Effects Of Baicalin Magnesium Salt On Proliferation,Migration And Apoptosis Of Hep G2 Cells

Liver cancer is one of the malignant tumors that pose a grave threat to human health.The early symptom of hepatocellular carcinoma is not obvious and the optimum treatment chance is lost when diagnosed.In the middle and late stages,radiation therapy and chemotherapy were primarily employed.The effect of chemotherapy drugs on tumor cells did not achieve the expected results and the chemotherapy drugs’side effect can reduce patients’quality of life and caused the complications such as irreversible liver and kidney damage-severe emesis,fever,weakened immunity,etc.Therefore,novel anti-cancer drugs that are less harmful to humans are needed.Traditional Chinese medicine has very unique benefits in the treatment of liver cancer.Chinese medicine will not generate drug tolerance and has less antagonistic role,which can improve the survival rate among patients.Clinical experiments had demonstrated that the combined adjuvant treatment of liver cancer with Chinese medicine had remarkable curative effects.The newly rewritten cancer treatment guidelines of NCCN also endorsed the combined application of TACE and Chinese medicine.However,the role of TCM in cancer therapeutics had been underappreciated.This might be related to the fact that the mechanism of many herbs was not yet fully identified.Therefore,the hot spot of investigation at present is to investigate curative effect of herbal medicine in curing tumors and its mechanism to enhance the commercial application of Chinese herbs for clinical exploitation and treatment.Baicalin is commonly applied clinically as an adjunct to the treatment of hepatitis and many experiments had demonstrated its anti-cancer values.Scutellaria extracts include baicalin,wogoninside,baicalein,etc.Their clinical application is limited because these traditional compounds could not directly dissolve in water.In order to make baicalein extensively applicable in the field of clinical applications,Professor Liu from our School of Pharmaceutical Sciences found and synthesized new magnesium salt of baicalein（BA-Mg）and had applied for a patent.BA-Mg is very soluble in water,which compensates for the very poor solubility of baicalin in water.The aim of this study was to investigate the effects of baicalin magnesium salt（BA-Mg）on the proliferation,migration and apoptosis of through Hep G2 in vitro trials.Objective:Investigate the role of baicalin magnesium salt in the multiplication,emigration and apoptosis of Hep G2 for subsequent experimental application of BA-Mg.Methods:1.The CCK-8 method was performed to test the results of growth and proliferation of hepatocellular carcinoma Hep G2 cells by Mg scutellaria salt（drug purity 82.6%）at different levels at 150,175,200,225,250,275 and 300μg/m L in each group.Calculated the IC₅₀（half inhibition concentration）.2.Three drug concentrations near the half-inhibitory concentration of baicalin magnesium salt on liver cancer cells were selected to be the groups of low,medium and high density of baicalin mescaline salt were selected accordingly.The fourth group was the control drug baicalin group.Plus the control group for a total of 7 groups.It was divided into Low-dose scutellaria magnesium salt group（200μg/m L）,Mid-dose scutellaria magnesium salt group（250μg/m L）.Takayasu scutellaria magnesium salt group（300μg/m L）,baicalin Group（using DMSO as organic solvent）,DMSO group（excluding the influence of organic solvents in the baicalin group）,magnesium sulfate group(excluding the influence of Mg²⁺in BA-Mg)and blank control group.Add drugs to 7 dishes of cells respectively and select appropriate drug action time according to the results of CCK-8 for follow-up experiments.3.Community formation experiment was performed to see the changes in the clonogenic ability of cells after various drug treatments.4.Cytological analysis of cell cycle changes using flow cytometry Flow cytometry view of the cell cycle.5.Changes in apoptosis observation using flow cytometry.6.Detection of changes in the ability of each group of cells to migrate by cell scoring method.7.Western blot was used to investigate the variation in the manifestation of cell multiplication,migration and apoptosis related proteins ROCK-1,Bax,Bcl-2,CyclinE,Caspas-9,PCNA,etc.Results:1.Findings from the CCK-8 test revealed that the inhibitory