The Role And Mechanism Of AMPK Signal Pathway And Autophagy In 3-chloro-1,2-propanediol-induced Renal Cell Apoptosis | Posted on:2019-06-27 | Degree:Master | Type:Thesis | Country:China | Candidate:C N Jin | Full Text:PDF | GTID:2531305687973659 | Subject:Engineering | Abstract/Summary: | PDF Full Text Request | 3-chloro-1,2-propanediol(3-MCPD)is a food contaminant mainly produced during heat processing.3-MCPD has been reported to have toxicity with the kidney and the male reproductive system as the main target organs.Based on these studies,3-MCPD was classified as group 2B,“possibly carcinogenic to humans” by IARC.Our previous studies have found that mitochondrial-dependent apoptosis pathway is one of the major mechanisms of 3-MCPD-induced human embryonic kidney cell injury.3-MCPD exposure caused a decline of mitochondrial membrane potential,mitochondrial respiratory chain disorders,especially ATP synthase(ATP5,ATP6,ATP8)down-regulated expression and a decrease of intracellular ATP levels,suggesting that 3-MCPD may induce renal cell apoptosis may be associated with energy regulation.The AMP-activated protein kinase(AMPK)is an energy regulator that can participate in the process of apoptosis.And there are complex interactions between autophagy and apoptosis.Based on the previous sthdy,our study explored the role and mechanism of AMPK signal pathway and autophagy in apoptosis induced by 3-MCPD.This study can provide a stong scientific basis for explaining the molecular mechanisms of human cells to 3-MCPD stress response and corresponding food safety issues.The main research contents and results are as follows:1.3-MCPD induces renal cell apoptosis via AMPK signal pathwayHEK293 cells were treated with different concentrations of 3-MCPD(0,2.5,5,10,and20 m M)for 24 h.The expression of AMPK,liver kinase B1(LKB1)and Caspase 3 were detected by western blotting.The results showed that 3-MCPD treatment increased the ratio of p-AMPKα/AMPKα,and the protein expression of p-LKB1 and cleaved caspase 3,suggesting that 3-MCPD could activate the expression of p-LKB1,up-regulate the expression of AMPK and induce cell apoptosis.HEK293 cells were pretreated with5-aminoimidazole-4-carboxamide ribonucleotide(AICAR,AMPK activator)for 3 h,followed by treatment with 3-MCPD for 24 h.The results showed that AMPK activator AICAR co-treatment activated AMPK expression promoted 3-MCPD-evoked cell death,LDH leakage and apoptosis rate,mitochondrial dysfunction,up-regulation the expression of caspase 3 and increase the enzyme activities of caspase.Dorsomorphin(DOR,AMPK inhibitor)co-treatment could inhibit AMPK expression and alleviate 3-MCPD-induced renal cell apoptosis.The results showed that AMPK was involved in the apoptosis of renal cells induced by 3-MCPD,the activation of AMPK may promote apoptosis while the inhibition of AMPK expression can inhibit apoptosis.2.3-MCPD induces autophagy in HEK293 cellsReal-time PCR was used to detect the expression of autophagy-related marker proteins,LC3 and Beclin1.The results showed that within the concentration range of 0-10 m M3-MCPD,the m RNA expression of LC3-II/LC3-I and Beclin1 were significantly increased.At the same time,western blotting results indicated that LC3-II/LC3-I protein expression showed an upward trend,indicating that 3-MCPD could induce autophagy.Western blotting detected the protein expression of mammalian target of rapamycin(mTOR)and p70 ribosomal protein S6 kinase(p70S6K)and found that 3-MCPD could activate AMPK,down-regulate the phosphorylation expression of mTOR and its downstream molecule p70S6 K and induce autophagy.The detected results also showed that AMPK activator AICAR co-treatment could significantly reduce the up-regulated expression of LC3-II protein induced by 3-MCPD,while AMPK inhibitor DOR co-treatment could significantly enhance 3-MCPD-induced the up-regulated expression of LC3-II protein,indicating that AMPK was involved in the process of 3-MCPD-induced autophagy.3.Autophagy plays a protective role in the 3-MCPD-induced renal cell apoptosisHEK293 cells were pretreated with 3-methyladenine(3-MA,autophagy inhibitor)or rapamycin(RAP,autophagy activator)for 3 h,followed by treatment with 3-MCPD for 24 h.The results showed that autophagy inhibitor 3-MA co-treatment could inhibit3-MCPD-evoked autophagy,which promoted decrease in renal cell viability,increase in the apoptosis rate,mitochondrial dysfunction,up-regulation caspase 3 protein expression and increase in caspase enzyme activities induced by 3-MCPD,suggesting that the autophagy inhibitor 3-MA had the effect of enhancing 3-MCPD-evoked renal toxicity.However,the autophagy activator RAP co-treatment enhanced 3-MCPD-induced autophagy and alleviated mitochondrial dysfunction,down-regulated caspase 3 protein expression and decreased the enzymatic activities of caspase,suggesting that RAP had a protective effect on3-MCPD-induced renal cell apoptosis.In summary,cell autophagy acted as a stress-protective agent under the stress of 3-MCPD.Inhibiting autophagy could enhance apoptosis while promoting autophagy may inhibit apoptosis.Autophagy may increase the survival time of renal cells to some extent. | Keywords/Search Tags: | 3-chloro-1,2-propanediol (3-MCPD), human embryonic kidney 293 cells(HEK293 cells), AMP-activated protein kinase(AMPK), apoptosis, autophagy | PDF Full Text Request | Related items |
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