Effect Of Gastrodin In Treating Imidacloprid-induced Liver Injury And Its Underlying Mechanism In Cats Poisoning

In recent years,with the increase in the number of pet families,people are paying more and more attention to the environmental hygiene of pet keeping.Commercial insecticides with imidacloprid（IMI）as the main ingredient are widely used because they can effectively kill vector organisms such as mosquitoes,flies,termites and fleas in the environment and protect humans and pets from their infestation.However,pets have living habits such as lying down and licking,and insecticides in the environment are easy to cause pet poisoning and even death through skin contact or oral ingestion.At present,there are few clinical reports on the metabolic distribution of IMI in pets and the toxicity（toxic dose,poisoning symptoms and lethal dose）.Due to the low activity of glucuronidase in the cat’s liver,it is easy to cause liver damage after poisoning.Clinically,there is no effective drug treatment for IMI-induced cat poisoning and liver damage except supportive care.A large number of studies have found that Gastrodin（GAS）plays an important role in protecting the liver and nerves in humans,but there have been no reports on GAS in the treatment of liver damage in cats.In this study,in order to investigate the toxic dose of IMI commercial insecticide induced by cat poisoning and the therapeutic effect and mechanism of GAS after poisoning on liver damage,the following tests were designed:1.The acute oral toxicity test of the Up-and-down procedure（UDP）was used to calculate the half lethal dose(LD₅₀)of the cat after oral ingestion of the IMI commercial insecticide,and the pharmacokinetic test determined its absorption,distribution and metabolism in the cat,while observing the clinical manifestations of cat poisoning and assessing the degree of liver damage.The results showed that the oral LD₅₀ of IMI commercial insecticide cat was 1033 mg/kg,and the 95%confidence interval was 490-2000 mg/kg.Different doses of commercial IMI insecticides cause cats to produce different degrees of poisoning,including vomiting,salivation,muscle tremor,ataxia,pupil dilation,hyperesthesia,and increased body temperature.According to the toxic dose and poisoning manifestations,it is divided into mild poisoning,moderate poisoning and severe poisoning.Among them,the dose of IMI commercial insecticide is 200-400 mg/kg can cause mild poisoning,400-900 mg/kg can cause moderate poisoning,and 900-2000 mg/kg can cause severe poisoning and even death.The results of routine blood and blood biochemical tests showed that the blood glucose level of the ingested IMI commercial insecticide increased in a short period of time,and the total number of white blood cells,neutrophils,albumin,total protein,potassium ions,alanine aminotransferase（ALT）and aspartate aminotransferase（AST）exceeded the normal cut-off value in the high-dose ingested patients,and the pathological tissue sections of the high-dose ingestants observed diffuse congestion in the liver,the hepatic lobular boundaries were not clear,accompanied by inflammatory cell infiltration,showing obvious liver damage.A high performance liquid chromatography（HPLC）analysis method for IMI commercial insecticides was established,the mass fraction of IMI commercial insecticide species IMI was quantitatively analyzed,and the concentration of IMI in plasma after oral administration of IMI commercial insecticide in cats was measured,and the results showed that the peak time(T_max)of IMI in cats was 1 h,the half-life(T_1/2)was 10-14 h,and the average residence time（MRT）was14-19 h.2.In order to explore the therapeutic effect of GAS on cats poisoned by IMI commercial insecticides,acute or chronic poisoning of cats induced by the administration of a single high dose(LD₅₀)or multiple low doses(1/10LD₅₀)of IMI commercial insecticides,the poisoned cats were treated with GAS orally,and clinical symptoms,blood routine and blood biochemical examinations,liver histopathological tests and related inflammatory factor expression levels were measured.The results showed that compared with the group without GAS treatment,the symptoms of acute poisoned cat poisoning decreased significantly after GAS treatment,and the blood routine and blood biochemical abnormalities returned to the normal reference range after 3 h.Chronic poisoned cats had decreased liver inflammatory cell infiltration,and the expression level of liver inflammatory factors（TNF-α,IL-1β,IL-6）m RNA decreased.At the same time,it was found that the blood routine and blood biochemical abnormalities of chronically poisoned cats were restored to the normal reference range after 3 days of GAS treatment.The above results show that GAS plays the role of alleviation of poisoning symptoms and liver protection after cat IMI poisoning.3.In order to further explore the alleviation mechanism of GAS on liver damage after IMI commercial insecticide poisoning,6-8 weeks old C57BL/6 mice were selected and divided into blank group,GAS control group,IMI group and IMI+GAS low/medium/high dose group for drug gavage,and the death and weight changes of each group were recorded.Liver tissue was collected after the test for H&E,IHC,RT-PCR,and WB tests.The results showed that the mortality rates of mice in the IMI group and the low-dose GAS group were40%and 20%,respectively,while the mortality rate of the remaining groups was 0.The histopathological results showed that a large number of inflammatory cell infiltrates and diffuse congestion were seen around the central vein and duct area of the liver in the IMI group.The RT-PCR results showed that compared with the blank group and the GAS high-dose group,the m RNA expression levels of genes related to inflammatory factors（TNF-α,IL-1β,IL-6,NLRP3,NF-κB,COX-2）and apoptosis（Bax）in the IMI group were significantly increased（P<0.05）,and the m RNA expression levels of genes related to oxidative stress（OH-1,Nrf2）were significantly reduced（P<0.05）.The expression levels of inflammatory factor,apoptosis,and oxidative stress-related proteins in liver tissue detected by IHC and WB were consistent with the trend of RT-PCR assays.It has been shown that GAS reduces liver damage by antagonizing IMI-induced inflammation,apoptosis,and oxidative stress.This study found that IMI commercial insecticides have high fat solubility and fast absorption speed,which is easy to cause cat poisoning and liver damage.GAS alleviates liver damage by antagonizing inflammation,apoptosis and oxidative stress caused by IMI,and provides new ideas for veterinarians to diagnose and treat cat poisoning and liver damage in clinical practice.