人抵抗素通过激活PPARγ的抗炎机制研究^*

Anti-inflammatory Mechanisms of Human Resistin Through Activation of Peroxisome Proliferator-Activated Receptor γ

摘要

目的 研究抵抗素通过激活过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor, PPAR)抑制脂多糖（lipopolysaccharide, LPS）诱导白细胞介素（interleukin, IL）-1β和肿瘤坏死因子α（tumor necrosis factor-α, TNF-α）表达的调控作用及分子机制。方法 以5 mg/kg LPS腹腔注射C57BL/6J小鼠制备内毒素炎症模型，检测小鼠血清和脾组织抵抗素表达水平。以抵抗素处理RAW264.7细胞和腹腔注射C57BL/6J小鼠为模型，采用实时荧光定量、免疫印迹和ELISA，检测抵抗素预处理联合PPARγ激活剂罗格列酮或者抑制剂GW9662干预条件下，LPS诱导的RAW264.7细胞内PPARγ、p65总蛋白、p-P65、核因子κB抑制蛋白α（inhibitor of NF-κB alpha, IκBα）、IL-1β、TNF-α mRNA和蛋白表达水平。以20 mg/kg腹腔注射LPS的C57BL/6J小鼠制作内毒素休克模型，检测抵抗素对小鼠生存和血清IL-1β、TNF-α水平的影响。结果 腹腔注射LPS后，小鼠脾脏抵抗素mRNA和蛋白水平降低超过80%，血清浓度下降58%，与PBS对照组相比差异有统计学意义（P<0.001）。经20 ng/mL抵抗素预处理后，LPS刺激的RAW264.7细胞PPARγ mRNA和相对蛋白表达水平分别由0.58±0.07和0.34±0.04升高至0.95±0.03和1.78±0.28，并在抵抗素和罗格列酮联合作用进一步升高至2.03±0.16和2.82±0.08，3组间差异均有统计学意义（P<0.05）。抵抗素使LPS联合GW9662作用的RAW264.7细胞内PPARγ mRNA和相对蛋白表达水平分别由0.44±0.06和0.43±0.03升高至1.19±0.08和0.55±0.04，两组间差异均有统计学意义（P<0.05），同时细胞内IL-1β mRNA由12.31±1.50下降至7.45±0.83，IL-1β蛋白由（179.62±9.27） pg/mL降至（108.59±17.32） pg/mL，TNF-α mRNA由3.41±0.11降至2.50±0.04，蛋白水平由（1 265.30±154.78） pg/mL下降至（1 025.96±45.41） pg/mL，且两组比较差异均有统计学意义（P<0.05），表明GW9662的促LPS诱导的IL-1β、TNF-α表达效应可部分受Retn抑制。检测发现，抵抗素作用下细胞内IκBα蛋白水平增加、p-P65/P65表达降低；同时IL-1β和TNF-α mRNA水平分别降低24.7%和26.9%，蛋白表达水平分别降低36.3%和37.2%，与LPS对照相比差异均有统计学意义（P<0.05）。25 μg/kg抵抗素预处理可使小鼠血清IL-1β水平由（1 616.66±96.68） pg/mL降至（1 277.04±27.73） pg/mL，TNF-α由（617.80±145.54） pg/mL降至（322.91±129.50） pg/mL，IFN-γ由（12944.50±90.69） pg/mL降至（9 222.84±268.83） pg/mL，与单用LPS处理组相比差异均有统计学意义（P<0.05）；同时小鼠7 d存活率由20%提高到80%，与LPS组相比差异有统计学意义（P<0.05）。结论 腹腔注射LPS可致小鼠脾脏和血清中抵抗素表达降低，人抵抗素可通过激活PPARγ、抑制NF-κB信号通路而降低LPS诱导的IL-1β、TNF-α表达和LPS诱导的休克小鼠死亡率。

