Interventional Effects Of Astaxanthin On Ochratoxin A-induced Subacute Kidney Injury In Mice

Mycotoxins are a kind of antinutritional factors existing in feedstuffs and compound feeds.They are secondary toxic metabolites produced by fungi of different genera.In China,mycotoxins not only pollute animal feeds,but also cause serious economic losses in animal husbandry.Among them,Aspergillus ochratorum is the most important one.Ochratoxin(OT)is a secondary metabolite produced by Penicillium and Aspergillus.There are seven types of ochratoxin A,B,C,etc.among them,ochratoxin A(OTA)has the widest distribution and the strongest toxicity.It has been proved to have reproductive toxicity in recent years.Ota is considered to be an important indicator of human and animal diseases and pollution in food processing.It can enter the human body along with the food chain,which is seriously harmful to human health.At present,the mechanism of Ota induced reproductive damage in male mice is not clear.Astaxanthin is a kind of carotene,which has been paid more and more attention because of its unique chemical structure of nine consecutive ππ conjugated double bonds.These bioactive compounds have the functions of anti-oxidation,cell repair,anti proliferation,anti inflammation and anti-aging.The latest research found that astaxanthin has significant clinical therapeutic effect on atherosclerosis,Alzheimer’s disease,Parkinson’s disease,cirrhosis,diabetes and pulmonary fibrosis.However,the protective effect and molecular mechanism of Ota induced subacute renal injury in mice are not clear.The C57BL/J mice were weighed and randomly divided into four groups(n = 20 per group): group I(Control),group II(OTA),group III(ASX),and group IV(ASX + OTA).All treatments were administered orally.Group I received 0.1 ml of olive oil and then gavage with 0.1 ml Na HCO3 after 2 hours.Group II received OTA at 5 mg/kg body weight(BW)/day and then 0.1 ml olive oil after 2 hours.Group III received ASX at 100 mg/kg BW/day and then 0.1 ml Na HCO3 after 2 hours.Group IV received 100 mg/kg BW/day of ASX and 2 hours later they received 5 mg/kg BW/ day of OTA.The treatments were administered for 7 days a week and then discontinued for 2 days,repeated three times.Dietary intake and BW were measured every day over the course of the study period.TUNEL staining,TEM,Western blot and real Time PCR were used in the experiment and other advanced molecular biological techniques were used to investigate the effects of subacute OTA exposure on apoptosis,proliferation,inflammation,oxidation-related gene expression of mouse kidney cells and the role of Nrf2 / Keap1 signaling pathway in this period,so as to explore whether ASX can intervene subacute renal injury and its intervention effect by influencing apoptosis,inflammation,proliferation,oxidation-related genes.The results of feed consumption,average body weight and organ coefficient showed that after 27 days of OTA exposure and ASX intervention,feed consumption,average body weight and kidney organ coefficient of OTA group were significantly lower than those of control group(P < 0.01).After ASX treatment,feed consumption and average weight of mice increased significantly(P < 0.01),but there was no significant difference in renal organ coefficient between the two groups.Compared with OTA group,feed consumption and average body weight of mice in ASX group and OTA group increased significantly(P < 0.01),but there was no significant difference in renal organ coefficient.The results of serum biochemical indexes showed that: after 27 days of OTA exposure and ASX intervention,Compared with the control group,the levels of UA and BUN in the serum of mice treated with OTA were significantly increased(p < 0.01 and p < 0.05);however,the level of CRE was not significantly affected.By contrast,the levels of UA,BUN,and CRE in serum were not affected by ASX compared with those in the control.In addition,the UA and the BUN levels in the ASX group were significantly lower than those in the OTA group(p < 0.01 and p < 0.05),whereas the CRE level was not significantly changed.In the ASX + OTA group,the serum UA and BUN levels were significantly decreased(p < 0.01 and p < 0.05,respectively)compared with those in the OTA group.There was no significant change in CRE levels in the ASX + OTA group.The results of morphology and ultrastructure showed that: after 27 days of OTA exposure and ASX intervention,In the control cells,the glomerular structure was intact,the renal small cystic lumen was uniform in size,the proximal tubule epithelial cells were closely arranged,the diameter of the lumen was large,the lumen was irregular,and the cell body was large.The cytoplasm was stained deeply with H&E.The nucleus was large and round,and located at the base of the cell.The distal tubule lumen was large and obvious,the cells were lightly colored,and the nucleus was round and located on the proximal cavity surface.In contrast to the control cells,after OTA treatment,the renal tubules were swollen,and the tubular capillaries became narrow and even disappeared.Renal tubular epithelial cells showed granule degeneration,vesicular degeneration,nuclear condensation,and nuclear lysis.No gross morphological changes were seen after ASX treatment.In the OTA + ASX group,glomerular swelling was relieved,the renal cystic space was restored,and tubular epithelial