocompounds on LPS-stimulated THP-1 cells

在人類免疫系統中，單核球 (monocyte) 參與體內第一線的防禦機制，並被認為是具有可分化為樹突狀細胞 (dendrite cell) 或巨噬細胞 (macrophages) 能力的前驅細胞。單核球平時在血液系統中移動，一旦受到外來的刺激，一方面會分泌許多調節免疫功能的細胞激素（cytokines），同時亦會移動到目標組織中，進而分化為巨噬細胞。在先天免疫反應中單核球或巨噬細胞都扮演重要的角色，它們所分泌出來的細胞激素如： TNF-α、IL-1、IL-6 ，可以造成急性期反應 (acute-phase response)，並啟動後續與發炎反應有關的訊息傳導路徑。目前在我們實驗室中已經有許多植物化合物具有抗發炎的活性；例如在紫草中的紫草素 (shikonin) 被證實可以抑制 TNF-α mRNA 的表現；紫錐菊粗萃物中的正丁醇層（Butanol fraction）似乎在樹突狀細胞之微矩陣實驗中具有抗發炎的活性；大黃中的大黃素 (emodin) 則可以降低樹突細胞中有關於發炎反應的表面抗原的生成；咸豐草中的萃取物(cytopiloyne) 在單核球細胞株中，似乎具有降低TNF-α及IL-6 mRNA表現的活性。目前我們對於這些植化物如何調節影響免疫反應的機制並不清楚，因此我們意圖經由利用單核球細胞株（THP-1）在脂多醣體（LPS）刺激下，利用由228個與免疫相關的基因組成DNA晶片，在不同的時間點中去測定這些基因mRNA的表現。期能以系統性的方式來檢視免疫反應的機轉。在本次實驗中我們發現紫草素在0.5小時的時候可以降低在LPS刺激下會表現的某些與發炎反應有關基因之mRNA的表現，例如：TNF-α、IL-1β、IL-8、CCL4、CCL5以及PTGS1、2。而大黃素則在0.5、2、24、48小時也具有抑制一些與發炎有關的基因的表現。在cytopiloyne的處理下， TNF-α、IL-8 、CCL3、CCL4、CCL5、CD14、STAT1、IL-1β在後期的表現量增加外，其它的時間點都觀察到與發炎反應有關的某些特殊基因的表現量降低。至於紫錐菊粗萃物中的正丁醇層，除了CCL3、STAT1、IL-1β、IFNAR1在八小時或48小時基因表現有增加外，其它的時間點都觀察到與發炎反應有關的基因的表現量降低。此外我們也發現紫草素及大黃素對於NFATC基因具有很強的抑制作用，而NFATC是IL-2的轉錄因子，IL-2可以刺激B細胞與T細胞的增生，因此紫草素及大黃素可能在活體中具有調節B細胞與T細胞增生的功能。Cytopiloyne對於WNT1基因具有很強的抑制作用，而前人曾研究指出WNT1過度表現時會造成染色體數目異常，是一種oncogene。我們希望在未來能夠作更進一步的分析，去確定這些植化物在動物體內對單核球是否具有相同的活性，期望能藉此更進一步評估這些植化物是否具有醫療的效果。In human systems, monocytes can differentiate into dendritic cells (DCs) or macrophages, and respond dramatically to a number of immune-modulation activities. Both monocytes and macrophages are known as important cell types that play a key role in innate immunity and produce a number of cytokines, including tumor necrosis factor alpha (TNF-α), interleukine-1β (IL-1β) and interleukine-6 (IL-6). These cytokines can induce “systemic acute-phase immune responses” which are often involved in pro-inflammatory signaling pathways. In our laboratory, we have accumulated a series of phytocompounds that confer anti-inflammatory activities. For example, shikonin can decrease TNF-α mRNA expression. Cytopiloyne can decrease monocytes’ TNF-α and IL-6 mRNA expression in RT-PCR assay. Emodin can substantially decrease specific DC surface marker that is associated with inflammation. The Echinacea purpura butanol fraction seems to confer in anti-inflammatory response in DCs. Based on the above observations, we evaluated the immune responses in terms of differential gene expressions in LPS-stimulated THP-1 cells, a monocyte cell line, by use of home-made mini DNA microarray chips which contains 228 genes. In this study, shikonin can decrease expression of genes encoding some LPS-induced signaling pathways, including TNF-α, IL-1β, IL-8, cysteine-cysteine motif chemokine ligand (CCL4), CCL5 and prostaglandin synthase (PTGS) gene at 0.5 hr. Emodin also can decrease TNF-α, IL-1β, IL-8, CCL4, CCL5 and PTGS gene expression at 0.5, 2, 24 and 48 hrs post treatment. Cytopiloyne can decrease expression of genes encoding LPS-induced signaling pathways at all time points tested, TNF-α, IL-8, CCL3, CCL4, CCL5, CD14, STAT1 and IL-1β were increased in mRNA expression at 24 and 48 hrs. Echinacea can decrease the expression of genes that encode LPS-induced signaling pathways at all time points tested, CCL3, STAT1, IL-1β and IFNAR1 were increased in mRNA expression at 8 and 48 hrs. Besides, we found shikonin and emodin conferred strong effect on NFATC gene expression. NFATC gene is a transcriptional factor of IL-2, a cytokine that can stimulate T and B cells proliferation. Therefore, shikonin and emodin may be able to effect T and B cell proliferation. Cytopiloyne had a strong effect on oncogene, WNT1 gene, via down-regulation. In future studies, we intend to use applicable in vivo system to study the effect of these phytocompounds using animal models, and hope to employ these phytocompounds as useful food supplements or alternative herbal medicines.ABSTRACT i 中文摘要 ii INTRODUCTION 1 MATERIALS AND METHODS Cell culture and monocyte preparation 8 Preparation of phytocompounds 8 MTT cell viability assay 8 RNA isolation 9 RNA electrophoresis 9 Primer design and RT-PCR condition 10 Reverse transcription and first strand cDNA labeling with amino allyl-dUTP(AA-dUTP) 11 Labeling of amino allyl-modified cDNA with CyDye 12 DNA Hybridization 12 Post-hybridization washing and data analysis 13 RESULTS Determination of optimal dosage for test phytocompounds 15 Evaluation of anti-inflammatory activities of phytocompounds by RT-PCR 15 Comparison of gene expression patterns between RT-PCR and DNA microarray 16 Analysis on of gene expression by hierarchical and non-hierarchical classification 17 Evaluation of the effect of phytocompounds on LPS-induced gene expression in TLRs signaling pathways 18 Analysis of gene expression by functional grouping classification 20 Three responsive genes that are most affected by test phytovompounds 22 DISCUSSION The advantage of functional grouping to analyze data from focused-mini microarray 25 Effect of phytocompounds in LPS-induced signaling pathways and anti-inflammatory activities 26 Other effect of test phytocompounds 28 FIGURES 30 REFERENCES 52 APPENDIX Appendix I- gene list with functional classification 5

