Studying The Effects Of Low Doses Of Fumonisin B1 On Ochratoxin A-induced Nephrotoxicity And The Protective Effects Of Pig Heat Shock Protein 70

Fumonisin B1(FB1)is a secondary metabolite produced by Fusarium moniliforme Sheld,which widely exists in corn,wheat and other crops.Because the maximum allowable content of FB1 in feed ingredients specified in the national standard is significantly higher than other mycotoxins,and the actual content of FB1 in various crops is significantly lower than this regulation,it is often ignored in practical production.However,although these low doses of FB1 themselves are not likely to be toxic,it has not been investigated whether they exacerbate the toxicity of other mycotoxins.Ochratoxin A(OTA)is one of the toxic compounds by a variety of Aspergillus and Penicillium,widely distributed in different crops,feed and raw materials.According to some research reports,FB1 mainly affects the liver and kidney functions in animals,and can also cause equine leukoencephalomalacia(ELEM),porcine pulmonary edema,and esophageal cancer in humans and so on.The primary target organ of OTA is the kidney,which mainly causes nephrotoxicity.Heat shock protein 70(Hsp70)is an intracellular "chaperone" protein,which has cytoprotective effects and can also protect animals from damage caused by external stimuli,but whether it can alleviate the toxicity of mycotoxins has not been reported.This thesis mainly studies the effects of low-dose of FB1 on the nephrotoxicity of OTA and its underlying mechanisms.At the same time,a recombinant eukaryotic plasmid and a recombinant Lactococcus lactis expression system were constructed based on the porcine Hsp70 gene,and the protective effects and mechanisms of Hsp70 on renal injury and cell apoptosis induced by co-exposure of low doses of FB1 and OTA were researched in vitro and in vivo.Experiment 1.The effects of low doses of FB1 on OTA-induced renal mitochondrial damage and apoptosis in p K-15 cells and miceThe purpose of this study was to investigate the effects of low doses of FB1 on OTAinduced mitochondrial damage and apoptosis in renal epithelial cells in vitro and in vivo.Firstly,the porcine kidney epithelial cells were used as an in vitro model and treated with different concentrations of FB1 or/and OTA for 48 h.MTT and LDH results showed that1～8 μM FB1 did not significantly affect cell viability and cell membrane integrity,while5 μM OTA reduced cell viability and caused cell membrane damage significantly,which increased the LDH release,and 8 μM FB1 significantly aggravated the cell viability decreasing and cell membrane damage induced by 5 μM OTA.To verify the effects of low doses of FB1 on OTA-induced mitochondrial damage and cell apoptosis,the p K-15 cells were treated with 8 μM FB1 or/and 5 μM OTA for 48 h,respectively.The results showed that the mitochondrial membrane potential(MMP)was decreased and the mitochondrial ROS(mt ROS)was increased in p K-15 cells,and the effects in the dual mycotoxins group were more significant.The results of cytochrome C(Cyto C)co-localized with mitochondria showed that FB1 increased OTA-induced the production of Cyto C in mitochondria and the amount of leakage from mitochondria to cytoplasm significantly,indicating that the integrity of the mitochondrial membrane was broken and the oxidative stress was exacerbated.Further detection of the renal epithelial cell apoptosis found that the m RNA and protein levels of proapoptosis genes were also significantly up-regulated.Secondly,male ICR mice were used as the in vivo models,and mice were randomly divided into 5 groups(n=12 each),and then cochallenged with different doses of FB1(0,0.2,0.5,1.0 mg/kg bw)and 0.4 mg/kg bw OTA respectively for 16 d.The results showed that with the increase of FB1 concentrations,the decrease in body weight and increase in kidney index in mice were more and more obvious,and the activity of superoxide dismutase(SOD)was decreased in serum,and the levels of malondialdehyde(MDA),creatinine(Cr)and blood urea nitrogen(BUN)were increased significantly.The hematoxylin-eosin staining(H&E)results of kidney tissue showed that with the increase in FB1 concentrations,the glomerular swelling became more serious.And the IFA of kidney tissue showed that the number of Cyto C in tubules and glomerulus increased significantly.RT-q PCR and western blotting results showed that the m RNA and protein levels of pro-apoptosis genes were up-regulated in a concentration-dependent manner.Taken together,low doses of FB1 aggravated OTA-induced mitochondrial damage and renal epithelial cell apoptosis in p K-15 cells and mice.Experiment 2.The mechanism of low doses of FB1 aggravates OTA-induced renal injury and cell apoptosisThe purpose of this study was to investigate the effects of low doses of FB1 on renal injury and cell apoptosis induced by OTA exposure and its mechanism.The p K-15 cells were used as the research object and were treated with 8 μM FB1 or/and 5 μM OTA for 48 h,respectively.The results showed that low doses of FB1 increased the m RNA levels of damage factors in p K-15 cells induced by OTA.Further detection of the effects of FB1 on OTAinduced oxidative stress and apoptosis of p K-15 cells found that low doses of FB1 aggravated OTA-induced oxidative stress and apoptosis