Study On The Mechanism Of The Gemmanane-type Sesquiterpenoid TMJ-12 In Inhibiting Benign Prostatic Hyperplasia

Objectives:Compound TMJ-12 is a germacrane-type sesquiterpenoid compound extracted from the plant Carpesium carnuum.Its effect on BPH1 cells and the mechanism of action have not been revealed.This paper studies the mechanism of TMJ-12 inhibiting the proliferation of BPH1 cells,provide a basis for the treatment of benign prostatic hyperplasia with natural molecule compounds.Methods:Screen the inhibitory rate of TMJ-12 at different concentrations and time on BPH1 cells by MTT method,and calculate the IC₅₀.Cell behavioral photography shows the effect of TMJ-12 on the morphology of BPH1 cells.A cytotoxicity assay indicated that TMJ-12inhibited BPH1 cell proliferation and invasion.Hoechst33258 staining method confirms whether TMJ-12 induces apoptosis of BPH1 cells.Flow cytometry detects the effect of TMJ-12 on the apoptosis of BPH1 cells,and obtains the status of early and late cell apoptosis.JC-1 mitochondrial membrane potential staining to observe whether TMJ-12 induces early apoptosis of BPH1 cells through the mitochondrial pathway.Simultaneous detection of BPH1 cell cycle arrest by TMJ-12 by flow cytometry.The potential targets of the disease BHP were obtained from the Dis Ge NET,Gene Cards,and Gen CLip databases;while the potential targets of the compound TMJ-12 were obtained from the Pharm Mapper database.input the intersection target of compound TMJ-12 and disease of BPH into STRING database for KEGG enrichment pathway analysis,Screening out the top 20 pathways with high enrichment targets.Western bot were used to detect apoptosis and cell cycle proteins,also detect mitochondrial pathway and PI3K/Akt pathway proteins.Run Autodock4.2 molecular docking technology to dock the compound TMJ-12 with target proteins.Results:MTT inhibition rate showed the cell proliferation,after stimulated different concentrations and times of TMJ-12 with BPH1 cells,The IC₅₀of compound TMJ-12 stimulated cells for 24,48,and 72 hours were 18.81,10.53,8.02?M,respectively.It can be seen that the compound TMJ-12 has a proliferation inhibitory effect on BPH1 cells,and it is concentration and time dependent.The results of the cytotoxicity assay showed that as the concentration of compound TMJ-12 increased,the BPH1 cell colonies gradually decreased.The results of flow cytometry and Hoechst33258 staining showed that compound TMJ-12can induce BPH1 cells apoptosis.At the same time,with the increase of the concentration and time of compound TMJ-12,BPH1 cells changed from early apoptosis to late apoptosis.JC-1 mitochondrial membrane potential staining results show that the compound TMJ-12 can induce apoptosis of BPH1 cells through the mitochondrial pathway.The effect of flow cytometry on the cell cycle shows that the compound TMJ-12 can induce G2/M phase arrest in BPH1 cells.Western blot results show that compound TMJ-12 can regulate the expression of several apoptosis and cell cycle-related proteins,including Bcl-2,Bax,Bad,Caspase-9,Caspase-3,CDK1,Cyclin B1,CDC25C and c-Myc et al.At the same time,the compound TMJ-12 can inhibit the activation of the PI3K/Akt pathway;molecular docking results show that the compound TMJ-12 can inhibit the PI3K/Akt pathway by binding to the PTEN protein,thereby causing apoptosis of BPH1 cells.Conclusion:In summary,it can be concluded that compound TMJ-12 extracted from the plant Carpesium carnuum.can effectively inhibit the proliferation of BPH1 cells,at the same time cause apoptosis of BPH1 cells and G2/M cycle arrest.With the increase of TMJ-12stimulation concentration and time,BPH1 cells changed from early apoptosis to late apoptosis.The compound TMJ-12 can also inhibit the activation of the PI3K/Akt pathway to promote the programmed apoptosis of BPH1 cells.This study lays the foundation for the treatment of benign prostatic hyperplasia with natural molecule compounds.