Abstract

Objective To study the molecular mechanism and regulatory effect of resistin on lipopolysaccharide (LPS)-induced interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) expression through activation of peroxisome proliferator-activated receptor γ (PPARγ). Methods An endotoxin-induced inflammation model was established by intraperitoneal injection of 5 mg/kg LPS in C57BL/6J mice, and the expression levels of resistin in mouse serum and spleen tissue were measured. Using resistin-treated RAW264.7 cells and C57BL/6J mice administered intraperitoneal injections as models, real-time quantitative PCR, Western blot, and ELISA were used to assess the mRNA and protein expression of PPARγ, total p65, phosphorylated p65, inhibitor of NF-κB alpha (IκBα), IL-1β, and TNF-α in LPS-induced RAW264.7 cells under conditions of resistin pretreatment or combined intervention with the PPARγ activator rosiglitazone or the inhibitor GW9662. Using C57BL/6J mice with intraperitoneal injection of 20 mg/kg LPS as a model, the effects of resistin on survival and serum IL-1β and TNF-α levels were evaluated. Results After intraperitoneal injection of LPS, the mRNA and protein levels of resistin in the spleen of mice decreased by more than 80%, and the serum concentration dropped by 58%, which was statistically significant compared with the PBS control group (P < 0.001). Under pretreatment with 20 ng/mL resistin, the mRNA and relative protein expression levels of PPARγ in LPS-stimulated RAW264.7 cells increased from 0.58 ±0.07 and 0.34 ± 0.04 to 0.95 ± 0.03 and 1.78 ± 0.28, respectively, and further increased to 2.03 ± 0.16 and 2.82 ± 0.08 under the combined action of resistin and rosiglitazone, with statistically significant differences among the three treatments ( P < 0.05). Resistin increased PPARγ mRNA and relative protein expression levels in LPS- and GW9662-treated RAW264.7 cells from 0.44 ± 0.06 and 0.43± 0.03 to 1.19± 0.08 and 0.55 ± 0.04, respectively, with statistically significant differences between the two groups (P < 0.05). Meanwhile, intracellular IL-1β mRNA levels decreased from 12.31 ± 1.50 to 7.45 ± 0.83, and cell culture supernatant protein levels dropped from (179.62 ± 9.27) pg/mL to (108.59 ± 17.32) pg/mL. Additionally,TNF-α mRNA levels declined from 3.41 ± 0.11 to 2.50 ± 0.04, while supernatant protein levels decreased from (1 265.30 ± 154.78) pg/mL to (1025.96±45.41) pg/mL. Statistical comparisons between the two groups showed significant differences (P < 0.05), indicating that Retn partially inhibits GW9662's effect on promoting LPS-induced IL-1β and TNF-α expression. It was found that under the action of resistin, intracellular IκBα protein levels increased and p-p65/p65 expression decreased; meanwhile, IL-1β and TNF-α mRNA levels decreased by 24.7% and 26.9%, and protein expression levels decreased by 36.3% and 37.2%, respectively, with statistically significant differences compared with the LPS group (P < 0.05). Compared with the LPS-only treatment group, pretreatment with 25 μg/kg resistin reduced mouse serum IL-1β levels from (1 616.66 ± 96.68) pg/mL to (1 277.04 ± 27.73) pg/mL, TNF-α from (617.80 ± 145.54) pg/mL to (322.91 ± 129.50) pg/mL and IFN-γ from (12 944.50 ± 90.69) pg/mL to (9 222.84 ± 268.30) pg/mL, with all differences being statistically significant ( P < 0.05); meanwhile, the 7-day survival rate of mice increased from 20% to 80%, which was statistically significant compared with the LPS group ( P < 0.05). Conclusion Intraperitoneal injection of LPS can lead to decreased resistin expression in the mouse spleen and human resistin can reduce LPS-induced IL-1β and TNF-α expression, as well as the mortality of LPS-induced shock mice by activating PPARγ and inhibiting NF-κB signaling.

关键词

抵抗素 / 脂多糖 / PPARγ / IL-1β / TNF-α

Key words

Resistin / LPS / PPARγ / IL-1β / TNF-α

参考文献

[1] Steppan C M, Bailey S T, Bhat S, et al. The hormone resistin links obesity to diabetes[J]. Nature, 2001, 409(6818): 307. DOI: 10.1038/35053000.

[2] Patel S D, Rajala M W, Rossetti L, et al. Disulfide-dependent multimeric assembly of resistin family hormones[J]. Science, 2004, 304(5674): 1154. DOI: 10.1126/science.1093466.

[3] Juan C C, Kan L S, Huang C C, et al. Production and characterization of bioactive recombinant resistin in Escherichia coli[J]. J Biotechnol, 2003, 103(2): 113. DOI: 10.1016/s0168-1656(03)00099-3.

[4] Li X, Li X, Huang P, et al. Acetylation of TIR domains in the TLR4-Mal-MyD88 complex regulates immune responses in sepsis[J]. EMBO J, 2024, 43(21): 4954. DOI: 10.1038/s44318-024-00237-8.

