Lactobacillus plantarum MYL26 induces endotoxin tolerance phenotype in Caco-2 cells

Background: Crohn's disease and ulcerative colitis are the major types of chronic inflammatory bowel diseaseoccurring in the colon and small intestine. A growing body of research has proposed that probiotics are able toattenuate the inflammatory symptoms of these diseases in vitro and in vivo. However, the mechanism of probioticactions remains unclear.Results: Our results suggested Lactobacillus plantarum MYL26 inhibited inflammation in Caco-2 cells throughregulation of gene expressions of TOLLIP, SOCS1, SOCS3, and IκBα, rather than SHIP-1 and IRAK-3.Conclusions: We proposed that live/ heat-killed Lactobacillus plantarum MYL26 and bacterial cell wall extracttreatments impaired TLR4-NFκb signal transduction through Tollip, SOCS-1 and SOCS-3 activation, thus inducing LPStolerance. Our findings suggest that either heat-killed probiotics or probiotic cell wall extracts are able to attenuateinflammation through pathways similar to that of live bacteria

嗜熱鏈球菌ATCC 19258、保加利亞桿菌ATCC 11842、保加利亞桿菌MYL 2與龍根雙歧桿菌MYL 6抗氧化及抗動脈粥狀硬化之探討

乳酸菌被應用在食品發酵上，已有一段深遠的歷史，而醱酵乳品和人類生活形成緊密關係。尤其近年來，許多文獻相繼提出乳酸菌的保健益生功能，其中，特別提及的即是乳酸菌抗氧化能力在疾病預防上可能扮演重要角色。大部分的研究都專注在活菌體的益生功能，不過，在1993年乳酸菌胞內物抗氧化能力首度被日本學者Kaizu證實。本研究第一部分即利用兩種脂質過氧化作用之抑制作用模式系統，包括游離脂肪酸（亞麻油酸）系統與生理性脂質（血漿脂質）系統，來評估優格菌株胞內物的抗氧化能力。Streptococcus salivarius ssp. thermophilus ATCC 19258 和Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 對亞麻油酸過氧化作用的抑制率分別為61%和57%，而S. salivarius ssp. thermophilus ATCC 19258 和 L. delbrueckii ssp. bulgaricus ATCC 11842 對血漿脂質過氧化作用的抑制率分別為57%和41%，由這兩種脂質過氧化作用模式系統，證實這兩株優格菌株具有高度的抗氧化能力。本研究再進一步利用 Intestine 407 細胞株模式系統，來探討優格菌株的抗氧化能力對降低氧化劑過氧化氫所引起氧化傷害之效果，結果顯示優格菌株胞內物確實可以降低氧化劑過氧化氫的氧化性傷害，雖然 L. delbrueckii ssp. bulgaricus ATCC 11842 對降低DNA 損傷的效果較弱，然而 S. salivarius ssp. thermophilus ATCC 19258 則表現對保護 Intestine407 對抗氧化劑過氧化氫所引起的基因毒性良好之能力。同時，這兩株優格菌株也發揮抑制過氧化氫細胞毒性的能力，S. salivarius ssp. thermophilus ATCC 19258 和 L. delbrueckii ssp. bulgaricus ATCC 11842所表現的細胞毒性抑制率分別為71%和48%。 由於國人的十大死因已轉變為以慢性疾病為主要的型態，其中以心血管相關疾病增加最為明顯。氧化傷害與心血管相關疾病的發生息息相關，例如氧化傷害在動脈粥狀硬化的形成過程扮演重要角色。本研究第二部分即比較兩株乳酸菌Bifidobacterium longum MYL2和 Lactobacillus delbrueckii ssp. bulgaricus MYL6其活菌體及胞內物的抗氧化能力。結果顯示，B. longum MYL2和 L. delbrueckii ssp. bulgaricus MYL6在109cfu/mL 時，菌體及胞內物具有清除α-diphenyl-β-picrylhydrazyl (DPPH)自由基能力(63~75%)、抑制liposome 過氧化(25~31%)以及顯著降低Intestine 407腸細胞脂質過氧化物 (malondialdehyde, MDA) 的生成。此外，本研究再進一步利用這兩株具有抗氧化能力乳酸菌的活菌體及其胞內物，探討對腦中風病人(cerebrovascular accident, CVA)和健康受試者血液中低密度脂蛋白(low density lipoprotein, LDL)氧化作用的影響。LDL的氧化作用是以監測LDL conjugated dienes 形成的起始時間(lag time)為指標。當B. longum MYL2和 L. delbrueckii subsp. bulgaricus MYL6以109cfu/mL 的菌數分別處理LDL時，LDL 氧化作用的起始時間顯著被延長，健康受試者血液中LDL氧化被抑制效果優於腦中風病人，而當以B. longum MYL2和 L. delbrueckii ssp. bulgaricus MYL6 109cfu/mL 菌數的胞內物處理腦中風病人及健康受試者的LDL時，LDL 氧化作用的起始時間顯著被延長超過180分鐘，此結果表示，抑制LDL氧化作用以乳酸菌的胞內物優於菌體。推論乳酸菌的胞內物具有效的抑制因子。 上述結果證實乳酸菌具有抗氧化傷害能力，抗氧化能力以乳酸菌的胞內物優於菌體。Lactic acid bacteria have long been used in fermented foods which are likely to be more desirable for daily lives. Recently, lactic acid bacteria are reported to have health-promoting characteristics in many studies. Above all, the anti-oxidative effects of lactic acid bacteria have been proven to play an important role in disease prevention. However, most of the researches focus on the probiotic effects of viable cell. Yet, it was first demonstrated anti-oxidative effects of intracellular extract of lactic acid bacteria by Kaizu et al in 1993. The first part of this study aims to confirm such anti-oxidative activities of yogurt bacteria. The anti-oxidative activity of intracellular extracts of yogurt bacteria was measured based on the inhibition of lipid peroxidation of two model systems, namely a free fatty acid (linoleic acid) system and a biological lipid (plasma lipid) system. The inhibitory rate on linoleic acid peroxidation was 61 and 57% for Streptococcus salivarius ssp. thermophilus ATCC 19258 and Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842, respectively. The inhibitory rate on plasma lipid peroxidation was 57 and 41% for S. salivarius