日本血吸虫成虫可溶性蛋白和重组半胱氨酸蛋白酶抑制蛋白抑制小鼠结肠炎的研究（英文）
|
摘要
目的探讨日本血吸虫可溶性成虫蛋白(SjSWP)和重组半胱氨酸蛋白酶抑制蛋白(rSjcystatin)对模拟炎症性肠病的小鼠结肠炎的作用。方法 24只BALB/c雌鼠分为健康对照组、磷酸盐缓冲液组(PBS组)、rSjcystatin治疗组(rSjcystatin组)、 Sj SWP治疗组(SjSWP组)。PBS、 rSjcystatin、 SjSWP组用0.1 ml 5%2,4,6-三硝基苯磺酸(TNBS)灌肠诱导结肠炎,灌肠后6、 24 h分别腹腔注射100μl PBS、 50μg r Sjcystatin、 50μg SjSWP;健康对照组在相应时间点用等量的PBS灌肠及腹腔注射。TNBS灌肠72 h后无痛处死各组小鼠,检测宏观评分、结肠长度、髓过氧化物酶活性和显微镜下微观评分来评估炎症参数和病理改变。流式细胞仪检测小鼠脾脏中Th1和Th2及T调节细胞(Tregs)的比例。ELISA检测小鼠结肠组织匀浆上清中γ干扰素(IFN-γ)、白细胞介素-4 (IL-4)、 IL-13、 IL-10等炎性细胞因子的水平。多组计量资料分析采用单因素方差分析,多组均数间的两两比较用Student-Newman-Keuls检验。结果 TNBS诱导的结肠炎小鼠表现出明显的炎症和病理学特征,表现为体质量下降,便血、腹泻、结肠缩短、溃疡,以及炎性细胞因子IFN-γ、炎性细胞浸润和髓过氧化物酶活性的增加。与PBS组相比, rSjcystatin和SjSWP的干预治疗可以显著改善TNBS诱导的结肠炎症,表现为宏观评分(改善结肠糜烂、黏连、溃疡和缩短)和微观评分(减少组织损伤和炎症)的降低。健康对照组、 PBS组、 rSjcystatin组和SjSWP组小鼠结肠的宏观评分分别为0、 9.8±0.8、 4.3±1.0和4.5±1.4,微观评分分别为0、 6.0±0.4、 2.2±0.5和2.8±0.6,rSjcystatin组、 SjSWP组与PBS组相比差异均有统计学意义(F=57.8、 34.1, P <0.01), rSjcystatin组与SjSWP组间差异均无统计学意义(P> 0.05)。健康对照组、 PBS组、 rSjcystatin组和SjSWP组髓过氧化物酶分别为0.3±0.0、 1.4±0.1、 0.6±0.1和0.6±0.1 (F=32.5, P <0.01);脾脏Tregs比例分别为(4.4±0.7)%、(8.2±3.1)%、(18.4±2.1)%和(13.4±1.9)%(F=8.3, P <0.01);结肠组织匀浆上清中IFN-γ、 IL-10水平分别为(952.0±61.1)、(1 684.6±158.3)、(1 092.0±81.9)、(1 001.6±111.2) pg/ml,(322.8±27.4)、(399.3±77.9)、(860.6±153.6)、(892.0±113.8) pg/ml (F=9.6、 8.3, P <0.05);与PBS组相比, rSjcystatin与SjSWP组的INF-γ、髓过氧化物酶活性明显减低, Tregs应答增强, IL-10分泌增多,差异均有统计学意义(P <0.01), rSjcystatin组与SjSWP组间差异均无统计学意义(P> 0.05)。结论 SjSWP及rSjcystatin均可通过上调小鼠Treg和Th2介导的免疫应答,减轻小鼠实验性结肠炎,抑制导致结肠炎发病的Th1免疫应答。
Objective To explore the potential therapeutic effect of Schistosoma japonicum soluble worm protein(SjSWP) and recombinant cystatin(rSjcystatin) on 2,4,6-trinitrobenzene sulfonic acid(TNBS)-induced colitis in a mouse model that mimics human inflammatory bowel diseases(IBDs). Methods Twenty-four female BALB/c mice were divided into four groups: non-treated control group(control), phosphate buffer(PBS) treated group,rSjcystatin treated group(rSjcystatin) and SjSWP treated group(SjSWP). The PBS, rSjcystatin, SjSWP treated groups were respectively intraperitoneally injected with 100 μl PBS, 50 μg rSjcystatin and 50 μg SjSWP at 6 h and 24 h after being induced with 0.1 ml of 5% TNBS by enema for colitis, while the control group was injected with the same amount of PBS at corresponding time without colitis induced. After 72 hours of TNBS enema, mice in each group were sacrificed under anesthesia. The pathological and inflammatory parameters were determined by measuring macroscopic score, myeloperoxidase(MPO) activity, colon length and microscopic score. The Th1 to Th2 cells and T regulatory cells(Tregs) in the spleen of mice were measured by flow cytometry, and the immune responses induced by