| Ochratoxin A(OTA)is a class of secondary metabolites produced by fungi such as Aspergillus Ochraceus and Penicillium verrucosum.OTA is widely distributed in nature and has stable chemical properties,which causes great harm to biological safety and livestock and poultry production.The kidney is the main target organ of OTA.It has been proven that OTA exposure can induce kidney damage in pigs and poultry.OTA is also suspected to be related to Balkan endemic nephropath(BEN).However,the research on OTA nephrotoxicity mainly focuses on the toxic effects of OTA exposure on renal tubules and renal tubule-related cells,while the toxicological effects of OTA exposure on the glomeruli are still unclear.The chronic kidney disease model established by Cyclosporin A(CSA)is a widely accepted and stable kidney disease model.HK-2 cells are the main in vitro model cells used to simulate OTA and CSA-induced kidney injury.In the current experiments on the toxicological effects of mycotoxins,studies are mainly carried out on healthy model animals.However,the research on the toxicity mechanism of exposure to mycotoxins when the body is under stress and weakened immunity is still unclear.The trace elements selenium and zinc are the essential trace elements of the body,which play an important role in the process of the body’s anti-oxidation and scavenging oxygen free radicals.However,the combined antioxidant effect of the two and the protective effect of OTA-induced renal cell fibrosis are still unclear.This thesis first explored the toxic effects of OTA exposure on glomeruli and its mechanism,and then used CSA-induced HK-2 cell damage as an in vitro model to explore the toxic effects of non-toxic doses of OTA exposure on CSA renal cell damage.Finally,the protective effect of selenium and zinc supplementation on renal cell fibrosis induced by OTA was discussed.Test 1 Research of OTA induces glomerular damage in mice and its mechanismThe main purpose of this experiment is to explore the toxicological effects of OTA exposure on the glomeruli.In the in vivo experiment,24 C57bl/6 mice were randomly divided into 4 groups(1)control group(2)OTA low-dose group(0.5 mg/kg intraperitoneal injection every other day)(3)OTA middle-dose group(every day Intraperitoneal injection of 1.5 mg/kg)and(4)OTA high-dose group(intraperitoneal injection of 2.5 mg/kg every other day).The challenge was continued for 3 weeks,the mice were sacrificed,and their blood and kidneys were collected for subsequent experiments.The results showed that as the concentration of OTA treatment increased,the growth performance and kidney index of mice decreased significantly(P <0.05),and the serum levels of renal biochemical indicators creatinine(Cre)and urea nitrogen(BUN)increased significantly(P <0.05).The results of H&E staining and Masson staining showed that as the concentration of OTA treatment increased,the glomeruli of mice split,atrophy,and collagen fiber deposition increased.Fluorescence quantitative PCR test and Western blot test showed that OTA exposure induced the expression of inflammatory factors(IL-6,COX-2 and TNF-α)and fibrotic factors(Vimentin,TGF-βand α-SMA)in mouse kidneys increase.The above results indicate that OTA exposure can induce glomerular damage in C57/BL/6 mice.In the in vitro experiment,human glomerular mesangial cells(HMC)were used as the carrier and treated with OTA(0-8 μM)for 48 hours.Cell viability(MTT)and lactate dehydrogenase(LDH)tests show that the toxic effects of OTA on HMC cells are doseand time-dependent.Compared with the control group,OTA exposure significantly increased the expression of inflammatory factors and fibrosis factors in HMC cells(P<0.05).In addition,Western blot experiments showed that OTA exposure can significantly up-regulate the expression of P-ERK1/2,P-P65 and P-IκB-α proteins in vivo and in vitro,and the laser confocal colocalization test showed that OTA exposure can induce P65 The base is transferred from the cytoplasm to the nucleus.After adding FR180240 and BAY11-7028,inhibitors of ERK1/2 and NF-κB pathways,the expression of inflammatory factors and fibrotic factors induced by OTA was significantly down-regulated(P <0.05).The above results indicate that OTA can induce glomerular damage in mice by activating the ERK/NF-κB signaling pathway.Test 2 Nontoxic concentration of OTA treatment aggregated cyclosporin A-induced fibrosis in HK-2 cellsThe main purpose of this experiment is to explore the toxicological effects and mechanism of non-toxic doses of OTA exposure on the CSA-induced epithelialmesenchymal transition and fibrosis of HK-2 cells.First,determine the non-toxic dose of OTA concentration and the toxic dose of CSA concentration through MTT and LDH tests.After treating HK-2 cells with 2.5μM CSA for 24 h to establish an in vitro injury model,they continued to be exposed to 2.5μM OTA for 24 h.Compared with the CSA treatment group alone,fluorescence quantitative PCR test,western blot test and immunofluorescence test showed that the expression of epithelial-mesenchymal transition and fibrosis factors(E-cadherin,Vimentin and α-SMA)increased.And the non-toxic dose of OTA exposure aggravated the expression of endogenous TGF-β in HK-2 cells induced by CSA,and further activated its downstream signaling pathway Smad2,which turned into TGF-β factor m RNA,protein and immunofluorescence intensity increased,p-Smad2/Smad2 protein expression and Smad2 immunofluorescence intensity increased.After adding the specific TGF-β inhibitor SB431542,the epithelial-mesenchymal transition and fibrosis of HK-2 cells induced by OTA and CSA were weakened.In addition,non-toxic dose of OTA exposure reduced the expression of PI3K/AKT signaling pathway in HK-2 cells induced by CSA,which was manifested as a decrease in the expression of P-AKT/AKT protein.After adding Insulin,a specific activator of AKT,the protein and fluorescence expression of Vimentin and α-SMA decreased significantly.And the addition of Insulin can also inhibit the TGF-β/SMAD signaling pathway.The above results indicate that non-toxic doses of OTA can aggravate the CSA-induced epithelial-mesenchymal transition and fibrosis of HK-2 cells by activating the AKT/TGF-β/SMAD signaling pathway.Test 3 Research of the protective effect of zinc and selenium on OTA-induced HK-2 cells fibrosisThe purpose of this experimental study is to explore the protective effect of the combined application of trace elements zinc and selenium on the cytotoxicity and fibrosis of HK-2 cells induced by OTA and explore its potential mechanism.Cell viability MTT and LDH test results showed that OTA induced HK-2 cell damage in a dose-dependent manner,and treatments with 25-50μM zinc and 4-8μM selenomethionine could significantly inhibit the HK-2 cytotoxicity induced by OTA(P < 0.05),and the combined application of the two can achieve better results;fluorescence quantitative PCR test,western blot test and immunofluorescence test prove that the combined application of zinc and selenium can significantly down-regulate OTAinduced fibrosis factors(E-cadherin,Vimentin,Α-SMA,Collagen I,Fibronectin I)expression.In addition,OTA dose-dependently increases the accumulation of intracellular ROS and activates the expression of autophagy-related proteins(ATG5 and LC3B).However,the addition of zinc and selenium can significantly inhibit the above effects.After using H2O2 to reactivate intracellular ROS levels,the protective effects induced by zinc and selenium were abolished,and autophagy could be activated and reactivated in cells.Finally,after adding Rapamycin(specific autophagy activator),the protective effects of zinc and selenium were inhibited compared with the zinc +selenomethionine + OTA group.Then the level of ROS in the cell did not decrease.The results of this experiment show that OTA can induce cytotoxicity,oxidative stress and fibrosis of HK-2.Zinc and selenium can reduce the cytotoxicity and fibrosis induced by OTA through ROS-mediated autophagy,thereby alleviating the role of OTA nephrotoxicity. |
PDF Full Text Request