Metabolomic Study On The Protective Effect Of Low-Dose Calcium Supplementation On Liver Injury Induced By Cadmium Exposure In Rats

Cadmium（Cadmium,Cd）is a common heavy metal pollutant.With the rapid development of modern industry,a large amount of cadmium has been discharged into the air,water,and soil,which is a serious threat to human health.Cadmium has a long biological half-life,slow metabolism,easy to accumulate in the body,highly toxic,and can cause damage to a variety of tissues and organs.As a substantial digestive organ and detoxification organ of the body,the liver is the main organ of cadmium damage and accumulation after entering the body.It is reported that increasing calcium intake can reduce the absorption of cadmium,reduce the concentration of cadmium in tissue,and then reduce the liver damage caused by cadmium exposure to a certain extent.Therefore,it is of great health significance to study the effect of calcium supplementation on reducing liver injury induced by cadmium.However,the dose of calcium supplementation in existing studies is generally high,which may lead to toxic side effects（such as hypercalciuria,kidney stones,vascular calcification,increased risk of cardiovascular disease,etc.）.At the same time,at present,the way of exposure to cadmium in animals is mainly focused on drinking water,intraperitoneal injection and oral administration,these methods are difficult to simulate the actual situation of human exposure to cadmium mainly through diet.Therefore,it is of scientific value and practical significance to explore the protective effect of low-dose calcium supplementation on dietary cadmium-induced hepatotoxicity and its possible mechanism,and can provide a basis for follow-up research to find a safe and reasonable calcium supplement dose.According to the procedure and method of toxicological Evaluation of Food Safety （GB15193.13-2015）,the rat model of dietary cadmium exposure was established by adding cadmium（1 mg/kg）to the diet（calcium content is 5.0 g/kg）,and then the toxic effects of different levels of calcium supplementation（1.5,4.0,6.0 g/kg）on rats were observed.After 90 days of oral toxicity test,the toxic effects of different calcium supplementation levels on rats were compared.the results showed that the minimum visible damage level of calcium intake of rats exposed to dietary cadmium in general population（（lowest observed adverse effect level,LOAEL）was 9.0g/kg.At the same time,according to the literature report,the rats were fed with low（1.0 mg/kg）,medium（5.0 mg/kg）and high（50 mg/kg）levels of cadmium in diet for 13 weeks to simulate the dietary cadmium exposure levels of the general population（about 10μg/day）,the high exposure population（about 50μg/day）and the population in the polluted area（about 500μg/day）.Based on this,this experiment used the calcium intake LOAEL obtained from the previous study as the calcium supplement dose to intervene the rats exposed to different levels of dietary cadmium,and to explore the protective effect of low-dose calcium supplementation on rat liver injury induced by dietary cadmium exposure and its possible mechanism.The research content includes the following two parts:1.Protective effects of low-dose calcium supplementation on liver injury induced by dietary cadmium exposure in rats.A total of 70 clean-grade 4-week-old female Sprague-Dawley（SD）rats weighing 60-80 g were selected in the experiment.Rats were randomly divided into 7 groups（n=10）,including control group,low,middle,and high dose dietary cadmium exposure group and corresponding calcium supplement group.Among them,the rats in the cadmium exposure group were given different dietary cadmium levels（1.0,5.0,50.0 mg/kg）to simulate the dietary cadmium exposure levels of the general population（about 10μg/day）,the high exposure population（about 50μg/day）and the polluted area population（about 500μg/day）.The calcium supplement dose was based on the calcium intake LOAEL（9 g/kg）obtained in the previous study.The specific groups are as follows:A:normal control（NC）;B:low-dose cadmium exposure group（Cd₁）;C:low-dose cadmium exposure calcium supplement group（Cd₁+Ca）;D:medium-dose cadmium exposure calcium supplement group）（Cd₅）;E:medium-dose cadmium exposure calcium supplement group（Cd₅+Ca）;F:high-dose cadmium exposure calcium supplement group)(Cd₅₀);G:high-dose cadmium exposure calcium supplement group(Cd₅₀+Ca).Among them,the rats in group A were fed with conventional diet（Ca,5 g/kg）,the rats in group B were fed with low cadmium（Cd,1 mg/kg;Ca,5g/kg）,the rats in group C were fed with low cadmium plus calcium（Cd,1 mg/kg;Ca,9 g/kg）,and the rats in group D were fed with medium cadmium（Cd,5 mg/kg;Ca,5 g/kg）.Rats in group E were fed with Cd,5 mg/kg;Ca,9 g/kg,rats in group F were fed with high cadmium（Cd,50 mg/kg;Ca,5g/kg）,and rats in group G were fed with high cadmium plus calcium（Cd,50 mg/kg;Ca,9 g/kg）.After 13 weeks of experiment,blood samples were taken from the inferior vena cava and all rats were killed to collect liver samples.The levels of liver functional enzymes in serum,biochemical indexes of blood lipids,the contents of cadmium,antioxidant enzymes and malondialdehyde