Effects Of Vitex Scutellarin On Proliferation And Apoptosis Of Monocyte Macrophages

Fructus viticis has been recorded in Shennong's Herbal Classic,the earliest pharmacy monograph in China.Traditional Chinese Medicine clinic often uses it to treat wind-heat fever headache,painful gums,red eyes,obscure eyes,dizziness,etc.Modern pharmacological studies have shown that fructus viticis has anti-tumor,antipyretic analgesic,anti-inflammatory,antibacterial,antioxidant effects.Vitexicarpin is one of the effective components of fructus viticis,belonging to polymethoxylated flavonoids.In recent years,studies on anti-tumor effects showed that vitexicarpin has a strong proliferation inhibitory activity against a variety of tumor cells,making it possible to become a plant-derived anti-tumor drug candidate.The immunomodulatory effects of vitexicarpin have also been noticed earlier.Some studies have reported that vitexicarpin can inhibit the proliferation of mouse T lymphocytes and B lymphocytes.Recent studies have shown that vitexicarpin has anti-inflammatory effects and may improve the degree of foot swelling in adjuvant arthritis mice by regulating inflammatory cytokines.The above studies show that vitexicarpin may have good application prospects for immune related diseases.However,up to now,no studies have been reported on the role of vitexicarpin in mononuclear macrophages belonging to immune cells.Mononuclear-macrophages are important immune cells in the body.They not only participate in non-specific immunity,but also are important antigen-presenting cells and effector cells in specific immunity.They participate in numerous physical and pathological processes of the body.Therefore,it is necessary to study the effect of vitexicarpin on mononuclear macrophages.Research purposeThis paper studied the effect of vitexicarpin on mononuclear macrophages,and explored its effect on immune cells.These provide more pharmacological basis for the TCM clinical use of fructus viticis in the treatment of immune related disorders.Research contentIn this study,mouse mononuclear macrophage leukemia cell RAW264.7 and human monocytic leukemia cell THP1 were investigated,to investigate the effect of vitexicarpin on proliferation and apoptosis of monocyte-macrophages.The mechanism of cell apoptosis was explored from the level of reactive oxygen species and mitochondrial membrane potential.In order to objectively reflect the effects of drugs on organisms,cell activity experiments also used mouse peritoneal macrophages as research objects.Research method1.Cell activity experiments:RAW264.7 cells,mouse peritoneal macrophages and THP1 cells were treated with different concentrations of vitexicarpin for 24,48,and 72 hours,respectively,and CCK8 assay was used to measure cell activity.2.Nucleus fluorescence staining was used to observe the apoptosis cell morphology:the morphological changes of apoptosis in RAW264.7 cells and THP1 cells were observed using nuclear fluorescent double staining.3.Flow cytometry was used to detect the apoptosis rate:the apoptosis rate of RAW264.7 cells was detected by flow cytometry using PI staining.4.Observation and determination of cellular reactive oxygen levels:the reactive oxygen species in RAW264.7 cells and THP1 cells were labeled with the fluorescent probe DCFH-DA,qualitatively observed under a fluorescence microscope,and quantitatively detected fluorescence intensity by a microplate reader.5.Observation and determination of cell mitochondrial membrane potential:mitochondria in RAW264.7 cells and THP1 cells were stained with JC-1 for qualitative observation under a fluorescence microscope or quantitatively detected by a microplate reader.Research result1.The effects of vitexicarpin on proliferation and apoptosis in RAW264.7 cells.The results of cell activity experiments show that vitexicarpin inhibited the proliferation of RAW264.7 cells in a dose-and time-dependent manner.The IC50(50%inhibitory concentration)of vitexicarpin on RAW264.7 cells at 24,48,and 72 hours was 6.202,2.628,and 3,064 ?mol/L,respectively.The results of nuclear fluorescence staining showed that RAW264.7 cells exhibited apoptotic morphology after being treated with a concentration of 5?mol/L and 10?mol/L of vitexicarpin.The results of flow cytometry showed that the percentage of apoptotic cells in the Sub-G1 phase of RAW264.7 cells induced by vitexicarpin increased(P<0.05).The results of reactive oxygen species(ROS)detection showed that intracellular ROS levels were increased after vitexicarpin acted on RAW264.7 cells.The results of JC-1 staining showed that vitexicarpin could reduce the mitochondrial membrane potential of RAW264.7 cells.2.The effects of vitexicarpin on proliferation of mouse peritoneal macrophagesAfter 24 hours and 48 hours of exposure to various concentrations of vitexicarpin in mouse peritoneal macrophages,the dose of 50 ?mol/L vitexicarpin was only weakly inhibited,while the inhibitory effects of other concentrations were not obvious.After 72 hours,all concentrations of drugs showed different degrees of inhibition.With the increase of drug concentration,the inhibitory effect gradually increased,showing dose-dependent effects.Among them,50 ?gmol/L of vitexicarpin had the strongest inhibitory effect and the inhibition rate was 26.4%.3.The effects of vitexicarpin on proliferation and apoptosis in THP1 cells.The results of cell activity experiments show that vitexicarpin inhibited the proliferation of THP1 cells in a dose-and time-dependent manner.The results of nuclear fluorescence staining showed that,compared with the normal group,the number of THP1 cells exhibiting apoptotic morphology increased after being treated with 15 2,5,and 10?mol/L of vitexicarpin.The results of ROS detection showed that,as the concentration of vitexicarpin increased,the level of ROS in THP1 cells increased significantly.The levels of ROS in the vitexicarpin groups with concentrations of 1,2,5,and 10 ?mol/L were significantly higher than those in the normal control group,showing concentration-dependent effects.The results of JC-1 staining showed that with the increase of vitexicarpin concentration,the red fluorescence of the group with 2,5,10 ?mol/L of vitexicarpin decreased,and the green fluorescence increased,indicating that the mitochondrial membrane potential decreased and these cells may be in an apoptotic state.Research conclusionVitexicarpin can inhibit the cell activity of RAW264.7 cells,mouse peritoneal macrophages and THP1 cells.Especially,vitexicarpin have stronger inhibitory activity against the cell activity of RAW264.7 cells and THP1 cells with proliferative ability,and vitexicarpin can promote its apoptosis.Its mechanism is related to the increase of intracellular ROS levels and the decrease of mitochondrial membrane potential.The inhibition of proliferation and promotion of apoptosis of monocyte-macrophage cells showed that it may play an important role in immune function and provide more pharmacological basis for the clinical use of fructus viticis in the treatment of immune related disorders.The innovation of this study1.In this study,it was found that vitexicaipin can inhibit the proliferation of RAW264.7 cells,mouse peritoneal macrophages and THP1 cells,and can promote apoptosis of RAW264.7 cells and THP1 cells.2.This study revealed that the mechanism of apoptosis of RAW264.7 cells and THP1 cells promoted by vitexicarpin was related to the increase of intracellular reactive oxygen species and the decrease of mitochondrial membrane potential.3.The study on the proliferation and apoptosis of mononuclear macrophage by vitexicarpin provides more pharmacological basis for the clinical use of fructus viticis.