Effects Of Zinc Imbalance On Liver Oxidative Damage And The Protective Effect Of Fisetin

Zinc,as one of the essential trace elements in the animal body,plays an important role in the process of growth and development of the body.Zinc deficiency can cause negative effects such as reduced immunity,impaired reproductive system and reduced growth performance.However,prolonged exposure to high zinc can cause damage to the organism.As the largest gland in the organism,the liver has various functions such as metabolizing protein,fat,carbohydrates and secreting bile,and its large number of mitochondria is the main site for generating reactive oxygen species(ROS),so the liver is vulnerable to oxidative damage caused by ROS attack when the organism is damaged.The phenolic hydroxyl structure of plant polyphenols can scavenge free radicals and is a natural antioxidant.Fisetin(3,3’,4’,7-tetrahydroxyflavone),as a bioactive flavonol molecule,has good antioxidant activity.In this experiment,piglets and mice were selected to investigate the effects of zinc deficiency and zinc excess diets on the liver in terms of oxidative stress,apoptosis and autophagy,and to establish a zinc imbalance model for AML12 cells to investigate the oxidative damage caused by zinc imbalance on hepatocytes and the effects on apoptosis and autophagy.We further investigated the protective effect of Fisetin on zinc imbalance-induced liver injury and its related mechanism.The main findings were as follows:1.Effect of diets with different levels of zinc on the liver of pigletsOne hundred and twenty 38-day-old Duroc×Landrace×Yorkshire piglets were fed a basal diet without any additional zinc source(low Zn group),a normal Zn diet with 100 mg/kg ZnSO4(calculated by Zn)(normal Zn group),and a high Zn diet with 2250 mg/kg ZnO(calculated by Zn)(high Zn group).The pre-test was conducted for 3 days and the formal test for 35 days,and liver tissues were taken at the end of the test to determine the relevant indexes,and the test results were as follows:(1)T-AOC,T-SOD activity and CAT activity were not significantly different in the low Zn group compared with the normal Zn group;T-AOC was significantly higher in the high Zn group(P<0.05),and T-SOD activity and CAT activity had a tendency to increase.(2)Compared with the normal Zn group,the liver LDH activity of piglets in the low Zn group was significantly higher(P<0.05),and the LDH activity in the high-dose zinc oxide group had a tendency to increase.(3)Compared with the normal Zn group,the expression level of pro-apoptotic protein Bax was significantly higher in the low Zn group(P<0.05),and the protein expression level in the high Zn group had a tendency to increase.(4)The expression level of autophagy-related protein Beclin-1 was elevated in the low Zn group,while it was inhibited in the high Zn group.The above results indicated that the prolonged effect of high Zn could increase liver T-AOC in piglets,but also caused damage to the organism;feeding low or high Zn diets caused apoptosis in hepatocytes,and feeding low Zn diets induced autophagy in liver cells,while prolonged high Zn exposure inhibited autophagy.2.Effects of diets with different levels of zinc on liver damage in miceForty-eight male C57BL/6 mice at 3 weeks of age were fed a basal diet without additional zinc(17 mg/kg Zn in the basal diet)(Zn-deficient group),a normal diet with 30 mg/kg ZnCO3(calculated by Zn)(normal Zn group),and a high Zn diet with 150 mg/kg ZnCO3(calculated by Zn)(high Zn group).To further verify the effect of zinc imbalance on liver damage,pre-feeding for 3 days and formal test days for 28 days,the results of the test were as follows:(1)Feeding the zinc-deficient diet or the high-zinc diet significantly increased the liver LDH activity in mice compared to the normal Zn group(P<0.01).(2)Compared with the normal Zn group,the liver ROS level was significantly increased in the Zn-deficient group(P<0.01),the liver T-SOD activity and T-AOC were significantly decreased(P<0.05),the GSH content was decreased and the MDA content was significantly increased(P<0.05);the ROS level and MDA content were significantly increased in the high Zn group(P<0.05),and the T-SOD,GSH,and T-AOC had a decreasing trend.In addition,the expression of endoplasmic reticulum stress-related protein CHOP was significantly increased in the high Zn group(P<0.05),and the Zn-deficient group had an increasing trend.(3)Compared with the normal Zn group,the expression of anti-apoptotic protein Bcl-2 was significantly inhibited in the deficiency and high Zn groups(P<0.05),while the expression level of Bax was significantly increased in the deficiency and high Zn groups(P<0.05).