Study On The Mechanism Of Anti-hepatocarcinoma Effect Of Curcumae Rhizoma And Sparganii Rhizoma Based On NF-κB Pathway

On the basis of pharmacodynamic study of Curcumae Rhizoma and Sparganii Rhizoma in vitro and in vivo,this study further explored the influence of their compound compatibility on vital signs,serum cytokines,inflammatory factors and rel cell protein family of tumor bearing mice,and combined with in vitro experiments to explore their regulation and intervention on binding proteins in NF-κB signaling pathway,so as to clarify the effect of their compound compatibility on liver cancer stem cells The mechanism of pre proliferation and apoptosis.First.Pharmacodynamic study of Curcumae Rhizoma and Sparganii Rhizoma in vivo and in vitro.1.To construct and evaluate H₂₂ hepatoma subcutaneously transplanted tumor model in vivo.The results showed that on the 15th day after inoculation,the volume and weight of subcutaneous transplanted tumor in each group were more than 100mm³ and 400mg respectively,and the growth was even.The nuclei of transplanted tumor were dark blue and the ratio of cell nucleus to cytoplasm was high by HE staining.The success rate of subcutaneous tumor model was 100%,only 2%of mice died.2.To explore the anti hepatoma effect of Curcumae Rhizoma and Sparganii Rhizoma in vivo.The body weight of mice in each group was observed:The weight loss of mice in each group was more than 10%to the blank group in the middle and later period of modeling;The volume of transplanted tumor in the high dose group was significantly reduced（P<0.01）to the model group;The thymus weight of the positive drug group was smaller（P<0.05）,and the thymus index was lower（P<0.05）to the model group;The thymus weight of each drug group was larger（P<0.05）,and the thymus index was higher（P<0.05）to the positive drug group,.Results:The average tumor weight of Curcuma high dose group,middle dose group and high dose group was significantly lower than the model group（P<0.01）to the model group,and the tumor inhibition rates were 56.71%,44.63%and 42.62%.The average tumor weight of positive drug group was the lowest.The average tumor weight was the largest and the tumor inhibition rate was the lowest（7.38%）.3.To explore the effect of effective components of Curcumae Rhizoma and Sparganii Rhizoma on liver cancer in vitro.MTT method was used to study the effect of Curcumol and Sparstolonin B on the proliferation of Hep G2 hepatoma cells.The results showed that Curcumae Rhizoma alcohol and Sparstolonin B inhibited the proliferation of Hep G2 cells,and IC₅₀was 216.53μg/ml and301.48μg/ml.The results showed that Curcumol and Sparstolonin B could directly inhibit the proliferation of hepatoma cells and inhibit the development of tumor.Second.Pharmacodynamic study on the compatibility of curcuma zedoary and Sparganii Rhizoma in the treatment of liver cancer.1.To explore the antitumor effect of Curcumae Rhizoma and Sparganii Rhizoma on H₂₂hepatoma bearing mice.The body weight of mice in each group was observed:There was no significant difference between the two groups to the model group;The body weight of each group was significantly higher than that of the positive group after the 12th day（P<0.01）to the positive group.Results:The volume of transplanted tumor in the compound high dose group decreased significantly after the 9th day（P<0.01）to the model group,the volume of transplanted tumor in the compound medium dose group decreased significantly after the 12th day（P<0.01）,and the volume of transplanted tumor in the compound low dose group decreased significantly after the 15th day（P<0.05）.The thymus weight of the positive drug group was smaller（P<0.05）,and the thymus index was lower（P<0.05）to the model group;The thymus weight of each compound drug group was larger（P<0.05）,and the thymus index was higher（P<0.05）to the positive drug group.The average tumor weight of high and medium dose compound group was significantly lower than that of the model group（P<0.01）,and the tumor inhibition rates were 64.26%and 40.33%to the model group.The average tumor weight of positive drug group was the lowest,and the inhibition rate was 89.51%.2.To explore the inhibitory effect of the effective components of Curcumae Rhizoma and Sparganii Rhizoma on the proliferation of hepatoma in vitro.The inhibition rate of cell proliferation was 76.47%in 1000.00μg/ml group,74.51%in 500.00μg/ml group,67.65%in 250.00μg/ml group,60.78%in 125.00μg/ml group and 62.50μg/ml group respectively The inhibition rate of cell proliferation was 42.16%.It is suggested that Curcumol and Sparstolonin B have antitumor effect in vitro,and the inhibition rate increases with the increase of drug concentration.3.To explore the effect of the compatibility of Curcumae Rhizoma and Sparganii Rhizoma compound on the related inflammatory factors in NF-κB pathway.The levels of TNF-α,IL-1αand IL-6 were measured by ELISA.The results showed that the content of TNF-αand IL-6 could be reduced by zedoary Sparganii Rhizoma compound（P<0.01）;There was no significant effect on the content of IL-1α.Third.Study on the Mechanism of the Effect of the Combination of Curcuma and Trigonism on the Expression of Related Proteins in NF-κB.1.To explore the anti hepatoma mechanism of Curcumae Rhizoma and Sparganii Rhizoma compound in vivo.The levels of NF-κBp65,Bcl-2 and Bax were measured by immunohistochemistry.The results showed that Curcumae Rhizoma and Sanling compound could reduce the content of NF-κBp65,Bcl-2 and increase the content of Bax,suggesting that the anti hepatoma effect of Curcumae Rhizoma and Sanling compound might be related to the decrease of TNF-α,IL-6,NF-κBp65,Bcl-2 and the increase of Bax,and the anti-tumor mechanism of Curcumae Rhizoma and Sanling compound might be related to NF-κB signaling pathway.2.To explore the anti hepatoma mechanism of Curcumae Rhizoma and Sparganii Rhizoma in vitro.Western blotting was used to detect the expression of IKK-α/βand IκB-αin Hep G2 cells after the intervention of Curcumol and Sparstolonin B,which is helpful to further explore the mechanism of action of Curcumol and Sparstolonin B on Hep G2 cells.The gray values of IKK-α/βprotein blank group,combined 500.00μg/ml group,combined 250.00μg/ml group and combined125.00μg/ml group were as follows:1.47±0.10,0.49±0.02,0.74±0.06,0.85±0.07;the gray values of IκB-αprotein were as follows:0.53±0.04,0.98±0.09,0.62±0.07,0.47±0.02.The results showed that compared with the blank group,the expression of IKK-α/βprotein decreased in each combination group,suggesting that Curcumol combined with Sparstolonin B can inhibit the expression of IKK-α/βprotein in Hep G2 cells,and the inhibition increased with the increase of the concentration of the drug;The expression of IκB-αprotein increased in each combination group to the blank group,suggesting that Curcumol combined with Sparstolonin B can promote the expression of IκB-αprotein The expression in Hep G2 cells increased with the increase of drug concentration.