| Objective:1.Screening by network pharmacology of gene targets and signaling pathways related to the immunomodulatory effect of curcumin active ingredients of turmeric on acute promyelomyeloid leukemia.2.To explore the effect and efficacy of the combined administration of three curcumin active ingredients of Mongolian turmeric on mice with acute promyetic leukemia model.3.To study the effects of the main active ingredient of Mongolian turmeric to inhibit tumors through immunomodulation and treat human acute promyelomyeloid leukemia(APL).Method:1.Use Pub Chem,Swisstargetprediction,Pharm Mapper,SEA,Uniport and other databases to screen the active ingredient targets of three curcuminoids.APL disease targets were obtained through the above databases of GEO,Gene Card,and OMIM.The Venny database was used to obtain three intersection genes of curcumin and APL.The found intersection targets were introduced into Cytoscape3.7.2 to construct an active ingredient-target-signaling pathway interaction network,and then the protein-protein interaction(PPI)analysis was constructed by using the STRING database,and the found intersection targets were introduced into the active ingredient-target-signaling pathway interaction network.Finally,the common targets of three curcumins and acute promyelocytic leukemia(APL)were used to perform GO(gene ontology)functional analysis and KEGG(Kyoto encyclopedia of genes and genomes)signaling pathway enrichment analysis using the Metascape platform.2.2.72 male NOD/SCID mice were divided into normal control group,model group,drug administration group,and positive control group according to the random number table method,and each group was 8.In addition to the blank group,each group of mice was injected with 5×10~6/0.2m L of human promyelogenous leukemia HL-60 cells in the tail vein,and on the day of inoculation,the cultured cells were counted and the cell concentration was adjusted.Each group of mice were inoculated with 0.2ml cell suspension,after 4 weeks of inoculation,orbital blood collection,blood analyzer detection,the number of white blood cells,lymphocytes and platelets,neutrophil count in the peripheral blood of the model mice,and the change of serum CD33 positive expression rate of each group of mice was detected by flow cytometry,whether the modeling was successful,and the tumor growth was observed,and each tail vein in the normal control group was injected with the same volume of 0.2ml sodium carboxymethylcellulose(CMC-Na).From the 2nd day after successful modeling,normal group:CMC-Na,administration group:curcumin(Cur),demethoxycurcumin(DMC),bisdemethoxycurcumin(BDMC),CUR:DMC:BDMC=1:1:1(combined group),CUR:DMC:BDMC=12:4:1(combined group),turmeric water extract,gavage at 100 mg/kg,positive control group:cyclophosphamide 0.1～0.3ml/10g,each gavage for 14d,model group CMC-Na.The activity of each group of mice was observed daily.The mice were given the finished drug to remove the eyeballs of the mice,and the blood,spleen and liver tissue samples of the mice were collected for examination;(HE)staining to observe tumor morphological changes in the spleen and liver;The expression of TNF-a(tumor necrosis factor-α)and MMP2(matrix metalloproteinase 2(MMP2))in the serum of mice in each group was detected by enzyme-linked immunosorbent assay(ELISA),and the antitumor effect of the main active components of Mongolian turmeric on acute promyelocytic leukemia was analyzed by judging the change trend of these cytokines in each group.3.Western Blot(WB)was used to detect the expression levels of MAPK8(JNK)and STAT3 proteins in the liver and spleen of mouse tumor tissues;The expression levels of MAPK8(JNK)and STAT3 in the liver and spleen of mouse tumor tissues were measured by real-time PCR(RT-PCR).Results:1.Through network pharmacological analysis,31 three curcumin-APL intersection targets were obtained,and the results of GO enrichment analysis showed972 signaling pathways,including inflammation and immune-related factors;The results of KEGG pathway enrichment analysis showed 59 signaling pathways,among which the most significant signaling pathways were:AGE-RAGE,JAK-STAT and MAPK signaling pathway as research objects,and STAT3,MAPK8(JNK),TNF-a and MMP2 were screened as the main research targets.The molecular docking results showed that the three curcumins and MAPK8(JNK)core target proteins could bind spontaneously,and the target proteins had good correlation and could form stable complexes with proteins,and their mechanism of action may exert anti-tumor effects by inhibiting the MAPK signaling pathway and the JAK-STAT signaling pathway.2.HE staining results:Model group:severe abnormality of tissue structure,a large number of dense and fine vacuoles can be seen in the tissue,necrosis in some areas can be seen,the