rSjcystatin and SjSWP were investigated. The levels of cytokines including interferon-gamma(IFN-γ), interleukin-4(IL-4), IL-13, IL-10 in the colon tissue of mice were detected by enzyme-linked immunosorbent assay(ELISA).Multiple groups of measurement data were analyzed by one-way analysis of variance, and pairwise comparisons between multiple groups of mean were tested by Student-Newman-Keuls. Results The mice with TNBS-induced colitis showed significant inflammation and pathology characterized by the weight loss, rectal bleeding, diarrhea, colon shrinking and ulceration, as well as induced inflammatory cytokine INF-γ, inflammatory cell infiltration and increased MPO activity. In this study we identified that treatment with rSjcystatin and SjSWP significantly reduced the TNBSinduced colon inflammation characterized by reduced disease manifestations in macroscopic score(improved colon erosion, adhesion, ulceration and colon shrinking) and microscopic scores(less tissue damage and inflammation),compared to the group of mice received PBS only. The macroscopic scores of colon in the control group, PBS group, rSjcystatin group and SjSWP group were 0, 9.8 ± 0.8, 4.3 ± 1.0 and 4.5 ± 1.4; the mean microscopic scores of mice were 0, 6.00 ± 0.4, 2.2 ± 0.5 and 2.8 ± 0.6, respectively. Among the above results, rSjcystatin and SjSWP had significant differences compared with PBS group( F = 57.8, 34.1, P < 0.01). There was no significant difference between rSjcystatin group and SjSWP group(P > 0.05). MPO values in control group, PBS group, rSjcystatin group and SjSWP group were(0.3 ± 0.0),(1.4 ± 0.1),(0.6 ± 0.1) and(0.6 ± 0.1)(F = 32.5, P < 0.01); the Tregs in spleen were(4.4 ± 0.7),(8.2 ± 3.1),(18.4 ± 2.1) and(13.4 ± 1.9)%(F = 8.3, P < 0.01); the IFN-γ and IL-10 in colon tissue homogenate supernatants of colon tissue were(952.0 ± 61.1),(1 684.6 ± 158.3),(1 092.0 ± 81.9) and(1 001.6 ± 111.2) pg/ml;(322.8 ± 27.4),(399.3 ± 77.9),(860.6 ± 153.6) and(892.0 ± 113.8) pg/ml respectively(F =9.6, 8.3, P < 0.05). The results showed that, in rSjcystatin group and SjSWP group, the inflammatory cytokine INF-γ,MPO activity in colon tissue decreased, combined with boosted Tregs response and the secretion of regulatory cytokine IL-10, compared to the group of mice received PBS only with statistical difference( P < 0.01). There was no significant difference for the therapeutic efficacy on inflammatory colitis between rSjcystatin group and SjSWP group(P > 0.05). Conclusions SjSWP and rSjcystatin could alleviate experimental colitis in mice via stimulating Tregs and Th2 mediated responses and, correspondingly, suppressing Th1 pro-inflammatory response related to the pathogenesis of colitis.
