Study On The Synergistic Effect Of PRMT1 And PRMT5 In LPS-induced Hepatic Inflammation

Liver is one of the most important organs of human and animals,and it plays an important role in metabolism and detoxification.In poultry production,pathogenic microorganism invasion and toxin secretion infection are the most common causes of liver injury.LPS(lipopolysaccharide)has been proved to be one of the main components of the cell wall of Gram-negative bacteria.The negative bacteria enter the body will be dissolved in the intestinal tract and release a large amount of LPS into the blood or portal vein into the liver.LPS will cause damage to the liver and spleen,damage the immune system,and affect the growth performance and immune function of animals(Protzer et al.,2012,Schnabl and Brenner,2014).Epigenetic modification plays an important role in inflammation and other diseases.Protein arginine methylation dominated by the protein arginine methyltransferase family(protein arginine methyl-transferase,PRMTs)is one of the most important post-translational modifications.As the main enzymes of the PRMTs family,PRMT1 and PRMT5 have been proved to play important roles in the regulation of inflammation.Recent studies have shown that PRMT1 and PRMT5 play a synergistic role in leukemia,small cell lung cancer and other cancers,but the synergistic mechanism of PRMT1 and PRMT5 in inflammatory regulation is still unknown.Does the expression of PRMT1 and PRMT5 change in liver inflammation? Is there a synergistic mechanism between PRMT1 and PRMT5 in liver inflammation? In view of these problems,our team carried out the research work.Experiment 1 Expression of PRMT1 and PRMT5 in LPS liver inflammation model.The LPS mice inflammation model was constructed with 15 mg/kg dose of LPS,and the expression of PRMTs family proteins was detected.The liver HE stained sections of LPS mice showed increased cell volume and inflammatory cell infiltration,the infiltration of inflammatory cells increased significantly,while cytokines were significantly increased and the protein expression of PRMT1 and PRMT5 decreased significantly(P < 0.05).The hepatocyte inflammation model was established by using LPS at the dose of 1000 ng/m L.The results showed that the cytokines increased significantly,and the protein expression of PRMT1 and PRMT5 decreased significantly under the stimulation of LPS(P < 0.01).In this chapter,the in vivo and in vitro models of liver inflammation induced by LPS were successfully constructed.PRMT1 and PRMT5 decreased in isotropy in LPS-induced liver inflammation.Experiment 2 Regulatory mechanisms of PRMT1 and PRMT5 in LPS liver inflammation.When LO2 cells were stimulated with 10 μM NF-κB inhibitor(PDTC)and 1000 ng/m L LPS,the expression of P-p65 protein was significantly decreased in PDTC-LPS group,but PRMT1 and PRMT5 were still decreased with LPS stimulation.In LPS inflammation model,the expression of P-STAT1 protein increased at first and then decreased,while the expression of P-STAT3 protein decreased continuously.In order to explore whether PRMT1 and PRMT5 play a role as methyltransferases,LO2 cells were treated with si-PTMT1,si-PRMT5 and PRMTs inhibitor(AMI-1)respectively.The results showed that there was no significant change in the protein of P-STAT3 after specific knockdown of PRMT1 and PRMT5,but the expression of P-STAT3 protein in AMI-1 group was significantly down-regulated,which was consistent with the change in LPS inflammation model.The protein expression of ADMA and SDMA decreased gradually in LPS inflammation model.This chapter proved that NF-κB,JAK-STAT1 and JAK-STAT3 signal pathways were activated in LPS-induced liver inflammation,and PRMT1 and PRMT5 played a regulatory role as protein arginine methyltransferase in LPS-induced liver inflammation.Experiment 3 Synergistic mechanism of PRMT1 and PRMT5.When LO2 cells were stimulated by Type I PRMTs and PRMT5 inhibitors,the results showed that the cells in the LPS group were significantly smaller,deformed and reduced,and the cells in the combined inhibitors group showed the same change.But there was no significant change in the single inhibitor group,and the results of cytokines showed that it was significantly increased in the LPS group(P < 0.001).There was no change in the single inhibitor group,and the significantly increased was found in the combined inhibitors group(P < 0.001).The protein detection results of ADMA and SDMA showed that ADMA significantly decreased and SDMA compensation increased under PRMTI inhibitor,while SDMA significantly decreased and ADMA compensation increased under PRMT5 inhibitor,only combined inhibitors group could decrease ADMA and SDMA.These results proved that there was a compensatory mechanism between PRMT1 and PRMT5.At the same time,Co-IP(co-immunoprecipitation)technique was used to verify the interaction between endogenous PRMT1 and PRMT5 in LO2 cells.12 candidate common substrates of PRMT1 and PRMT5 were screened from relevant literature and databases based on the amino acid sequences of PRMTs family methylation modified substrate proteins,and the molecular weight range of substrate protein was determined by IP(immunoprecipitation)and silver staining.The candidate substrate expression plasmid was transferred into LO2 cells and stimulated by Type I PRMTs and PRMT5 inhibitors.The Co-IP results showed that there was a compensatory mechanism in the immunoprecipitation products of hnRNPK group,so hnRNPK was the shared substrate of PRMT1 and PRMT5.In order to determine the methylation substrate site,9 arginine point mutation plasmids were constructed according to the RG and RGG sites of hnRNPK,and the Co-IP results showed that there was no compensation mechanism in hnRNPK-232RK(arginine mutation to lysine at position 232)group,so the 232 th arginine of hnRNPK was the shared methylation substrate site of PRMT1 and PRMT5.Experiment 4 Hn RNPK regulates cell proliferation through arginine methylation modification.In this chapter,Flag-pc3.1,Flag-hnRNPK and Flag-hnRNPK-232 RK plasmids were transfected into LO2 cells.48 hours later,cell samples were collected to detect cell viability and the expression of cell proliferation and apoptosis markers.The results showed that the m RNA expression of p53 was significantly decreased in point mutation group and the m RNA expression of c-Myc in hnRNPK group was significantly decreased(P < 0.05).The viability of CCK8 cells was consistent with that of RT-q PCR.Hn RNPK methylation could promote cell apoptosis.In conclusion,this study found the synergistic mechanism of PRMT1 and PRMT5 in LPS-induced liver inflammation.In LPS-induced liver inflammation,PRMT1 and PRMT5 were synergistically down-regulated,activating NF-κB,JAK-STAT1 and JAK-STAT3 and other inflammatory signaling pathways.Meanwhile,the decrease of PRMT1 and PRMT5 also weakens the methylation of the 232 nd arginine of hnRNPK,which is the shared substrate protein of PRMT1 and PRMT5.Hn RNPK methylation deficiency inhibited the transcriptional activity of p53 and enhanced the transcriptional activity of c-Myc,thus promoting the proliferation of hepatocytes.These results enriched the synergistic action between different types of PRMTs and provided a theoretical basis for the treatment of common bacterial infectious diseases and the prevention and prevention of animal liver injury in the process of livestock production.