Protective Effects Of Quercetin On Cadmium Induced Oxidative Damage And Apoptosis Of Hepatocytes

Objective:Cadmium(Cd)is a toxic heavy metal,which is widely used in industrial and agricultural production.With the application of Cd,the environmental pollution caused by it is becoming more and more serious.Cd can cause serious toxic damage to the liver,kidney and reproductive system of humans and animals.The liver is one of the main target organs of Cd poisoning,and its damage is more serious.In order to reduce the damage of Cd to the liver,quercetin(QE),an antioxidant,was selected in this experiment.By studying the effects of QE on oxidative stress and apoptosis of hepatocytes induced by Cd in rats and BRL-3A cells,we can judge its protective effect on Cd induced hepatocyte injury.Methods:In vitro test:BRL-3A cells were selected and divided into 4 groups.Control group:normal medium treatment for 24 h;group Cd:12.5 μmol/L CdCl2 treatment for 24 h;group Cd+QE:12.5 μmol/L CdCl2 and 5 μmol/L QE treatment for 24 h;group QE:5 μmol/L QE treatment for 24 h.After treatment,collect samples.In vivo test:36 5-week-old clean SD rats were randomly divided into 6 groups.Each group was treated as follows every day:group Ⅰ:intraperitoneal injection(ip)of normal saline and drinking water;group Ⅱ:ip 1 mg/kg b.w.of CdCl2;group Ⅲ:ip 2 mg/kg b.w.of CdCl2;group Ⅳ:ip 1 mg/kg b.w.of CdCl2 and oral administration 100 mg/kg b.w.of QE;group Ⅴ:ip 2 mg/kg b.w.of CdCl2 and oral administration 100 mg/kg b.w.of QE;group Ⅵ:oral administration 100 mg/kg b.w.of QE.The test cycle was 28 days.Blood and liver tissue samples were collected at the end of the test.The content of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)in serum and cell culture supernatant,the activity or content of superoxide dismutase(SOD),catalase(CAT),glutathione(GSH)and malondialdehyde(MDA)in liver tissue and BRL-3A cells were detected by enzyme labeling instrument;The level of reactive oxygen species(ROS)in BRL-3A cells was detected by flow cytometry;The mitochondrial membrane potential and apoptosis of BRL-3A cells were detected by inverted fluorescence microscope;The changes of blood parameters were detected by blood routine instrument;Using HE staining and TUNEL method to observe the pathological changes and apoptosis of liver tissue;The mRNA level and protein expression of Nrf2,NQO1,Keap1,CytC,caspase-9,caspase-3,Bax and Bcl-2 in liver tissue and BRL-3A cells were detected by fluorescence quantitative PCR and Western blot.Result:In vitro test:(1)The concentration of CdCl2is 12.5 μmol/L and QE is 5μmol/L by MTT test.Compared with the control group,the contents of ALT,AST and LDH in the medium of the Cd group were significantly increased(P<0.05),and compared with the Cd group,the contents of ALT,AST and LDH in the medium of the Cd+QE group were significantly decreased(P<0.05).(2)Compared with the control group,the activities of SOD and CAT in BRL-3A cells in the Cd group were significantly decreased(P<0.05),the content of GSH was significantly decreased(P<0.05),and the content of MDA was significantly increased(P<0.05).Compared with the Cd group,the activities of SOD and CAT and the content of GSH in BRL-3A cells in the Cd+QE group were significantly increased(P<0.05),and the content of MDA was significantly decreased(P<0.05).Compared with the control group,the mRNA and protein expression levels of Nrf2,NQO1 and Keapl genes in BRL-3 A cells in the Cd group were significantly decreased(P<0.05),after QE intervention,compared with the Cd group,the mRNA and protein expression levels of Nrf2,NQO1,and Keapl genes in the Cd+QE group were significantly increased(P<0.05),and the Nrf2 signaling pathway was activated.(3)By Hoechst 33258 assay,it can be concluded that compared with the control group,Cd leads to increased apoptosis of BRL-3A cells.JC-1 method showed that Cd caused the decrease of mitochondrial membrane potential,after adding QE,compared with the Cd group,the apoptosis of the Cd+QE group was alleviated,and the mitochondrial membrane potential increased.Compared with the control group,the mRNA and protein expression levels of CytC,caspase-9,caspase-3,and Bax in the Cd group were significantly increased(P<0.05),and the mRNA and protein expression levels of Bcl-2 were significantly decreased(P<0.05),compared with the Cd group,the mRNA and protein expression levels of CytC,caspase-9,caspase-3,and Bax in the Cd+QE group were significantly decreased(P<0.05),and the mRNA and protein expression levels of Bcl-2 were significantly increased(P<0.05).In vivo test:(1)Cd-induced rat body weight increased slowly,and after the 7th day,the body weight of group Ⅲ was significantly lower than that of group Ⅰ(P<0.05),after QE intervention,compared with group Ⅱ and group Ⅲ,the weight of group Ⅳ and Ⅴ increased.Blood routine test showed that Cd caused inflammation and anemia in rats,and QE could reduce inflammation and anemia in rats.Compared with group Ⅰ,ALT,AST and LDH in group Ⅱ and Ⅲ were significantly increased(P<0.05),Compared with group Ⅱ and Ⅲ,the contents of ALT,AST and LDH in groups Ⅳ and Ⅴ decreased significantly(P<0.05).By observing the tissue sections,Cd-induced pathological phenomena such as cell vacuolization and cell membrane damage in rat liver tissue.QE can improve the pathological symptoms.(2)Compared with group I,the activities of SOD and cat in group Ⅱ and groupⅢ decreased significantly(P<0.05),the content of GSH decreased significantly(P<0.05),and the content of MDA increased significantly(P<0.05).QE significantly increased the activity or content of SOD,CAT and GSH in liver,and significantly decreased the content of MDA(P<0.05).Cd treatment significantly decreased the mRNA and protein expression levels of Nrf2,NQO1 and Keapl genes in rat liver(P<0.05),after QE intervention,the mRNA and protein expression levels of Nrf2,NQO1,and Keap1 genes were significantly increased(P<0.05),and the Nrf2 signaling pathway was activated.(3)The results of TUNEL method showed that Cd caused the increase of hepatocyte apoptosis in rats,compared with group Ⅰ,the mRNA and protein expression levels of CytC,caspase-9,caspase-3 and Bax in group Ⅱ and group Ⅲincreased significantly(P<0.05),The protein expression level of cleaved caspase-3 was significantly increased(P<0.05),and the mRNA and protein expression levels of Bcl-2 were significantly decreased(P<0.05)by fluorescence quantitative PCR and Western blot detection,after adding QE,TUNEL test showed that QE intervention inhibited apoptosis,according to the detection of mRNA and protein levels,the mRNA and protein expression levels of CytC,caspase-9,caspase-3 and Bax in groupⅣ and Ⅴ decreased significantly compared with group Ⅱ and Ⅲ respectively(P<0.05),the expression level of cleaved caspase-3 protein decreased significantly(P<0.05),and the expression levels of Bcl-2 mRNA and protein increased significantly(P<0.05).Conclusion:This study found that Cd exposure led to slow weight loss,inflammation and anemia,pathological changes of liver tissue,hepatocyte injury,oxidative stress and apoptosis.QE inhibits Cd-induced slow growth,inflammation,anemia and liver injury in rats.By activating the Nrf2 signaling pathway,scavenging ROS,increasing the activity or content of antioxidants,QE alleviates the oxidative damage caused by Cd exposure,inhibits the caspase apoptosis pathway,and reduces cell apoptosis,thereby exerting a protective effect on Cd-induced hepatocytes.