| Liver is the largest digestive gland,the largest metabolic and detoxifying organ in livestock and poultry.It also plays a very important role in bile production and excretion,coagulation,im-munity,heat production and regulation of water and electrolytes.It can be said that the liver is a huge "chemical plant" of the body.Mildew of livestock and poultry feed,abuse of antibiotics,excessive use of minerals,excessive addition of protein,severe infection of microorganisms,etc.,bring a great burden to the liver,so that liver activity is inhibited or damaged,the liver is in a state of poisoning.Long-term accumulation of toxins can induce hepatocyte degeneration necr-osis,inflammatory swelling,fibrous tissue hyperplasia and other liver lesions,and then liver tumors.Caffeic acid phenylethyl ester(CAPE)is one of the main active ingredients in propolis.It is a flavonoid compound with o-diphenol hydroxyl group.CAPE has strong anti-tumor,anti-oxid ation,anti-inflammatory,immune regulation and other pharmacological activities.Especially its anti-lung cancer effect has aroused widespread concern.It affects cells by influencing the cell.The antitumor mechanisms include redox state,regulating the expression of apoptosis-related factors,inhibiting the proliferation of tumor cells,and inhibiting the proliferation of nuclear transcription factor NF-KB.Caffeic acid p-nitrophenylethyl ester(CAPE-NO2)is a new compou-nd designed and synthesized by the Department of Pharmacology,College of Pharmacy,Southwest University.According to the structural characteristics of CAPE,CAPE-NO2 has stron-ger bioactivity than caffeic acid phenylethyl ester through metabolic,immune and cardiovascul-ar tests.Hepatocellular carcinoma HepG-2 cells are often used as ideal cell lines for in vivo and in vitro hepatocyte metabolism or genotoxicity testing,they are often used as tool cells to screen hi-ghly active anti-hepatoma compounds.On the basis of optimizing the synthesis of CAPE-NO2the anti-hepatoma effect of the compound was studied in vitro and in vivo.1.Synthesis and characterization of CAPE-NO2Caffeic acid p-nitro phenylethyl ester(CAPE-NO2)was synthesized by one-pot method with caffeic acid and 4-nitro phenylethyl bromide under a little EDCI and DMAP.By melting point determination,infrared spectroscopy and nuclear magnetic resonance analysis,the structure of the compound was confirmed to be the same as that of the standard compound.The synthetic process is simple,mild and yield is 92%,which is suitable for industrial production.2.Inhibitory effect of CAPE-NO2 on hepatocellular carcinoma HepG-2 cell xenografts in nude miceBALB/C nude mice transplanted with hepatoma HepG-2 cell line were used to establish the tumor model,the tumor inhibition effect was observed,the thymus and spleen index,peritoneal macrophage phagocytosis,splenic T-lymphocyte proliferation,NK cell activity,serum NO,IL-1 and TNF-alpha levels were measured.The results showed that:(1)The tumor inhibition rates of CAPE-NO2(3mg/kg,6mg/kg,12mg/kg)mice were 50.67%,55.58%and 71.31%respectively,which were significantly higher than those of CAPE(3mg/kg,6mg/kg,12mg/kg),which were 38.84%,41.38%and 48.89%,and the relative tumor proliferation rates of CAPE-NO2(3mg/kg,6mg/kg,12mg/kg)were 47.03%,36.270%and 21.18%respectively.It was significantly lower than that of CAPE(3mg/kg,6mg/kg,12mg/kg)in 60.90%,46.16%and 30、11%,indicating that CAPE-NO2 had a good inhibitory effect on transplanted tumor of HepG-2 cell line in nude mice.(2)Compared with CAPE,CAPE-NO2 significantly increased the index of thymus and spleen in tumor-bearing mice(P<0.01),indicating that CAPE-NO2 had a protective effect on thymus and spleen,which was enhanced by the decrease of hydrophobic parameter Clog P.(3)Compared with the model group,CAPE-NO2 significantly promoted the phagocytosis of neutral red by mouse peritoneal macrophages(P<0.01),and the phagocytosis was dose-dependent.The lower the concentration.the stronger the phagocytosis.(4)Compared with CAPE,CAPE-NO2 significantly decreased the elevated NO level in tumor-bearing mice(P<0.01),and showed a dose-dependent relationship,indicating that CAPE-NO2 may play an anti-tumor role by reducing the amount of NO.(5)Compared with the model group,CAPE-NO2 significantly increased NK cell killing activity in tumor-bearing mice(P<0.01),showing a dose-dependent relationship.