Difference Of Nephrotoxicity And Mechanism Between Cinnabar And Mercury Chloride Under Serum Deprivation

Objective:To study the difference of nephrotoxicity and its mechanism between cinnabar and mercury chloride under serum deprivation.Methods:Basal medium?serum deprivation?was given to induce the cells in a state of stress.10%FBS group was used as the negative control group.HK-2 cells were divided into control group,HgCl₂ group,cinnabar group and 10%FBS group.The concentrations of HgCl₂ and cinnabar were 4 nM in all experiments.The control group was given equal volume of basic medium,and the 10%FBS group was given equal volume of complete medium containing 10%fetal bovine serum.After 4 days treatment,cell viability was measured by MTT assay and the release of cytosolic LDH was detected by an LDH assay kit.The flow cytometry was used to estimate cell apoptosis and intracellular ROS contents.Then,the intracellular content of GSH was detected using the GSH/GSSG ratio detection assay kit.The mRNA expressions levels of mitochondrial apoptosis pathway,death receptor apoptosis pathway and ER apoptosis pathway relative genes were detected by real-time fluorescence quantitative PCR?RT-PCR?.Furthermore,the expressions of CHOP and PERK were detected by Western blot.2.Under the condition of serum deprivation,HK-2 cells were stimulated by 100?M hydrogen peroxide?H₂O₂?with drugs of the above groups simultaneously.After 4 days incubation,flow cytometry was used to detect the apoptosis rate and the contents of intracellular ROS.Real time fluorescent quantitative PCR?RT-PCR?was used to detect the mRNA expression level of CHOP and PERK.Western blot was used to detect the expression of CHOP and the phosphorylation of PERK level.3.RNA-Seq-Technology was employed to detect the transcriptome of each treatment groups,and omicshare analysis platform was used to cluster the inflammation related genes.RT-PCR was adopted to validate the mRNA level of the inflammation related genes IL-1?,IL-6,MMP1,MMP3 and MMP10.Under the condition of no serum,HK-2 cells were given LPS+control,LPS+HgCl2,LPS+cinnabar at the same time,the concentration of LPS was 100 ng/ml.Four days later,the cells were collected and the total RNA was extracted.Then,the mRNA levels of IL-1?and IL-6 were measured by RT-PCR.Results:The results indicatived that compared with the normal growing cells?10%FBS?,serum deprivation causes stress to cells such as the decrease of cell viability and the intercellular GSH and the increase of apoptotic rate and intercellular ROS contents.After treatment with HgCl₂ and cinnabar,the cell viability was decreased significantly and the depletion of GSH was ascended than serum-free control group.Compared with the HgCl₂ group,the cell viability was remarkably increased;LDH release rate,apoptosis rate,intracellular ROS content,and intracellular GSH depletion were conspicuously reduced in cinnabar group;in addition,cinnabar can dramatically inhibit the mRNA expression of apoptosis-endoplasmic reticulum pathway related genes CHOP,GRP78,PERK,ATF6,ATF4,and ERN1;at the same time,cinnabar can reduce the protein expression levels of CHOP and PERK.Under the dual stimulation of serum deprivation and H₂O₂,cinnabar can still observably reduce the cell apoptotic rate and ROS content which is still mediated by inhibit the expression of CHOP and PERK.In the results of RNA-Seq,we found that a large number of inflammation-related genes were differentially expressed between cinnabar and HgCl₂ group,and cinnabar prominently down-regulated the mRNA level of the pro-inflammatory factors IL-1?,IL-6 and metal matrix peptidase?MMPs?;at the same time,Under the dual stimulation of serum deprivation and LPS,cinnabar can still reduce Il-?and IL-6transcription levels.Conclusion:The results of this study showed that cinnabar and HgCl₂ have great differences in the toxicity to HK-2 cells under stress.The cytotoxicity of cinnabar is significantly lower than that of HgCl₂.The mechanism may be related to cinnabar improving the stress of endoplasmic reticulum,inhibiting the expression of CHOP,PERK and inflammation related genes.