effect of baicalin magnesium salt on Hep G2 cell proliferation was positively correlated with administration time and drug concentration.The half-life concentration(i.e.IC₅₀)of the drug at 82.6%purity was 297.9μg/m L at 24 h and 237.9μg/m L at 48h.The inhibition rate was higher at 48 h than at 24 h for the simultaneous drug levels;Less drug was required at 48 h for the simultaneous inhibition rate.The results indicated that the drug was more effective at 48 h.Thus,48 h was picked as the follow-up experiment time.200μg/m L,250μg/m L and 300μg/m L were taken as the follow-up laboratory levels.2.The results of the colonization experiment were as follow.The colonization rates of the first four drug groups treated with Hep G2 cells were（36.50±2.52）%,（27.94±2.52）%,（11.17±0.67）%and（23.56±0.54）%of colonization inhibition by each.All four drug groups were remarkably dissimilar compared with the control group（P<0.05）.The discrepancy among the three dose groups of baicalin magic salt was found to be of statistical value（P<0.05）.At the same dose,magnesium salts could inhibit cell promotion more markedly（P<0.05）.DMSO and magnesium sulfate did not significantly affect the ability to form cell colonies（P>0.05）.3.The flow cycle results suggested that the ratio of the G2 phase in the first four experimental groups is clearly less than the ratio in the control group（P<0.05）.The ratio of cycles influenced by normal cells subjected to DMSO in the fifth group and normal cells affected by magnesium sulfate in the sixth group was not significant（P>0.05）.The discrepancies among the 200μg/m L baicalin magnesium salt group（low dose group）,the 300μg/m L baicalin magnesium salt high dose group were remarkable,the discrepancy between the medium dose group（250μg/m L）and the high dose group was significant.The group（300μg/m L）difference was considered visually meaningful（P<0.05）.A statistically considerable amount of variance was found between the medium dose group（250μg/m L）and the high dose group（300μg/m L）.The changes were highly significant in the baicalin magic salt group compared with the same dose of baicalin.（P<0.05）.4.The findings of apoptosis detection by flow cytometry suggested that each drug group manifested obvious apoptosis when compared to the control group（P<0.05）.The DMSO and Mg²⁺groups showed no significant effect on Hep G2 cell apoptosis（P>0.05）.Significant differences were observed when the groups were compared between lower,moderate and hyper-concentrated levels of magnesium salts.（P<0.05）.Under a same dosage,baicalin magnesium salt significantly promoted Hep G2 cell apoptosis（P<0.05）compared with baicalin.5.Scratch test results were as follows:Scratch tests at each time period showed that both scutellaria magnesium salt and baicalein markedly depressed the ability of Hep G2 to migrate.The results were most pronounced after 12 hours after scoring.The cure rate in the control group was five more times than that in the high-dose baicalin magnesium salt treatment group（P<0.05）.In contrast,the cell mobility was not obviously curtailed in the DMSO and MAGS groups（P>0.05）.At 12 h,24 h,and 36h.There was a major different among the different drug concentrations（P<0.05）.At 12 h,24 h,and 36 h.There was a remarkable differences observed between the group with high dose of baicalin magnesium salt and the group with the same dose of baicalin（P<0.05）.The baicalin salt group had the lowest cell scratch healing rate.At 48 h,no significant variation was observed between the high-dose baicalin magnesium salt group and baicalin group.（P>0.05）.6.Results of WB indicated that magnesium salts of Scutellaria baicalensis depressed the levels of CyclinE,ROCK-1 and PCNA.Lowered the level of Bcl-2 protein presentation and raised the level of Bax and Caspase-9production in Hep G2 cells（P<0.05）.Baicalin magnesium salt was pro-apoptotic,cell cycle arrested and reduced emigration.Summary:1.Both BA and BA-Mg satins unfavorably influenced the cell growth and migrating power of Hep G2 cells,contributed to the apoptosis and cell cycle arrest of Hep G2 cells.2.The suppressive role of baicalin magnesium salt on cell proliferation,migrations,cell cycle arrest and pro-apoptosis might be related to the low level of expression of Bcl-2,ROCK-1,PCNA,CyclinE and high level of exposure to Bax and Caspase-9.3.At the same dose,baicalin magnesium salt was more effective than baicalin.