| Objective:The leech extract was prepared by water extraction method,and the content of hirudin in the extract was indirectly detected by thrombin(according to pharmacopoeia 2015)and the components of the extract were detected by high resolution liquid-mass spectrometry(QE)technology,respectively,so as to determine the main components of the leech extract.By observing the effects of leech extract on the activity,cycle,apoptosis and invasion of human Retinoblastoma RB weri-rb-1 cells,the inhibitory effect of leech extract on the growth and invasion of human Retinoblastoma RB weri-rb-1 cells was verified.Vascular endothelial growth factor VEGF,hypoxia inducible factor-1a hif-1a,and Matrix metalloproteinase-9 were expressed in weri-rb-1 cells by leeches extract The effect of mmp-9 on the Phosphatidylinositol 3kinase/Protein Kinae B PI3K/AKT pathway,which elucidated the possible molecular mechanism of leech extract inhibiting weri-rb-1 cells.The potential of leech extract to inhibit tumor angiogenesis was studied by observing the effect of leech extract on the activity of Human Vascular endothelial cells(HUVEC)and the expression of VEGF and Vascular endothelial Growth Factor Receptor 2(VEGFR2)..Methods:1.Preparation and detection of leech extract: The leech extract was prepared by water extraction,and the content of hirudin was determined indirectly by thrombin.The main components of leech were analyzed by-QE technique.2.Effects of leech extract on activity,cycle,apoptosis and invasion of weri-rb-1 cells:(1)weri-rb-1 cells were co-cultured with leech extracts of different concentrations(0.02 u /ml,0.04 u /ml,0.08 u /ml,0.16 u /ml)for 0,24,48,and 72 hours.The optimal drug intervention concentration and time point were selected by cck-8.(2)The concentration of two leech extracts(0.04 u /ml and 0.08 u /ml)and 48 h were used as the drug intervention conditions,and the effect of the drug on the cell cycle was detected by flow cytometry.(3)The effect of drugs on apoptosis was detected by flow cytometry with 2 experimental concentrations and 48 h as the drug intervention conditions.(4)The transwell invasion assay examined the effect of two experimental concentrations on the invasion of weri-rb-1 cells.The cells of the control group were cultured in medium only,and no drugs were added.3.The leech extract expressed VEGF in weri-rb-1 cells Effects and related molecular mechanisms:(1)the concentration of two leech extracts(0.04 u /ml and 0.08 u /ml)interfered with weri-rb-1 cells for 48 h.The effect of leech extracts on the expression of VEGF in weri-rb-1 cells was detected by Elisa.(2)Leech extract concentration was used to interfere weri-rb-1 cells for 48 h in the experimental group.Rt-pcr was used to detect the effect of leech extract on the expression levels of hif-1a and mmp-9 m RNA in weri-rb-1 cells.(3)Leech extracts were used in the experimental group to interfere with weri-rb-1 cells for 48 h,and Western Blot analysis was performed to determine the effect of leech extracts on the expression of hif-1a,mmp-9,PI3 K and Human phosphorylated AKT protein p-akt in weri-rb-1 cells.The cells of the control group were cultured in medium only,and no drugs were added.4.Effect of leech extract on the activity and expression of VEFG and VEGFR2 in HUVEC cells:(1)HUVEC cells were co-cultured with leech extracts of different concentrations(0.02 u /ml,0.04 u /ml,0.08 u /ml,0.16 u /ml)for 0,24,48,and 72 hours.The optimal drug intervention concentration and time point were selected by cck-8.(2)After the concentration of two leech extracts(0.04 u /ml and 0.08 u /ml)interfered with HUVEC cells for 48 h,rt-pcr was used to detect the effect of leech extracts on the expression levels of VEGF and VEGFR2 m RNA in HUVEC cells.