| Background and Objects:Swertia plants have been used as herbal medicine for thousands of years.It is widely used in treating fever,malaria,anemia,bronchial asthma,liver disease,hepatitis,gastritis,constipation,dyspepsia,skin parasites,epilepsy,ulcer,oligoshia,hypertension,depression,certain types of mental disorders,jaundice,diabetes and other diseases all over the world.The original plant of s.mileensis is s.mileensis,which is a special traditional Chinese medicine in Yunnan.It has many pharmacological activities,such as liver protection,antisepsis,hypoglycemic,anticholine and spasmolysis,especially for viral hepatitis.Anti-tumor studies have been reported on various plants of familiar genus Swertia in Gentianaceae.Based on this,we took Swertiamarin,a Secoiridoid component extracted from s.mileensis,as an object to explore:1.Whether s.mileensis has therapeutic effect on human hepatocellular carcinoma(HCC);2.Whether swertiamarin(STM)extract from s.mileensis can inhibit human hepatocellular carcinoma cells;3 Specific mechanism of effects of swertiamarin(STM)on human hepatocellular carcinoma cells.Methods:1 in vitro cell phenotype detection and IC50 screeningSwertiamarin acted on three human liver cancer cells HepG2,Huh7 and sk-hep-1,and one normal human cell hl-7702,then the changes of cell proliferation,invasion and apoptosis were detected and IC50 concentration was determined.2 mRNA gene chip detection and bioinformatics analysisThe mRNA gene chip was used to detect the interference of Swertiamarin(STM)in HepG2 cells,and the results were analyzed by multi-database informatics to predict the specific mechanism of the effect of Swertiamarin(STM)on human liver cancer cells.3 Verification of cell signal transduction mechanismQ-PCR and western blot were used to verify whether Swertiamarin(STM)could affect the expression of CDKN1A(P21)TNF-α expression and AKT in human liver cancer cells HepG2,as well as whether the regulation tendency of these three factors was up-regulated or down-regulated.Moreover,immunofluorescence assay was used to more directly show the effect of Swertiamarin(STM)on CDKN1A(P21)in human liver cancer cells HepG2.siDKN1A(P21):CCK8 flow cytometry,q-PCR and western blot were used to detect the proliferation and apoptosis changes of HepG2 cells after the intervention of Swertiamarin(STM)in the presence of CDKN1A(P21)expression interference,as well as the changes of CDKN1A(P21)AKT and TNF-α expression in HepG2 cells.Overexpress CDKN1A(P21):CCK8 flow cytometry,q-PCR and western blot were used to detect the effect of increased CDKN1A(P21)expression on the anticancer ability of Swertiamarin(STM)and the regulation of AKT and TNF-α.Vivo experiment:Swertiamarin(STM)was injected into the subcutaneous sk-hep-1 implanted tumor model of nude mice,and the volume and weight of the implanted tumor were compared.Then use q-PCR,and immunofluorescence staining to determine the effect of Swertiamarin(STM)on CDKN1 A(P21)AKT and TNF-αin the implanted tumor cells.Results:1 in vitro cell phenotype detection and IC50 screeningWe first calculated that the IC50 inhibitory concentration of Swertiamarin(STM)on HepG2 was 87.57 g/ml.The IC50 inhibitory concentration of huh7 was 63.035 g/ml.The IC50 inhibitory concentration of sk-hepl was 55.302 g/ml.According to this concentration,the effect of Swertiamarin(STM)on HepG2 huh7 sk-hepl cells can significantly inhibit the proliferation and invasion of HepG2 huh7 sk-hep1 and promote the apoptosis of HepG2 huh7 sk-hepl,indicating that Swertiamarin(STM)has an obvious inhibitory effect on human liver cancer cells.2 mRNA gene chip detection and bioinformatics analysisHepG2 cells after Swertiamarin(STM)intervention were selected for mRNA gene chip detection.Among the 33,655 mRNA detected,3,563 genes were found to be different from the control group.Using multiple databases to analyze the results of the microarray,we predicted that Swertiamarin(STM)extract of s.mileensis was involved in the regulation of PI3K-akt pathway and TNF-α pathway at least in part by influencing the expression of CDKN 1 A(P21).Thus,it can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and promote the apoptosis of human hepatocellular carcinoma cells.3 Verification of cell signal transduction mechanism(1)Results of q-PCR,western blot and immunofluorescence①After the intervention of Swertiamarin(STM),HepG2 showed a significant increase in CDKN1A(P21)TNF-α expression and a significant decrease in AKT compared with the control group;② After the intervention of Swertiamarin(STM),the amount of AKT in HepG2 cells decreased while the amount of TNF-α protein increased:③Immunofluorescence test showed that the fluorescence staining intensity of CDKN1A(P21)in the control group was lower than that in the normal hepatocytes.Compared with the control group,the fluorescence staining intensity of CDKN1A(P21)in the four cell lines in the experimental group was enhanced,especially in the three liver cancer cells in the experimental group.