Hypothesis / aims of study
To investigate the effects of Histamine H3 receptor on the inflammatory response during C2C12 striated myogenesis induced by alpha tumor necrosis factor (α-TNF), which discloses the role of H3 receptor when exogenous inflammatory stimulation occurs in urethral sphincter during the development and the repair process from the molecular aspect.
Study design, materials and methods
The C2C12 myogenesis (striated differentiation) was induced to mimic the development and repair process of urethral sphincter and three timepoints at the differentiation day 1,3,6 (D0, D3, D6) were recorded. Histamine H3 receptor agonist Methimepip (Met) and antagonist Cirpoxifan (CPX) stimulated the cells and the cell count kit-8 (CCK 8) method and flow cytometry (FCM) was used to detect the cell growth and apoptosis induced by α-TNF. The inflammasome marker nucleotide-binding oligomerization domain like receptor family, pyrin domain-containing 3 (NLRP3) and the three differentiation markers (early, middle and late markers) MyoD1, Myogenin and myosin-2, were detected by real-time quantitative Polymerase Chain Reaction (Q-PCR) method. The secretion of inflammatory cytokines interleukin-1 beta (IL-1β) was detected by enzyme-linked immuno-sorbent assay (ELISA).
During C2C12 myogenesis, α-TNF inhibited the cells growth by CCK8 and only α-TNF+CXP induced 30% apoptosis of D6 cells by FCM. Alpha-TNF induced inflammasome gene NLPR3 mRNA expression up to 201% vs.190% in D3 and D6, respectively; Met reduced the NLRP-3 by α-TNF as 33%vs.37%, while CPX increased the effect of α-TNF on NLRP-3 as 23%vs.24% in D3 and D6. Alpha-TNF reduced the expression of myogenesis marker MyoD1 in D3 and D6 by 45 % and 43 %, Myogenin by 29%vs.35%, and Myosin-2 by 42%vs.33%, respectively; Met increased the inhibition effect of α-TNF on differentiated markers by 23%vs.31%，23%vs.26%，31%vs.36%; while CPX reduced the inhibitory effect of α-TNF on differentiated markers by 40%vs.25%，35%vs.31%，50%vs.21%. The secretion of IL-1β was 49.7 pmol/ml by α-TNF on D6 cells, and Met reduced the IL-1b secretion by α-TNF to 37.9 pmol/ml (T=12.5, P<0.001), while CPX increased α-TNF effects up to 64.4 pmol/ml (T=29.5, P<0.001).
Fig. 1 Effects of α-TNF on apoptosis of C2C12 cells during myogenesis.
Notes: D0, undifferentiated cells, mainly myoblasts; D3, myoblasts differentiated for 3 days, around half myoblasts and half myotubes; D6, myoblasts differentiated for 6 days, mainly myotubes.
Fig. 2 The effects of α-TNF on the mRNA expression of myogenesis markers and inflammasome key element NLRP-3 gene during myogenesis
Notes: D0, undifferentiated cells, mainly myoblasts; D3, myoblasts differentiated for 3 days, around half myoblasts and half myotubes; D6, myoblasts differentiated for 6 days, mainly myotubes. Ctrl, control group; T, α-TNF group; T-M, α-TNF +Methimepip (Met, H3 receptor agonist) group; T-C, α-TNF + Ciproxifan (H3 receptor blocker) group. ＊, there are statistic differences compared with the control group (P<0.05); ※: statistic differences compared with α-TNF group (P<0.05). Repeat samples, n=9.
Interpretation of results
Histamine is a biogenic amine widely existing in plants and animals, and involved in the immune and biochemical functions of the body. Up to now, there are 4 subtypes of receptors, histamine H1, H2, H3, H4 receptor reported. In our previous research, we found histamine receptors subtype of H1, H2 and H3 receptors in striated C2C12 myogenesis and adult mid-urethral striated muscles . The expression of H3 receptor is increasing amazingly during myogenesis, and H3 receptor is also found to diminish cytoplasma calcium peak of the differentiated C2C12 myotubes under electrical stimulation . It has been reported that inflammasome key element NLRP3 protein produced in mouse C2C12 myoblasts when inflammation occurs, and proper concentration of α-TNF can induce inflammation, autophagy and apoptosis in mouse C2C12 cells; NLRP3 inflammasome, which is involved in innate immunity and in most inflammatory reactions, was found in C2C12 inflammation . We hypothesized that C2C12 myogenesis can be modeled as for the development and repair of urethral sphincter. We attempted to induce the inflammatory response of C2C12 cells during myogenesis by exogenous stimulation of α-TNF and checked whether H3 receptor is functional there, which would give some references about whether H3 receptor functions when exogenous inflammatory stimulation occurs in the process of the development and repair of the urethral sphincter. From our pilot and incomplete results at present, we find that histamine H3 receptor agonist Met might reduce the inflammation induced by α-TNF, while CPX might increase the effects of α-TNF during the myogenesis day 3,6 (D3, D6),by detecting inflammation related gene NLRP-3 and secretion of the cytokine IL-1β.