Identification And Mechanism Of Intestinal GPCR SER-6 In Response To Graphene Oxide In Caenorhabditis Elegans

With the mass production and extensive application of nanomaterials,more and more people pay attention to their adverse effects and the underlying molecular mechanisms.Graphene oxide(GO),a new carbon nanomaterial,can induce the production of active oxygen species and oxidative stress,inhibit the normal function of tissues,and affect the lifespan in organisms.But the mechanisms of GO toxicity are still largely unclear.We have used the model organism Caenorhabditis elegans to study the effects of GO toxicity,and explained the molecular mechanisms of some mRNAs and ncRNAs in response to GO toxicity.G protein coupled receptors(GPCRs)are seven-pass transmembrane receptors widely present in organisms.GPCRs are important molecules that mediate extracellular signal-induced cellular responses and are often used as drug targets in biomedical field;however,the research on GPCRs in nanotoxicology is still insufficient.GPCRs are involved in the regulation of various important life activities such as behavior and innate immunity in C.elegans.GPCRs are expressed in many tissues,but many studies are currently focused on the neuronal GPCRs.Our previous studies have shown that the intestine is the primary target organ for the GO toxicity.In the present study,the functions of GPCRs in the intestine were identified in nematodes exposure to GO at the environmental relevant concentrations,and the molecular mechanism of certain intestinal GPCR was elucidated during the regulation of GO toxicity.Based on the transcriptomics data,GPCRs that may participate in the regulation of GO toxicity in the intestine were screened and identified.The expressions of GPCR dop-1,ser-6and srh-2 were decreased,but the expressions of GPCR mgl-2 and sra-14 were increased in GO-exposed nematodes.Among them,dop-1 or ser-6 RNAi knockdown nematodes were sensitive to GO toxicity,and mgl-2 or sra-14 RNAi knockdown nematodes were resistant to GO toxicity.Moreover,dop-1,mgl-2,ser-6 and sra-14 were further confirmed to respond to GO toxicity in the intestine.Next,the molecular mechanisms of SER-6 encoding the octopamine receptor in response to GO in the intestine were studied.Octopamine is an endogenous ligand of SER-6,which is often used as a neurotransmitter to transmit signals and is synthesized by tyramine?-hydroxylase TBH-1.In the nematodes exposed in GO,the expression of tbh-1 was decreased,and the tbh-1 RNAi knockdown nematodes were sensitive to GO toxicity.Our results indicated that TBH-1,as an upstream molecule of SER-6,is involved in the response of GO toxicity.Therefore,we hypothesize that the changing expression of tbh-1 induced by GO exposure will affect the function of SER-6,and further regulate the downstream molecules and signaling pathways.The insulin signaling pathway and the p38 MAPK signaling pathway are known to regulate the GO toxicity in the intestine of C.elegans.In GO-exposed nematodes with the ser-6 overexpression in the intestine,the expression of daf-2 encoding insulin receptor was down-regulated,daf-16 encoding FOXO transcription factor,and sod-3 encoding superoxide dismutase were up-regulated in insulin signaling pathway.pmk-1 encoding mitogen-activated protein kinase(MAPK),skn-1 encoding Nrf protein and gst-4 encoding glutathione S-transferase 4 were also up-regulated in the p38 MAPK signaling pathway.SER-6 acted upstream of DAF-2 and DAF-16 in the insulin signaling pathway,and also acted upstream of PMK-1 in p38 MAPK signaling pathway to regulate the GO toxicity.Therefore,we screened and identified four intestinal GPCRs that play a role in response to GO toxicity in C.elegans,and revealed the molecular mechanisms of SER-6 in regulating the GO toxicity.For the underlying mechanism,our results indicated that the tyramine?-hydroxylase TBH-1 in neurons regulated the GO toxicity by affecting the function of intestinal GPCR SER-6.Insulin and p38 MAPK signaling pathways acted downstream of SER-6 to be involved in the regulation of GO toxicity.Our studies provide an important insight into understanding the molecular basis for the intestinal GPCRs in regulating in vivo toxicity of GO,and present new potential valuable molecular endpoints for the evaluation and detection of nanotoxicity.