枯草芽孢杆菌肽聚糖对β-伴大豆球蛋白诱导的鲤幼鱼肠上皮细胞损伤的保护作用
|
摘要
为观察不同浓度枯草芽孢杆菌肽聚糖对β-伴大豆球蛋白(β-conglycinin)诱导的鲤幼鱼肠上皮细胞炎性损伤的保护作用,将原代培养72 h(26°C、6%的CO2)的24孔鲤幼鱼IECs培养板随机设为6组,每组4个重复。其中1组为阴性对照,其余5组用浓度为1.0 mg/m L的枯草芽孢杆菌肽聚糖诱导24 h后,分别更换浓度为0(阳性对照)、0.15、0.30、0.45和0.60 mg/m L枯草芽孢杆菌肽聚糖培养液,相同条件培养12、24和36 h,分别检测细胞的抗超氧阴离子(ASA)和抗羟自由基(AHR)能力、蛋白羰基(PC)含量、抗氧化酶(SOD、CAT和GPx)活性和细胞因子IL-1β、IL-8、IL-10、TNF-α1、TGF-β基因表达。结果显示,β-conglycinin上调细胞炎性因子表达,降低细胞抗氧化性能。添加枯草芽孢杆菌肽聚糖后,随PG浓度增加和处理时间延长,细胞抗氧化性能增加,呈显著的量效关系,且36 h呈二次曲线相关。IL-1β、IL-8、TNF-α1 m RNA表达呈二次曲线递减,IL-10、TGF-βm RNA表达呈线性递增。研究表明,不同浓度枯草芽孢杆菌肽聚糖均可保护由β-conglycinin诱导引起的鲤幼鱼IECs氧化损伤,提高IECs抗氧化能力;高浓度PG通过抑制致炎因子和促进抗炎细胞因子的基因表达,提高IECs的抗炎能力。
This experiment was conducted to investigate the protective effects of different concentration of Bacillus subtilis peptidoglycan on the β-conglycinin-induced inflammatory lesions in intestinal epithelial cells of juvenile carp(Cyprinus carpio). In 24-cell microplates, the intestinal epithelial cells(IECs) of juvenile carp were primarily cultured for 72 h at 26 °C and 6% CO2, and then the IECs were randomly divided into 6 groups with 4 replicates per group. One of the 6 groups was set as negative control group, and the other groups were all supplemented with 1.0 mg/m L β-conglycinin in culture medium in order to establish inflammatory lesions. After 24 h further culture in the same conditions, the culture medium was changed into the B. subtilis peptidoglycan culture medium with 0(positive control group), 0.15, 0.30, 0.45 and 0.60 mg/m L, respectively. The samples were collected to measure the antioxidant and anti-inflammatory indices after 12, 24 and 36 h B. subtilis peptidoglycan culture. The results showed as follows: β-conglycinin exposure significantly decreased the activity of ASA, AHR,SOD, CAT, GPx, and increased the content of PC and the m RNA expression of inflammatory cytokines(IL-1β,IL-8, TNF-α1, IL-10 and TGF-β). The 12, 24 and 36 hours after the peptidoglycan were given, the activity of ASA,AHR, SOD, CAT, GPx and the content of PC in the cells were decreased in a dose-dependent manner; and the m RNA levels of IL-1β, IL-8 and TNF-α1 were down-regulated and IL-10 and TGF-β were up-regulated. Different concentration of B. subtilis peptidoglycan could significantly improve the anti-oxidative activity, and increase the m RNA levels of anti-inflammation cytokine, and decrease the pro-inflammation cytokine, and inhibit the inflammatory response of the carp IECs induced by β-conglycinin.
This experiment was conducted to investigate the protective effects of different concentration of Bacillus subtilis peptidoglycan on the β-conglycinin-induced inflammatory lesions in intestinal epithelial cells of juvenile carp(Cyprinus carpio). In 24-cell microplates, the intestinal epithelial cells(IECs) of juvenile carp were primarily cultured for 72 h at 26 °C and 6% CO2, and then the IECs were randomly divided into 6 groups with 4 replicates per group. One of the 6 groups was set as negative control group, and the other groups were all supplemented with 1.0 mg/m L β-conglycinin in culture medium in order to establish inflammatory lesions. After 24 h further culture in the same conditions, the culture medium was changed into the B. subtilis peptidoglycan culture medium with 0(positive control group), 0.15, 0.30, 0.45 and 0.60 mg/m L, respectively. The samples were collected to measure the antioxidant and anti-inflammatory indices after 12, 24 and 36 h B. subtilis peptidoglycan culture. The results showed as follows: β-conglycinin exposure significantly decreased the activity of ASA, AHR,SOD, CAT, GPx, and increased the content of PC and the m RNA expression of inflammatory cytokines(IL-1β,IL-8, TNF-α1, IL-10 and TGF-β). The 12, 24 and 36 hours after the peptidoglycan were given, the activity of ASA,AHR, SOD, CAT, GPx and the content of PC in the cells were decreased in a dose-dependent manner; and the m RNA levels of IL-1β, IL-8 and TNF-α1 were down-regulated and IL-10 and TGF-β were up-regulated. Different concentration of B. subtilis peptidoglycan could significantly improve the anti-oxidative activity, and increase the m RNA levels of anti-inflammation cytokine, and decrease the pro-inflammation cytokine, and inhibit the inflammatory response of the carp IECs induced by β-conglycinin.
