| Objective:To investigate the anti-tumor effect and mechanism of Laminaria japonica polysaccharide(LJP)on hepatocellular carcinoma based on senescence marker protein 30(SMP-30).This study is expected to provide valuable information for the prevention and treatment of liver cancer and utilization of kelp resources.Methods:(1)Human hepatocellular carcinoma cell lines(Bel-7404/HepG2)in logarithmic growth phase were inoculated with 5×10~3 cells per well in a 96-well culture plate,and the supernatant were discarded after 24 hours of culture.We set the Bel-7404/HepG2 cells into different groups:without LJP intervention as the control group,DMEM blank control group and LJP group(5,10,15,20,25,30,35,40 mg/mL concentration gradient),and 3 replicates were set in each group.After 24 hours,48 hours,and 72 hours,the morphological changes of the hepatoma cells in each group were observed by using an inverted microscope.The inhibition of proliferation of hepatoma cells was detected by the CCK-8 method.(2)The effect of LJP on the expression of hepatoma-associated antigen SMP-30 in human normal liver cells(LO2)and human hepatoma cells(Bel-7404/HepG2)was detected by qRT-PCR and Western blot methods.(3)Mouse hepatocarcinoma Hepa 1-6 cell lines were used to construct a mouse model of hepatocellular carcinoma xenografts.The model of C57BL/6 mice were randomLy divided into 4 groups:normal saline group,LJP low concentration group(200 mg/kg·d),LJP medium concentration group(400 mg/kg·d),LJP high concentration group(800 mg/kg·d).Each group has 6 mice.LJP was injected through caudal vein regularly.The dynamic changes of mouse weight and tumor volume was observed in each group.After four weeks,the experiment was terminated.The mice were sacrificed and the tumor masses were removed.The size of each mass was measured and weighed.The inhibition rate of each group was calculated.Results:(1)The Bel-7404/HepG2 cells grew well in control group.After24 hours,48 hours,and 72 hours,the cell volume of Bel-7404/HepG2 cells gradually became smaller and pyknosis.It was more obvious deformation in 35mg/mL LJP group.It showed that LJP inhibited the proliferation of Bel-7404/HepG2 cells in CCK-8 method,and the inhibition was positively correlated with the dose of LJP.With 35mg/mL LJP and acted 72h,it was more obvious inhibition in Bel-7404/HepG2 cells(P<0.05).Their inhibition rate was89%and 87%,respectively.The results showed that the optimal concentration and the action time of LJP for inhibiting the proliferation of liver cancer cells was 35 mg/mL and 72h,respectively.(2)After 35mg/mL LJP and 72h treatment,the SMP-30 mRNA expression in Bel-7404/HepG2 cells was higher than that in control group(P<0.05).It showed that the LJP can upregulate the expression of SMP-30 in liver cells.At the same time,we also found that the growth rate of SMP-30 in LO2,Bel-7404 and HepG2 was 100%,161%and 128%,respectively.It showed the effect of LJP on Bel-7404 cells is the most obvious(P<0.05).On the other hand,after LJP treatment,the SMP-30 protein expression in Bel-7404/HepG2 cells was increased.Both of them were high than that in LO2cells(P<0.05).(3)After LJP treatment,the C57BL/6 mice with hepatoma xenografts were given increased body weight.It showed that the higher the concentration of LJP,the greater the weight gain of mice(P<0.05).In addition,with the higher concentration and longer time treatment,the inhibitory effect of tumor growth are more obvious(P<0.05).Conclusion:The LJP has an effect of inhibiting the growth of hepatocellular carcinoma.Up-regulation of hepatoma-associated antigen SMP-30 may be one of the mechanisms of its function. |
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