Study On The Apparent Mechanism Of Salvianolic Acid B Combined With Stem Cells In The Treatment Of Acute Liver Failure

ObjectiveAcute liver failure(ALF)is the main cause of death in patients with liver disease.At present,the mortality rate of patients with liver disease is as high as 90% at home and abroad.At present,the first choice of western medicine in the treatment of acute liver failure is orthotopic liver transplantation,but a variety of immune reactions after liver transplantation and the difficulty and high price of liver transplantation donor source seriously hinder its wide application.At present,the search for new methods for the treatment of acute liver failure is a key clinical problem.Bone mesenchymal stem cells(BMSCs)transplantation has become a new research hotspot in the treatment of acute liver failure in recent years because of its advantages of small trauma,strong proliferation and differentiation ability,as well as suitability for autologous transplantation.However,the low survival rate of BMSCs after transplantation and the insufficient proliferation activity seriously limit its application,therefore,how to improve the survival rate of BMSCs after transplantation has become the focus of BMSCs transplantation in the treatment of ALF.Salvia miltiorrhiza has certain antioxidant and anti-cell death effects,and its main component,salvianolic acid B,can significantly inhibit hepatic and renal mitochondrial peroxidation and reduce cell injury and apoptosis.In recent years,our group has accumulated a wealth of work basis by exploring the effects of small molecules of traditional Chinese medicine on various types of stem cells.Previous studies have found that salvianolic acid B has antioxidant and stem cell differentiation promoting effects.Therefore,in the present study,we investigated the apparent mechanism of salvianolic acid B combined with stem cells in the treatment of acute liver failure on the basis of previous work.Methods1.To study the anti-ferroptosis effect of salvianolic acid B on bone mesenchymal stem cells by constructing an Erastin-induced ferroptosis cell model.(1)An ferroptosis model of BMSCs cells was constructed,and the model was successfully constructed as verified by flow cytometry,iron ion detection,reactive oxygen species ROS detection and lipid peroxide detection.(2)Salvianolic acid B was added to BMSCs cells,and salvianolic acid B antiferroptosis of bone mesenchymal stem cells was verified from the cellular level by flow cytometry,iron ion detection,reactive oxygen species ROS detection,lipid peroxide detection and Western blot.2.To find target genes and pathways by microarray and bioinformatics analysis.(1)To analyze the differential expression of salvianolic acid B on BMSCs by miRNA microarray technology and find the differentially expressed genes of interest;(2)To predict the target genes of this gene using bioinformatics methods;(3)To analyze its related functions and pathways using biological GO enrichment analysis and KEGG pathway analysis and analyze their involvement in ferroptosis.3.Experimentally verify target gene expression and its targeting and correlation with ferroptosis.(1)using the BMSCs cell Erastin induction model,the expression differences of Mir-582-3p after salvianolic acid B treatment were verified by RT-qPCR;(2)BMSCs ferroptosis and NSUN5 target protein expression after regulating Mir-582-3p were verified by iron ion detection,reactive oxygen species ROS detection,lipid peroxide detection and Western blot;and(3)Nmir-582-3p targeting NSUN5 was verified by dual luciferase reporter gene.4.Further mechanistic studies:(1)using the BMSCs cell Erastin induction model,the differentially expressed mRNAs and proteins of ferroptosis in BMSCs were verified by RTqPCR and western blotting screening;(2)the expression signal and location of the target protein were detected by immunofluorescence;(3)the overexpression or knockdown of NSUN5 in BMSCs cells was confirmed by lentiviral vector overexpression or knockdown of NSUN5 by western blotting;(4)after