| Objective:To study the role of HSF2by method of interference and overexpression on the human colonic epithelial cell lines-Caco-2cell in regulating LPS-induced inflammation.Methods:Caco-2cells were transfected with HSF2siRNA and recombinant plasmid (pCMV-HSF2-FLAG) using Lipofectamine. Cytotoxicity was estimated by the MTT (3-[4,5-dimelhyl-2-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. After LPS stimulated the different treatment cells, Nitric oxide (NO) were measured by Griess reagent.The protein and mRNA expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2), tumor necrosis factor-a (TNF-a) and interleukin-8(IL-8) were analysed by Western Blot, ELLISA and RT-PCR, respectively. Phosphorylation levels of extracellular signal-regulated kinase1/2(ERK1/2), c-Jun N-terminal protein kinase (JNK) and p38mitogen-activated protein kinase (p38MAPK), inhibitor kB-a (IκB-a) and NF-κB p65were measured by Western Blot.Results:1The viability of the transfected siRNA and recombinant plasmid Caco-2cells groups were no difference compared with the negative siRNA group,Blank vector group and the control group (P>0.05).2NO secretion of cell supernate in LPS-stimulated cell groups (control group、negative siRNA group、siRNA group、blank vector group、overexpression group) was significantly higher than the free stimulated groups (28.690±2.929vs29.530±0.974vs71.230±2.974vs29.010±1.741vs17.380±0.738vs8.126±0.749μm), siRNA group increased the most obvious, and overexpression group increased weaker among these five groups (P<0.05). 3TNF-α and IL-8expression of cell supernate in LPS-stimulated cell groups (control group、negative siRNA group、siRNA group、blank vector group、overexpression group) were significantly higher than the free stimulated groups (TNF-α:57.010±6.148vs51.960±3.515vs438.800±55.140vs77.180±9.763vs56.200±8.863vs10.030±0.190pg/ml; IL-8:41.810±1.736vs41.570±2.346vs74.250±7.490vs41.220±1.545vs30.940±4.114vs17.620±0.957pg/ml), siRNA group increased the most obvious, and overexpression group increased weaker among these five groups (P<0.05).4iNOS, COX-2, TNF-α and IL-8mRNA expression in LPS-stimulated cell groups (control group, negative siRNA group、siRNA group、blank vector group、overexpression group) were significantly higher than the free stimulated group (iNOS:6.415±0.451vs5.439±0.652vs13.860±1.295vs5.076±0.338vs3.169±0.341vs0.844±0.084; COX-2:5.087±0.239vs5.098±0.295vs37.860±1.635vs4.324±0.965vs3.885±0.432vs0.977±0.228; TNF-a:5.459±0.750vs4.997±0.595vs13.120±1.489vs5.482±0.650vs2.921±0.532vs0.447±0.074;IL-8:7.703±1.084vs6.037±0.925vs28.690±3.777vs7.741±2.502vs2.026±0.258vs0.948±0.237), siRNA group was most apparent, overexpression group increased weaker among these five groups (P<0.05)5COX-2protein expression in LPS-stimulated cell groups (control group、negative siRNA group、siRNA group、blank vector group、overexpression group) were significantly higher than the free stimulated groups,siRNA group was most apparent, and overexpression group increased weaker among these five groups (P<0.05)6Silenced HSF2significantly increased the LPS-induced phosphorylation of IκB-a, NF-κB,JNK and p38MAPK,but attenuated ERK1/2.Over-expressed HSF2is quite the contrary, respectively.Conclusions:1. Silenced HSF2significantly attenuated the LPS-induced expression of NO, iNOS, COX-2, TNF-a and IL-8in Caco-2cells. Over-expressed HSF2is quite the contrary, respectively.the study suggests HSF2can weaken the expression of the proinflammatory enzymes and proinflammatory cytokines;2. In addition, It increased phosphorylation of IκB-a, NF-κB,JNK and p38MAPK,but decreased ERKl/2.Over-expressed HSF2is quite the contrary, respectively. These findings suggest that HSF2inhibited LPS-induced inflammatory mediators via up-regulation ERKl/2, but, down-regulation phosphorylation of JNK, p38MAPK and NF-κB pathways. These studies reveal, in part, HSF2has a potential anti-inflammatory properties, might be a new clue to reveal the pathogenesis of UC and a new target for intervention in the inflammatory response. |
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