摘要
目的观察糖尿病肾病(diabetic nephropathy DN)大鼠肾组织中TOLL受体4(Toll-like receptor4 TLR4)的表达变化情况,并对TLR4在DN发生发展中所起的可能作用进行初步的探讨。
方法试验分两组-模型组和对造组。模型组大鼠应用腹腔内一次性大剂量注射链脲佐菌素(streptozoticin STZ)溶液制作糖尿病模型,而对造组仅给予腹腔注射不含STZ的溶液。于造模成功后2w、4w、6w、8w、12w分别测定模型组和对造组24h尿蛋白排泄量、血肌酐(Cr)、尿素氮(BUN)、肾重/体重(KW/BW)、尿白蛋白/肌酐比值(A/C)、血清CRP及TNF-α滴度;观察肾脏损伤后病理形态学变化并测定细胞外基质增生程度;RT-PCR法测定肾组织TLR4的mRNA表达;免疫组化法检测肾组织TLR4、核因子кB(NF-кB)、生长转化因子-β1(transforming growth factor-β1 TGF-β1)、纤维连接蛋白(fibronectin FN)的蛋白表达。
结果糖尿病大鼠2w后即发现24h尿蛋白排泄量增加等早期肾脏病征象,此时肾组织TLR4以及NF-кB表达即开始增加,与对照组相比,差异具有显著性,并随病变程度的加重呈持续性增加。12w时试验大鼠相关炎症介质的血清滴度明显增高,并出现肾组织纤维化及肾功能损伤。相关性分析提示TLR4的表达与炎症反应程度、肾组织纤维化程度及肾功能损伤程度均呈显著正相关。
结论TLR4及其信号传导通路可能通过激活免疫炎症反应参与了糖尿病肾病的发生和发展。
目的观察氯沙坦对糖尿病肾病大鼠的炎症抑制作用。
方法采用链脲佐菌素(streptozotocin STZ,65mg·kg-1)腹腔注射建立糖尿病大鼠模型。试验分3组:对照组、模型组、治疗组,后一组于成模后一周给予氯沙坦20mg·kg-1·d-1灌胃。分别测定对照组和模型组4、8、12周24h尿蛋白、尿白蛋白/肌酐比值(A/C)、血CRP及TNF-α水平;观察肾脏病理形态学变化;应用免疫组化法检测肾组织TOLL样受体4(TLR4)、核因子-кB(NF-кB)的蛋白表达;应用RT-PCR法测定肾组织TLR4的核酸表达;12周后测定治疗组的上述指标。
结果模型组与对照组相比:模型组大鼠24h尿蛋白、A/C、血CRP及TNF-α含量显著增高;肾组织TLR4、NF-кB的表达明显增加。相关性分析提示TLR4的表达与NF-кB的表达、24h尿蛋白、A/C、血CRP及TNF-α含量均呈正相关关系。氯沙坦治疗组大鼠较模型组大鼠24h尿蛋白、A/C、血CRP、TNF-α含量下降,肾组织TLR4、NF-кB的表达亦减弱,治疗前后比较存在差异(P<0.05)。
结论氯沙坦对糖尿病肾病具有保护作用,部分作用机制可能是通过下调NF-кB、TLR4的表达,降低血CRP、TNF-α含量,抑制炎症反应而实现的。
Objective To investigate the changes of Toll-like receptor4(TLR4) expression in renal tissue and regulating possible mechanism in rats with diabetic nephropathy. Methods Male SD rats were randomly divided into two groups as follows: control group and model group . Model group were induced to be diabetic nephropathy by intraperitoneal injection with streptozoticin(STZ). 24h urine protein, creatinine, urea nitrogen, profile of kidney hypertrophy(KW/BW), urine albumen/ creatinine value(A/C), the plasma concentration of CRP and TNF-αwere determined at week 2, 4, 6, 8 and 12 respectively. The transmutation of renal pathomorphology was observed and the hyperplastic degree of extracellular matrix was evaluated. mRNA expression of TLR4 were detected by reverse transcript(RT) -PCR. Renal protein expression of TLR4, nuclear factor-кB( NF-кB), transforming growth factor-β1 (TGF-β1) and fibronectin (FN) were detected by immunohistochemical method.
