Molecular Mechanism Study Of Tanshinone Analogues Against Erythroleukemia

Objective:To investigate the in vivo anti-erythroleukemia effectiveness of tanshinone analogue(TB-3)obtained from the chemical library of Guizhou traditional Chinese medicine with anti-erythroleukemia activity,to identify their anti-erythroleukemia molecular targets and to elucidate their mechanisms of action.Methods:MTT assay to detect the inhibitory effect of tanshinone analogue on the growth of erythroleukemia cells(HEL and K562);flow cytometry and cell staining to detect the apoptosis-inducing effect of TB-3 on erythroleukemia cells on erythroleukemia cells;JC-1 staining to detect the effect of TB-3 on the mitochondrial membrane potential of erythroleukemia cells;Western blot to detect the effect of TB-3 on the expression of proliferation and apoptosis-related genes in erythroleukemia cells were examined by Western blot;the molecular targets of the TB-3 against erythroleukemia were studied by cellular thermal shift assay and molecular docking;the in vivo effectiveness of TB-3 in the treatment of erythroleukemia was studied in an animal model of erythroleukemia mice constructed with Friend Murine leukemia viruse(F-Lu MV),and the histopathological effects of the compounds on the spleen of mice were examined by H&E staining.Results:1.Tanshinone analogue TB-3 inhibited the growth of both erythroleukemia cells HEL and K562 in a concentration-and time-dependent manner(p < 0.001),and the growth inhibition of HEL cells was significantly stronger than that of K562;TB-3treated with HEL cells caused chromatin and nuclei agglutination,TB-3 caused chromatin and nucleus condensation,DNA fragmentation,and significantly induced late apoptosis in both cells(p < 0.001);both natural products caused a decrease in mitochondrial membrane potential in HEL cells.2.After F-Lu MV was injected into newborn mice,the results showed that the animal model of erythroleukemia mice was successfully constructed by detecting blood routine,spleen viral load determination and Wright-Giemsa staining of peripheral blood and bone marrow after five weeks;by intraperitoneal injection of TB-3 into erythroleukemic mice on every other day for a total of 7 injections,the results showed that TB-3 could effectively delay the survival of erythroleukemia mice The results showed that TB-3 could effectively delay the survival time,increase the hematocrit and slow down the splenomegaly of erythroleukemia mice,and could effectively treat erythroleukemia in mice in vivo.In F-Lu MV-induced erythroleukemic mice,compared with the normal group,the erythroleukemic mice showed reduced hemoglobin count,significantly lower hemoglobin and erythrocyte pressure volume(p < 0.001),and slightly lower platelets and lymphocytes(p < 0.05).The results of spleen viral load assay showed that F-Lu MV product was detected in the spleen of erythroleukemic mice;using Wright-Gimza staining of peripheral blood and bone marrow,pathological nuclear division and Howell-Jolly were seen in blood smears of mice in the erythroleukemic group,nonerythroid primitive cells(NEC)≥ 0.30 and nucleated erythrocytes ≥ 0.50 in bone marrow smears,and prokaryotic and mononuclear cells in the red lineage,with intermediate.After successful modeling,TB-3 was injected intraperitoneally into mice with erythroleukemia,and the results showed that tanshinone analogues could effectively delay the survival time of mice with erythroleukemia(p < 0.01),increase the hematocrit(p < 0.01),and slow down splenomegaly(p < 0.001),and could effective treatment of murine erythroleukemia.3.TB-3 treatment of both erythroleukemia cells resulted in increased expression of Bax,a key protein in the mitochondria-dependent apoptotic pathway,decreased expression of Bcl-2,and activated expression of caspase-8 and caspase-3 at the transcriptional and protein levels;TB-3 treatment of both erythroleukemia cells downregulated expression of WRN and PARP,key proteins for DNA damage repair;TB-3significantly regulated expression of P38 protein.3 significantly regulated the expression of P38 protein and the phosphorylation levels of ERK1/2 and P38,which indicated that TB-3 could regulate the mitochondria-dependent and DNA damageinduced apoptotic pathway and P38-MAPK signaling pathway in erythroleukemia cells.Analysis of the effect of TB-3 on Fli-1 expression in HEL and K562 cells showed that TB-3 significantly inhibited Fli-1 expression in HEL cells,and the results of cellular thermal shift assays showed that TB-3 was significantly enriched in live HEL cells,while Fli-1 expression was not detected in K562,suggesting that Fli-1 expression may be involved in the regulatory effect of TB-3 on This suggests that Fli-1 expression may be involved in the regulatory role of TB-3 on HEL,resulting in a greater sensitivity of TB-3 to HEL cells compared to K562 cells.The combination of bioinformatics and molecular target prediction showed that Bcl-2 might be the target of TB-3;the results of cellular thermodynamic assay showed that TB-3 could significantly enrich Bcl-2protein in live cells(short treatment time and change of treatment temperature),which confirmed that TB-3 could directly target and bind Bcl-2 protein.Conclusions:1.Both natural products can effectively inhibit the growth of erythroleukemia cells in vitro and treat F-Lu MV-induced erythroleukemia in mice in vivo.2.TB-3 targets Bcl-2 protein and regulates the mitochondria-dependent and DNA damage-induced apoptotic pathway and p38 MAPK signaling pathway in erythroleukemia cells.