ssp. thermophilus ATCC 19258 and L. delbrueckii ssp. bulgaricus ATCC 11842, respectively. Results from both systems demonstrated that these two yogurt strains were potentially highly antioxidative. Further experiments were done on Intestine 407 cells to determine the antioxidative effects of yogurt bacteria on reducing oxidative damage of oxidant H2O2. Intracellular extracts of yogurt bacteria reduced the genotoxicity of H2O2. Although L. delbrueckii ssp. bulgaricus ATCC 11842 had only minor effects on reducing DNA damage, S. salivarius ssp. thermophilus ATCC 19258 showed excellent potential for protecting the Intestine 407 cells from the genotoxicity of oxidant H2O2. Both yogurt bacteria demonstrated high cytotoxicity inhibitory ability for oxidant H2O2. The cytotoxicity inhibition rate was 71 and 48% for S. salivarius ssp. thermophilus ATCC 19258 and L. delbrueckii ssp. bulgaricus ATCC 11842, respectively. According to the top ten domestic mortality reports, the chronic diseases are the major causes nowadays in which cardiovascular-related disease has increased dramatically. Furthermore, oxidative stress and cardiovascular-related disease are highly correlated which for example, oxidative damage plays a vital role in the progression of atherosclerosis. The second part of this study aims to compare the anti-oxidative ability of intact cells and intracellular extracts of two lactic acid bacterial strains, Bifidobacterium longum MYL2 and Lactobacillus delbrueckii ssp. bulgaricus MYL6. Results showed that both intact cells and intracellular extracts of 109 cells of B. longum MYL2 and L. delbrueckii ssp. bulgaricus MYL6 had the ability to scavenge α-diphenyl-β-picrylhydrazyl (DPPH) free radical by 70.4-75.1%, to inhibit liposome peroxidation by 25-31%, and to decrease significantly the malondialdehyde (MDA) production in Intestine 407 cells. The effect of intact cells and intracellular extracts of these two bacterial strains on the oxidation of low density lipoprotein (LDL) isolated from cerebrovascular accident (CVA) patients and healthy subjects were also compared. Oxidation of LDL was monitored by measuring the lag time for the formation of conjugated dienes in isolated LDL particles. When LDL was treated respectively with 109 intact cells of B. longum MYL2 and L. delbrueckii ssp. bulgaricus MYL6, the lag time of oxidation of LDL was prolonged significantly. The extent of inhibition was greater on LDL isolated from healthy subjects than from CVA patients. When LDL from either CVA patients or healthy subjects was treated with intracellular extracts of 109 cells of B. longum MYL2 and L. delbrueckii ssp. bulgaricus MYL6, respectively, the copper-mediated oxidation was extensively inhibited with a lag time exceeding 180 min. Results from this study show a greater inhibitory effect on LDL oxidation exerted by the intracellular extract than intact cells, suggesting the presence of effective inhibitory factors in the intracellular extract. In summary, lactic acid bacteria are though to be a promising candidate to diminish oxidative stress. In addition, intracellular extracts of lactic acid bacteria have exhibited higher antioxidant activity than that of intact cells.Contents Acknowledgments.................... i Chinese Abstract....................iii English Abstract....................vi Contents.......................ix Abbreviations.....................x Overallintroduction..................1 1.Probiotics.........................1 2.Oxidative stress, atherosclerosis and antioxidant........33 3.Antioxidative effects of lactic acid bacteria...........46 4.Objectives and hypothesis................49 Chapter I Antioxidative Effects of Intracellular Extracts of Yogurt Bacteria on Lipid Peroxidation and Intestine Cells............................................64 Chapter II Antioxidative Effect of Lactic Acid Bacteria: Intact Cells vs. Intracellular Extracts...........88 Overall discussion and future perspectives.................................114 Appendix Resume and publication list........................................12

