Study On The Cytotoxicity And Mechanism Of Paraquat On CIK Cells

Paraquat?PQ?is a water-soluble herbiside widely used in the world,especially in developing countries and it belongs to the bipyridine organic heterocyclic compound.PQ can easily enter the aquatic ecosystems via surface runoff or underground drainage with extensive used in agricultural production due to its high water solubility and low volatility,resulting in natural water pollution,which is a threat to the animals and human beings.Due to its high toxicity and high mortality rate,PQ has attracted wide attention from researchers in many countries.There have been many studies on the mechanism of PQ toxicity,however,little is known about the toxic effects of PQ on aquatic organisms,especially on fish cells.In the present study,the effect of PQ on the viability of CIK cells was firstly determined by MTT assay,and then the cellular morphology and nuclear morphology of CIK cells were observed under inverted fluorescent microscope.Moreover,flow cytometry was used to detect cell cycle and apoptosis,and ELISA and qPCR techniques were used to detect the antioxidant enzyme activity,inflammatory cytokines,cell cycle and apoptosis-related gene or protein of the cells following PQ exposure for 24 or 48 hours.The aim of this study was to elucidate the cytotoxicity mechanism of PQ and provide theoretical support for the water ecosystem protection.The main results of this study are as follows:?1?The viability of CIK cells was determined by MTT,and the cellular shape was observed under inverted microscope so as to determine and evaluate the cytotoxicity of PQ on CIK cells.The result of MTT test showed that the 24,48,and 72 h IC₅₀ of PQ on CIK cells were 252.65,9.84 and 2.63?M,respectively.Additionally,our result also showed that PQ exposure could significantly influence cellular shape and cause injury to CIK cells,indicting that PQ had remarkable cytotoxicity on fish CIK cells.?2?The result of flow cytometry detection showed that PQ exposure caused G₀/G₁ phase arrest of CIK cell cycle.Meanwhile,the results of qPCR showed that PQ-exposure at various concentrations?1-50?M?altered the expression of cell cycle related factors such as p53,p21,mdm2,gadd45,e2f4,cyclin b,cyclin e,and CDK4,suggesting that these factors may be involved in PQ caused cell cycle arrest.?3?The biochemical analysis result showed that the activities of SOD and CAT in CIK cells were totally suppressed after PQ exposure,and the T-AOC content in the CIK cells after PQ exposure was initially increased while subsequently decreased,suggesting that PQ exposure might affect the antioxidant system of CIK cells.Moreover,the MDA level in the PQ-treated CIK cells was significantly higher than that of control group,suggesting that PQ exposure may cause lipid peroxidation in the treated cells.These results indicate that PQ exposure causes oxidative stress and promotes lipid peroxidation in CIK cells.?4?The result of ELISA detection showed that PQ exposure promoted expressions of pro-inflammatory cytokines,including TNF-?and IL-1?,and altered the IFN-?content.However,the contents of anti-inflammatory cytokines TGF-?and IL-10 in the PQ treated cells were initially increased,but subsequently decreased.These results suggest that up-regulation of pro-inflammatory cytokines and down-regulation of anti-inflammatory cytokines might favour the inflammation response of CIK cells caused by PQ exposure.?5?Hoechst 33342 staining revealed that PQ exposure at concentrations of 5,15,30 and 50?M significantly increased the nuclei of apoptotic bodies in CIK cells.Moreover,the result of flow cytometric analysis with annexin V/PI staining showed that PQ-exposure induced the apoptosis of CIK cells in a concentration-dependent pattern.Furthermore,the expression of apoptosis-related gene bax and bcl-2 was detected by qPCR,in which bax in the PQ treatment groups present a obvious up-regulation of expression in comparison with the control group.However,bcl-2 transcription in the PQ-treated CIK cells was basically suppressed.In addition,PQ-exposure generally promoted the transcriptions or enzymic activities of caspase-3 and caspase-9 in CIK cells,suggesting that PQ-induced apoptosis of CIK cells may be mediated via mitochondrial pathway.The results of this study showed that PQ exposure caused cell cycle arrest,oxidative damage,inflammatory reaction,and apoptosis of CIK cells.Our results demonstrated that PQ exerted cytotoxicity on CIK cells via MTT assay.The present study further enriches and perfects the mechanism of PQ cytotoxicity on fish CIK cells,which may provide theoretical support for the safe use of PQ and the water ecosystem protection.