in liver were measured,and the pathological changes were observed.The results showed that there was no significant difference in the levels of ALT,AST,TG,TCHO,LDL,HDL,SOD,CAT,GSH and MDA between the low and middle dose cadmium exposure groups and the normal control group.At the same time,the pathological results showed that the liver structure was normal and no histopathological changes were observed in the low and middle dose cadmium exposure groups,and no histopathological changes were observed after low dose calcium supplementation.The levels of ALT,AST,TG,TCHO,LDL and MDA in the high dose cadmium exposure group were significantly higher than those in the control group（P<0.05）,and the levels of TG,TCHO and MDA in the low dose calcium supplement group were significantly lower than those in the control group（P<0.05）.The levels of),GSH in the low dose calcium supplementation group were significantly higher than those in the control group（P<0.05）.The levels of TG,TCHO and MDA in the high dose cadmium exposure group were significantly higher than those in the control group.However,the levels of ALT,AST,LDL,SOD,CAT and HDL did not change significantly.At the same time,the pathological results showed that the liver structure of rats in the high-dose cadmium exposure group was abnormal,vacuolar degeneration,liver trabeculae crowded,disordered;after low-dose calcium supplement,the pathological results showed that the liver structure of rats returned to normal.It is worth noting that the concentration of cadmium in the liver of rats in each cadmium exposure group was significantly higher than that in the control group（P<0.05）in a dose-dependent manner.However,after calcium supplementation,the concentration of cadmium in rat liver had no significant change（P>0.05）.These results suggest that high-dose cadmium dietary exposure can cause abnormal changes of liver structure（vacuolar degeneration,hepatic trabecular crowding,disarrangement）,increase of liver cadmium concentration,liver injury（increased activity of ALT and AST）,decrease of antioxidant capacity（abnormal levels of SOD,GSH,CAT,MDA）and disorder of lipid metabolism（abnormal levels of TCHO,LDL,TG,HDL）.After low dose calcium supplementation,the pathological results of the liver were normal,the antioxidant capacity was partially improved（the levels of GSH and MDA were adjusted）,and the indexes of lipid metabolism were partially adjusted（the levels of TG and TCHO were adjusted）.The above results suggest that low dose calcium supplementation can improve the hepatotoxicity caused by dietary cadmium exposure to some extent.However,this protective effect is not caused by calcium by reducing the concentration of cadmium in tissues.On this basis,metabolomics methods were further used to explore the possible mechanism of the protective effect of low-dose calcium supplementation on dietary cadmium-induced hepatotoxicity in rats.2.Metabolomic study on the protective effect of low-dose calcium supplementation on liver injury in rats exposed to dietary cadmium.In the first chapter,the serum and liver samples of normal control group（NC）,high-dose cadmium exposure group(Cd₅₀)and high-dose cadmium exposure calcium intervention group (Cd₅₀+Ca)were determined by UPLC-Q-TOF/MS.After extracting,optimizing and identifying the original data,the differences of metabolites in serum and liver samples of rats were analyzed by principal component analysis（PCA）,partial least square discriminant analysis（PLS-DA）and orthogonal partial least square discriminant analysis（OPLS-DA）.Combined with online metabonomic database analysis,the possible mechanism of protective effect of low dose calcium supplementation on liver of rats exposed to cadmium was explored.The results showed that after exposure to high-dose dietary cadmium,the levels of malic acid and lysophosphatidylcholine in serum increased,while the levels of arginine and citrate decreased.The levels of malic acid,fumaric acid,prostaglandin,lysophosphatidylcholine and phosphatidylethanolamine in liver tissue samples of rats exposed to high dose cadmium diet increased,while the levels of phenylalanine,citric acid,arachidonic acid and arginine decreased.After giving calcium supplementation,it was found that the levels of the above differential metabolites were adjusted back.The related metabolic pathways are energy metabolism （tricarboxylic acid cycle）,lipid metabolism（glycerol phospholipid metabolism,arachidonic acid metabolism）and amino acid metabolism（phenylalanine metabolism,arginine,and proline metabolism）.These results suggest that high-dose cadmium dietary exposure can cause abnormal changes of small molecular substances in rats,and after low-dose calcium supplementation,the levels of some differential compounds（arginine,citrate,malic acid,lysophosphatidylcholine,phenylalanine, fumaric acid,citric acid,arachidonic acid,prostaglandin,phosphatidylethanolamine,etc.）tend to return to normal.The changes of these substances suggest that the protective effect of low-dose calcium supplementation on dietary cadmium-induced hepatotoxicity in rats may be related to energy metabolism,lipid metabolism and amino acid metabolism.