Heat-Killed Lactic Acid Bacteria Enhance Immunomodulatory Potential by Skewing the Immune Response toward Th1 Polarization

Heat-killed lactic acid bacteria not only possess immunomodulatory functions but also provide the advantages of longer product shelf life, easier storage, and more convenient transportation. To establish appropriate heat treatments for the industrial preparation of probiotics with immunomodulatory effects, 4 different heat treatments were used to kill 11 strains of lactic acid bacteria. Comparisons among the strains and with viable forms were carried out in terms of immunomodulatory activity and adhesion to Caco-2 cells. Field-emission scanning electron microscope (FE-SEM) was employed to observe morphological changes in bacteria after heating. Among the 11 viable strains, Lactobacillus gasseri AI-88 was the strongest inducer of interferon-gamma (IFN)-gamma and interleukin (IL)-12p70 production. However, after heat treatments its stimulatory ability was attenuated. Heat-killed Enterococcus faecalis YM-73 and Lactobacillus salivarius AP-32 strains showed enhanced stimulation of IFN-gamma and IL-12p70 secretion and coincidental decrease in IL-13 production. The adhesion of lactic acid bacteria to Caco-2 cells decreased with increases in temperature. However, heat exposure did not influence immunomodulatory activity. With rising temperature, roughness and unevenness of bacterial cell surfaces increased significantly. The results indicated that heat-killed E. faecalis YM-73 and L. salivarius AP-32 have immunomodulatory ability via increased Th1-associated cytokines and reduced Th2-associated cytokines, switching the immune response from a Th2 toward a Th1 response. These 2 heat-killed strains have the potential for development as commercial products

Hepatoprotective+Effect+of+Lactic+Acid+Bacteria+in+the+Attenuation+of+Oxidative+Stress+from+tert-Butyl+Hydroperoxide

The pathogenesis and progression of liver disease are associated with free radical injury and oxidative stress, which can be partially attenuated by antioxidants and free radical scavengers. Lactic acid bacteria, which have been traditionally used in the production of various fermented foods, are important intestinal microflora and natural antioxidants. The hypothesis that lactic acid bacteria can prevent or decrease tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in HepG2 cells was investigated. Intracellular extracts and heat-killed cells of Lactobacillus acidophilus La12, Lactobacillus delbrueckii ssp. bulgaricus Lb23, Bifidobacterium longum Bl36 and Streptococcus salivarius ssp. thermophilus St28 were used in this study. Lactate dehydrogenase (LDH), alanine aminotransferase (ALT), reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GRd), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) were determined to explore the influence of lactic acid bacteria intervention on cell damage and antioxidative status. Toxic damage to hepatocytes. by t-BH P was attenuated by lactic acid bacteria (which exerted protective effects by decreasing the risk of accumulated ROS and by reactivating antioxidant enzymes) in HepG2 cells treated with lactic acid bacteria before t-BHP exposure. The results of this study provide new insights into the mechanisms by which lactic acid bacteria with antioxidative properties can help to protect the liver

Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells

Background: Epigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. Methods: H1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and β-actin, were performed by western blot analysis. Results: Our data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. Conclusion: Chidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest

Protective Effect of D-Methionine on Body Weight Loss, Anorexia, and Nephrotoxicity in Cisplatin-Induced Chronic Toxicity in Rats

D-methionine is a sulfur-containing amino acid that can act as a potent antioxidant. Anorexia and nephrotoxicity are side effects of cisplatin. The protective effects of D-methionine on cisplatin-induced anorexia and renal injury were investigated. The model of chronic cisplatin administration (5 mg/kg body weight) involved intraperitoneal injection on days 1, 8, and 15 and oral D-methionine (300 mg/kg body weight) coadministration daily for 20 days. On the 21st day of treatment, food intake and body weight in the cisplatin-treated group significantly decreased by 52% and 31%, respectively, when compared with a control group. D-methionine coadministration with cisplatin decreased food intake and body weight by 29% and 8%, respectively. In cisplatin-treated rats, white blood cell, mean corpuscular volume, and platelet values significantly decreased, while mean corpuscular hemoglobin concentration significantly increased by 8.6% when compared with control rats. Cisplatin administration resulted in significantly decreased feeding efficiency, elevated renal oxidative stress, and reduced antioxidative activity. Leukocyte infiltration, tubule vacuolization, tubular expansion, and swelling were observed in the kidneys of cisplatin-treated rats. Oral D-methionine exhibited an antianorexic effect, with improvement in food intake, feeding efficiency, and hematological toxicities, as well as a protective effect against nephrotoxicity by elevated antioxidative activity. D-methionine may serve as a chemoprotectant in patients receiving cisplatin as part of a chemotherapy regimen