Results 24 hours urine protein excretion was increased, mng symptom of renal disease, at 2 week in model group. At same time, the TLR4 expression in renal tissue was markedly increased compared with control group. To follow the aggravation of the disease, the expression of TLR4 was durative increased. At 12 week, the plasma concentration of CRP and TNF-αof the rats which with diabetic nephropathy were raised up obviously and it emerged nephridial tissue fibrosis and injury of renal function .Positive correlation is found between TLR4 expression and nephridial tissue fibrosis, the same correlation also existed between TLR4 expression and injury degree of renal function.
Conclusion TLR4 possibly via adjustment inflammatory reaction to play a pivotal role in the pathogenesis of renal damage in diabetic nephropathy.
Objective To observe the inflammation depressant effect of losartan on experimental diabetic nephropathy in rat.
Methods SD rats were randomly divided into three groups: control group, model group, losartan-treatment group. The latter two groups were induced to be diabetic nephropathy by intraperitoneal injection with 65 mg/kg streptozoticin(STZ). Then, 20 mg·kg-1·d-1 losartan was continuously given 1 week after STZ injection in losartan-treatment group. The 24h urine protein, urine albumen/ creatinine value (A/C), plasma concentration of CRP and TNF-αwere detected at week 4, 8 and 12 respectively. Renal protein expression of TLR4 and NF-кB were detected by immunohistochemical method. TLR4 mRNA expression in the renal was detected by reverse transcript (RT)–PCR. All of these were detected in losartan-treatment group at week 12.
Results The 24h urine protein,A/C,plasma concentration of CRP and TNF-α,the renal expression of TLR4 and NF-кB increased in diabetic rats, compared with control group. Furthermore, TLR4 expression has positive correlation with NF-кB expression, 24h urine protein, A/C, plasma concentration of CRP and TNF-α. The plasma concentration of creatinine , CRP and TNF-αdecreased in losartan-treatment group , and the renal expression of TLR4 and NF-кB were also down-regulated (P<0.05).
Conclusions Losartan can lessen the diabetic nephropathy impairment through down-regulation of the TLR4 and NF-кB expression in renal, reduction of CRP and TNF-αplasma concentration.
引文
1. Shankland SJ,Scholey JW, Ly H et al. Expression of transforming growth factor-beta 1 during diabetic renal hypertrophy[J]. Kidney Int, 1994, 46(2):42-43.
2. Pichup JC, Mattock MB, Chusney GD, Burt D: BIDDM as a disease of the innate immune system: association of acute phase reactants and interleukin-6 with metabolic syndrome X. Diabeto logia 1997,40:1286-1292.
3. Festa A, D’Agostino R, Tracey RP, Haffner SM: Elevated levels of acute-phase proteins and plasminogen activator inhibitor-1 predict the development of type 2 diabetes: the Insulin Resistance Atherosclerosis Study. Diabetes 2002, 51:1131-1137.
4. Akira S, Takeda K, Kaisho T: Toll-like receptors: Critical protein linking innate and acquired immunity. Nat Immunol 2001, 2: 675-680.
5. Tsuboi N, Yoshikai Y, Matsuo S, Kikuchi T, Iwami K et al: Role of Toll-like receptors in C-C chemokine production by renal tubular epithelial cells. J Immunol 2002, 169: 2026-2033.
6. Medzhiotov R, Preston- Hurlburt P, Janeway CA: A human homologue of the Drosopltila Toll protein signals activation of adaptive immunity. Nature 1997, 388(6640):394-397.
7. Johnson GB, Brunn GJ, Kodaira Y, Platt JL: Receptor-mediated monitoring of tissue wellbeing via detection of soluble heparan sulfate by Toll-like receptor4. J Immunol 2002, 168: 5233-5239.
8. Termeer C, Benedix F, Sleeman J, Fieber C et al: Oligosaccharides of Hyaluronan activate dendritic cell via Toll-like receptor4. J Exp Med 2002, 195: 99-111.
9. Ohashi K, Burkart V, Flohe S, Kolb H: Heat shock protein 60 is a putative endogenous ligand of the Toll-like receptor 4 complex. J Immunol 2000, 164: 558-561.
10. Hofmann MA, Schierkofer S, Isermann B, et al: Peripheral blood mononuclear cells isolated from patients with diabetic nephropathy show increased activation of the oxidative stress sensitive transcription factor NF-кB. Diabetologia, 1999, 42 (2): 222-232.
