Original Article Expression of S100 Family Proteins in Neonatal Rats with Sepsis and Its Significance
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Int J Clin Exp Pathol 2015;8(2):1631-1639 www.ijcep.com /ISSN:1936-2625/IJCEP0004338 Original Article Expression of S100 family proteins in neonatal rats with sepsis and its significance Haiying Huang, Luoyang Tu Department of Critical Care Medicine, Sir Run Run Shaw Hospital affiliated School of Medicine, Zhejiang Univer- sity, 3 East Qingchun Rd. Jianggan District, Hangzhou, Zhejiang Province, People’s Republic of China Received December 1, 2014; Accepted January 28, 2015; Epub February 1, 2015; Published February 15, 2015 Abstract: Objective: This study aims to study the expression changes of S100 family proteins in neonatal rats with sepsis and investigate the effect and significance of S100 family proteins in pathogenesis and development of sepsis. Methods: The functions of S100 family proteins were analyzed with bioinformatics. The immune-associated proteins were chosen as the candidate proteins. Twenty neonatal SPF SD rats were randomly divided into two groups: sepsis model group and control group. The liver sample was stained with HE to evaluate the establishment of sepsis model. The expression amount of proinflammatory factor IL-1, IL-6 and TNF-α was detected with ELISA. The expression changes of S100A8, S100A9, S100A11 and S100A12 in sepsis model rats were detected with real-time PCR and Western blotting. After shRNA plasmid was transfected into THP-1 cells and the expression of S100A12 was silenced, the expression changes of proinflammatory factor IL-1, IL-6 and TNF-α in LPS-induced inflammation were studied in order to investigate the S100A12 mediated inflammatory process. Results: IL-1, IL-6 and TNF-α in the serum of rats with sepsis induced by LPS were 55.79 ± 3.80 ng/l, 48.76 ± 1.03 ng/l and 29.98 ± 2.27 ng/l respectively. S100A8, S100A9, S100A11 and S100A12 detected with real-time PCR in sepsis model group were 14.4 ± 1.37, 10.23 ± 1.81, 5.5 ± 1.64 and 9.97 ± 1.82 respectively. Compared with the control group, S100A8, S100A9, and S100A12 were significantly up-regulated. The shRNA silenced the expression of S100A12 which re- duced the expression of proinflammatory factors after LPS stimulated the cells (P < 0.05). Conclusion: Compared with the control group, S100A8, S100A9, and S100A12 were significantly up-regulated in rat sepsis model group. After the expression of S100A12 in propylene glycol monomethyl ether acetate (PMA) induced human macrophages was silenced, the expression of proinflammatory factor IL-1, IL-6 and TNF-α was down-regulated. Keywords: Sepsis, S100 family, LPS, THP-1 Introduction contraction. S100 protein family, one of its larg- est subfamilies, has more than twenty mem- Sepsis is a systemic inflammatory response bers. The proteins of this family have similar syndrome caused by pathogenic microorgan- structure and functions and a high degree of isms invading into circulatory system, growing homology and can play multiple biological and reproducing and producing endotoxin and effects by combining with Ca2+ and changing exotoxin which can induce the injury of multiple their conformation. Recent studies have dem- organs and is a high-mortality disease in clinic. onstrated that multiple members of S100 pro- The routine use of antibiotics is unable to treat tein family are involved in the occurrence and sepsis effectively indicating that sepsis may be development of inflammation, especially acute a complex process in which a variety of immune- inflammation. Therefore, in this experiment, we associated molecules act collaboratively. Th- will use the bioinformatics database to screen erefore, in-depth study of its pathogenic mech- out the inflammation-associated proteins in the anism is a difficult medical problem demanding S100 family and investigate the mechanism of prompt solution currently [1, 2]. S100 family proteins in the occurrence and development of sepsis by establishing the sep- Calcium binding protein is a large protein family sis models in neonatal rats in the hope of pro- with the functions of controlling cell cycle, cell viding a train of thought for disclosing the differentiation, enzyme activation and muscle pathogenesis of sepsis [3, 4]. S100 family proteins and neonatal rats with sepsis Materials and methods concentration of 10 μg/ml). The saline was injected in the control group. Experimental animals and cells Detection of related proinflammatory factors Twenty male or female SPF SD rats, aged one with ELISA week and weighing 25 ± 5 g, were purchased from Slac Laboratory Animal LLC. They were IL-1, IL-6 and TNF-α in rat serum were detected randomly divided into two groups with 10 rats quantitatively using IL-1, IL-6 and TNF-α ELISA in each group: sepsis model group and control kits. The