nucleus structure disappears,there are a large number of red blood cells in the intercellular space,and a large number of inflammatory cells can be seen infiltrated in the intercellular space.Drug administration group:mild abnormality of tissue structure,a small number of dense and fine vacuoles can be seen in the tissue,no obvious hepatocyte hyperstaining,degeneration,a small amount of red blood cell infiltration in the intercellular space,a small amount of inflammatory cell infiltration in the intercellular space,the effect of the 12:4:1 group and the 1:1:1 group is more obvious.Some of the same ELISA statistical analysis results were obtained in the determination of MMP2 and TNF-αlevels in each group by ELISA detection,i.e.,the levels of MMP2 and TNF-αin the APL model group were significantly increased compared to the blank group,demonstrating the presence of an inflammatory state.Compared with the model group,the expression of MMP2 and TNF-αin each drug group was downregulated to different degrees(P<0.01,P<0.05),among which the regulatory effect was the most obvious in the combined drug group(12:4:1)and the combined drug group(1:1:1),and there was no significant difference in the levels of MMP2 and TNF-αbetween the drug group and the cyclophosphamide positive group(P>0.05).Enhance cellular immune function and exert anti-tumor effect.3.The Western-Blotting method detected the effect of each drug group on the protein expression of the relevant proteins MAPK8(JNK)and STAT3 in mouse liver.Compared with the blank group,the protein expression of MAPK8(JNK)and STAT3in the model group was modulated to varying degrees(P<0.01,P<0.05).Compared with the model group,the protein expression of MAPK8(JNK)and STAT3 in the CUR,DMC,BDMC group,combined drug group(1:1:1)and combined drug group(12:4:1)were downregulated to varying degrees(P<0.01,P<0.05),among which the regulatory effect was the most obvious in the combined drug group(12:4:1)and the combined drug group(1:1:1).The Western-Blotting method examines the effect of each drug group on the protein expression of MAPK8(JNK)and STAT3,the relevant proteins of mouse spleen.Compared with the blank group,the protein expression of MAPK8(JNK)and STAT3 in the model group was modulated to varying degrees(P<0.01,P<0.05).Compared with the model group,the protein expression of MAPK8(JNK)and STAT3 in the CUR,DMC,BDMC group,combined drug group(1:1:1),combined drug group(12:4:1)and turmeric water extract group were downregulated to different degrees(P<0.01,P<0.05),among which the regulatory effect was the most obvious in the combined drug group(12:4:1)and the combined drug group(1:1:1).The results of RT-PCR experiments showed that the expression changes of MAPK8(JNK)and STAT3 mRNA in the liver of mice stimulated by each experimental group were explored,and the expression of MAPK8(JNK)and STAT3mRNA in the model group was adjusted to different degrees(P<0.01,P<0.05)compared with the blank group.Compared with the model group,the mRNA expression of MAPK8(JNK)and STAT3 in the CUR group,DMC group,BDMC group,combined drug group(1:1:1),combined drug group(12:4:1)and turmeric water extract group were downregulated to varying degrees(P<0.01,P<0.05),among which the regulatory effect was the most obvious in the combined drug group(12:4:1)and the combined drug group(1:1:1).The results of RT-PCR experiments showed that the expression changes of MAPK8(JNK)and STAT3 mRNA in the spleen of mice stimulated by each experimental group were explored,and the expression of MAPK8(JNK)and STAT3 mRNA in the model group was regulated to different degrees(P<0.01,P<0.05)compared with the blank group.Compared with the model group,the mRNA expression of MAPK8(JNK)and STAT3 in the CUR group,DMC group,BDMC group,combined drug group(1:1:1),combined drug group(12:4:1)and turmeric water extract group were downregulated to varying degrees(P<0.01,P<0.05),among which the regulatory effect was the most obvious in the combined drug group(12:4:1)and the combined drug group(1:1:1),thereby improving the anti-tumor immunomodulatory effect.Conclusion:1.Three kinds of curcumin can inhibit the growth of tumors in APL mice to varying degrees,and when the three curcumins are combined,the anti-tumor effect on APL mice is further improved.2.Curcumin can significantly inhibit the cell proliferation of tumor tissues of APL mice,significantly reduce the expression of MAPK8(JNK)and STAT3(P<0.05),and promote apoptosis of tumor cells.3.It was confirmed that the three curcumins had inhibitory effects on MAPK8(JNK)and STAT3 in the MAPK signaling pathway and JAK-STAT pathway,and proved that the anti-tumor effect of the three combined curcumins was better than that of a single drug APL anti-tumor. |
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