(6)Compared with CAPE,CAPE-NO2 significantly increased the proliferation of T lymphocytes induced by Con A in nude mice bearing tumor(P<0.1).With the increase of dose,the proliferation activity increased and returned to normal level,indicating that CAPE-NO2 can improve the response ability of T cells in tumor-bearing mice.(7)Compared with the model group,CAPE group and CAPE-NO2 group could significantly increase the level(?)secretion in tumor-bearing mice(P<0.01)and sigaificanty inhibit the level of TNF-α(P<0.01),showing a dose-dependent relationship,while the 12 mg/kg CAPE-NO2 group returned to nearly normal level.Conclusion:CAPE-NO2 has a good inhibitory effect on transplanted tumor of HepG-2 cell line,which is closely related to the improvement of immune function of tumor-bearing mice.3.Effect of CAPE-NO2 on apoptosis of hepatocellular carcinoma HepG-2 cellsBy studying the effect of CAPE-NO2 on apoptosis of hepatocellular carcinoma HepG-2 cell-s,the possible mechanism of CAPE-NO2 inhibiting hepatocellular carcinoma was explored.The proliferation inhibition rate of hepatocellular carcinoma cells was observed.The morphological changes,apoptosis rate,cell cycle distribution,apoptosis-related protein expression and apop-tosis signal pathway-related protein expression of apoptotic cells were detected.The results showed that:(1)Compared with CAPE,CAPE-NO2 significantly inhibited the proliferation of HepG-2 cells(P<0.01).With the increase of concentration,the inhibition rate increased,and the IC50 of CAPE-NO2 on HepG-2 cells decreased significantly(14.36±1.36)(P<0.01),indicating that CAPE-NO2 had a strong inhibitory effect on the proliferation of HepG-2 cells.(2)After treated with CAPE-NO2 under fluorescence microscope,the cells showed enhanced staining,brighter fluorescence,pyknosis of nucleus,and some nuclei fragmented into spherical granules,which were apoptotic or necrotic cells.Compared with CAPE,the cells with multiple visual fields were counted under fluorescence microscope.Statistics showed that CAPE-NO2 had obvious effect on apoptosis of HepG-2 cells,and the apoptosis rate increased significantly with the increase of concentration(P<0.01),showing a concentration-dependent manner,indicating that CAPE-NO2 could induce apoptosis of HepG-2 cells.(3)The apoptotic rates of CAPE-NO2 with 5,10 and 20 uM concentrations were 18.03%,20.66%and 20.47%respectively,which were significantly higher than those of the control group(P<0.01).Compared with CAPE-NO2 with the same concentration,the apoptotic rate of HepG-2 cells was significantly increased(P<0.01),indicating that CAPE-NO2 could induce apoptosis of HepG-2 cells.(4)Compared with CAPE,the cell cycle distribution of CAPE-NO2 on hepatocellular carcinoma HepG-2 cells changed significantly.The number of hepatocellular carcinoma cells in G0/G1 phase decreased,but the number of hepatocellular carcinoma cells in G2/M phase increased,and there was no significant change in S phase cells.After CAPE-NO2 treatment,the cell cycle of HCC was blocked at G2/M phase.and the cell number increased with the concentration of CAPE-NO2(P<0.01),in a dose-dependent manner.(5)Compared with CAPE,CAPE-NO2 could significantly enhancerbe cleavage of Caspase-3 protein,up-regulate the expression of Bax and down-regulate the expression of Bcl-2 protein in HepG-2 cells in a dose-dependent manner(P<0.01).(6)Compared with CAPE,CAPE-NO2 significantly decreased the expression of PI3K/Akt/mTOR signaling pathway-related protein(P<0.01),and the proportion of PI3K/p-PI3K,p-Akt/Akt and p-mTOR/mTOR decreased in a dose-dependent manner.It is suggested that the apoptosis of hepatoma cells induced by CAPE-NO2 may be related to the inhibition of Akt phosphorylation.4.Study on structure-activity relationship between CAPE-NO2 and anti hepatoma HepG-2Chemoffice 8.0 was used to analyze the structure-activity relationship between the structure-al parameters of CAPE-NO2 and IC50 of anti-hepatoma HepG-2 cells.The results showed that compared with CAPE,the activity of CAPE-NO2 against HepG-2 cells increased with the decree-se of ClogP,the decrease of molecular total energy and energy difference(△E),the increase of dipole moment and the change of charge.Conclusion:Caffeic acid p-nitrophenylethyl ester(CAPE-NO2)is a compound obtained by introducing electron-withdrawing group nitro(-NO2)into the benzene ring of CAPE.The compound has good anti-hepatoma effect,which may be related to enhancing immune function and inducing apoptosis. |
PDF Full Text Request