Dendrobium Nobile Active Ingredient Dendrobin A Against Hepatocellular Carcinoma

Objective:To study the effect of Dendrobium nobile active ingredient Dendrobin A on hepatocellular carcinoma in vitro and in vivo,and to explore its related mechanism.Method:（1）Hepatocellular carcinoma cell（HCC）lines Hep G2,SK-HEP-1 and normal liver cell line LO-2 were routinely cultured.（2）The effect of Dendrobin A on the proliferation of LO-2,Hep G2 and SK-HEP-1 was detected by CCK8 and plate cloning assay,and the expression of proliferation-related indicator PCNA was detected by q RT-PCR,western blot and immunofluorescence staining.（3）The effects of Dendrobin A on the migration and invasion of SK-HEP-1 and Hep G2 cell lines were detected by Wound healing and Transwell assay.（4）The changes of the cycle and apoptosis of SK-HEP-1 and Hep G2 induced by Dendrobin A were detected by flow cytometry,and the expression of related proteins was detected by Western blot.（5）The effects of Dendrobin A on Epithelial-mesenchymal transition（EMT）of hepatoma cells SK-HEP-1 and Hep G2 were detected by q RT-PCR and Western blot.（6）The SK-HEP-1 cells before and after treatment with Dendrobin A were subjected to screen differentially expressed genes by mRNA sequencing,and the GO terms and KEGG pathway related differentially expressed genes.（7）NF-κB,a typical KEGG signal enriched by RNA sequencing,was verified.（8）Molecular docking of Dendrobin A with proteins p65 and p50 was performed by Auto Dock software.（9）SK-HEP-1 cells were subcutaneous injected to BALB/c nude mice.After tumorigenesis and when the tumor volume was 300 mm³,mice were randomly divided into DMSO control group and Dendrobin A treatment group（8 mg/kg,once every three days）.At the end of 3 weeks after drug administration,the mice were killed,tumor weight was measured,and the expression of Ki67 protein was detected by immunohistochemistry staining.Results:（1）The results of CCK-8 experiment showed that the activity of Hep G2,SK-HEP-1 and LO-2 cells decreased with the increase of drug concentration in a certain range.Finally,80μmol/L Dendrobin A is selected and used as the administration concentration.The results of plate cloning showed that the cell clone formation rate of Dendrobin A treatment group was significantly reduced compared to DMSO control（P<0.05）,the expression of proliferating cell nuclear antigen（PCNA）was down-regulated at the mRNA and protein levels（P<0.05）,and the expression intensity and positive rate of PCNA immunofluorescence were also down-regulated.（2）The results of Wound hearing showed that Dendrobin A significantly inhibited the migration of SK-HEP-1 and Hep G2 cells（P<0.01）;Transwell experiments results showed that the number of cells migrated to the lower chamber in drug-treated SK-HEP-1 and Hep G2 cell group was significantly lower than that in the control group（P<0.01）.（3）Dendrobin A arrested SK-HEP-1 and Hep G2 cells at G₂/M phase;Dendrobin A significantly induced SK-HEP-1 and Hep G2 cell apoptosis,and the apoptosis rate increased by 2.25 and 3.45 times compared to DMSO control,respectively.（4）Dendrobin A up-regulated the expression of E-cadherin and down-regulated the expression of N-cadherin,Vimentin and Snail in SK-HEP-1 and Hep G2 cells（P<0.05）.（5）After treating SK-HEP-1 cells with Dendrobin A,the differentially expressed genes were screened,of which 475 were down-regulated and 355 were up-regulated.In GO function,there are 4337 biological processes,417 cell components and 709molecular function terms;There are 47 KEGG pathways,including NF-κB is one of the typical pathways enriched（Q=0.003358）.（6）The protein expression levels of p-IκB,p-p65 and p-P50 in hepatocellular carcinoma cells treated with Dendrobin A were down-regulated（P<0.05）.After treated with 1000 nmol/L concentration of NF-κB agonist PMA,the expression levels of p-IκB,p-p65 and p-p50 were up-regulated（P<0.05）.The results of cell function test also showed that the SK-HEP-1 and Hep G2 cells treated with PMA recovered the Dendrobin A-suppressed tumor cell activity,migration and invasion（P<0.05）.（7）Dendrobin A has good binding ability to protein p65 and p50,the binding energy is-6.0 kal/mol and-6.3 kal/mol,respectively.（8）The animal experiment results showed that the tumor volume and weight of the Dendrobin A treatment group were significantly smaller than that of the control group at the end of the experiment（P<0.05）,and the Ki67 proteins positive rate of the Dendrobin A treatment group was lower than that of the control group（P<0.01）.Conclusion:（1）The active component Dendrobin A of Dendrobium nobile has significant anti-hepatocellular carcinoma effect,and its mechanism is related to inhibiting tumor cell proliferation,migration,invasion and EMT,blocking cell cycle progression and inducing cell apoptosis.（2）The HCC cells treated by Dendrobin A involve a variety of changes in functions and signal pathways,including inhibition of the classical NF-κB signal pathway.