in p K-15 cells,manifested as the total cellular ROS production and the level of MDA were significantly increased,and the activity of SOD was significantly decreased,the cell apoptosis rate was increased,and the genes expression of Bax,Casp9,and Casp3 was up-regulated.After the ROS scavenger of N-acetyl-L-cysteine(NAC)was added,the levels of apoptosis genes were down-regulated,indicating that increased ROS production promotes cell apoptosis.The further study we also found that the co-exposure of low doses of FB1 and OTA up-regulated the expression of the p-JNK protein.After adding the JNK inhibitor of SP600125,the expression of the p-JNK protein was inhibited and the apoptosis level was decreased,suggesting that co-exposure to low doses of FB1 and OTA activated the JNK/MAPK signaling pathway and induced cell apoptosis.Finally,p K-15 cells were pretreated with NAC and SP600125,respectively,and then coexposure of FB1 and OTA for 48 h,and detected the levels of ROS and p-JNK protein.The results showed that NAC inhibited the expression of the p-JNK protein,while SP600125 did not affect ROS levels,indicating that ROS can mediate the activation of the JNK/MAPK signaling pathway.Taken together,this research demonstrated that low doses of FB1 aggravated OTA-induced renal injury and cell apoptosis through activation of the ROSmediated JNK/MAPK signaling pathway.Experiment 3.The alleviation effects of Hsp70 on renal injury and renal cytotoxicity induced by co-exposure to low doses of FB1 and OTA and its mechanismThe purpose of this study was to investigate the protective effects of Hsp70 on renal epithelial cells and its underlying mechanism under the co-exposure of low doses of FB1 and OTA.In vitro experiments,p K-15 cells were transfected with eukaryotic plasmid p EGFPC3-Hsp70 for 24 h or pretreated with Hsp70 activator ML346 for 4 h,and then treated with dual mycotoxins for 48 h.The results showed that when the eukaryotic plasmid was transfected or pretreated with ML346,the MMP was restored to a certain extent,the production of mt ROS was significantly inhibited,and the content of Cyto C leaked from mitochondria to the cytoplasm was also reduced.The m RNA and protein levels of proapoptosis genes were also down-regulated.In addition,we also found that the phosphorylation level of JNK protein was inhibited when the Hsp70 level was up-regulated.In vivo experiments,ICR mice were randomly divided into 3 groups(n=12 each),including(0.4 mg/kg bw OTA + 1.0 mg/kg bw FB1)group,p EGFP-C3-Hsp70(20 μg)+(0.4 mg/kg bw OTA + 1.0 mg/kg bw FB1)group and blank control group,for 16 d.The results showed that after intraperitoneal injection of eukaryotic plasmids,the average body weight change and abnormal renal index of mice were alleviated,and the levels of BUN,Cr,MDA in serum and pro-apoptosis genes were lower than mycotoxins exposure group,and the Cyto C in tubules and glomerulus were also reduced,and glomerular swelling was significantly mitigated,suggesting that up-regulation of Hsp70 enhanced the tissues antioxidant capacity and lightened the renal injury in mice.Besides,we also found that the phosphorylation level of JNK protein was also inhibited in kidney tissue.To explore the mechanism of the protective effects of Hsp70 upregulation,p K-15 cells were transfected with p EGFP-C3-Hsp70 for 24 h and then JNK activator of anisomycin for 1 h,and finally,cells were treated with dual mycotoxins for 48 h.The results showed that the protective effects of p EGFP-C3-Hsp70 were significantly weakened after the JNK was activated.Taken together,Hsp70 can alleviate renal injury and cell apoptosis through reducing oxidative stress and inhibiting the JNK/MAPK signaling pathway.Experiment 4.Construction of porcine Hsp70 recombinant Lactococcus lactis expression system and its protective effects on mice model were co-exposed by low doses of FB1 and OTAIn this experiment,the DNA homologous recombination technology was used for constructing the p NZ8149-Hsp70 expression vector,which was introduced into the host strain NZ3900 by electrotransformation.The recombinant strain was identified by PCR and plasmid DNA sequencing,and named NZ3900/p NZ8149-Hsp70.The protein expression of Hsp70 was induced with Nisin at a final concentration of 25 ng/m L.In addition,the recombinant Lactococcus lactis expression system NZ3900/p NZ8149-Hsp70 did not change the original characteristics of NZ3900,including growth curve,acid production capacity,acid accumulator resistance,bacterial hydrophobicity,autoagglutination capacity and coagglutination capacity.Finally,the protective effects of NZ3900/p NZ8149-Hsp70 on mice model were co-exposed by FB1 and OTA was evaluated.The results showed that compared with the mycotoxins-challenged group,NZ3900/p NZ8149-Hsp70 decreased the levels of MDA in serum,and SOD activity was improved,it suggested that NZ3900/p NZ8149-Hsp70 improved the antioxidant capacity of mice.Besides,the levels of BUN and Cr in serum were decreased,and glomerular swelling was eased,it suggested that NZ3900/p NZ8149-Hsp70 lightened renal injury in mice.And the protective effects were more significant than NZ3900.In conclusion,the recombinant L.lactis expression system of NZ3900/p NZ8149-Hsp70 possesses good protective effects on the mycotoxins-exposed mice model.