[5] Brown M A, Jones W K. NF-kappaB action in sepsis: the innate immune system and the heart[J]. Front Biosci, 2004, 9: 1201. DOI: 10.2741/1304.

[6] Zhu Y, Kan L, Qi C, et al. Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR[J]. J Biol Chem, 2000, 275(18): 13510. DOI: 10.1074/jbc.275.18.13510.

[7] Széles L, Töröcsik D, Nagy L. PPARgamma in immunity and inflammation: cell types and diseases[J]. Biochim Biophys Acta, 2007, 1771(8): 1014. DOI: 10.1016/j.bbalip.2007.02.005.

[8] Schmidt M V, Brüne B, Von Knethen A. The nuclear hormone receptor PPARγ as a therapeutic target in major diseases[J]. Scientific World Journal, 2010, 10: 2181. DOI: 10.1100/tsw.2010.213.

[9] Zapolska-Downar D, Naruszewicz M. Propionate reduces the cytokine-induced VCAM-1 and ICAM-1 expression by inhibiting nuclear factor-kappa B (NF-kappaB) activation[J]. J Physiol Pharmacol, 2009, 60(2): 123.

[10] Kim J H, Song J, Park K W. The multifaceted factor peroxisome proliferator-activated receptor γ (PPARγ) in metabolism, immunity, and cancer[J]. Arch Pharm Res, 2015, 38(3): 302. DOI: 10.1007/s12272-015-0559-x.

[11] Jang J C, Li J, Gambini L, et al. Human resistin protects against endotoxic shock by blocking LPS-TLR4 interaction[J/OL]. Proc Natl Acad Sci U S A, 2017, 114(48): E10399. https://doi.org/10.1073/pnas.1716015114.

[12] Han B, Li R, Wang M Z, et al. A novel increase in resistin levels induced by TLR4 deficiency inhibits lipopolysaccharide-induced IL-1β and TNF-α expression in C57BL/6J mice[J]. Int J Clin Exp Med, 2019, 12(9): 11460.

[13] Pirvulescu M M, Gan A M, Stan D, et al. Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine, MCP-1 and activation of TLR4 and Gi/o proteins signaling[J]. Int J Biochem Cell Biol, 2014, 50: 29. DOI: 10.1016/j.biocel.2014.01.022.

[14] 李思成. 符合代谢综合征的糖尿病患者血清抵抗素与微量白蛋白尿相关性及罗格列酮干预研究[D]. 遵义: 遵义医学院, 2008: 20. DOI: 10.7666/d.y1308303.

[15] Li S C. Correlation of Serum Resistin, Microal buminuria in Diabetic Subjects with Metabolic Syndrome and Clinic Effects of Rosiglitazone Therapy[D]. Zunyi: Zunyi Medical College, 2008: 20. DOI: 10.7666/d.y1308303.

[16] 包红丽. 抵抗素通过AMPK/SLC7A11信号通路诱导H9C2心肌细胞肥大的机制研究[D]. 呼和浩特: 内蒙古医科大学, 2025: 36. DOI: 10.27231/d.cnki.gnmyc.2025.000372.

[17] Bao H L. Mechanism of Resistin induced hypertrophy of H9c2 cardiomyocytesthrough AMPK/SLC7A11 signaling pathway[D]. Hohhot: Inner Mongolia Medical University, 2025: 36. DOI: 10.27231/d.cnki.gnmyc.2025.000372.

[18] 张梅芳, 李晓丽, 薛刚, 等. 瓜薤香丹方联合比索洛尔对稳定型心绞痛患者心肌缺血总负荷及血清抵抗素水平的影响[J]. 世界临床药物, 2025, 46(2): 158. DOI: 10.13683/j.wph.2025.02.007.

[19] Zhang M F, Li X L, Xie G, et al. Effects of Guaxie Xiangdan recipe combined with bisoprolol on total ischemic burden and serum resistin level in patients with stable angina pectoris[J]. World Clinical Drug, 2025, 46(2): 158. DOI: 10.13683/j.wph.2025.02.007.

[20] Li B, Fang J, He T, et al. Resistin up-regulates LPL expression through the PPARγ-dependent PI3K/AKT signaling pathway impacting lipid accumulation in RAW264. 7 macrophages[J]. Cytokine, 2019, 119: 168. DOI: 10.1016/j.cyto.2019.03.016.