Objective To explore the potential therapeutic effect of Schistosoma japonicum soluble worm protein(SjSWP) and recombinant cystatin(rSjcystatin) on 2,4,6-trinitrobenzene sulfonic acid(TNBS)-induced colitis in a mouse model that mimics human inflammatory bowel diseases(IBDs). Methods Twenty-four female BALB/c mice were divided into four groups: non-treated control group(control), phosphate buffer(PBS) treated group,rSjcystatin treated group(rSjcystatin) and SjSWP treated group(SjSWP). The PBS, rSjcystatin, SjSWP treated groups were respectively intraperitoneally injected with 100 μl PBS, 50 μg rSjcystatin and 50 μg SjSWP at 6 h and 24 h after being induced with 0.1 ml of 5% TNBS by enema for colitis, while the control group was injected with the same amount of PBS at corresponding time without colitis induced. After 72 hours of TNBS enema, mice in each group were sacrificed under anesthesia. The pathological and inflammatory parameters were determined by measuring macroscopic score, myeloperoxidase(MPO) activity, colon length and microscopic score. The Th1 to Th2 cells and T regulatory cells(Tregs) in the spleen of mice were measured by flow cytometry, and the immune responses induced by rSjcystatin and SjSWP were investigated. The levels of cytokines including interferon-gamma(IFN-γ), interleukin-4(IL-4), IL-13, IL-10 in the colon tissue of mice were detected by enzyme-linked immunosorbent assay(ELISA).Multiple groups of measurement data were analyzed by one-way analysis of variance, and pairwise comparisons between multiple groups of mean were tested by Student-Newman-Keuls. Results The mice with TNBS-induced colitis showed significant inflammation and pathology characterized by the weight loss, rectal bleeding, diarrhea, colon shrinking and ulceration, as well as induced inflammatory cytokine INF-γ, inflammatory cell infiltration and increased MPO activity. In this study we identified that treatment with rSjcystatin and SjSWP significantly reduced the TNBSinduced colon inflammation characterized by reduced disease manifestations in macroscopic score(improved colon erosion, adhesion, ulceration and colon shrinking) and microscopic scores(less tissue damage and inflammation),compared to the group of mice received PBS only. The macroscopic scores of colon in the control group, PBS group, rSjcystatin group and SjSWP group were 0, 9.8 ± 0.8, 4.3 ± 1.0 and 4.5 ± 1.4; the mean microscopic scores of mice were 0, 6.00 ± 0.4, 2.2 ± 0.5 and 2.8 ± 0.6, respectively. Among the above results, rSjcystatin and SjSWP had significant differences compared with PBS group( F = 57.8, 34.1, P < 0.01). There was no significant difference between rSjcystatin group and SjSWP group(P > 0.05). MPO values in control group, PBS group, rSjcystatin group and SjSWP group were(0.3 ± 0.0),(1.4 ± 0.1),(0.6 ± 0.1) and(0.6 ± 0.1)(F = 32.5, P < 0.01); the Tregs in spleen were(4.4 ± 0.7),(8.2 ± 3.1),(18.4 ± 2.1) and(13.4 ± 1.9)%(F = 8.3, P < 0.01); the IFN-γ and IL-10 in colon tissue homogenate supernatants of colon tissue were(952.0 ± 61.1),(1 684.6 ± 158.3),(1 092.0 ± 81.9) and(1 001.6 ± 111.2) pg/ml;(322.8 ± 27.4),(399.3 ± 77.9),(860.6 ± 153.6) and(892.0 ± 113.8) pg/ml respectively(F =9.6, 8.3, P < 0.05). The results showed that, in rSjcystatin group and SjSWP group, the inflammatory cytokine INF-γ,MPO activity in colon tissue decreased, combined with boosted Tregs response and the secretion of regulatory cytokine IL-10, compared to the group of mice received PBS only with statistical difference( P < 0.01). There was no significant difference for the therapeutic efficacy on inflammatory colitis between rSjcystatin group and SjSWP group(P > 0.05). Conclusions SjSWP and rSjcystatin could alleviate experimental colitis in mice via stimulating Tregs and Th2 mediated responses and, correspondingly, suppressing Th1 pro-inflammatory response related to the pathogenesis of colitis.
引文
[1]Strachan DP.Hay fever,hygiene,and household size[J].BMJ,1989,299(6710):1259-1260.
[2]Driss V,El Nady M,Delbeke M,et al.The schistosome glutathione S-transferase P28GST,a unique helminth protein,prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils[J].Mucosal Immunol,2016,9(2):322-335.
[3]Allen JE,Maizels RM.Diversity and dialogue in immunity to helminths[J].Nat Rev Immunol,2011,11(6):375-388.
[4]Hasby EA,Hasby Saad MA,Shohieb Z,et al.FoxP3+Tregulatory cells and immunomodulation after Schistosoma mansoni egg antigen immunization in experimental model of inflammatory bowel disease[J].Cell Immunol,2015,295(1):67-76.
[5]Heylen M,Ruyssers NE,Gielis EM,et al.Of worms,mice and man:an overview of experimental and clinical helminth-based therapy for inflammatory bowel disease[J].Pharmacol Ther,2014,143(2):153-167.
[6]Schierack P,Lucius R,Sonnenburg B,et al.Parasite-specific immunomodulatory functions of filarial cystatin[J].Infect Immun,2003,71(5):2422-2429.
