Histamine H3 receptor inhibited the inflammatory response during striated myogenesis induced by exogenous alpha-tumor necrosis factor

Abstract

Objective \nTo explore the effects of histamine H3 receptor on inflammatory response during C2C12 striated myogenesis induced by exogenous alpha-tumor necrosis factor (TNF-α) and elucidate the resistant mechanism of exogenous inflammatory stimulation in pediatric urinary sphincter during development and repair process from a molecular prospective. \n \n \nMethods \nTNF-α stimulated C2C12 cells under striated myogenesis (D0/D3/D6) for mimicing exogenous inflammatory stimulation in pediatric urinary sphincter during development and repair process. Cell count kit-8 (CCK 8) method and flow cytometry (FCM) were utilized for detecting cell growth and apoptosis induced by TNF-α, and histamine H3 receptor agonist methimepip (Met) and antagonist cirpoxifan (CPX). The inflammatory marker NLRP3 and three differentiation markers (early, middle & late) MyoD1, myogenin and myosin-2 were detected by real-time quantitative polymerase chain reaction (Q -PCR). The secretion of interleukin-1 beta (IL-1b) was detected by enzyme-linked immunosorbent assay (ELISA). \n \n \nResults \nDuring C2C12 myogenesis, TNF-α and NF-α+ CXP inhibited cell growth by CCK-8 (P 0.05). TNF-α and TNF-α+ Met did not induce apoptosis or necrosis while TNF-α+ CXP induced 29.9% apoptosis and 31.4% necrosis of D6 cell. The mRNA expression of NLRP3 increased 101% vs. 90% by TNF-α in D3 and D6 respectively; Met reduced the NLRP3 mRNA induced by TNF-α as 33% vs. 37%, while CPX increased NLRP-3 mRNA induced by TNF-α as 23% vs. 24%. TNF-α reduced the expression of myogenesis marker MyoD1 mRNA in D3 and D6 by 45% and 43%, Myogenin mRNA by 29% vs. 35% and Myosin-2 mRNA by 42% vs. 33% respectively; Met decreased the TNF-α-reduced differentiation marker mRNA again by 23% vs. 31%, 23% vs. 26%, 31% vs. 36%; while CPX increased the TNF-α-reduced differentiation marker mRNA by 40% vs. 25%, 35% vs. 31%, 50% vs. 21%. The secretion of IL-1β was 49.7 pmol/ml by TNF-α on D6 cells and Met decreased the IL-1β secretion by TNF-α at 37.9 nmol/L (T=12.5, P<0.001) while CPX increased the IL-1β secretion by TNF-α at 64.4 nmol/L (T=29.5, P<0.001). \n \n \nConclusions \nTNF-α can induce inflammatory response during C2C12 striated myogenesis and also inhibit this differentiation process while histamine H3 receptor can inhibit the inflammatory response induced by TNF-α and control to reduce the differentiation process. So when exogenous inflammatory stimulation affects urinary sphincter during the development and postoperative repair process, activating H3 receptor may antagonize the effects of exogenous inflammatory stimulus and maintain normal cellular development and repair process. \n \n \nKey words: \nChild;\xa0Sphincter of urethra;\xa0Histamine H3 receptor;\xa0Myogenic differentiation;\xa0Inflammatory response