引文
[1]Zhao Y,Qin G X,Sun Z W,et al.Effects of glycinin andβ-conglycinin on enterocyte apoptosis,proliferation and migration of piglets[J].Food and Agricultural Immunology,2010,21(3):209-218.
[2]West X Z,Malinin N L,Merkulova A A,et al.Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands[J].Nature,2010,467(7318):972-976.
[3]Bhor V M,Raghuram N,Sivakami S.Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats[J].International Journal of Biochemistry&Cell Biology,2004,36(1):89-97.
[4]Baltac?o?lu E,Akal?n F A,Alver A,et al.Protein carbonyl levels in serum and gingival crevicular fluid in patients with chronic periodontitis[J].Archives of Oral Biology,2008,53(8):716-722.
[5]Zhang J X,Guo L Y,Feng L,et al.Soybeanβ-conglycinin induces inflammation and oxidation and causes dysfunction of intestinal digestion and absorption in fish[J].PLo S One,2013,8(3):e58115.
[6]Wu Z,Pan D D,Guo Y X,et al.Structure and anti-inflammatory capacity of peptidoglycan from Lactobacillus acidophilus in RAW-264.7 cells[J].Carbohydrate Polymers,2013,96(2):466-473.
[7]Christensen H R,Fr?kiaer H,Pestka J J.Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells[J].Journal of Immunology,2002,168(1):171-178.
[8]李丹,李艾黎,季晓梅,等.乳杆菌肽聚糖对β-乳球蛋白致敏小鼠脾细胞Th1/Th2及Treg/Th17失衡的体外影响[J].微生物学通报,2014,41(7):1334-1341.Li D,Li A L,Ji X M,et al.Effect of whole peptidoglycan from lactobacilli on the imbalance of Th1/Th2 and Treg/Th17 in lymphocyte of bovineβ-lactoglobulin-sensitized mice in vitro[J].Microbiology China,2014,41(7):1334-1341(in Chinese).
[9]Frodermann V,Chau T A,Sayedyahossein S,et al.Amodulatory interleukin-10 response to staphylococcal peptidoglycan prevents Th1/Th17 adaptive immunity to Staphylococcus aureus[J].Journal of Infectious Diseases,2011,204(2):253-262.
[10]Mantis N J,Rol N,Corthésy B.Secretory Ig A's complex roles in immunity and mucosal homeostasis in the gut[J].Mucosal Immunology,2011,4(6):603-611.
[11]Yin H C,Guan J J,Yang D H,et al.Effects of dietary protein level on growth performance,muscle composition and nitrogen excretion in juvenile Ancherythroculter nigrocauda(Yih&Wu 1964)[J].Asian Journal ofScience and Technology,2014,5(3):208-213.
[12]Sun P,Li D F,Li Z J,et al.Effects of glycinin on Ig E-mediated increase of mast cell numbers and histamine release in the small intestine[J].The Journal of Nutritional Biochemistry,2008,19(9):627-633.
[13]Turan A,Mahmood A.The profile of antioxidant systems and lipid peroxidation across the crypt-villus axis in rat intestine[J].Digestive Diseases and Sciences,2007,52(8):1840-1844.
[14]Kohen R,Nyska A.Oxidation of biological systems:oxidative stress phenomena,antioxidants,redox reactions,and methods for their quantification[J].Toxicologic Pathology,2002,30(6):620-650.
[15]Koven W,Rojas-García C,Finn R,et al.Stimulatory effect of ingested protein and/or free amino acids on the secretion of the gastro-endocrine hormone cholecystokinin and on tryptic activity,in early-feeding herring larvae,Clupea harengus[J].Marine Biology,2002,140(6):1241-1247.
[16]Urán P A,Gon?alves A A,Taverne-Thiele J J,et al.Soybean meal induces intestinal inflammation in common carp(Cyprinus carpio L.)[J].Fish&Shellfish Immunology,2008,25(6):751-760.
[17]Komatsu K,Tsutsui S,Hino K,et al.Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout,Oncorhynchus mykiss,in response to bacterial infection[J].Developmental&Comparative Immunology,2009,33(4):499-506.
[18]Fu C L,Ye Y L,Lee Y L,et al.Effects of overexpression of IL-10,IL-12,TGF-βand IL-4 on allergen induced change in bronchial responsiveness[J].Respiratory Research,2006,7(1):72.
[19]Fernandez E M,Valenti V,Rockel C,et al.Anti-inflammatory capacity of selected lactobacilli in experimental colitis is driven by NOD2-mediated recognition of a specific peptidoglycan-derived muropeptide[J].Gut,2011,60(8):1050-1059.
