Association between ihha and shha gene downregulation and swim bladder inflation impairment in zebrafish exposed to the combination of morphine and disulfiram

Recent evidence suggests that disulfiram may serve as a useful adjunct in chronic morphine treatment by mitigating opioid-induced hyperalgesia, withdrawal, and dependence. However, exposure to disulfiram produces abnormalities in swim bladder inflation in zebrafish, which points to the possibility of interference with Hedgehog signaling - a key regulator of swim bladder formation. Therefore, this study aimed to find the association between the expression of markers present in the developing zebrafish swim bladder (sox2, fgf10a, pbx1a, acta2, anxa5) and the Hedgehog pathway genes (ptch1, ptch2, smo, ihha, and shha) in zebrafish embryos treated with the morphine-disulfiram combination. For this purpose, zebrafish were treated for 54 or 117h with disulfiram alone or in combination with morphine. Swim bladder inflation was assessed under a light microscope, and gene expression was quantified with RT-qPCR assay using SYBRGreen dye. Morphine alone upregulated ptch2, smo, and all of the other markers present in the swim bladder except for the ihha gene; while disulfiram alone downregulated all Hh pathway genes in the zebrafish embryos along with fgf10a and pbx1a markers. When combined with morphine, the expression of shha and ihha further decreased, while ptch1 increased considerably. An upregulation of sox2 and fgf10a swim bladder markers was also present. Our investigation has shown that the synergistic disruption in swim bladder inflation in zebrafish treated with the morphine-disulfiram combination could be associated with ihha and shha downregulation, triggering a compensatory upregulation of acta2 and anxa5 expression.