cell degeneration and necrosis were reduced.Under transmission electron microscopy,the kidney cells were closely arranged and intact in the control group and the ASX group.The mitochondria had a clear structure,the mitochondrial cristae were neatly arranged,and uniform chromatin distribution was observed in the nucleus.In contrast,in the OTA treatment group,the mitochondrial structure was disordered,the mitochondrial cristae disappeared,the nuclear chromatin was dispersed,the basement membrane was thickened,the nuclear membrane had disappeared,and the endoplasmic reticulum was swollen.In the ASX + OTA group,the normal appearance of the mitochondria was restored,and the structure of the mitochondrial cristae and the nucleus was nucleus was intact.The results showed that the renal cell apoptosis of mice after OTA exposure and the effect of ASX intervention:(TUNEL)staining results showed that compared with the control group,the number of apoptotic cells increased significantly after OTA treatment(P < 0.01).There was no significant difference between ASX group and ASX + OTA group.The number of apoptotic cells in ASX group and ASX + OTA group was significantly lower than that in OTA group(P < 0.01).Compared with the control group,the expression of p53,Caspase-3,Bax and bcl-2 m RNA in OTA group increased significantly(P < 0.01).After 27 days of ASX intervention,compared with OTA group,bcl-2 m RNA expression dose increased significantly(P < 0.01).The other indexes decreased significantly(P < 0.01).Compared with the control group,the expression of bcl-2 m RNA in ASX group increased significantly(P < 0.01).It is suggested that the inhibition of apoptosis gene and the promotion of apoptosis gene expression may be an important molecular mechanism for ASX to intervene the apoptosis of mouse kidney cells induced by OTA subacute exposure.After 27 days of OTA exposure and ASX intervention,compared with the control group,the expression of TNF-α m RNA and NF-? B protein in the kidney of OTA group decreased significantly(P < 0.05 or P < 0.01),and the expression of NF-? B protein,TGF-β,IL-1 β,IL-6 and NF-? B m RNA in the nucleus increased(P < 0.01).After 27 days of ASX intervention,compared with the OTA group,the expression of TNF-α,TGF-β m RNA and cytoplasmic NF-? B protein in the ASX+OTA group increased(p<0.05 or p<0.01),and the expression of NF-? B protein,IL-1 β,IL-6,NF-? B m RNA in the nucleus decreased(p<0.05 or p<0.01).Compared with the control group,the expression of TNF-α and TGF-β m RNA in ASX group increased(P < 0.01)and NF-? B m RNA decreased significantly(P < 0.05).There was no significant difference in other indexes.It is suggested that ASX may interfere with the proliferation of renal inflammatory nuclear cells induced by OTA subacute exposure by promoting the expression of cell proliferation genes and inhibiting the expression of pro-inflammatory genes.The results showed that: after 27 days of OTA exposure and ASX intervention,the results showed that: Compared with the control group,significantly lower levels of total superoxide dismutase(T-SOD),glutathione(GSH),and catalase(CAT)were detected in the kidney tissue of the OTA group(p < 0.01),whereas the malondialdehyde(MDA)levels increased significantly(p < 0.01).In contrast,CAT levels increased significantly in the ASX group(p < 0.01).There were no statistical differences in MDA,GSH,and T-SOD levels compared with those in the control group.In addition,compared with those in the OTA group,the GSH,T-SOD,and CAT levels in the ASX and the ASX + OTA groups were increased significantly(p < 0.01),whereas a significant decrease in MDA was noted(p < 0.01).The results of nrf2-keap1 signal pathway related gene expression showed that After OTA treatment,the m RNA levels of Nrf2,Nqo1,Ho1,Gclc,and Gpx1 were significantly decreased compared with those in the control group(p < 0.05 or p < 0.01).The Keap1 m RNA level increased significantly(p < 0.01).By contrast,ASX treatment significantly upregulated the m RNA levels of Nrf2,Nqo1,Ho1,Gclc,and Gpx1 compared with those in control group(p < 0.05 or p < 0.01)and significantly downregulated the m RNA level of Keap1(p < 0.05).Treatment with ASX + OTA significantly upregulated the m RNA levels of Nrf2,Nqo1,Ho1,Gclc,and Gpx1 compared with those in the OTA group(p < 0.05 or p < 0.01),and significantly downregulated the m RNA level of Keap1(p < 0.01).The expression of nrf2-keap1 signaling pathway related proteins showed that,after OTA treatment,the levels of NRF2-related target proteins NQO1,HO-1,γ-GCS,and GSH-Px decreased compared with those in the control group(p < 0.05 or p < 0.01);however,the levels of KEAP1 increased(p < 0.01).After ASX treatment,the levels of NRF2-related target proteins increased in the ASX group and the ASX + OTA group compared with those in the OTA group(p < 0.05 or p < 0.01),while the level of KEAP1 decreased(p < 0.05);the protein levels of NRF2 in the nuclear was significantly increased,and the level of NRF2 in the cytoplasm was significantly down-regulated(p < 0.05 or p < 0.01).The results show that ASX can promote cell proliferation,inhibit cell apoptosis,necrosis,inflammation,improve the antioxidant capacity of the body to protect the renal injury induced by OTA,and reveal the important mechanism of ASX to achieve the protection of renal injury induced by OTA through nrf2-keap1 signal pathway.