(4)LC3Ⅱ/Ⅰ decreased in the zinc-deficient group and LC3Ⅱ/Ⅰ increased in the high Zn group,and the protein expression of p62 showed the opposite trend.The above results indicated that:zinc imbalanced diets caused endoplasmic reticulum stress and decreased antioxidant properties in mice;both zinc-deficient or high-zinc diets induced increased apoptosis in mouse hepatocytes;high-zinc feeding promoted autophagy in mouse hepatocytes and zinc deficiency feeding inhibited autophagy.3.Damaging effects of zinc imbalance on mouse hepatocytesIn this experiment,mouse hepatocytes AML12 was used as the study target,and an in vitro zinc imbalance model was established by 50 μM TPEN(zinc ion specific chelator)or 250 μM ZnSO4 treatment to further investigate its effect on liver cell damage and its mechanism.The results were as follows:(1)The results of AML12 cells treated with different concentrations of ZnSO4(150 μM,200 μM,250 μM,300 μM,350 μM)for 2,4 and 6 h showed that the cell activity decreased with the increase of ZnSO4 concentration under the same treatment time conditions,and the cell activity decreased with the increase of treatment time when the cells were treated with the same concentration of ZnSO4.The cell activity was close to 50%after 4 h of treatment with 250 μM ZnSO4.With a 4 h treatment time,the cell activity was close to 50%after 50 μM TPEN treatment.In the experiment,250 μM ZnSO4(high Zn group)and 50 μM TPEN(Zn-deficient group)treatments were selected to establish the zinc imbalance model.(2)Zinc imbalance significantly increased LDH activity in the culture medium(P<0.05).(3)Cellular ROS production was highly significantly increased(P<0.01),MDA level was increased,and T-AOC and GSH levels were highly significantly decreased(P<0.01)in the zinc imbalance treated group.(4)Compared with the control group,Annexin V-EGFP green fluorescence signal and PI red fluorescence were enhanced and Bax protein expression was significantly increased in the zinc imbalance model group(P<0.05).(5)Compared with the control group,LC3Ⅱ/Ⅰ was significantly decreased(P<0.05)and p62 protein expression was increased in the zinc-deficient group,and LC3IⅡ/Ⅰ was highly significantly increased(P<0.01)and p62 protein expression was significantly decreased(P<0.05)in the high Zn group compared with the control group.The above results suggest that zinc imbalance can reduce AML12 cell activity,cause oxidative cell damage and induce apoptosis,and zinc deficiency inhibits cell autophagy,while high zinc treatment promotes cell autophagy.4.Protective effect of Fisetin on oxidative damage of hepatocytes caused by zinc imbalanceThe above research indicated that oxidative stress caused by zinc imbalance is the main cause of organism injury.The aim of this experiment was to investigate the protective effect of Fisetin on liver injury induced by zinc imbalance and its related mechanism.The results were as follows:(1)20 μM,40 μM and 60 μM Fisetin together with 250 μM ZnSO4 significantly increased cell viability,and the difference in cell viability between the latter two groups was not significant;therefore,Fisetin at a concentration of 40 μM was selected for the subsequent research.The difference in cell viability between 40 μM Fisetin+50 μM TPEN and Fisetin alone treatment groups was not significant.(2)Fisetin suppressed the increase of intracellular ROS level caused by zinc overload highly significantly(P<0.01),and Fisetin+ZnSO4 had a tendency to increase T-AOC and decrease MDA compared with ZnSO4 group,while cellular GSH content was highly significantly decreased in ZnSO4-treated group compared with the control group,and the combined effect of Fisetin and ZnSO4 could increase cellular GSH content highly significantly(P<0.01).Compared with ZnSO4 alone,Fisetin+ZnSO4 further increased the expression of p-Nrf2 significantly and HO-1 expression had an increasing trend.(3)The difference in intracellular Zn2+ concentration between the Fisetin and control groups was not significant,and the difference between the intracellular Zn2+ content in the ZnSO4 and Fisetin+ZnSO4 groups was not significant.The above results suggest that Fisetin can improve cell activity and alleviate high Zn-induced oxidative damage in a dose-dependent manner,and its protective ability is related to the regulation of Nrf2/HO-1 signaling pathway.In summary,in vitro and in vivo tests showed that either low or high zinc treatment caused liver damage and apoptosis in piglets and mice,and cellular tests further showed that zinc deficiency inhibited cellular autophagy,while high zinc treatment promoted cellular autophagy.Fisetin had a good protective effect on alleviating high zinc-induced oxidative damage,and its protective ability was related to the regulation of Nrf2/HO-1 signaling pathway.