| BackgroundOrganotin compounds are a kind of important organometallic compounds,which have been widely used in industry,agriculture,marine industry and so on since the middle of the 20th century.Tributyltin(TBT)is the most widely used organotin compound.TBT is widely used in marine antifouling agents,wood preservatives,industrial fungicides,plastic products stabilizers,agricultural fungicides.Because of its biocidal effect,TBT was added to ship painting as an antifouling agent in the early 1960s to prevent the attachment and parasitism of aquatic organisms to ships,docks,docks and so on.Due to the continuous diffusion and migration of TBT into the surrounding water body and settling into the sediment of the water body,it exists widely in the aquatic system.A large.number of aquatic ecological investigations and experimental studies have shown that TBT is an endocrine disruptor that can cause biological aberrations in the marine gastropods,leading to ecological disasters in the aquatic environment,thus attracting the attention of many researchers.Based on the serious harm of TBT to the ecological environment,the International Maritime Organization promulgated the International Convention on the Control of Harmful Antifouling Systems for Ships in 2003,which prohibited the use of TBT in ship antifouling agents since 2008.After the ban on TBT,although the application of TBT has been greatly reduced,some developing countries and countries that have not joined the International Maritime Organization still use paints containing TBT due to the lack of corresponding substitutes.In addition,TBT continues to be used in plumbing materials and wood preservatives.As a result,there are still a wide range of new sources of pollution in TBT.In addition to new pollution sources,the problem of TBT residues caused by heavy use cannot be ignored.Like many other environmental endocrine disruptors,the pollution of TBT is persistent.Its half-life is long,although the half-life in water is only about 6 weeks-5 months,but in sediments it can be as long as several decades..There are still a lot of TBT residues in the low silt of offshore,harbor and inland ports,and the continuous emission of TBT,for a long time is the continuous pollution source of TBT.Therefore,to sum up,TBT pollution will still exist widely for a long time in the future.The characteristics of TBT allow it to enter the human body through the food chain.TBT has a strong lipophilicity and accumulation,easy to enrich through the food chain,through biomagnification,so as to achieve a higher concentration in the human body.Human beings can be exposed to TBT by eating seafood,drinking water and beverages contaminated by TBT,inhaling indoor dust,and contacting industrial products containing TBT.Among them,oral intake through the food chain is the most important way.A large number of studies have shown that TBT has endocrine disrupting effects not only on aquatic organisms,but also on mammals.TBT is a typical environmental obesity factor.At nanomole concentration,it can induce PPAR γ,which is the key factor of adipose differentiation,and then induce adipose hyperplasia and the progression of obesity.Some previous studies have shown that TBT can lead to obesity and metabolic disorders in experimental animals,but there are few studies on its mechanism.There is evidence that obesity is closely related to chronic inflammation.In addition,oxidative stress and endoplasmic reticulum stress(ER stress)play an important role in the development of metabolic diseases.Nuclear Factor-κB(NF-κB)signal pathway,as the key pathway of inflammation,is closely related to ER stress and can be activated by oxidative stress,thus playing a role in the development of metabolic disorders.Therefore,the main purpose of this experiment is to study whether TBT interferes with NF-κB signal pathway through ER stress and oxidative stress,promotes the development of inflammation,and leads to obesity and metabolic disorder in rats.ObjectivesThe toxic effects of chronic long-term low-dose exposure of environmental endocrine disruptors on the growth and development of animals and humans have become the focus of the entire society.However,in the process of studying the health risks and ecological studies of these pollutants,it is more worthy of in-depth study to clarify the specific mechanism of their toxic effects and how their endocrine disruptive effects affect the ecological environment.In this paper,the mechanism of the effect of TBT on metabolism in rats was studied to explore the chronic toxic effects of TBT,which is helpful to deepen the study of the harmful effects of environmental endocrine disruptors such as TBT on human health,thus providing a possible direction for solving the health hazards caused by TBT and the increasingly serious endocrine disruptors pollution.The purpose of this study is to explore the effects and related mechanisms of long-term low-dose TBT exposure on metabolism in rats from the following angles:1)to monitor the body weight and visceral fat(epididymal fat,perirenal fat)of rats exposed to TBT;to detect the contents of blood lipids,lipid metabolic intermediates and