The Effect Of DBP And DEHP On Apoptosis And Inflammatory Factors Secretion In Grass Carp Hepatocytes By NOD2/RIP2/NF-?B Pathway

Plasticizer is a kind of polymer material assistant which can improve the flexibility of polymer.They are mainly used in the production of flexible PVC,plasticized PVC can be used in hospital equipment,food packaging and many other industrial and commercial products.However,plasticizer,as a new environmental pollutant,widely exists in different ecological environments such as water,silt and soil,and is difficult to degrade.As a new environmental pollutant,the research on the toxic mechanism of plasticizer to fish is far from enough.The researches showed that the nuclear binding oligomerization domain-2(NOD2)signal pathway can be used as a potential target pathway to regulate liver injury.NOD2 activated the downstream nuclear factor kappa B(NF-?B)signal pathway through receptor interacting protein 2(RIP2).Activation of NF-?B signaling pathway was related to chronic inflammatory diseases,and it was a key regulatory factor involved in infection and inflammatory response.Apoptosis was also promoted by activation of NF-?B pathway and expressions of its downstream inflammatory factors.Apoptosis is a kind of programmed cell death,which plays an important role in toxicology.Many studies also found that when two harmful stimuli combine,there was a synergistic relationship between them,which strengthened their harmful effects.Therefore,we chose two plasticizers: diethylhexyl phthalate(DEHP)and dibutyl phthalate(DBP)as the research objects and exposed them to grass carp hepatocytes.CCK8 assay,fluorescence staining,flow cytometry,RT-PCR and Western blotting were used to study the toxic effects of mixed plasticizers on grass carp hepatocytes.In this study,we established a model of plasticizers exposure to grass carp hepatocytes,analyzed the toxic mechanism of NOD2/RIP2/NF-?B signaling pathway in grass carp hepatocytes exposed to mixed plasticizers,and identified the role of NOD2/RIP2/NF-?B signaling pathway in grass carp hepatocytes apoptosis after mixed plasticizers exposure.The test results showed that:(1)According to the results of CCK8 test,it can be found that there is a dose-dependent relationship between DBP and DEHP exposure on the viability of grass carp hepatocytes,that is,the higher the concentration of DBP and DEHP,the lower the viability of grass carp hepatocytes.It should be noted that the viability of hepatocytes exposed to DBP and DEHP was lower than that exposed to DBP or DEHP alone.These results indicated that exposure to mixed plasticizers could aggravate the decrease of sell viability in grass carp hepatocytes.(2)Based on the establishment of grass carp hepatocyte subculture model,the model of DBP and DEHP exposure and the inhibition of NOD2/RIP2/NF-?B signaling pathway was successfully constructed.The results showed that DBP and DEHP exposure could activate nod2-rip2-nf-? B signaling pathway in grass carp hepatocytes,which was manifested by the changes of mRNA expression levels of NOD2/RIP2/NF-?B signaling pathway related genes(NOD2,RIP2,TAK1,IKK?,NF-?B,IL-6,IL-8,IL-1 ? and iNOS).It is worth noting that the activation level of nod2-rip2-nf-? B signaling pathway was higher after the mixed exposure of DBP and DEHP.Specifically,the change of mRNA expression level of NOD2/RIP2/NF-?B signaling pathway related genes(NOD2,RIP2,TAK1,IKK ?,NF-? B,IL-6,IL-8,IL-1? and iNOS)was more obvious.(3)Next,we used fluorescence staining,flow cytometry,RT-PCR and Western blotting to detect the apoptosis level in grass carp hepatocytes.The results showed that DBP and DEHP could induce the apoptosis in grass carp hepatocytes,which was manifested by the increase of the number and proportion of apoptotic cells,the change of mRNA expression level of apoptosis related genes(Bcl2,Bcl-xl,Bax,Cytc,Caspase9 and AIF)and the increase of protein expression levels of apoptosis related genes(Bax and Caspase 9).(4)In this study,we used NOD2/RIP2/NF-?B signaling pathway inhibitor(NOD2-).The results showed that NOD2-could significantly inhibit the activation of NOD2/RIP2/NF-?B signaling pathway induced by DBP,DEHP and the mixture of DBP and DEHP,but it can not achieve complete inhibition.Specifically,the mRNA expression levels of NOD2/RIP2/NF-?B signaling pathway related genes(NOD2,RIP2,TAK1,IKK?,NF-?B,IL-6,IL-8,IL-1? and iNOS)were significantly decreased.To sum up,DBP or/and DEHP induce apoptosis and increased levels of inflammatory factors in grass carp hepatocytes by activating NOD2/RIP2/NF-?B pathway.Among them,the increase of inflammatory factors is related to apoptosis.Compared with DEHP,DBP has stronger hepatotoxicity,indicating that grass carp hepatocytes are more sensitive to DBP.The combined exposure of DBP and DEHP could aggravate the apoptosis and increased levels of inflammatory factors in grass carp hepatocytes.