The Regulation Mechanism Of CHOP On Liver Damage Induced By Endoplasmic Reticulum Stress And The Intervention Effects Of Earthworm Active Components

Objective:To explore the upstream and downstream genes and related pathways of C/EBP homologous protein(CHOP)in endoplasmic reticulum stress(ERS)and the intervention effects of earthworm active components(EWPs)on it.Methods:(1)Protective effect of EWPs against ERS induced L-02 hepatocyte apoptosisThe liver damage models were induced by tunicamycin(TM)in L-02 cells,the L-02 cell apoptosis in different groups was detected by flow cytometry,the protein and mRNA expressions of ERS related signal molecules,such as GRP78,PERK,e IF2α,ATF4,CHOP,Bax and Bcl-2 were detected by qPCR and Western blot.(2)Constructed the shRNA eukaryotic expression vectors targeting the gene of CHOPThe 3 specificity shRNA eukaryotic expression vector were designed and constructed targeted CHOP gene mRNA,named shRNA-1,sh RNA-2 and shRNA-3,and one non homologous negative control expression vector named sh RNA-NC.These shRNA recombinant vectors were identified by PCR and DNA sequencing analysis.Then the CHOP expression was determined by Western blot and the plasmid with the best inhibitory effect on CHOP expression was screened.(2)RNA-seq high-throughput sequencingThe cell samples of different groups were extracted total RNA with Trizol reagents.The quality of total RNA was detected by Agilent 2100 systerm.The mRNA library preparations were sequenced on BGIseq500 platform.The results of RNA-seq were analysis by evaluating,gene expression annotation,analysis of differentially expressed genes(DEGs)and enrichment of the GO/KEGG function analysis to find the upstream and downstream genes and related pathways of CHOP and the intervention effects of earthworm active components(EWPs)on it.(3)In view of the sequencing results RNA-seq,the related pathways or targets were validated by q PCR.Results:(1)TM could obviously reduce the survival rate of L-02 cells in a time-dependent and dose-dependent manner.Compared with normal group,the apoptosis rate in model group was significantly increased(P<0.05 or P<0.01).The protein and mRNA expressions of ERS related signal molecules,such as GRP78,PERK,e IF2α,ATF4,CHOP and Bax,was significantly up-regulated(P<0.05 or P<0.01),while Bcl-2 was significantly down-regulated(P<0.05 or P<0.01).After the administration of EWPs of different concentrations,compared with model group,EWPs could significantly increase L-02 hepatocyte survival rates and decrease cell apoptosis rates.It could also reduce the protein and mRNA expressions of ERS related signal molecules doesdependently(P<0.05 or P<0.01),such as GRP78,PERK,e IF2α,ATF4,CHOP and Bax,and increased the protein and mRNA expressions of Bcl-2(P<0.05 or P<0.01).(2)Four shRNA expression vectors were constructed successfully according to the results of PCR and DNA sequencing analysis.After treatment with 0.075 g/L tunicamycin for 24 h,the protein expression levels of CHOP decreased significantly in L-02 hepatocytes transfected with shRNA-1,shRNA-2 and shRNA-3 compared with control group(P<0.05).The interfering effect of shRNA-2 on CHOP protein expression was the best.(3)A total of 8 samples were detected by RNA-seq technique,resulting in an average of 24137262 original reads.After removing low quality reads,the remaining average number of clean reads was 23932318.The gene expression level changed significantly when CHOP was knockdown.The main biological processes involved in the differential of genes were cell apoptosis,endoplasmic reticulum stress,protein phosphorylation regulation,cell proliferation,protein misfolding reaction,and so on.Molecular function was mainly enriched in protein binding,nucleic acid binding transcription factor activity,protein kinase activity,transaminase activity,etc.Through the enrichment analysis of KEGG metabolic pathway,we found that the differential gene was mainly enriched in the pathways of endoplasmic reticulum protein processing,p53 signaling pathway,oxidative phosphorylation,axon orientation,Fox O signaling pathway and amino acid biosynthesis.Among them,CLGN,LCN2,LAMP3 and FAM129 A increased with the up-regulated of CHOP,when CHOP was knockdown,they were down-regulated,while IFI27 and TARP were contrary to them.These phenomena indicated that these factors had close relationship with CHOP.(3)As the results of qPCR and Western blot shown: CLGN,LCN2,LAMP3 and FAM129 A were significantly up-regulated in model group compared with normal control(P<0.05),and they were down-regulated after we knocked down CHOP.IFI27 and TARP shown the exact opposite situation,they were significantly down-regulated in model group compared with normal controls(P<0.05),and they were up-regulated after we knocked down DDIT3(P<0.05).Compared with model group,CLGN,LCN2,LAMP3 and FAM129 A were significantly down-regulated after treatment with EWPs,while IFI27 and TARP were contrary to them(P<0.05).Conclusion:EWPs had a significantly protective effect on hepatocyte apoptosis induced by ERS in L-02 cells.Its mechanism may be related to down-regulation the mRNA and proteins expressions of GRP78,PERK,ATF4,e IF2α,CHOP and Bax,and upregulation Bcl-2.The upstream and downstream genes and related pathways of CHOP in ERS were selected by RNA-seq,these results will contribute to the further study of CHOP targets and mechanism as well as the mechanism of EWPs.