[7]Hartmann S,Kyewski B,Sonnenburg B,et al.A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production[J].Eur J Immunol,1997,27(9):2253-2260.
[8]Magister S,Kos J.Cystatins in immune system[J].J Cancer,2013,4(1):45-56.
[9]Wang SS,Xie YY,Yang XD,et al.Therapeutic potential of recombinant cystatin from Schistosoma japonicum in TNBS-induced experimental colitis of mice[J].Parasit Vectors,2016,9:6.
[10]Liu F,Cheng WS,Pappoe F,et al.Schistosoma japonicum cystatin attenuates murine collagen-induced arthritis[J].Parasitol Res,2016,115(10):3795-3806.
[11]Wynn TA,Chawla A,Pollard JW.Origins and hallmarks of macrophages:development,homeostasis,and disease[J].Nature,2013,496(7446):445.
[12]Yang X,Liu J,Yue Y,et al.Cloning,expression and characterisation of a typeⅡcystatin from Schistosoma japonicum,which could regulate macrophage activation[J].Parasitol Res,2014,113(11):3985-3992.
[13]He BH.Schistosoma japonicum:the molecular characteristics of cystatin and it’s immune regulation effects on mice[D].Wuhan:Wuhan University,2014.
[14]Ratcliffe EC,Wilson RA.The magnitude and kinetics of delayed-type hypersensitivity responses in mice vaccinated with irradiated cercariae of Schistosoma mansoni[J].Parasitology,1991,103(1):65-75.
[15]Menachem Y,Trop S,Kolker O,et al.Adoptive transfer of NK1.1+lymphocytes in immune-mediated colitis:a proinflammatory or a tolerizing subgroup of cells?[J].Microbes Infect,2005,7(5/6):825-835.
[16]Obermeier F,Kojouharoff G,Hans W,et al.Interferon-gamma(IFN-γ)-and tumour necrosis factor(TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium(DSS)-induced colitis in mice[J].Clin Exp Immunol,1999,116(2):238-245.
[17]Elliott DE,Weinstock JV.Nematodes and human therapeutic trials for inflammatory disease[J].Parasite Immunol,2017,39(5).
[18]Maloy KJ,Powrie F.Intestinal homeostasis and its breakdown in inflammatory bowel disease[J].Nature,2011,474(7351):298-306.
[19]MacDonald TT,Monteleone I,Fantini MC,et al.Regulation of homeostasis and inflammation in the intestine[J].Gastroenterology,2011,140(6):1768-1775.
[20]Ip WKE,Hoshi N,Shouval DS,et al.Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages[J].Science,2017,356(6337):513-519.
[21]Denning TL,Kim G,Kronenberg M.Cutting edge:CD4+CD25+regulatory T cells impaired for intestinal homing can prevent colitis[J].J Immunol,2005,174(12):7487-7491.
[22]Elliott DE,Li J,Blum A,et al.Exposure to schistosome eggs protects mice from TNBS-induced colitis[J].Am J Physiol Gastrointest Liver Physiol,2003,284(3):385-391.
[23]Schnoeller C,Rausch S,Pillai S,et al.A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages[J].J Immunol,2008,180(6):4265-4272.
[24]Khatri V,Amdare N,Tarnekar A,et al.Brugia malayi cystatin therapeutically ameliorates dextran sulfate sodiuminduced colitis in mice[J].J Dig Dis,2015,16(10):585-594.
[25]Leung JM,Loke P.A role for IL-22 in the relationship between intestinal helminths,gut microbiota and mucosal immunity[J].Int J Parasitol,2013,43(3/4):253-257.
[26]Giacomin P,Zakrzewski M,Jenkins TP,et al.Changes in duodenal tissue-associated microbiota following hookworm infection and consecutive gluten challenges in humans with coeliac disease[J].Sci Rep,2016,6:36797.
[2]Driss V,El Nady M,Delbeke M,et al.The schistosome glutathione S-transferase P28GST,a unique helminth protein,prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils[J].Mucosal Immunol,2016,9(2):322-335.
[3]Allen JE,Maizels RM.Diversity and dialogue in immunity to helminths[J].Nat Rev Immunol,2011,11(6):375-388.
[4]Hasby EA,Hasby Saad MA,Shohieb Z,et al.FoxP3+Tregulatory cells and immunomodulation after Schistosoma mansoni egg antigen immunization in experimental model of inflammatory bowel disease[J].Cell Immunol,2015,295(1):67-76.