11.丁鹤华,黎峰,徐明彤:抑制核因子кB对糖尿病肾病的作用。中华内科学杂志,2002,4(9): 605-609.
1何冰,韩萍,吕先科. 2型糖尿病患者急性时相蛋白与糖尿病肾病的关系[J].中华内分泌杂志, 2003,19: 260- 262.
2 Liu B, Yang Y, Dial J, et al. TLR4 up-regulation at protein or gene level is pathogenic for lupus-like autoimmune disease. J Immunol, 2006, 177:6880-6888.
3 Anders HJ, Banas B, Schlondorff D. Signaling danger: toll-like receptors and their potential roles in kidney disease. J Am Soc Nephrol, 2004, 15:854-867.
4 Pichup JC, Mattock MB, Chusney GD, et al. BIDDM as a disease of the innate immune system: association of acute phase reactants and interleukin-6 with metabolic syndrome X. Diabeto logia, 1997, 40:1286-1292.
5 Hansson L, Lindholm LH, Niskanen L, et al. Effect of angiotensin“converting”enzyme inhibition compared with conventional therapy on cardiovascular morbidity and mortality in hypertension the captopril prevention project(CAPPP) randomized trial [J]. Lancet, 1999, 353: 611- 616.
6 Dol F, Martin G, Stals B, et al. Angiotensin AT1 receptor antagonist irbesartan decreased lesion size, chemokine expression and macrophage accumulation in apocipoprotein E deficient mice [J], J Cardiovasc Pharmacol, 2001, 38: 395- 405.
7 Aronson D, Bartha P, Zinder O, et al. Association between fasting glucose and C-reactive protein in middle-aged subjects. Diabet Med, 2002, 21:39- 44.
8 Siragy HM, Awad A, Abadir P, et al. The angiotensinΠtypeΙreceptor mediates renal interstitial content of tumor necrosis factor- alpha in diabetic. Endocrinology, 2003, 144: 2229-2233.
9 Feterowski C, Emmanuilidis K, Miethke T, et al. Effects of functional Toll-like receptor-4 mutations on the immune response to human and experimental sepsis. Immunology, 2003, 109: 426–431.
10 Ando M, Shibuya A, Tsuchiya K, et al. Reduced expression of Toll-like receptor 4 contributes to impaired cytokine response of monocytes in uremic patients. Kidney Int, 2006, 70:358-362.
11高苹,贾汝汉,王学玉.厄贝沙坦对2型糖尿病大鼠肾组织中核因子-кB的调节.中华肾脏病杂志, 2002,18: 364- 368.
1 Akira S, Takeda K, Kaisho T: Toll-like receptors: Critical proteins linking innate and acquired immunity. Nat Immunol 2: 675–680,2001.
2 D’Alessandro AM, Pirsch JD, Knechtle SJ, Odorico JS, Van der Werf WJ, Collins BH, Becker YT, Kalayoglu M, Armbrust MJ, Sollinger HW: Living unrelated renal donation: the University of Wisconsin experience. Surgery 124: 604–610, 1998.
3 Ozinsky A, Underhill DM, Fontenot JD, Hajjar AM, Smith KD, Wilson CB, Schroeder L, Aderem A: The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci USA 97: 13766–13771, 2000.
4 Beg AA: Endogenous ligands of Toll-like receptors: Implications for regulating inflammatory and immune responses. Trends Immunol 23: 509–513, 2002.
5 Matzinger P: The danger model: A renewed sense of self. Science 296: 301–305, 2002.
6 Wang L, Smith D, Bot S, Dellamary L, Bloom A, Bot A: Noncoding RNA danger motifs bridge innate and adaptive immunity and are potent adjuvants for vaccination. J Clin Invest 110: 1175–1184, 2002.
7 Lu CY, Penfield JG, Kielar ML, Vazquez MA, Jeyarajah DR: Hypothesis: Is renal allograft rejection initiated by the response to injury sustained during the transplant process? Kidney Int 55: 2157–2168, 1999.
8 Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev Immunol 21: 335–376, 2003.
9 Barton GM, Medzhitov R: Toll-like receptor signaling pathways. Science 300: 1524–1525, 2003.
10 Horng T, Barton GM, Medzhitov R: TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol 2: 835–841, 2001.
11 Takeuchi O, Kawai T, Sanjo H, Copeland NG, Gilbert DJ, Jenkins NA, Takeda K, Akira S: TLR6: A novel member of an expanding toll-like receptor family. Gene 231: 59–65, 1999.