overexpression or knockdown of NSUN5,the effect of NSUN5 on BMSCs cell ferroptosis was observed by iron ion detection,reactive oxygen species detection,peroxidase detection and Western blot;(5)in order to study the deep mechanism of NSUN5 inhibiting ferroptosis,the binding of NSUN5 to FTH1 and FTL in BMSCs was analyzed by RIP and RT-qPCR;(6)by The expression of FTH1 and FTL in BMSCs after regulation of NSUN5 was detected by blotting;(7)The co-localization of NSUN5,FTH1 and FTL in the cytoplasm of BMSCs was detected by immunofluorescence assay:(8)To determine whether FTH1 and FTL mediated the inhibitory effect of NSUN5 on ferroptosis in BMSCs,the ferroptosis phenotype after overexpression of FTH1 and FTL was detected by iron ion detection,reactive oxygen species ROS detection,and lipoperoxide;(9)The 5m C level after NSUN5 gene knockdown or overexpression was detected by dot blot hybridization assay;the 5m C expression level was analyzed by immunofluorescence;(10)To analyze the functional significance of NSUN5-mediated 5m C modification of FTH1 and FTL mRNA,the enrichment of FTH1 and FTL mRNA by anti-5m C antibody and the change of their levels after regulation of NSUN5 was analyzed by RIP and RT-qPCR assays;(11)The methylation status of FTH1 and FTL genes in BMSCs was assessed by bisulfite sequencing using FTH1 and FTL-specific primers;C-to-T transformation of methylation sites was determined;(13)to determine whether other proteins were involved in the process of FTH1 and FTL modification by NSUN5,theRNA-binding protein TRAP1 was found by mass spectrometry;(14)anti-NSUN5 and TRAP1 enrichment was analyzed by RIP;(15)the interaction between real TRAP1 and FTH1 or FTL was confirmed by co-IP and western blotting;(16)co-localization of NSUN5 and TRAP1 protein was confirmed by immunofluorescence analysis;(17)the expression of FTH1/FTL was observed by knocking down NSUN5;and(18)TRAP1 mRNA and protein levels were observed by silencing NSUN5.5.Salvianolic acid B combined with BMSCs was used to treat ALF in rats through animal experiments.(1)To study and construct a rat model of D-gal/LPS acute liver failure,serological tests such as liver function were used to observe the liver function of rats such as TP,TBA,IRON2 and AMON;(2)The histopathological conditions of rats were detected by HE staining;(3)The expression of NSUN5 and FTH1/FTL in rat tissues was detected by immunohistochemistry.Results1.After the success of construction of the ferroptosis Erastin-induced model,salvianolic acid B was added,and salvianolic acid B was verified from the cellular level to resist ferroptosis in BMSCs cells.2.miRNA microarray analysis revealed that salvianolic acid B acted on mir-582-3p,a differentially expressed gene of BMSCs,and bioinformatics methods were used to predict the target gene of this gene,it revealed that mir-582-3p targeted NSUN5,and GO and KEGG pathway analysis revealed that mir-582-3p targeted NSUN5 might be associated with ferroptosis.3.RT-qPCR was used to verify that the expression of Mir-582-3p was up-regulated after salvianolic acid B treatment,Mir-582-3p regulated NSUN5 target protein expression and affected ferroptosis occurrence,and dual-luciferase reporter gene demonstrated that mir-582-3p targeted NSUN5.4.Further mechanistic studies revealed that(1)RT-qPCR and western blotting screening validation revealed that NSUN5 mRNAs and protein expression were downregulated in BMSCs;(2)Immunofluorescence assay revealed that the target protein NSUN5 fluorescence signal was weaker than that of control cells;(3)By lentiviral vector overexpression or knockdown of NSUN5,western blotting confirmed the overexpression or knockdown of NSUN5 in BMSCs cells;(4)After overexpression or knockdown of NSUN5,iron ion detection and Western blot revealed that NSUN5 inhibited ferroptosis in BMSCs;(5)To study the deep mechanism of NSUN5 inhibiting ferroptosis,RIP and RT-qPCR analysis revealed that NSUN5 neutralized the binding of FTH1 and FTL in BMSCs;(6)Blotting revealed that NSUN5 overexpression increased the expression