[5]Heylen M,Ruyssers NE,Gielis EM,et al.Of worms,mice and man:an overview of experimental and clinical helminth-based therapy for inflammatory bowel disease[J].Pharmacol Ther,2014,143(2):153-167.
[6]Schierack P,Lucius R,Sonnenburg B,et al.Parasite-specific immunomodulatory functions of filarial cystatin[J].Infect Immun,2003,71(5):2422-2429.
[7]Hartmann S,Kyewski B,Sonnenburg B,et al.A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production[J].Eur J Immunol,1997,27(9):2253-2260.
[8]Magister S,Kos J.Cystatins in immune system[J].J Cancer,2013,4(1):45-56.
[9]Wang SS,Xie YY,Yang XD,et al.Therapeutic potential of recombinant cystatin from Schistosoma japonicum in TNBS-induced experimental colitis of mice[J].Parasit Vectors,2016,9:6.
[10]Liu F,Cheng WS,Pappoe F,et al.Schistosoma japonicum cystatin attenuates murine collagen-induced arthritis[J].Parasitol Res,2016,115(10):3795-3806.
[11]Wynn TA,Chawla A,Pollard JW.Origins and hallmarks of macrophages:development,homeostasis,and disease[J].Nature,2013,496(7446):445.
[12]Yang X,Liu J,Yue Y,et al.Cloning,expression and characterisation of a typeⅡcystatin from Schistosoma japonicum,which could regulate macrophage activation[J].Parasitol Res,2014,113(11):3985-3992.
[13]He BH.Schistosoma japonicum:the molecular characteristics of cystatin and it’s immune regulation effects on mice[D].Wuhan:Wuhan University,2014.
[14]Ratcliffe EC,Wilson RA.The magnitude and kinetics of delayed-type hypersensitivity responses in mice vaccinated with irradiated cercariae of Schistosoma mansoni[J].Parasitology,1991,103(1):65-75.
[15]Menachem Y,Trop S,Kolker O,et al.Adoptive transfer of NK1.1+lymphocytes in immune-mediated colitis:a proinflammatory or a tolerizing subgroup of cells?[J].Microbes Infect,2005,7(5/6):825-835.
[16]Obermeier F,Kojouharoff G,Hans W,et al.Interferon-gamma(IFN-γ)-and tumour necrosis factor(TNF)-induced nitric oxide as toxic effector molecule in chronic dextran sulphate sodium(DSS)-induced colitis in mice[J].Clin Exp Immunol,1999,116(2):238-245.
[17]Elliott DE,Weinstock JV.Nematodes and human therapeutic trials for inflammatory disease[J].Parasite Immunol,2017,39(5).
[18]Maloy KJ,Powrie F.Intestinal homeostasis and its breakdown in inflammatory bowel disease[J].Nature,2011,474(7351):298-306.
[19]MacDonald TT,Monteleone I,Fantini MC,et al.Regulation of homeostasis and inflammation in the intestine[J].Gastroenterology,2011,140(6):1768-1775.
[20]Ip WKE,Hoshi N,Shouval DS,et al.Anti-inflammatory effect of IL-10 mediated by metabolic reprogramming of macrophages[J].Science,2017,356(6337):513-519.
[21]Denning TL,Kim G,Kronenberg M.Cutting edge:CD4+CD25+regulatory T cells impaired for intestinal homing can prevent colitis[J].J Immunol,2005,174(12):7487-7491.
[22]Elliott DE,Li J,Blum A,et al.Exposure to schistosome eggs protects mice from TNBS-induced colitis[J].Am J Physiol Gastrointest Liver Physiol,2003,284(3):385-391.
[23]Schnoeller C,Rausch S,Pillai S,et al.A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10-producing macrophages[J].J Immunol,2008,180(6):4265-4272.
[24]Khatri V,Amdare N,Tarnekar A,et al.Brugia malayi cystatin therapeutically ameliorates dextran sulfate sodiuminduced colitis in mice[J].J Dig Dis,2015,16(10):585-594.
[25]Leung JM,Loke P.A role for IL-22 in the relationship between intestinal helminths,gut microbiota and mucosal immunity[J].Int J Parasitol,2013,43(3/4):253-257.
[26]Giacomin P,Zakrzewski M,Jenkins TP,et al.Changes in duodenal tissue-associated microbiota following hookworm infection and consecutive gluten challenges in humans with coeliac disease[J].Sci Rep,2016,6:36797.