| Objective:Usenamine(C18H17NO6)is a patented compound separated and synthesized by our research group.The genes and signal pathways affecting Usenamine’s anti-liver cancer effect were analyzed by mRNAseq sequencing,and the sites of action of key genes were further analyzed by molecular docking technology,and the anti-tumor mechanism of the compound was explored from the protein sites of gene expression and drug action.Through acute toxicity test,genetic toxicity test and 30-day feeding test,we understand the toxic effect intensity and toxic properties of Usenamine,and preliminarily evaluate the safety of the compound,which will provide the basis for pre-clinical research and application development of the compound.Method:C18H17NO6 was synthesized by chemical reaction,and then purified by silica gel column chromatography,and its purity was determined by HPLC.The mRNA sequencing was used to analyze the effect of Usenamine on gene expression and signal pathways of liver cancer HepG2 cells.The molecular docking technology was used to analyze the sites of action of Usenamine with PRKACA protein and PRKCB protein.Flow cytometry was used to detect the effect of Usenamine on the cell cycle of HepG2.Acute toxicity test,genetic toxicity test(in vivo and in vitro chromosome aberration test,bone marrow micronucleus test and sperm abnormality test),30-day repeated exposure test were used to preliminary explore its toxicity.Result:1.D-lichenic acid chloroform solution and hexamethyldisilazane can be reacted to synthesize Usenamine.HPLC experiment results show that the purity of the prepared Usenamine reaches 97%.2.HepG2 cells were treated with 0.45μmol/L,0.9μmol/L,1.8μmol/L usronolide, and differentially up-regulated coding genes were 297,132,and 136,and down-regulated genes were 236,135,and 151,respectively.3.The genes with dose-effect relationship and anti-cancer effects reported in the literature are CCRL2(up)and GPR78,CLEC2D,HOTAIR(down).4.0.45μmol/L,0.9μmol/L,and 1.8μmol/L Usenamine significantly up-regulated 1,1,and 6 apoptosis pathways in HepG2 cells.5.Usenamine significantly up-regulates the TNF signaling pathway,down-regulates the WNT and non-small cell lung cancer signaling pathways,affects the MAPK signaling pathway,and down-regulates the WNT\non-small cell lung cancer\MAPK pathway network.6.Usenamine has a strong affinity for both PRKACA protein and PRKCB protein.It binds to PRKACA protein through HIS142,THR299,THR300,GLU311,ASP301 these five amino acids,and it binds to PRKCB protein through THR404,ASN471,ASP470.All of the amino acids are disabled by the formation of hydrogen bonds to stabilize the combined state.7.Usenamine can block cells in G1 and S phases at doses of 0.25,0.5,and 1 μmol/L,and increases with the increase of the dose.8.In the acute toxicity test,when the mice were given 15000mg/kg orally,there was no abnormal performance and no animal death.9.The results of the genetic toxicity test showed:Under intragastric administration of 0,2000,4000,8000 mg/kg Usenamine,the chromosomal aberration rates of bone marrow cells in Kunming mice were:female 3.2±1.10%,4.4±1.67%,4.0±1.41%,2.8±1.09%,male 3.2±1.10%,3.2±1.79%,4.0±1.10%,3.6±1.41%,there is no statistical difference between each dose group and the negative control(0mg/kg)(P>0.05).Usenamine is effective in Kunming mice The bone marrow cells do not cause chromosomal aberrations.B2B cells were cultured in vitro,and the chromosomal aberration rates were 2.4±0.89%,2.8±2.28%,4.4±1.67%,3.2±1.79%at doses of 0,25,50,and 100 μmol/L,respectively.Each dose group and the negative control(Omg/kg)there was no statistical difference(P>0.05),and Usenamine had no chromosomal aberration effect on B2B cells.Gavage at doses of 0,2000,4000,8000,15000 mg/kg Usenamine.The micronucleus rates of bone marrow polychromatic erythrocytes in Kunming mice were 1.40 ± 0.65‰,1.24 ± 0.68‰,1.68±0.48‰,1.1 6±0.88‰,1.1 6±0.43‰,male 1.92±0.83‰,3.04±0.43‰,2.00±0.92‰,2.52±0.69‰,2.12±0.88‰,there was no statistical difference between each dose group and the negative control(0mg/kg)(P>0.05).Usenamine did not cause bone marrow polychromatic red blood cells Micronucleus effect.The chromosomal aberration rates of Kunming mice at doses of 0,2000,4000,and 8000 mg/kg were 3.2±1.10,4.4±1.67,4.0±1.41,and 2.8±1.09 for females,and 3.22±1.10,3.22±1.79,4.0±1.10 and 3.6±1.41 for males,there is no statistical difference(P>0.05)from the negative control(0mg/kg),indicating that Usenamine has no chromosomal aberration effect on the bone marrow cells of Kunming mice.Gavage at doses of 0,2000,4000,8000,15000 mg/kg Usenamine The sperm deformity rates of Kunming male mice were 1.6±0.22%,1.72±0.30%,1.82±0.47%,1.76±0.50%,2.30±0.36%,and there was no statistical difference between each dose group and the negative control(0mg/kg)The difference(P>0.05),Usenamine did not cause sperm deformity.10.Kunming mice were given 2000,4000,and 8000mg/kg Usenamine by oral gavage every day for 30 days.The red blood cell counts of female mice in the 8000mg/kg group and 4000mg/kg group were significantly lower than those in the Omg/kg group(P<0.05).The red blood cell count and hemoglobin concentration of the 4000 mg/kg group of male mice were significantly lower than those of the 0 mg/kg group(P<0.05).It shows that Usenamine can affect the production of red blood cells.The aspartate aminotransferase level of male mice in the 4000mg/kg and 8000mg/kg dose groups was significantly lower than that in the 0mg/kg group(P<0.05),and Usenamine could damage the liver function of the mice.The serum creatinine and urea levels of male mice in the 8000mg/kg dose group were significantly lower than those in the Omg/kg group(P<0.05),and Usenamine could affect the renal function of the mice.The serum triglyceride,low-density lipoprotein,and very low-density lipoprotein levels of male mice in the 8000 mg/kg dose group were significantly higher than those in the 0 mg/kg group(P<0.05);Usenamine can affect the lipid metabolism of mice Features.The glucose level of female mice in the 8000mg/kg group was significantly lower than that in the Omg/kg group(P<0.05),and Usenamine can affect the glucose metabolism of mice.Pathological examinations showed no obvious pathological changes in the liver,kidney,and testis of the mice.Conclusion:1.Usenamine can be synthesized by chemical means and is easy to purify.2.C18H17NO6 can significantly regulate HepG2 cell genes and signal pathways.3.CCRL2,GPR78,CLEC2D,and HOTAIR are the target genes of Usenamine in anti-liver cancer.4.Usenamine significantly affects the TNF signaling pathway,WNT pathway and non-small cell lung cancer pathway.The regulation of these pathways is conducive to anti-tumor effects.5.C18H17NO6 binds tightly with the PRKACA protein and PRKCB protein of the WNT pathway and exerts an anti-cancer effect.6.C18H17NO6 acute oral toxicity is non-toxic.7.C18H17NO6 has no obvious genotoxicity in this study.8.In the short-term repeated administration test,Usenamine can affect the liver and kidney function and blood glucose and lipid metabolism of mice,and there are gender differences.No obvious pathological changes were seen in liver,kidney,and testis. |
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