levels of FTH1 and FTL;(7)Immunofluorescence assay revealed that NSUN5,FTH1 and FTL colocalized in the cytoplasm of BMSCs:(8)In order to determine whether FTH1 and FTL mediated the inhibitory effect of NSUN5 on ferroptosis in BMSCs,experiments such as iron ions revealed that the increase in total iron concentration was reduced,ROS levels were decreased,and peroxidation accumulation was decreased after overexpression of FTH1 and FTL compared with the model group;(9)Dot blot hybridization assay revealed that NSUN5 gene knockdown could reduce 5m C levels,while NSUN5 overexpression was the opposite;immunofluorescence revealed that 5m C expression levels were the same as dot blot hybridization results;(10)To analyze the functional significance of NSUN5-mediated 5m C modification of FTH1 and FTL mRNA,RIP and RT-qPCR assays revealed that anti-5m C antibodies enriched FTH1 and FTL mRNA,and NSUN5 silencing decreased 5m C levels in these two transcripts;The '(FTH1)and 3'(FTL)untranslated regions(UTRs)were methylated;(12)NSUN5 down-regulation decreased the C-to-T transformation of FTH1 and FTL 5m C methylation sites by sequence analysis of sequencing results;(13)To determine whether other proteins were involved in the process of FTH1 and FTL modification by NSUN5,mass spectrometry was used to find theRNA-binding protein TRAP1;(14)AntiNSUN5 antibody was found to enrich TRAP1 by RIP;(15)The interaction between solid TRAP1 and FTH1 or FTL was confirmed by co-IP and western blotting;(16)Immunofluorescence analysis showed that NSUN5 and TRAP1 proteins were co-localized in the cytoplasm of BMSCs;(17)Silencing the expression of NSUN5,which reduced the precipitation of FTH1/FTL in the TRAP1 complex;(18)NSUN5 knockdown had no effect on the protein or mRNA levels of TRAP1.5.After salvianolic acid B combined with BMSCs treatment of ALF in rats,the general condition of rats was improved,death was reduced,liver appearance and blood stasis were significantly reduced,liver function ALT,AST,TBIL and plasma endotoxin ET and TP,TBA,IRON2 and AMON results were significantly improved compared with the model group,HE staining detected tissue disorders in rats were also milder than the model group,which indicate that salvianolic acid B combined with bone mesenchymal stem cell transplantation can treat acute liver failure.The decreased expression of NSUN5 and FTH1/FTL in rat tissues detected by immunohistochemistry suggested that the mechanism might be consistent with the cellular experimental mechanism and associated with the NSUN5-FTH1/FTL pathway.ConclusionSalvianolic acid B targeted NSUN5 against BMSCs ferroptosis,one of the mechanisms of which was targeted inhibition of NSUN5 expression by mir-582-3p and anti-BMSCs ferroptosis,and further mechanistic studies revealed that NSUN5 inhibited BMSCs ferroptosis by interacting with TRAP1 and 5m C modification of FTH1 and FTL mRNAs.Experimental animal studies have found that salvianolic acid B combined with BMSCs could treat ALF in rats,and the treatment in the combined group was more effective than that in the single group.The main results were as follows:1.The ferroptosis model of bone mesenchymal stem cells induced by erastin was successfully constructed,and salvianolic acid B had the potential to resist ferroptosis of bone mesenchymal stem cells.2.Differential gene mir-582-3p of BMSCs by salvianolic acid B of interest was found by microarray analysis,and further bioinformatics analysis revealed that mir-582-3p targeted NSUN5.3.The upstream mechanism of salvianolic acid B acting on BMSC anti-ferroptosis was that mir-582-3p targeted NSUN5,and Mir-582-3p inhibited NSUN5 by targeting the NSUN53 'UTR region.The deep mechanism of anti-BMSC ferroptosis was completed through the NSUN5-FTHH1/FTL pathway and recruitment of theRNA-binding protein TRAP1.4.Salvianolic acid B combined with BMSCs cell transplantation could treat acute liver failure in SD rats and improve the efficacy compared with single treatment.The mechanism might be that it exerted the anti-ferroptosis effect by NSUN5-FTH1/FTL.