The many faces of necrobiosis lipoidica: a report of three cases with histologic variations

Necrobiosis lipoidica (NL) is a granulomatous disease with unknown etiology and pathogenesis. Clinically, it is characterized by yellow-brown atrophic plaques with inflammatory rims on shins. Histologically, it shows diffused palisade and interstitial granulomatous dermatitis with focal connective tissue degeneration (necrobiosis). We described three non-diabetic NL cases with unusual histologic features. All patients presented erythematous to brownish plaque(s) on the shin(s). In Case 1, in addition to the characteristics of NL, the biopsy showed dense perineural infiltration, which may explain the pathogenesis of the anesthesia of NL lesions. In Case 2, well-formed tuberculoid granulomas were of special interest. In Case 3, there was conspicuous lobular panniculitis. These histologic features present difficulties in the diagnosis and represent the broad spectrum of NL lesions, histologically

Underrecognition and Undertreatment of Atherothrombotic Diseases: REACH Registry Taiwan Baseline Data

Atherothrombosis is a generalized disease affecting different vascular beds, making it the leading cause of death worldwide. To evaluate the long-term risk of atherothrombotic risk factors and determine the predictors for atherothrombotic events, an international, prospective, observational study was initiated, in which Taiwan was involved. Methods: The REduction of Atherothrombosis for Continued Health (REACH) Registry recruited outpatients with either symptomatic atherothrombotic diseases or multiple risk factors. Baseline data were collected using a universal standard case report form. All subjects were followed to document future outcomes. In this paper, we analyzed the baseline data of the participants from Taiwan. Results: In the REACH Registry, a total of 67,888 subjects from 44 countries were recruited. Among the 1062 Taiwanese participants, 971 were symptomatic subjects and 91 subjects were with risk factors only (RFO). In comparison with the global participants, the Taiwan patients were younger, with a higher prevalence of males, lower prevalence of hypertension, obesity, hypercholesterolemia, former smokers, and a greater prevalence of non-smokers. The baseline prevalence rates were: hypertension, 46.5%; fasting hyperglycemia, 38.4%; hypercholesterolemia, 45.8%; and hypertriglyceridemia, 42.8%. All these prevalence were higher than the global data, indicating an undertreatment status for the Taiwanese patients. Only 29 (2.7%) peripheral arterial disease (PAD) subjects were recruited in Taiwan, suggesting underrecognition of this disease. The RFO Taiwanese patients had fewer former smokers and more non-smokers than the symptomatic patients, suggesting that smoking may be an important factor contributing to atherothrombotic diseases. Conclusion: In Taiwan, atherothrombotic outpatients were generally undertreated and PAD was under-diagnosed

Porphyromonas gingivalis GroEL induces osteoclastogenesis of periodontal ligament cells and enhances alveolar bone resorption in rats.

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption

High Protein Diet and Huntington's Disease

<div><p>Huntington’s disease (HD) is a neurodegenerative disorder caused by the <i>huntingtin</i> (<i>HTT</i>) gene with expanded CAG repeats. In addition to the apparent brain abnormalities, impairments also occur in peripheral tissues. We previously reported that mutant Huntingtin (mHTT) exists in the liver and causes urea cycle deficiency. A low protein diet (17%) restores urea cycle activity and ameliorates symptoms in HD model mice. It remains unknown whether the dietary protein content should be monitored closely in HD patients because the normal protein consumption is lower in humans (~15% of total calories) than in mice (~22%). We assessed whether dietary protein content affects the urea cycle in HD patients. Thirty HD patients were hospitalized and received a standard protein diet (13.7% protein) for 5 days, followed by a high protein diet (HPD, 26.3% protein) for another 5 days. Urea cycle deficiency was monitored by the blood levels of citrulline and ammonia. HD progression was determined by the Unified Huntington’s Disease Rating Scale (UHDRS). The HPD increased blood citrulline concentration from 15.19 μmol/l to 16.30 μmol/l (<i>p</i> = 0.0378) in HD patients but did not change blood ammonia concentration. A 2-year pilot study of 14 HD patients found no significant correlation between blood citrulline concentration and HD progression. Our results indicated a short period of the HPD did not markedly compromise urea cycle function. Blood citrulline concentration is not a reliable biomarker of HD progression.</p></div