| Oridonin is a natural substance with multiple bioactivity separated from Rabdosia rubescens which is a small shrub of Isodon in Lamiaceae.Oridonin has obvious inhibitory effect on breast cancer,liver cancer,gastric cancer,colon cancer,pancreatic cancer and other malignant tumor cells without obvious toxicity to other organs.Nowadays,the studies of oridonin mainly focus on mitochondrial pathway,death receptor pathway,PI3K/Akt signaling pathway,MAPK signaling pathway,etc.The current literature on the mechanism of oridonin is fragmentary,which limits the development of drugs based on the oridonin.Network pharmacology is a new subject,which comprehensively and accurately analyzes the relationship among drugs,targets and diseases and finds effective targets for drug research based on public database,existing data,omics technology and other integrated approaches.It alleviates the limitations of drug research and brings dawn to new drug development.The previous studies of our group showed that oridonin had a significant proliferation inhibition effect on the gastric cancer SGC-7901 cells by blocking the cells in phase M,which showed that the inhibition of proliferation is related to the regulation of cell cycle.However,no other comprehensive reaches have been implemented.For the comprehensive study of oridonin,our group comprehensively and rapidly predicts and analyzes the signal transduction pathway related to the proliferation inhibition effect of oridonin on gastric cancer cells utilizing content related to the network pharmacology.However,the prediction results are not verified.The thesis studies the inhibitory effect of oridonin on the proliferation of human gastric cancer SGC-7901 cells through efficacy experiment based on the previous results of our group for the further understanding on the mechanism of oridonin in proliferation inhibition.Meanwhile,it verifies whether the mechanisms of proliferation inhibition and inducing cell apoptosis are related to the TNF-αsignaling pathway,AR signaling pathway and TGF-βsignaling pathway predicted by the network pharmacology.In this thesis,the inhibitory effect of oridonin on the growth of human gastric cancer SGC-7901 cells and the effect on the growth curve are determined by MTT method for observing the proliferation inhibition effect of oridonin on human gastric cancer SGC-7901cells.Morphological changes of cells are observed through inverted microscope and fluorescence microscope.The results show that the proliferation of SGC-7901 cells is inhibited by oridonin after 24,36 and 72 hours.The inhibition of proliferation is enhanced along with the increase of time and dose,which shows time-dose dependence.The IC50 of oridonin was52.38μM at 72 h.In inverted microscope,the cell morphology in the negative control group is normal in irregular polygon with high cell density and clear edge.A large number of suspended dead cells without obvious cell morphology,which had bright edge,appear in the culture medium with the increase of oridonin dosage.The cell body becomes smaller,the cytoplasm shrinks,the cell break,and cell fragments appear,which shows necrotic state.In fluorescence microscope,the cell morphology in the negative control group is round and complete with high cell density and uniform distribution showing the state with uniform dispersion and fluorescence.The number of cells decrease with the increase of oridonin dosage in the administration group in which the nucleus is compact and hyperchromatic and partial cells are broke in fragments with compact and hyperchromatic state and slightly white in color.There are apoptotic bodies formed by chromatin condensation.The results show that oridonin could significantly inhibit the proliferation of SGC-7901 cells.The thesis verifies the TNF-αsignaling pathway,AR signaling pathway and TGF-βsignaling pathway,which are predicted to be associated with the proliferation inhibition effect of oridonin on gastric cancer cells in network pharmacology,through Western Blotting method.TNF-αis a tumor necrosis factor,and TNF-αsignaling pathway includes caspase family,NF-κB signaling pathway and JNK signaling pathway.TNF-αcan activate the apoptosis promoter called caspase-8 by binding to the receptor TNFR.Then,caspase-8 activates the downstream apoptotic effectors called caspase-3 and caspase-7 through cascade amplification reaction after autoactivation,and the apoptosis was realized.The changes of TNF-αin cells stimulate the IKK kinase complex through which the IκBαis recognized by ubiquitin ligase after phosphorylation.IκBαcan be recognized and degraded by S26 protease complex after ubiquitination.The nuclear localization signal NLS of NF-κB,which enters the nucleus for transcription and induces the expression of multiple apoptosis inhibitors,is exposed.TNF-αactivates MAPKKK_Sby binding to receptor and starts the cascade reaction JNK.The process includes:MAPKKK_S→MAPKK_S→MAPK_S.MKK 7 is activated and JNK is specifically activated.So,the occurrence of tumor can be inhibited and the apoptosis of tumor cell apoptosis can be promoted.The expression of TNF-αwas increased with the increase of oridonin dosage during this experiment.Then,the expression of cleaved-caspase-8,cleaved-caspase-3 and cleaved-caspase-7 was activated,the expression of MEKK1,p-MEK7,p-JNK,p-IKBαwas increased and the expression of NF-κΒwas decreased in cells.It shows the proliferation inhibition effect of oridonin on the human gastric cancer SGC-7901 cells is related to TNF-αsignaling pathway.It works by promoting caspase family and JNK signaling pathway and inhibiting NF-κB signaling pathway.AR is an androgen receptor and a transcription factor of ligand induction.Androgen exerts the biological function by combining with AR.As the binding site of androgen and AR is mainly in the nucleus,the distribution and transfer of AR in cytoplasm and nucleus can also be used as the basis reflecting the activation and inhibition of AR signaling pathway.The downstream STEAP 1 protein of AR signaling pathway is usually located at the junction of cell membrane and participates in cell communication and other process.The research of AR signaling pathway is generally limited to prostate cancer,breast cancer and other sex hormone dependent diseases.Our group predicted that the proliferation inhibition effect of oridonin on the gastric cancer might be related to AR signaling pathway based on the analysis of network pharmacology.The experiment shows the increase of the expression of AR protein in cytoplasm,the decrease the expression of AR protein in nucleus and the decrease of the expression of STEP 1 protein in cells along with the increase of oridonin dosage.Oridonin works by inhibiting the migration of AR to the nucleus and the expression of downstream STEP 1protein,which indicats that the proliferation inhibition effect of oridonin is related to the inhibition of AR signaling pathway.TGF-βis a polypeptide cytokine.It realized the autophosphorylation by recognizing and binding to the receptor.Activated TβRI can specifically recognize and combine with downstream Smad 2 and Smad 3 which separate from TβRI and form heterologous complex with Smad 4 after activation.The heterologous complex transfers to the nucleus to regulate the expression of target genes.It is found that oridonin can lower the expression of TGF-βand p-smad 2/3 protein in cells,increase the expression of Smad 4 protein,which indicates that proliferation inhibition effect of oridonin on human gastric cancer SGC-7901 cells is related to the inhibition of TGF-βsignal pathway.All results show that oridonin inhibited the proliferation of human gastric cancer cells by regulating TNF-αsignaling pathways,AR signaling pathways and TGF-βsignaling pathways,which confirms the conclusion of network pharmacology prediction. |
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