1. Effects of blue shark cartilage alkaline extract on cytokine secretions in bronchoalveolar lavage fluid of ovalbumin-sensitized and -challenged mice Mei-Chun Ting 1, Hui-Hsiang Chang 2, Chia-Hsin Chang 1, Po-Chuen Shieh 1 * 1. Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Taiwan 2. Department of Food Technology, Tajen University, Taiwan The blue shark (Prionace glauca) is a species of requiem shark, family Carcharhinidae, which has cartilage axial skeleton. The main constituents of cartilage are collagen, glycosaminoglycan and a variety of proteins. The scientific literatures showed that the active substances of shark cartilage could inhibit angiogenesis, tumor formation and inflammation. Asthma is recognized as a T-helper type 2 (Th2)-skewed allergic disease. Th2-type cells are important in Humoral immunity and defense against extracellular pathogens via eosinophilic infiltration, as well as IgE and IgG1 production in vivo. Th2-type cytokines include interleukin (IL)-4, IL-5, IL-6, IL-9, IL-10, and IL-13. IL-4and IL-13 are implicated in isotype switching of B-cells to produce IgE. In contrast to the Th2 cells, Th1 cells are characterized by the production of IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Th1 cells promote cell-mediated immunity to destroy intracellular pathogens, inhibit eosinophilic infiltration, IgE and IgG1 production, and strengthen IgG2a production in vivo. Th1-skewed immune responses are generally pro-inflammatory and may result in autoimmune and chronic inflammatory diseases. However, Th2-skewed immune responses may cause asthma and allergy. Whether it is possible to maintain the Th1/Th2 balance through dietary modification is an important issue in immunology. An ovalbumin (OVA)-sensitized asthmatic murine model was used to evaluate the effects of blue shark cartilage alkaline extract (BSC-AE) on the allergic asthma. The aim of study is to evaluate the effects of BSC-AE on the cytokine secretions of bronchoalveolar lavage fluid (BALF) from OVA-sensitized mice. 1. Materials, experimental animals and feeds: The inbred female BALB/c mice (8 weeks old) were randomly divided into five groups, including non-sensitized control (code as PBS/DDW), dietary control (OVA/DDW), low dose BSC-AE supplement group (OVA/LD, 150 mg/kgBW/day), medium dose BSC-AE supplement group (OVA/MD, 450 mg/kgBW/day), and high dose BSC-AE supplement group (OVA/HD, 1350 mg/kgBW/day). Mice received 0.3 ml extra of deionized water or BSC-AE by tube gavage for five weeks. Mice were fed with experimental diets ad libitum for 35 consecutivedays. : 2. OVA-sensitized and -challenged allergic inflammation murine model: 3. Collection of BALF and assay of inflammatory mediators and cytokines in BALF: The level of protein in BALF were determined by BCA assay. The levels of cytokines (IL-1β, IL-2. IL-4. IL-5, IL-6, IL-10, IFN-γ and TNF-α) in BALF were determined by sandwich ELISA kits. The results showed that BSC-AE supplement significantly (P < 0.05) decreased the cytokine levels but did not markedly (P > 0.05) affect the cytokine ratios of Th1/Th2 and pro-/anti-inflammatory cytokines that secreted in BALF. It is suggested that BSC-AE supplement may not modulate the Th1/Th2 balance toward Th1 pole in the allergic asthmatic murine model, but benefit to diminish the Th2-skewed allergic airway inflammation.. Introduction Materials and Methods Results Conclusions Table 1 The Th1/Th2 cytokine levels of bronchoalvelor lavage fluid (BALF) from female BALB/c mice fed blue shark cartilage alkaline extract in an OVA-sensitized asthmatic murine model Groups Th-1 type cytokine (pg/ml)Th-2 type cytokine (pg/ml) IFN-γIL-2IL-4IL-5 OVA/DDW10.1±8.135.2±19.2102.8±44.4417.3±207.1 OVA/LD4.4±5.123.4±11.676.9±34.8320.0±179.7 OVA/MD4.1±4.424.0±12.174.9±23.5312.8±126.7 OVA/HD2.7±1.918.4±5.4*54.9±23.5*243.8±91.7* PBS/DDW1.2±1.414.8±3.7*48.6±8.6*173.4±36.8* Data are presented as mean ± SD (n = 9 -12). Data within the same column are analyzed using analysis of variance (ANOVA), followed by unpaired Student’s t-test. Asterisk ( ＊ ) means significantly (p ＜ 0.05) different from the dietary control group, OVA/DDW. The sensitivity of the ELISA kits used in this study was ＜ 15.6 pg/ml. Table 2 The Th2/Th1 cytokine ratios of bronchoalvelor lavage fluid (BALF) from female BALB/c mice fed blue shark cartilage alkaline extract in an OVA-sensitized asthmatic murine model Groups Th2/Th1 cytokine ratio (pg/pg) IL4/IL-2IL5/IL-2(IL4+IL5)/IL-2 OVA/DDW3.18±0.6312.41±1.7915.59±2.37 OVA/LD3.08±0.7711.99±2.5215.07±3.11 OVA/MD3.38±0.7713.59±2.9016.98±3.60 OVA/HD2.95±1.3612.10±6.4615.05±7.77 PBS/DDW3.24±0.4011.85±1.0415.10±1.37 Data are presented as mean ± SD (n = 9 -12). Data within the same column are analyzed using analysis of variance (ANOVA), followed by unpaired Student’s t-test. Table 3 The pro-/anti-inflammatory cytokine levels of bronchoalvelor lavage fluid (BALF) from female BALB/c mice fed blue shark cartilage alkaline extract in an OVA-sensitized asthmatic murine model Groups Pro-inflammation cytokine (pg/ml)Anti-inflammation cytokine (pg/ml) IL-1βTNF-αIL6IL-10 OVA/DDW21.1±10.0242.8±125.136.5±16.170.8±28.7 OVA/LD16.4±8.3148.6±64.621.9±11.1*47.8±16.0 OVA/MD15.9±6.6165.8±86.724.3±9.5*49.0±17.5* OVA/HD14.9±4.1149.9±47.1*17.0±6.5*39.7±12.2* PBS/DDW12.8±3.6*132.2±62.4*18.5±5.9*36.0±12.0* Data are presented as mean ± SD (n = 9 -12). Data within the same column are analyzed using analysis of variance (ANOVA), followed by unpaired Student’s t-test. Asterisk ( ＊ ) means significantly (p ＜ 0.05) different from the dietary control group, OVA/DDW. The sensitivity of the ELISA kits used in this study was ＜ 15.6 pg/ml. Table 4 The pro-/anti-inflammatory cytokine ratios of bronchoalvelor lavage fluid (BALF) from female BALB/c mice fed blue shark cartilage alkaline extract in an OVA-sensitized asthmatic murine model Groups pro-/anti-inflammtory cytokine ratio (pg/pg) IL6/IL10(IL-1β+ TNF-α+IL6)/IL10 OVA/DDW0.51±0.054.08±0.63 OVA/LD0.46±0.133.83±0.69 OVA/MD0.50±0.114.24±1.25 OVA/HD0.48±0.114.73±0.55* PBS/DDW0.56±0.174.23±1.00 Data are presented as mean ± SD (n = 9 -12). Data within the same column are analyzed using analysis of variance (ANOVA), followed by unpaired Student’s t-test. Asterisk ( ＊ ) means significantly (p ＜ 0.05) different from the dietary control group, OVA/DDW.