In this study, we were able to show that metamizole has a worse effect than paracetamol on colonic anastomosis. Metamizole also had higher antiproliferative and antimigration effects on colon fibroblast, but not collagen synthesis, than paracetamol. To the best of our knowledge, our study is the first report of comparison between metamizole and paracetamol on colonic anastomosis. Another novelty of our study is we showed the impact of metamizole and paracetamol on the fibroblast activities.
The role of the inflammatory process is very important in healing anastomosis wounds which are characterized by the formation of granulation tissue[6]. Our in vivo studies showed that metamizole inhibited the anastomosis process of rat colons. These results were in accordance with previous study that found metamizole tends to increase the incidence of anastomotic leakage[9]. In contrast, there was no significant inhibition in the anastomosis process of rat colon in the paracetamol group, which was supported by previous reports[10,11].
We also showed that metamizole has negative influence on the integration of the muscle layers of the rat colon wall. It might be related to the direct mechanism of non-selective resistance to COX–1 and COX–2 enzymes. If the enzyme activity was inhibited, then it will affect prostaglandin synthesis which is an important mediator in the inflammatory process[12].
In addition, metamizole also suppressed the process of granulation tissue formation in the anastomosis site. The inhibition of cyclooxygenase enzymes by metamizole might reduce prostaglandin synthesis which affected the process of granulation tissue formation by inhibiting vasodilation of blood vessels at the site of the wound so that the leukocyte migration process was reduced. There was also a decrease in leukocyte proliferation in the inflammatory area[12].
Our findings also revealed metamizole inhibited mucosal anastomosis healing, while paracetamol did not affect the process. These results were consistent with data from previous studies where the strength of rat colonic anastomosis joints was not affected by administration of both low and high doses of paracetamol[10]. This may be due to the central effect of paracetamol which was more dominant than peripheral in inhibiting prostaglandin synthesis[10].
Our in vitro findings revealed metamizole was more cytotoxic to fibroblasts compared to paracetamol. It has been reported that paracetamol, which has mild antiinflammatory effect[13], requires a larger dose to obtain the same proliferative inhibitory power as metamizole which has a higher anti-inflammatory effect[14]. In addition to the IC50 value of metamizole and paracetamol, we also need to know the maximum concentration (Cmax) of these drugs. The administration of 1 g of metamizole intravenously will obtain a value of Cmax 56.5 µg/mL[15]. Whereas the same dose of paracetamol will only produce Cmax 19–22 µg/mL[16,17]. When comparing the IC50 with Cmax of each treatment, the IC50metamizole (50.08 ± 6.73 µg / mL) value was below the Cmax so that the inhibition concentration could easily be reached in the blood. However, this does not apply to paracetamol, where the IC50 value (194.8 ± 48.1 µg / mL) was above the Cmax so it will be difficult to achieve inhibition concentration if the drug was given in therapeutic doses. Therefore, administration of paracetamol in therapeutic doses was very unlikely to give an adverse effect of anti-inflammation as can be caused by metamizole. This proliferation barrier was consistent with previous studies[18,19] about the antiproliferative effects of NSAIDs on rat and human fibroblasts. The antiproliferation effects of NSAIDs occur in direct barriers to the increase of cyclooxygenase enzymes in the inflammatory process[19,20]. The inflammatory response will activate the COX–2 enzyme thereby increasing the synthesis of PGE2 which can inhibit proliferation of fibroblasts[20–22]. The antiproliferative effects of NSAIDs are also accompanied by barriers to DNA synthesis[18]. Metamizole which has a cyclooxygenase non-selective inhibitor action will suppress COX–2 enzyme activation so that it can suppress the DNA synthesis process and proliferation of rat colon fibroblasts. Metamizole has more potent antiproliferative effect in pancreatic cell line, Panc–1, than paracetamol at the highest dose concentration of 250 μg/ml [23].
The effect of metamizole inhibition was also seen in the migration of fibroblasts. This inhibition effect of metamizole was dose dependent, and appears to be more dominant than paracetamol at the highest dose of treatment. According to Nicpon et al.[24], the effect of metamizole inhibition on cell function is dose dependent on the concentration. The higher the concentration, the more obstacles will occur. Our results showed similar results where the inhibition of fibroblast migration increased with increasing metamizole concentration. Paracetamol appeared to also have a negative effect on fibroblast migration activities. Even so, the effect did not increase with the addition of the treatment dose. The inhibition of fibroblast migration by paracetamol was still less compared to metamizole at the highest dose of 250 μg/ml. These results were in accordance with previous studies which showed that paracetamol was one of the NSAIDs which has the lowest anti-inflammatory effect. The inhibition of fibroblast migration by NSAIDs is to suppress the action of the cyclooxygenase enzyme. These effects can be restored by administration of exogenous prostaglandins [25]. In addition, the mechanism for the inhibition of fibroblast migration is through the matrix metalloproteinase enzyme pathway known as RECK (reversion-inducing-cysteine-rich protein with Kazal motifs)[26].
The activity of fibroblasts in synthesizing collagen will increase in the stages of anastomosis wound healing. However, our study showed that metamizole and paracetamol did not reveal any significant inhibitions of collagen synthesis compared to control. In addition, previous in vivo reports revealed that barriers due to metamizole and paracetamol to collagen synthesis are equivalent to the control group[27, 28].
It should be noted that the effect of NSAIDs on the activity of fibroblasts has not been able to explain the whole process of intestinal anastomosis, because of the important role of other cells such as mucosal epithelial cells, smooth muscle cells lining the intestinal wall, intestinal endothelial vessels, and various inflammatory cells in the healing process of intestinal anastomosis wounds. In addition, in this study NSAIDs treatment was given under normal fibroblast conditions so that it might be different from the inflammatory conditions in the wound healing process.
Further study is necessary to clarify the inhibitory and migration effects of metamizole and paracetamol on cyclooxygenase enzymes in rat and human fibroblast cells. Using in vitro methods with co-culture techniques will be able to know the effects of NSAIDs on the interaction between inflammatory cells and mucosal, endothelial and fibroblast epithelial cells in the process of wound healing.