A Comparative Study On The Proliferation And Invasion Of Hepatocellular Carcinoma HepG2 Cells By Curcumin And Turmeric Extracts Through Anticoagulant And Anti-inflammatory Pathways

Objective:Curcuma longa extract possessed anti-coagulant dominant,contrasted the curcumin of anti-inflammatory anti-hepatoma effects through the difference of them on anti-hepatoma Hep G2 cell proliferation and invasion effect,verify traditional Chinese medicine anti-inflammatory anti-hepatoma mechanism.Methods:?1?Using CCK8 method to observe the concentration gradient of Curcuma longa extract?171.5?343?686?1372?g/m L?and curcumin?Cur:14.7?29.5?58.9?117.8?g/m L?expose Hep G2 cells,we calculate the half of the drug effective concentration(IC₅₀)and detect the cells proliferation.Curcuma longa extract main content of curcumin in 8.59%conversion.Compared with the inhibition rate of 1/2IC₅₀ curcumin and 1/2IC₅₀ Curcuma longa extract and 1/2IC₅₀ concentration as a concentration for subsequent experiment.?2?1/2IC₅₀ Curcuma longa extract and the same dose of curcumin from8.59%contentconversionofit,aftertheconcentrationof lipopolysaccharide?LPS??0,3,6,12,24,48?g/m L?induced 24h,been tested the proliferation inhibiting rate of Hep G2 cells by CCK8 method.The preliminary judgment that Jiang Huang anti hepatoma effect leading role in anti-inflammatory oranticoagulation antihepatocarcinoma way??3?Thrombin?Th?5U/ml stimulated human umbilical vein endothelial cells?HUVECs?got HUVECs release solution.Experiment divided into blank control group,thrombin group,thrombin plus LPS group.We detected Tissue type plasminogen activator?t-PA?,plasminogen activator inhibitor?PAI-1?,the level of IL-6 of HUVECs release medium by ELISA.?4?Contrast detection of LPS and the HUVECs release solution induced Hep G2 cell proliferation effect by CCK8 method.?5?Contrast detection of curcumin and Curcuma longa extract exposed Hep G2 cell proliferation effect after the HUVECs release solution induced by CCK8 method.?6?Comparative on the invasion ability of curcumin and Curcuma longa extract on human hepatocellular carcinoma cell line Hep G2?7?RT-PCR detected of changes of IL-6,i NOS gene expression.Western blotting detected the expression of i NOS and IL-6 protein.Results:?1?LPS on the proliferation of Hep G2 cells play an important role in a time dose dependent.LPS more than 50?g/m L,with the concentration and time increasing,it inhibited Hep G2 cell proliferation,so LPS 50?g/m L as intervention concentration.?2?Curcuma longa extract and curcumin can inhibited proliferation of human hepatocarcinoma cell line Hep G2 in a time dose dependent?P<0.05?.The calculation of curcumin IC₅₀?24 h?value is 48.43 g/ml.In a follow-up experiment,we determined the concentration of curcumin treated Hep G2cells were 24?g/m L and 48?g/m L.Curcuma longa extract IC₅₀?24 h?value is 663.1?g/m L,We determined the concentration of turmeric extract treated Hep G2 cells were 331.5?g/m L and 663?g/m L.Calculation of curcumin in Curcuma longa extract of IC₅₀?24 h?value is 56.93?g/m L,and quite close with the IC₅₀ value of curcumin?24 h?48.43?g/m L.Clearly Curcuma longa extract mainly depended on Curcumin to inhibit hepatocellular carcinoma Hep G2 cells proliferation.After the translation of curcumincontained from the corresponding amount of Curcuma longa extract,the work quality concentration of Hep G2 cells treated with 57 g/ml and 28.5?g/m L.Following cell migration,invasion and expression of protein assays in turmeric extract 331.5?g/m L and the translation of curcumin 28.5?g/m L whichcorrespondingequivalentconversionforthecomparisonof concentration.?3?After LPS induced inflammatory,Curcuma longa extract against the inflammatoryreactionprocessbycurcumin,sothatincreasethe proliferation rate of Hep G2 cells.which The inhibition rate of anticoagulant components had no significant effect on the proliferation of Hep G2 cells.?4?Curcuma longa extract can effectively inhibited the release of t-PA,PAI-1 and IL-6 from HUVECs after thrombin stimulated.Curcuma longa extract,curcumin has double anti-inflammatory and anticoagulant functions.Curcuma longa extract possessed anti-coagulant dominant and the curcumin possessed anti-inflammatory anti-hepatoma effects.?5?In the HUVECs release medium,t-PA,PAI-1 and IL-6 factors have no significant changes for proliferation of the human Hep G2 cells.Curcumin on Hep G2 cells was slightly larger than the Curcuma longa extract.Further illustrated the effect of the Jiang Huang—Curcuma longa extract on Hep G2 cells with anticoagulant effect is dominant,and curcumin possessed anti-inflammatory anti-hepatoma effects.?6?The ratio ofno LPS group/LPS intervention group,compared with control group,1/2IC₅₀ and 1/4IC₅₀ Curcuma longa extract group after LPS intervention were able to made the number of the liver cancer cells through Matrigel adhesive increased,which invasion ability was significantly enhanced?P<0.05?.331.5?g/m L Curcuma longa extract LPS intervention group had no significant difference on HCC cell invasion ability?P>0.05?.?7?1/2IC₅₀ Curcuma longa extract in Hep G2 cells treated with 24 h,i NOS m RNA expression were significantly lowered significantly?P<0.05,0.01?.Curcuma longa extract group decreased obviously better than curcumin group in inflammatory condition,with significant differences between the twogroups?P<0.01?.WhentheLPSafter24htreatment,the pro-inflammatory effect of LPS on Hep G2 cells significantly?P<0.01?.For the Hep G2 cell inflammatory state after LPS intervention,the IL-6expression of Curcuma longa extract LPS intervention group and LPS intervention group of curcumin significantly inhibited?P<0.01?,the two groups had no significant difference in inhibition of equivalent?P>0.05?.?8?The results of Western blotting method detected the expression of i NOS protein showed that curcumin and Curcuma longa extract exposed Hep G2cell inflammatory under LPS intervention,which i NOS protein level were significantly inhibited.The Curcuma longa extract LPS intervention group decreased significantly stronger than curcumin LPS group.The difference between the two groups was significantly?P<0.01?.Detected the expression of IL-6 protein in Hep G2 cells showed that the inflammatory state intervention under LPS Curcuma longa extract LPS intervention group and curcumin LPS intervention group significantly inhibited the expression of IL-6?P<0.05?,the two groups showed no different effects?P>0.05?.Conclusion:1.Curcumin has excellent anti-inflammatory effects,which play antitumor effect mainly through anti-inflammatory pathways.2.Curcuma longa extract has excellent anticoagulant effect,although can significantly antagonize t-PA,PAI-1 clotting factor,the effect of against the proliferation and invasion of tumor cell is not significant.3.Curcumin and Curcuma longa extract mainly through down-regulation of IL-6,i NOS gene expression and the protein level of anti-inflammatory effect.And Curcuma longa extract the down-regulation of i NOS gene expression and protein level is lower,but its counter proliferation of tumor cells,invasion effect is not obvious.4.Jiang Huang play anti hepatocarcinoma effect mainly through anti-inflammatory pathways.