12 Krutzik SR, Ochoa MT, Sieling PA, Uematsu S, Ng YW, Legaspi A, Liu PT, Cole ST, Godowski PJ, Maeda Y, Sarno EN, Norgard MV, Brennan PJ, Akira S, Rea TH, Modlin RL: Activation and regulation of Toll-like receptors 2 and 1 in human leprosy. Nat Med 9: 525–532, 2003.
13 Ochoa MT, Legaspi AJ, Hatziris Z, Godowski PJ, Modlin RL, Sieling PA: Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue. Immunology 108: 10–15, 2003.
14 Takeuchi O, Kawai T, Muhlradt PF, Morr M, Radolf JD, Zychlinsky A, Takeda K, Akira S: Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 13: 933–940, 2001.
15 Li M, Carpio DF, Zheng Y, Bruzzo P, Singh V, Ouaaz F, Medzhitov RM, Beg AA: An essential role of the NF-kappa B/Toll-like receptor pathway in induction of inflammatory and tissue-repair gene expression by necrotic cells. J Immunol 166: 7128–7135, 2001.
16 Vabulas RM, Ahmad-Nejad P, Ghose S, Kirschning CJ, Issels RD, Wagner H: HSP70 as endogenous stimulus of the Toll/interleukin-1 receptor signal pathway. J Biol Chem 277: 15107–15112, 2002.
17 Alexopoulou L, Holt AC, Medzhitov R, Flavell RA: Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413: 732–738, 2001.
18 Tsuboi N, Yoshikai Y, Matsuo S, Kikuchi T, Iwami K, Nagai Y, Takeuchi O, Akira S, Matsuguchi T: Roles of toll-like receptors in C-C chemokine production by renal tubular epithelial cells. J Immunol 169: 2026–2033, 2002.
19 Biragyn A, Ruffini PA, Leifer CA, Klyushnenkova E, Shakhov A, Chertov O, Shirakawa AK, Farber JM, Segal DM, Oppenheim JJ, Kwak LW: Toll-like receptor 4-dependent activation of dendritic cells by beta-defensin 2. Science 298: 1025–1029, 2002.
20 Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science 282: 2085–2088, 1998.
21 Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, Malo D: Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). J Exp Med 189: 615–625, 1999.
22 Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 162: 3749–3752, 1999.
23 Feterowski C, Emmanuilidis K, Miethke T, Gerauer K, Rump M, Ulm K, Holzmann B, Weighardt H: Effects of functional Toll-like receptor-4 mutations onthe immune response to human and experimental sepsis. Immunology 109: 426–431, 2003.
24 Ohashi K, Burkart V, Flohe S, Kolb H: Heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 164: 558–561, 2000.
25 Johnson GB, Brunn GJ, Kodaira Y, Platt JL: Receptor-mediated monitoring of tissue well-being via detection of soluble heparan sulfate by Toll-like receptor 4. J Immunol 168: 5233–5239, 2002.
26 Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A: The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410: 1099–1103, 2001.
27 Gewirtz AT, Navas TA, Lyons S, Godowski PJ, Madara JL: Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol 167: 1882–1885, 2001.
28 Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S: A Toll-like receptor recognizes bacterial DNA. Nature 408: 740–745, 2000.
29 Shirota H, Sano K, Hirasawa N, Terui T, Ohuchi K, Hattori T, Shirato K, Tamura G: Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG-tagged antigen by dendritic cells. J Immunol 167: 66–74, 2001.
30 Bauer M, Redecke V, Ellwart JW, Scherer B, Kremer JP, Wagner H, Lipford GB: Bacterial CpG-DNA triggers activation and maturation of human CD11c-, CD123+ dendritic cells. J Immunol 166: 5000–5007, 2001.
31 Deng GM, Nilsson IM, Verdrengh M, Collins LV, Tarkowski A: Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis. Nat Med 5: 702–705, 1999.
32 Kadowaki N, Ho S, Antonenko S, Malefyt RW, Kastelein RA, Bazan F, Liu YJ: Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 194: 863–869, 2001.
33 Hornung V, Rothenfusser S, Britsch S, Krug A, Jahrsdorfer B, Giese T, Endres S, Hartmann G: Quantitative expression of toll-like receptor 1–10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol 168: 4531–4537, 2002.