their effects on glucose metabolism;to clarify the effects of long-term low-dose exposure of TBT on metabolism in rats.2)the changes of.hepatic macrophages and the effect of TBT exposure on the signal pathway of NF-κB,the key regulatory factor of inflammation in liver tissue,were detected by immunohistochemistry and Western Blotting.3)the enzymes and non-enzymatic substances that make up the antioxidant defense system and the biomarkers of endoplasmic reticulum stress were detected,and the oxidative stress and endoplasmic reticulum stress closely related to NF-κB were evaluated.4)To analyze the possible regulatory relationship between TBTCl exposure-induced obesity,metabolic disorders and NF-κB signaling pathway,oxidative stress,and endoplasmic reticulum stress.Methods1.Animal feeding and treatment21-day-old male SPF grade SD rats were randomly divided into 4 groups according to their body weight after adaptive feeding for 3 days,with 10 rats in each group.From PNDs 24,TBTCl dissolved in corn oil was given intragastrically every 3 days with the concentration and dose of 0.5 and 50 μg·kg-1,respectively.The body weight was measured and recorded before each intragastric administration,and the dose of the drug was adjusted according to the body weight of the day.After the last exposure to TBTCl,the rats were fed normally for another 10 days and were killed under PNDs 94 anesthesia.Then the blood was taken from the abdominal aorta and placed at room temperature for 1 hour.The serum was separated in 2500 rpm*10 minutes and stored at-80℃.The epididymal adipose tissue and perirenal adipose tissue were dissected and separated and weighed by balance.The liver tissue was separated,part of the liver was fixed with 4%paraformaldehyde solution,and the rest of the liver tissue was frozen in liquid nitrogen and stored in the refrigerator at-80℃.2.Glucose tolerance testGTT was performed on the third day before the rats were killed.Five rats were randomly selected from the four groups for GTT.After fasting for 12 hours and overnight,glucose(2g/kg body weight)was injected intraperitoneally.Before injection,the tail vein blood was taken,and the blood glucose concentration was detected by Roche blood glucose meter as the blood glucose level of 0 min.Blood samples were taken from the tail vein to determine the concentration of blood glucose at 15,30,60 and 120 minutes after injection.The AUC of glucose tolerance was used to evaluate the utilization ability of blood glucose in rats.3.Insulin tolerance testITT was also performed on the third day before the rats were killed.After the rats tested for GTT were randomly selected in each group,the remaining 5 rats were used in ITT,method:the rats were fasted for two hours,and then injected with insulin according to the dose of 1 U/kg body weight.Blood samples were taken from tail vein at 0,15,30,60 and 120 minutes to measure blood glucose level.Finally,AUC was calculated to evaluate insulin tolerance.4.Detection of serum-related indicatorsAfter the rat serum was obtained,triglyceride(TG),total cholesterol(TC),low density lipoprotein(LDL),and high density lipoprotein(HDL)were measure by Beckman Coulter AU480 automatic analyzer.Useing corresponding kits to detect serum free fatty acid(FFA),lipid peroxidation product malondialdehyde(MDA),and antioxidant-related indicators,including superoxide dismutase(SOD),peroxide Catalase(CAT),Glutathione(GSH)and Glutathione peroxidase(GSH-Px).5.Liver HELiver was fixed by 4%paraformaldehyde for 24 hours,and paraffin sections were made for hematoxylin-eosin staining(HE)to observe the liver tissue structure,and to observe the effect of TBT exposure on the liver tissue structure of rats.6.ImmunohistochemistryThe immunohistochemical method was used to detect the CD68 molecule,a classic marker of macrophages in the liver,to observe and analyze the effect of TBTCl exposure on rat liver tissue macrophages.7.Western Blotting to detect related proteinsWestern blotting was used to detect the expression of NF-κB 1(P105/P50),NF-κB 2(P100/P52),NF-κB P65 and their phosphorylated proteins in liver tissues,and to explore the effect of TBT exposure on NF-κB pathway.Western blotting was also used to detect the biomarkers of ER stress,including binding immunoglobulin protein(BiP),inositol-requiringenzyme1 α(IRE1 α)and endoplasmic reticulum oxidoreduclin1-L α(Ero1-L α).To explore whether TBT exposure caused unfolded protein response,resulting in ER stress.Results1.Increased epididymal fat and perirenal fat weight in TBTCI exposed ratsIn the course of the experiment,there was no abnormal activity and mental abnormality in all groups.At the end of the experiment,the weight of epididymal fat and perirenal fat increased in a dose-dependent manner.Compared with the control group,the weight of epididymal fat and perirenal fat in 50μg·kg-1 group increased by 49.3%and 64.4%respectively.Weight monitoring showed that there was no significant difference in body weight among the four groups.2.Effects of TBTC1 exposure on blood lipid levels in ratsThe serum of rats was extracted to determine the levels of TC,TG,HDL and LDL.The results showed that TBTCl treatment increased