| Diclofenac sodium is a third-generation non-steroidal anti-inflammatory drug(NSAID),which has good antipyretic,analgesic and anti-inflammatory pharmacological effects and is widely used in veterinary clinic.Recently,it was found that vultures could die of poisoning after eating animal carcasses through the food chain which contain sodium diclofenac residue.So the toxic effect of sodium diclofenac on birds has aroused widespread concern.In order to explore the toxic effects of diclofenac sodium in poultry,this study used the 30 days,817 chickens as experimental animals and constructed diclofenac sodium toxicity model by purfusing them 10 mg/kg and 20 mg/kg diclofenac sodium.Chicken duodenal tissue and intestinal contentsin chickens were collected,and the diclofenac sodium on duodenum in toxic injury to the organization and the intestinal flora was explored through the histopathological examination,urate stain,enzyme colorimetry,western bolt,16 S r RNA sequencing and high performance liquid chromatography and other methods.Studies have found that diclofenac sodium could increase the content of uric acid in the duodenal tissue and decrease the activity of XOD,resulting in dysfunction of the excretion of uric acid,which is deposited in the duodenal tissue in the form of urate,causing bleeding,intestinal villi rupture and shedding,intestinal epithelial cell edema and other pathological injuries.In addition,diclofenac sodium could up-regulate the expression of NRF2,Fas L,TNF-α and TNF-R1,which induced oxidative stress and apoptosis to cause intestinal damage.What’s more,diclofenac sodium could significantly increase the relative abundance of harmful bacteria,such as Proteobacteria and Bacteroidetes,while decrease the relative abundance of beneficial bacteria,Lactobacillales,which resulted in disturbance of bacterial community.In order to explore the relationship between the concentration of diclofenac sodium in the duodenum and toxic effects,a high performance liquid chromatography method was established to determine the content of diclofenac sodium in the duodenum.Using C18 chromatographic column(150×4.6 mm,5 μm),internal standard method determination(flurbiprofen is the internal standard),the mobile phase is: methanol: 1% glacial acetic acid(75:25,v/v),the flow rate is1.0 m L/min,column temperature 30℃,detection at 285 nm wavelength.Results The diclofenac sodium measured by this HPLC method had a good linear relationship in the concentration range of 0.1 ～ 6 μg/m L;the correlation coefficient r=0.9958;the precision and accuracy were good;the duodenal tissue sample solution was at room temperature 12 h,repeated freezing and thawing three times,and storage at-20 ℃ for 30 d,all remained stable;the extraction recovery rates of the three quality control concentrations of diclofenac sodium were(80.66±2.74),(81.83±3.56),(84.44±2.29).The extraction recovery rate of the internal standard flurbiprofen is:(82.25±3.12).This study successfully built the toxicity model of diclofenac sodium on chickens,and established the content detection method of diclofenac sodium in the duodenum of chickens by high performance liquid chromatography,which provided a theoretical basis for the toxicity effect of diclofenac sodium on the duodenum tissue and intestinal flora of poultry. |