(2)siDKN1A(P21)①The cell proliferation rate of HepG2 in the control group was the highest without interference.Cell proliferation in HepG2+STM group was significantly inhibited,while cell proliferation in HepG2+STM+siNC group was still significantly inhibited after siNC was added.When HepG2+STM was added to siP21,the inhibitory effect of Swertiamarin(STM)on HepG2 proliferation was significantly reduced;②The apoptosis rate of HepG2 in the control group was the highest without interference.Apoptosis was significantly increased in the HepG2+STM group,while apoptosis was still significant in the HepG2+STM+siNC group after the addition of siNC,which increased at a rate comparable to that of the HepG2+STM group.HepG2 apoptosis rate was the lowest in HepG2+STM+siP21 group;③Q-PCR:The increased expression of CDKN1A(P21)is accompanied by the decreased expression of AKT and TNF-α genes,while the decreased expression of CDKN1A(P21)is accompanied by the increased expression of AKT and TNF-α genes,suggesting that the level of CDKN1A(P21)can affect the expression of AKT and TNF-α genes.④western blot:Swertiamarin(STM)intervention resulted in increased levels of DKN1A(P21)and TNF-α in HepG2 cells,and decreased levels of AKT.The effect of Swertiamarin(STM)on CDKN1A(P21)AKT and TNF-α protein levels in HepG2 cells was not significantly changed after si-nc was added.When the protein replication of DKN1A(P21)is interfered with,the protein level of TNF-α antigen decreases,while the protein level of AKT increases.(3)overexpress CDKN1A(P21):①Proliferation of HepG2 cells decreased after overexpression of CDKN1A(P21);②Apoptosis of HepG2 cells was significantly increased after overexpression of CDKN1A(P21);③Q-PCR:CDKN1A(P21)was significantly up-regulated in HepG2 cells after overexpression of CDKN1A(P21),followed by down-regulation of AKT and up-regulation of TNF-α;④western blot:CDKN1A(P21)protein level was significantly increased in HepG2 cells after overexpression of CDKN1A(P21),followed by significantly decreased AKT protein and significantly increased TNF-αprotein.(4)vivo experiment:① After intratumoral injection of Swertiamarin(STM),tumor growth was significantly inhibited in nude mice,and tumor formation rate,volume and weight were significantly lower than those in the control group,indicating that Swertiamarin(STM)had a significant inhibitory effect on sk-hep-1 human hepatocellular carcinoma(HCC)in vivo;②Q-PCR:The expression of CDKN1A(P21)and TNF-gene increased with the intervention of Swertiamarin(STM),while the expression of AKT decreased with the intervention of Swertiamarin(STM);③Immunofluorescence staining:The green fluorescence of CDKN1A(P21)in the experimental group was significantly higher than that in the control group.The red fluorescence of AKT in the experimental group was significantly lower than that in the control group.The cyan fluorescence of TNF-α in the experimental group was significantly higher than that in the control group.The mixed fluorescence imaging in the control group was mainly red fluorescence on behalf of AKT,while the mixed fluorescence imaging in the experimental group was mainly green and cyan fluorescence on behalf of CDKN1 A(P21)and TNF-α.Conclusions:S.mileensis has a therapeutic effect on human hepatocellular carcinoma(HCC),and its anticancer effect is realized at least in part through Swertiamarin(STM),one of the main components in plants.At least part of the specific anticancer mechanism of Swertiamarin(STM)is the regulation of CDKN1A(P21)to participate in PI3K-akt pathway and TNF-α pathway,thus inhibiting the proliferation and invasion of human hepatocellular carcinoma cells and promoting the apoptosis of human hepatocellular carcinoma cells. |
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