[21] Reilly M P, Lehrke M, Wolfe M L, et al. Resistin is an inflammatory marker of atherosclerosis in humans[J]. Circulation, 2005, 111(7): 932. DOI: 10.1161/01.CIR.0000155620.10387.43.

[22] Oero M, Lago R, Gomez R, et al. Changes in plasma levels of fat-derived hormones adiponectin, leptin, resistin and visfatin in patients with rheumatoid arthritis[J]. Ann Rheum Dis, 2006, 65(9): 1198. DOI: 10.1136/ard.2005.046540.

[23] Van der Crabben S N, Blümer R M, Stegenga M E, et al. Early endotoxemia increases peripheral and hepatic insulin sensitivity in healthy humans[J]. J Clin Endocrinol Metab, 2009, 94(2): 463. DOI: 10.1210/jc.2008-0761.

[24] Xiang X, An W, Jiang C, et al. Lipopolysaccharide inhibits the expression of resistin in adipocytes[J]. J Mol Endocrinol, 2013, 51(3): 287. DOI: 10.1530/JME-13-0117.

[25] 李芳萍, 李志臻, 张淼, 等. 胰岛素、肿瘤坏死因子α和瘦素对抵抗素表达的影响[J]. 中山大学学报(医学科学版), 2009, 30(S2): 6. DOI: 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2009.0346.

[26] Li F P, Li Z Z, Zhang M, et al. Effects of Insulin, TNF-α and Leptin on Resistin mRNA Expression in 3T3-L1 Adipocytes[J]. Journal of Sun Yat-sen University (Medical Sciences), 2009, 30(S2): 6. DOI: 10.13471/j.cnki.j.sun.yat-sen.univ(med.sci).2009.0346.

[27] Propheter D C, Chara A L, Harris T A, et al. Resistin-like molecule Î 2 is a bactericidal protein that promotes spatial segregation of the microbiota and the colonic epithelium. Proc Natl Acad Sci U S A, 2017, 114(42): 11027. DOI: 10.1073/pnas.1711395114.

[28] Artis D, Wang M L, Keilbaugh S A, et al. RELMbeta/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract[J]. Proc Natl Acad Sci U S A, 2004, 101(37): 13596. DOI: 10.1073/pnas.0404034101.

[29] Tarkowski A, Bjersing J, Shestakov A, et al. Resistin competes with lipopolysaccharide for binding to toll-like receptor 4[J]. J Cell Mol Med, 2010, 14(6B): 1419. DOI: 10.1111/j.1582-4934.2009.00899.x.

[30] Von Knethen A, Brüne B . PPARgamma-an important regulator of monocyte/macrophage function[J]. Arch Immunol Ther Exp (Warsz), 2003, 51(4): 219.

[31] Marx N, Kehrle B, Kohlhammer K, et al. PPAR Activators as Antiinflammatory Mediators in Human T Lymphocytes: Implications for Atherosclerosis and Transplantation-Associated Arteriosclerosis[J]. Circ Res, 2002, 90(6): 703. DOI: 10.1161/01.res.0000014225.20727.8f.

[32] Joffe Y T, Collins M, Goedecke J H. The Relationship between Dietary Fatty Acids and Inflammatory Genes on the Obese Phenotype and Serum Lipids[J]. Nutrients, 2013, 5(5): 1672. DOI: 10.3390/nu5051672.

[33] Yao Q, Liu J, Zhang Z, et al. Peroxisome proliferator-activated receptor γ (PPARγ) induces the gene expression of integrin αVβ5 to promote macrophage M2 polarization[J]. J Biol Chem, 2018, 293(43): 16572. DOI: 10.1074/jbc.RA118.003161.

[34] Nencioni A, Wesselborg S, Brossart P. Role of peroxisome proliferator-activated receptor gamma and its ligands in the control of immune responses[J]. Crit Rev Immunol, 2003, 23(1/2): 1. DOI: 10.1615/critrevimmunol.v23.i12.10.

[35] Jung W K, Lee C M, Lee D S, et al. The 15-deoxy-δ12, 14-prostaglandin J2 inhibits LPS-stimulated inflammation via enhancement of the platelet-activating factor acetylhydrolase activity in human retinal pigment epithelial cells[J]. Int J Mol Med, 2014, 33(2): 449. DOI: 10.3892/ijmm.2013.1588.

[36] Lin Y, Wang Z, Liu L, et al. Akt is the downstream target of GRP78 in mediating cisplatin resistance in ER stress-tolerant human lung cancer cells[J]. Lung Cancer, 2011, 71(3): 291. DOI: 10.1016/j.lungcan.2010.06.004.