34 Muzio M, Bosisio D, Polentarutti N, D’amico G, Stoppacciaro A, Mancinelli R, van’t Veer C, Penton-Rol G, Ruco LP, Allavena P, Mantovani A: Differential expression and regulation of toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J Immunol 164: 5998–6004, 2000.
35 Jarrossay D, Napolitani G, Colonna M, Sallusto F, Lanzavecchia A: Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur J Immunol 31: 3388–3393, 2001.
36 Yi AK, Krieg AM: CpG DNA rescue from anti-IgM-induced WEHI-231 B lymphoma apoptosis via modulation of I kappa B alpha and I kappa B beta and sustained activation of nuclear factor-kappa B/c-Rel. J Immunol 160: 1240–1245, 1998.
37 Vos Q, Lees A, Wu ZQ, Snapper CM, Mond JJ: B-cell activation by T-cell-independent type 2 antigens as an integral part of the humoral immune response to pathogenic microorganisms. Immunol Rev 176: 154–170, 2000.
38 Vinuesa CG, Goodnow CC: Immunology: DNA drives autoimmunity. Nature 416: 595–598, 2002.
39 Stavnezer J: Antibody class switching. Adv Immunol 61: 79–146, 1996.
40 Cunningham PN, Wang Y, He G, Guo R, Quigg RJ: Role of toll-like receptor-4 in endotoxin-induced acute renal failure [Abstract]. J Immunol 172: 2629–2635, 2004.
41 Shahin RD, Engberg I, Hagberg L, Svanborg Eden C: Neutrophil recruitment and bacterial clearance correlated with LPS responsiveness in local gram-negative infection. J Immunol 138: 3475–3480, 1987.
42 Lehmann J, Retz M, Harder J, Krams M, Kellner U, Hartmann J, Hohgrawe K, Raffenberg U, Gerber M, Loch T, Weichert-Jacobsen K, Stockle M: Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection. BMC Infect Dis 2: 20, 2002.
43 Lameire N, Vanholder R: Pathophysiologic features and prevention of human and experimental acute tubular necrosis. J Am Soc Nephrol 12 [Suppl 17]: S20–S32, 2001.
44 Okusa MD: The inflammatory cascade in acute ischemic renal failure. Nephron 90: 133–138, 2002.
45 Anders HJ, Vielhauer V, Kretzler M, Cohen CD, Segerer S, Luckow B, Weller L, Grone HJ, Schlondorff D: Chemokine and chemokine receptor expression duringinitiation and resolution of immune complex glomerulonephritis. J Am Soc Nephrol 12: 919–931, 2001.
46 Hume DA, Underhill DM, Sweet MJ, Ozinsky AO, Liew FY, Aderem A: Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state. BMC Immunol 2: 11, 2001.
47 Pisetsky DS, Wenk KS, Reich CF 3rd: The role of cpg sequences in the induction of anti-DNA antibodies. Clin Immunol 100: 157–163, 2001.
48 Huck S, Deveaud E, Namane A, Zouali M: Abnormal DNA methylation and deoxycytosine-deoxyguanine content in nucleosomes from lymphocytes undergoing apoptosis. FASEB J 13: 1415–1422, 1999.
49 Richardson B, Scheinbart L, Strahler J, Gross L, Hanash S, Johnson M: Evidence for impaired T cell DNA methylation in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Rheum 33: 1665–1673, 1990.
50 Yung RL, Richardson BC: Role of T cell DNA methylation in lupus syndromes. Lupus 3: 487–491, 1998
51 Gilkeson GS, Pippen AM, Pisetsky DS: Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA. J Clin Invest 95: 1398–1402, 1995.
52 Krieg AM: A role for Toll in autoimmunity. Nat Immunol 3: 423–424, 2003.
53 Donnelly P, Henderson R, Fletcher K, Stratton A, Lennard T, Wilson R, Proud G, Taylor R: Specific and nonspecific immunoregulatory factors and renal transplantation. Transplantation 44: 523–528, 1987.
54 Land W, Schneeberger H, Schleibner S, Illner WD, Abendroth D, Rutili G, Arfors KE, Messmer K: The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants. Transplantation 57: 211–217, 1994.
55 Goldstein DR, Tesar BM, Akira S, Lakkis FG: Critical role of the Toll-like receptor signal adaptor protein MyD88 in acute allograft rejection. J Clin Invest 111: 1571–1578, 2003.