specific procedure is as follows. group. The rats were fed in standard animal cages with five rats in each cage. All rats freely Standard holes, sample holes and blank holes took food (breast-fed by mother rats) and drank were set respectively. The standard products of water during the experiment. The laboratory different concentration were added into seven was well ventilated and nature lighting day and standard holes respectively. The samples to be night was achieved. The temperature was main- measured were also added. The ELISA plate tained at 18~25°C. was covered with tectorial membrane. They were incubated for two hours at 37°C. 100 μl of THP-1 human macrophages purchased from test solution (biotinylated primary antibody) Shanghai Cell Bank of Chinese Academy of was added into each hole. The ELISA plate was Sciences were cultured in 1640 complete covered with tectorial membrane. They were medium (GIBIC), 15% fetal bovine serum (GIBIC) incubated for one hour at 37°C. The liquid in the holes was discarded. Each hole was washed and 5% CO2 incubator at 37°C. with 350 μl scrub solution, soaked with it for Reagents and instruments one to two minutes and washed repeatedly for three times. 100 μl of HRP labeled secondary Lipopolysaccharide (LPS) was purchased from antibody was added in each hole. The ELISA Sigma Company. IL-1, IL-6 and TNF-α ELISA kits plate was covered with tectorial membrane. were purchased from Wuhan Uscn Life Science They were incubated for thirty minutes at 37°C. Inc.. RNA extraction kit (RNeasy Plus Mini Kit) 90 μl of TMB zymolyte was added in each hole. was purchased from QIAGEN. Reverse tran- The ELISA plate was covered with tectorial scription kit (iScript cDNA Synthesis Kit) and membrane. They were colored for 15 to 25 min- real-time PCR fluorescent quantitative kit utes in dark place at 37°C. When gradient blue (SsoAdvanced SYBR Green Super mix) were appeared in the former three to four standard purchased from Bio-Rad. S100A8, S100A9, holes, the reaction was terminated. 50 μl of 2 S100A11 and S100A12 antibodies and S- M H2SO4 was added. OD value in each hole was 100A12 (Calgranulin-C) shRNA Plasmid (includ- measured immediately with ELIASA at 450 nm. ing Plasmid Transfection Reagent, Plasmid Transfection Medium and Control shRNA All data are expressed as the mean ± standard Plasmid) were purchased from Santa Cruz deviation (SD). The comparison between two Biotechnology. Horseradish peroxidase (HRP) groups was examined with t-test. Findings with labeled secondary antibody was purchased P < 0.05 were considered to be statistically dif- from Beijing Zhongshan Jinqiao Biotechnology ferent, and those with P < 0.01 were consid- Company Ltd.. ECL color kit and PVDF mem- ered to be statistically significant. brane (Polyvinylidene fluoride) were purchased from Millpore. Skimmed milk powder was pur- Paraffin embedding and HE staining chased from Oxoid. The liver tissue of the rats was cut into small pieces with a thickness of no more than 5 mm. CO2 constant temperature incubator was pur- chased from SANYO. MultiSkan FC ELIASA was These small pieces were marked, fixed in 10% purchased from Thermo Scientific. Fluorescent neutral formalin for more than 24 hours, rinsed quantitative PCR test system (CFX96 Touch) with tap water, on the next day dehydrated with was purchased from Bio-Rad. gradient ethanol, embedded in paraffin and sliced (thickness was 3 to 5 µm). Establishment of sepsis models in rats The paraffin slices were placed in haematoxylin LPS was injected intraperitoneally at a dosage and stained for fifteen minutes. Then the slices of 0.1 μg/g (LPS was dissolved in saline with a were rinsed with tap water for fifteen minutes 1632 Int J Clin Exp Pathol 2015;8(2):1631-1639 S100 family proteins and neonatal rats with sepsis Table 1. Molecular functions and subcellular localization of main members of S100 family proteins Gene Molecular function Subcellular location Associated disease S100A1 ATPase binding; identical protein binding; S100 protein binding; calcium ion binding; protein homodimerization activity Cytoplasm Cardiomyopathy S100A2 calcium ion binding; identical protein binding Breast cancer S100A3 calcium ion binding; zinc ion binding Cytoplasm S100A4 calcium ion binding; zinc ion binding; Cytoplasm Metastatic carcinoma S100A5 calcium ion binding; poly (A) RNA binding; RAGE receptor binding S100A6 calcium-dependent protein binding; calcium ion binding; ion transmembrane transporter activity; protein homodimeriza- Nucleus envelope. Cytoplasm. Cell membrane; melanoma tion activity; S100 protein binding; protein homodimerization activity; tropomyosin binding; zinc ion binding Peripheral membrane; Cytoplasmic side S100A7 calcium ion binding; RAGE receptor binding; zinc ion binding Cytoplasm; Secreted Immune related S100A8 arachidonic acid binding; calcium ion binding;