| Objective:In the in vivo and in vitro,to study the reversal effect of Huoxue Huayu Decoction of Curcuma wenyujin on human gastric cancer SGC-7901/VCR multidrug resistance and its possible mechanism.To provide the basis for expanding the use category of Curcuma wenyujin in clinic and a theoretical basis for the application of reversal of multidrug resistance of gastric cancer about promoting blood circulation and removing blood stasis method.Methods:With human gastric cancer multidrug resistance cell line SGC-7901/VCR,and establish the model of SGC-7901/VCR xenograft tumor.As a strong reversal of MDR activity with the generally acknowledged in vitro verapamil is regards as the control,based on the former research results,and to intervene with the different concentration of Huoxue Huayu Decoction of Curcuma wenyujin.after treating with Wen Yujin,to analyse proliferation,cell cycle and apoptosis by flow cytometry and MTT assay,expression of P-gp and LRP by immunohistochemistry and Western-blot,xenograft tumor ultrastructure by the electron microscope and expression of P-gp protein in tumor by immunohistochemistry and Western-blot.Results:?In vitro study:MTT assay showed that SGC-7901/VCR has strong resistance for VCR,ADM,5-FU,DDP,VCR,ADM,5-FU,DDP of SGC-7901 IC50 were 0.019±0 ug/ml,0.255 ±0.026 ?g/ml,3.335 ±0.267 ?g/ml,0.778 ±0.152 ?g/ml,IC50 of SGC-7901/VCR were 106.111±3.008,5.700±0.610,20.712 ±5.097,1.059±0.101,resistance index was 5584.789,22.353,6.21,1.361,SGC-7901/VCR with no drug culture proliferation activity increased significantly.The Alcohol extract Of Curcuma wenyujin IC5 95%interval 24h were 59.28(30.96,86.01).the three concentrations of Curcuma wenyujin are 20ug/ml,40ug/ml,80ug/ml,and verapamil is 10ug/ml with non-toxic concentration internationally recognized for subsequent testing.After treatment with different concentrations of Curcuma wenvujin and VP.we found that they all can reverse the resistance of SGC-7901/VCR to ADM and VCR.reverse index were 1.586.6.287.10.341.95.561 and 1.51.4.07.28.95.21.27.At the same time can promote SGC-7901/VCR apoptosis induced by VCR,early apoptosis and late apoptosis was increased in every groups,especially in the early apoptosis,the apoptosis index of control group.VCR group.W20ug/ml,W40ug/ml.W80ug/ml and VP group were 7.43± 0.81,21.4 ± 1.13,23.3 ±0.93,29.4 ±0.74,33.3± 1.01,37.4 ±0.96.compared with the control group,the apoptosis index of each groups was statistically significant.different concentrations of Curcuma wenyujin induced apoptosis in a concentration dependent manner with a significant difference between each concentration.Curcuma wenyujin and VP can promote VCR to retardant cells in G2/M phase significantly,decrease G1,G2/M were 7.25±0.95,24.32±2.33,44.16 ± 1.70,59.34± 13.19,70.59±6.63,64.31±5.86(P<0.05),increased Intracellular fluorescent expression of ADM 1H.Respectively were 0.2 ±0.12%,18.6± 10.28%,72.95 ± 11.14%,69.25±11.83%,71.75 ±3.52%,64.15 ±7.68%(P<0.05).P-gp and LRP protein were detected by immunohistochemistry and W-B,we found that Curcuma wenyujin and VP can down-regulate the expression of P-gp protein,Curcuma with the concentrations of 80?g/ml after cultivating 24 hours can reduce P-gp protein to level of resistance level of Non resistant strains.The expression of LRP is different between SGC-7901/VCR and SGC-7901(P<0.05),but in the curcuma wenyujin and VP treated groups,the expression of LRP had no significant change(P>0.05).?In the in vivo study:The success rate of transplant subcutaneous sarcoma of SGC-7901/VCR is 100%,divided into 6 groups,including the model group.VCR group,Curcuma wenyujin low dose group,high dose group,low dose of Curcuma wenyujin plus VCR group,VCR group of high dose of Curcuma wenyujin.We found that Curcuma wenyujin alone had no significant inhibitory effect on transplanted tumor,but Curcuma wenyujin combined with VCR can inhibit tumor growth,Curcuma wenyujin high dose plus VCR group has The strongest inhibitory rate with minimum volume and with statistical significance as compared with VCR group(P<0.05),the tumor inhibition rate was 51.56%,there were statistically significant with the model group and VCR group(P<0.05).With Electron microscopy we found that all groups except the model groups can induce chromatin margination.in Curcuma wenyujin plus VCR groups chromatin fragmentation and margination obviously,and with the increasing dose of Curcuma wenyujin.chromatin margination is obvious to a higher degree.By immunohistochemistry and WB.The expression of P-gp protein in Curcuma wenyujin combined VCR group down-regulated compared with the model group and VCR group with statistical significance(P<0.05).Conclusion:From in vitro to in vivo,Curcuma wenyujin of Huoxue Huayu Decoction can reverse multidr resistance in human gastric cancer SGC-7901/VCR,the mechanism may be related to inducing cell apoptosis,cell cycle arrest,improve chemical intracellular dr concentration,the expression of P-gp protein,but independent of LRP.The result extends the category of clinical use of Wen Yujin,further confirme that promoting blood circulation and removing blood stasis is one of the key principle in the treatment of tumors,and sgest prescription based on promoting blood circulation and removing blood stasis is the important direction of reversing the multidr resistance of gastric cancer. |
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