[37] Peeters L L, Vigne J L, Tee M K, et al. PPAR gamma represses VEGF expression in human endometrial cells: implications for uterine angiogenesis[J]. Angiogenesis, 2005, 8(4): 373. DOI: 10.1007/s10456-005-9027-4.

[38] Ma Z, Ji W, Fu Q, et al. Formononetin inhibited the inflammation of LPS-induced acute lung injury in mice associated with induction of PPAR gamma expression[J]. Inflammation, 2013, 36(6): 1560. DOI: 10.1007/s10753-013-9700-5.

[39] Di Paola R, Mazzon E, Maiere D, et al. Rosiglitazone reduces the evolution of experimental periodontitis in the rat[J]. J Dent Res, 2006, 85(2): 156. DOI: 10.1177/154405910608500208.

[40] Patel L, Buckels A C, Kinghorn I J, et al. Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators[J]. Biochem Biophys Res Commun, 2003, 300(2): 472. DOI: 10.1016/s0006-291x(02)02841-3.

[41] Kaplan J M, Denenberg A, Monaco M, et al. Changes in peroxisome proliferator-activated receptor-gamma activity in children with septic shock[J]. Intensive Care Med, 2010, 36(1): 123. DOI: 10.1007/s00134-009-1654-6.

[42] Lan Y, Guo W, Chen W, et al. Resistin as a potential diagnostic biomarker for sepsis: insights from DIA and ELISA analyses[J/OL]. Clin Proteomics, 2024, 21(1): 46. https://pmc.ncbi.nlm.nih.gov/articles/PMC11218185/. DOI: 10.1186/s12014-024-09498-1.

[43] Xu F, Luo J, Li W. Comparison of the diagnostic accuracy of resistin and CRP levels for sepsis in neonates and children: a systematic review and meta-analysis[J/OL]. Front Pediatr, 2025, 13: 1555671. https://doi.org/10.3389/fped.2025.1555671.

[44] Chen J, Liu Z, Zhou F, et al. Assessment of S100A8/A9 and resistin as predictive biomarkers for mortality in critically ill patients with sepsis[J/OL]. Front Cell Infect Microbiol, 2025, 15: 1555307. https://doi.org/10.3389/fcimb.2025.1555307.

[45] Chen H, Luo H, Tian T, et al. Integrated Analyses of Single-Cell Transcriptome and Mendelian Randomization Reveal the Protective Role of Resistin in Sepsis Survival in Intensive Care Unit[J/OL]. Int J Mol Sci, 2023, 24(19): 14982. https://doi.org/10.3390/ijms241914982.

[46] Macdonald S P J, Bosio E, Neil C, et al. Resistin and NGAL are associated with inflammatory response, endothelial activation and clinical outcomes in sepsis[J]. Inflamm Res, 2017, 66(7): 611. DOI: 10.1007/s00011-017-1043-5.

[47] Qiu L, Lin B, Lin Z, et al. Biochanin A ameliorates the cytokine secretion profile of lipopolysaccharide-stimulated macrophages by a PPARγ-dependent pathway[J]. Mol Med Rep, 2012, 5(1): 217. DOI: 10.3892/mmr.2011.599.

[48] Wang W M, Chen H, Zhong F, et al. Inhibitory effects of rosiglitazone on lipopolysaccharide-induced inflammation in a murine model and HK-2 cells[J]. Am J Nephrol, 2011, 34(2): 152. DOI: 10.1159/000329120.

[49] Hovsepian E, Penas F, Goren N B. 15-deoxy-Delta12, 14 prostaglandin GJ2 but not rosiglitazone regulates metalloproteinase 9, NOS-2, and cyclooxygenase 2 expression and functions by peroxisome proliferator-activated receptor gamma-dependent and -independent mechanisms in cardiac cells[J]. Shock, 2010, 34(1): 60. DOI: 10.1097/SHK.0b013e3181cdc398.

[50] Xu Y, Yao J, Zou C, et al. Asiatic acid protects against hepatic ischemia/reperfusion injury by inactivation of Kupffer cells via PPARγ/NLRP3 inflammasome signaling pathway[J]. Oncotarget, 2017, 8(49): 86339. DOI: 10.18632/oncotarget.21151.

基金资助

*国家自然科学基金(30760280)

国家自然科学基金(32360183)

江西省自然科学基金(20181BAB205064)