the content of TG in serum of rats,and the content of TG in 50 μg·kg-1 group was significantly higher than that in control group(P<0.05).There was no significant change in serum TC,HDL and LDL levels between each treatment group and the control group.The results of detection of FFA,the intermediate product of lipid metabolism in serum,showed that TBT exposure increased the level of FFA,and there was a significant difference between the highest dose group and the control group.3.Effects of TBTCl exposure on glucose metabolism in ratsThe results of GTT showed that the blood glucose concentration of 5 μg·kg-1 group was significantly higher than that of the control group at 30 min(P<0.05),and the blood glucose concentration of 5 μg·kg-1 group and 50 μg·kg-1 group was significantly higher than that of the control group at 60 min(both P<0.05),and the GTT-AUC of these two groups was also significantly higher than that of the control group(P<0.05).This suggests that TBT exposure decreased glucose utilization and led to impaired glucose tolerance in rats.In ITT test,the blood glucose concentration of 5μg·kg-1 group and 50μg·kg-1 group decreased slightly at 30 min,while at 60 min and 120 min,the blood glucose concentration of 5 μg·kg-1 group and 50 μg·kg-1 group was significantly higher than that of the control group(P<0.05),and the AUC of the two groups was also significantly higher than that of the control group(P<0.05).This shows that the insulin tolerance of rats is impaired and the insulin sensitivity is decreased under the condition of TBT exposure.4.TBTCl exposure caused abnormal liver tissue structure and increased macrophages in ratsHE section of rat liver tissue showed that there were different degrees of hepatocyte cord disorder,hepatic sinusoid dilatation and congestion,a large number of inflammatory cell infiltration in hepatic lobule and portal area,swelling of some hepatocytes,vesicular degeneration and necrosis,and eosinophilic degeneration and punctate necrosis of some hepatocytes in TBT exposure group.The immunohistochemical results of CD68,a marker of macrophages in rat liver,showed that the expression of macrophages in 50 μg·kg-1 group was significantly higher than that in control group(P<0.001),indicating that the number of macrophages increased after exposure to TBT.5.TBTCl exposure induced oxidative stress in ratsThe markers of oxidative stress in serum and liver of rats were detected.The results showed that the content of MDA.in serum and liver tissue increased in a dose-dependent manner,and there was significant difference between the highest dose group and the control group(P<0.05).In serum,the SOD activity of 5 μg·kg-1 group and 50μg·kg-1 group decreased significantly,and the CAT activity of 5 μg·kg-1 group and 50 μg·kg-1 group also decreased significantly(P<0.05).The GSH content of 0.5μg·kg-1 group decreased significantly(P<0.05).In liver,SOD activity decreased significantly in 50 μg·kg-1 group(P<0.05),CAT activity in three exposure groups decreased significantly(P<0.001,0.05,0.01),GSH content in the highest dose group decreased significantly(P<0.05),and GSH-PX activity in three exposure groups also decreased significantly(P<0.05,<0.01,<0.05,respectively).These results suggest that the antioxidant defense system of rats exposed to TBT is damaged,resulting in oxidative stress.6.Endoplasmic reticulum stress in rat liver induced by TBTCI exposureWestern blotting detected the markers of ER stress in liver tissue.BiP,IRE la and Erol-Lα in 0.5 μg·kg-1 group were significantly lower than those in control group(P<0.05),while IRE1α and Erol-Lα in 5 μg·kg-1 group and BiP,IREla and Ero1-Lα in 50 μg·kg-1 group were significantly increased(P<0.05).These results suggest that TBT exposure induces endoplasmic reticulum stress in rat liver.7.TBTCl exposure disrupted NF-κB signaling pathwayThe key molecules of NF-κB pathway were also detected by Western blotting.The ratios of phosphorylated NF-κB P65 and NF-κB P65 in the three dose groups were significantly higher than those in the control group(P<0.05),which indicated that the phosphorylated form of NF-κB P65 increased and the NF-κB signaling pathway was activation.We further tested NF-κB 1(P105/P50)and NF-κB 2(P100/P52),and the results showed that the ratio of P105/P50 in the 5 μg·kg-1 group and the 50 μg·kg-1 group was significantly reduced(P<0.05)compared with the control group.And the ratios of P100/P52 in the three exposure groups were significantly reduced compared with the control group(P<0.05).This indicates that TBT exposure not only activated the NF-κB classical signaling pathway involved in NF-κB 1(P105/P50),but also interfered with the NF-κB non-classical signaling pathway involved in NF-κB 2(P100/P52).ConclusionsLong-term low-dose exposure to TBT during puberty may disrupt the NF-κB signaling pathway by inducing oxidative stress and endoplasmic reticulum stress,resulting in liver tissue damage in rats and an increase in the number of liver macrophages,resulting in chronic inflammation,leading to obesity in rats,disorders of glucose metabolism and abnormal lipid metabolism. |
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