(3)After the concentration of leech extract in the experimental group was used to interfere with HUVEC cells for 48 h,the effect of leech extract on the expression of VEGF and VEGFR2 protein in HUVEC cells was detected by Western Blot.Results:1.Preparation and detection of leech extract: the concentration of hirudin in leech extract was determined by thrombin in the 2015 edition of Chinese pharmacopoeia.After repeated extraction and determination,the concentration of hirudin in leech extract remained relatively stable.The results of high resolution liquid-q analysis showed that the leech extract contained various components such as nucleosides,amino acids,inosine,purines and vitamins.2.Effects of leech extract on activity,cycle,apoptosis and invasion of weri-rb-1 cells:(1)the inhibition rates of leech extract in the group of 0.04 u /ml and 0.08 u /ml on RB cells at 48 h were 9.70% and 9.92%,respectively,showing statistically significant differences with the control group(OD value P <0.05).(2)The RB cells in the experimental group were mainly blocked in the G2/M phase.The positive cell rates(%)in G2/M phase were 3.00±2.32,12.59±5.36 and 14.79±4.12 in the control group,0.04U/ml leech extract group and 0.08U/ml leech extract group,respectively(P <0.01 in comparison with leech extract group).(3)the concentration of leech extract in the experimental group could increase the apoptosis rate of RB cells.The apoptosis rates of the above three groups were 4.64 ± 2.56,37.91 ± 3.44 and 33.05 ± 2.25,respectively(compared with the control group,P <0.01).(4)the number of cells in the Transwell chamber of the experimental group and in each high-power visual field was significantly reduced compared with the control group,and the OD values of the three groups were 1.21±4.36,0.44±2.08,and 0.53±3.42,respectively(compared with the control group,P <0.01).3.The leech extract expressed VEGF in weri-rb-1 cells Effects and related molecular mechanisms:(1)elisa results showed that the expression of VEGF in the medium of the extract concentrations of 0.04 u /ml and 0.08 u /ml of 2 leeches was decreased compared with the control group,which was statistically significant(P <0.05).(2)Rt-pcr results showed that compared with the control group,the m RNA levels of hif-1a and mmp-9 were decreased,both of which were statistically significant(P <0.05).(3)Western Blot results showed that compared with the control group,the protein contents of hif-1a,mmp-9,PI3 K and p-akt decreased,all of which were statistically significant(p <0.05).4.Effect of leech extract on the activity and expression of VEFG and VEGFR2 in HUVEC cells:(1)0.04 u /ml and 0.08 u /ml leech extracts were used to interfere with weri-rb-1 cells for 48 h,and the inhibition rates of the cells were 12.96 ± 1.59 and 14.81 ± 2.17,respectively,showing statistically significant differences with the control group(OD value P <0.05).(2)Rt-pcr results showed that m RNA levels of VEGF and VEGFR2 were decreased compared with the control group,both of which were statistically significant(P <0.05).(3)Western Blot results showed that the protein contents of VEGF and VEGFR2 were significantly lower than those of the control group(P <0.05).Conclusion:1 The content of hirudin in the leech extract by water extraction was stable.Leech extract contains nucleosides,amino acids,inosine,purines,vitamins and other components.2 Leech extract inhibited weri-rb-1 cells in retinoblastoma by inhibiting the activity of weri-rb-1,inducing cycle arrest,inducing apoptosis and inhibiting cell invasion.3 Leech extract inhibited weri-rb-1 cells through the VEGF/PI3K/AKT pathway,and at the same time inhibited the activity of hif-1a and mmp-9 in weri-rb-1 cells.4 Leech extract inhibited the activity of HUVEC cells and the expression of VEGF and VEGFR2 protein in HUVEC cells.To sum up,in vitro experiments,leech-derived extract may affect the cell cycle distribution,apoptosis and invasion of weri-rb-1 through the VEGF/PI3K/AKT pathway,hif-1a and mmp-9 factors to a certain extent,so as to inhibit RB.At the same time,it was found that the leech extract may inhibit the expression of VEGF and VEGFR2 in umbilical vein endothelial cells.Our results indicate that leech extract has an inhibitory effect on RB cells and may also have an inhibitory effect on RB blood vessels.The effect of promoting blood circulation and removing blood stasis,as well as exfoliating blood bath,has been further verified from the theory of TCM.The above results provide some preclinical data for the development of a new antitumor preparation of leech. |
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