Study On The Apoptosis Of HL-60 Cells And HepG2 Cells By Serum Containing Compound Huangdai Tablets

Purpose: Preparation of serum containing Compound Realgar Natural Indigo Tablet（CRNIT）according to serum pharmacological method.Study shows that the serum containing CRNIT induces apoptosis of HL-60 cells and Hep G2 cells.To study the effect of CRNIT on cancer cell line in vitro under the condition more close to the real absorption and metabolism in vivo.To confirm the reliability of the mechanism of action of the serum pharmacological of traditional Chinese medicine in vitro experiments.Method: CRNIT suspension was prepared,and SD rats of clean grade were gavaged twice a day for 3 consecutive days.Serum was extracted from abdominal aorta of rats after the last gastric perfusion.The concentration of arsenic in serum was measured by ICP-OES.Serum arsenic concentrations of 0,10,20 and 40 μg·L^-1 were selected to act on HL-60 cells.The morphological difference of HL-60 cells growth observed under light microscope.Hochest/Calcein-AM/PI staining and Annexin V-FITC/PI double staining were used to determine the effect on apoptosis of HL-60 cells,and Western Blot was used to detect the expression of apoptotic protein related to HL-60 cells.Serum arsenic concentrations of 0,10,20,40,80,and 160 μg·L^-1 were selected to act on HL-60 cells.The inhibition rate of HL-60 cells was determined by CCK-8 assay.Serum arsenic concentrations of 0,80,120 and 160 μg·L^-1 were selected to act on Hep G2 cells.The morphological difference of Hep G2 cells growth observed under light microscope.Hochest/Calcein-AM/PI staining and Annexin V-FITC/PI double staining were used to determine the effect on apoptosis of Hep G2 cells,and Western Blot was used to detect the expression of apoptotic protein related to Hep G2 cells.Serum arsenic concentrations of 0,40,80,120,160,and 200 μg·L^-1 were selected to act on Hep G2 cells.The inhibition rate of Hep G2 cells was determined by CCK-8 assay.Results: 1.The concentration of arsenic in CRNIT serum was 10 μg·L^-1 by ICP-OES.2.After the drug-containing serum acted on HL-60 cells and Hep G2 cells for 24 h,Observation under light microscope,the growth trend of both HL-60 cells and Hep G2 cells became looser and looser with the increase of drug-containing serum concentration,and the edges were blurred or even fragmented.3.CCK-8 was used to detect the effect of different concentrations of drug-containing serum on HL-60 cells and Hep G2 cells for 24 hours.The arsenic concentration of serum on HL-60 cells was 10,20,40,80,and 160 μg·L^-1,and the inhibition rate was 1.25%,8.74%,47.45%,68.11%,75.62%.The arsenic concentration of serum on Hep G2 cells was 40,80,120,160,and 200 μg·L^-1,and the inhibition rate was 1.70%,4.83%,37.55%,65.41%,93.21%.4.After Hochest/Calcein-AM/PI fluorescence staining,the number of viable cells decreased and the number of apoptotic cells increased in HL-60 cells and Hep G2 cells with the increase of drug-containing serum concentration.5.HL-60 cells and Hep G2 cells acted on different concentrations o f drug-containing serum,and the total apoptotic rate of cells increased in turn by flow cytometry.6.Western Blot assay was used to measure the expression of apoptotic proteins.With the increase of drug-containing serum concentration,the expression of apoptotic protein Bcl-2 was down-regulated and the expression of apoptotic protein Bax was up-regulated in HL-60 cells and Hep G2 cells.Conclusion: 1.The degree of apoptosis of HL-60 cells and Hep G2 cells was positively correlated with the concentration of CRNIT-containing serum.The increase of C RNIT-containing serum and its concentration enhance the inhibition of proliferation of HL-60 cells and Hep G2 cells,and the more significant the degree of inducing apoptosis.2.HL-60 cells are suspended cells belong to hematological tumors,Hep G2 cells are adherent cell belong to solid tumors,solid tumor cells compared with hematological tumor cells produce inhibition of cell growth,apoptosis results,need greater drug concentration.3.Traditional Chinese medicine uses serum pharmacology method,and the drug acts on the in vitro experiment after half-in-vivo experiment,which increases the reliability of the experiment,and provides a new way for Chinese medicine compound to act on the in vitro experiment in scientific research.