[20]Lilleeng E,Penn M H,Haugland?,et al.Decreased expression of TGF-β,GILT and T-cell markers in the early stages of soybean enteropathy in Atlantic salmon(Salmo salar L.)[J].Fish&Shellfish Immunology,2009,27(1):65-72.
[2]West X Z,Malinin N L,Merkulova A A,et al.Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands[J].Nature,2010,467(7318):972-976.
[3]Bhor V M,Raghuram N,Sivakami S.Oxidative damage and altered antioxidant enzyme activities in the small intestine of streptozotocin-induced diabetic rats[J].International Journal of Biochemistry&Cell Biology,2004,36(1):89-97.
[4]Baltac?o?lu E,Akal?n F A,Alver A,et al.Protein carbonyl levels in serum and gingival crevicular fluid in patients with chronic periodontitis[J].Archives of Oral Biology,2008,53(8):716-722.
[5]Zhang J X,Guo L Y,Feng L,et al.Soybeanβ-conglycinin induces inflammation and oxidation and causes dysfunction of intestinal digestion and absorption in fish[J].PLo S One,2013,8(3):e58115.
[6]Wu Z,Pan D D,Guo Y X,et al.Structure and anti-inflammatory capacity of peptidoglycan from Lactobacillus acidophilus in RAW-264.7 cells[J].Carbohydrate Polymers,2013,96(2):466-473.
[7]Christensen H R,Fr?kiaer H,Pestka J J.Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells[J].Journal of Immunology,2002,168(1):171-178.
[8]李丹,李艾黎,季晓梅,等.乳杆菌肽聚糖对β-乳球蛋白致敏小鼠脾细胞Th1/Th2及Treg/Th17失衡的体外影响[J].微生物学通报,2014,41(7):1334-1341.Li D,Li A L,Ji X M,et al.Effect of whole peptidoglycan from lactobacilli on the imbalance of Th1/Th2 and Treg/Th17 in lymphocyte of bovineβ-lactoglobulin-sensitized mice in vitro[J].Microbiology China,2014,41(7):1334-1341(in Chinese).
[9]Frodermann V,Chau T A,Sayedyahossein S,et al.Amodulatory interleukin-10 response to staphylococcal peptidoglycan prevents Th1/Th17 adaptive immunity to Staphylococcus aureus[J].Journal of Infectious Diseases,2011,204(2):253-262.
[10]Mantis N J,Rol N,Corthésy B.Secretory Ig A's complex roles in immunity and mucosal homeostasis in the gut[J].Mucosal Immunology,2011,4(6):603-611.
[11]Yin H C,Guan J J,Yang D H,et al.Effects of dietary protein level on growth performance,muscle composition and nitrogen excretion in juvenile Ancherythroculter nigrocauda(Yih&Wu 1964)[J].Asian Journal ofScience and Technology,2014,5(3):208-213.
[12]Sun P,Li D F,Li Z J,et al.Effects of glycinin on Ig E-mediated increase of mast cell numbers and histamine release in the small intestine[J].The Journal of Nutritional Biochemistry,2008,19(9):627-633.
[13]Turan A,Mahmood A.The profile of antioxidant systems and lipid peroxidation across the crypt-villus axis in rat intestine[J].Digestive Diseases and Sciences,2007,52(8):1840-1844.
[14]Kohen R,Nyska A.Oxidation of biological systems:oxidative stress phenomena,antioxidants,redox reactions,and methods for their quantification[J].Toxicologic Pathology,2002,30(6):620-650.
[15]Koven W,Rojas-García C,Finn R,et al.Stimulatory effect of ingested protein and/or free amino acids on the secretion of the gastro-endocrine hormone cholecystokinin and on tryptic activity,in early-feeding herring larvae,Clupea harengus[J].Marine Biology,2002,140(6):1241-1247.
[16]Urán P A,Gon?alves A A,Taverne-Thiele J J,et al.Soybean meal induces intestinal inflammation in common carp(Cyprinus carpio L.)[J].Fish&Shellfish Immunology,2008,25(6):751-760.
[17]Komatsu K,Tsutsui S,Hino K,et al.Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout,Oncorhynchus mykiss,in response to bacterial infection[J].Developmental&Comparative Immunology,2009,33(4):499-506.
[18]Fu C L,Ye Y L,Lee Y L,et al.Effects of overexpression of IL-10,IL-12,TGF-βand IL-4 on allergen induced change in bronchial responsiveness[J].Respiratory Research,2006,7(1):72.
[19]Fernandez E M,Valenti V,Rockel C,et al.Anti-inflammatory capacity of selected lactobacilli in experimental colitis is driven by NOD2-mediated recognition of a specific peptidoglycan-derived muropeptide[J].Gut,2011,60(8):1050-1059.
[20]Lilleeng E,Penn M H,Haugland?,et al.Decreased expression of TGF-β,GILT and T-cell markers in the early stages of soybean enteropathy in Atlantic salmon(Salmo salar L.)[J].Fish&Shellfish Immunology,2009,27(1):65-72.