Study On Synergy Of Compatibility Of Astragalus Mongholicus And Zedoray Rhizome And Mechanism Through Which The Compatibility Regulats Expression Of COX-2 By Influence PPARγ/NF-κB Single Pathway

AIM:1.Through study in vitro,we survey influence of Celecoxib,Rosiglitazone,Astragalus Mongholicus,Zedoray Rhizome and Compatibility of Astragalus Mongholicus and Zedoray Rhizome on growth and expression of AKP,LDH,VEGF in MKN-45 cell,and verify further that does Compatibility of Astragalus Mongholicus and Zedoray Rhizome has more effect than Astragalus Mongholicus and Zedoray Rhizome,meanwhile,observe influence to COX-2,NF-κB,PPARγin MKN-45 cell,and investigate the mechanism of action of regulating COX-2 by Chinese medicine again.2.we set up model of human gastric cancer original position orthotopic transplantation in BALB/c nude mice through tissue transfer method.Then we observe influence on growth and metastasis of human gastric cancer cell in BALB/c nude mice, and influence on expression of COX-2,NF-κB,PPARγ,and analysis that does Compatibility of Astragalus Mongholicus and Zedoray Rhizome has more effect than Astragalus Mongholicus and Zedoray Rhizome along,and synergistic action of Compatibility of Astragalus Mongholicus and Zedoray Rhizome,and machnism of regulating COX-2 expression.METHODS:Study in vitro:We cultivate human gastric cancer cell MKN-45 in vitro.Six groups are taken during logarithmic growth phase,Celecoxib group,Rosiglitazone group, Astragalus Mongholicus group,Zedoray Rhizome group,Compatibility group and Control group. Each medicine concentration is 100umol/L,20umol/L,10mg/L,5mg/L and 10mg/L.Collect cell after 24h,48h,72h.MTT method are taken to detect inhibition ratio to cell growth.LDH and AKP in MKN-45 are detected by spectrophotometric method.ELISA method are taken to detect suppression on expression of VEGF.And RT-PCR method are taken to detect expression-change of COX-2mRNA and NF-κBmRNA,PPARγmRNA,western blot method are used to detect expression-change of COX-2 proteinum.Study in vivo:MKN-45 cell is cultivated in vitro. Model of original position orthotopic transplantation human gastric cancer is set up in BALB/c nude mice through tissue transfer method.60 mouse is devided into 6 groups randomly: Celecoxib group,Rosiglitazone group,Astragalus Mongholicus group,Zedoray Rhizome group,Compatibility group and Control group.Three days later,after setting up model,we give medicine to nude mice(0.2ml/10g).CH(?)FRxLE grRXS:90P g·Ng^-1·G¹,Rosiglitazone grRXS:1.8P g·Ng^-1·G¹,A(?)DgDOXV 0 RngKR(?)XV grRXS:6.75g·Ng^-1·G¹,Zedoray Rhizome group: 4.5g·Ng^-1·G¹,CRP SD(?) grRXS:11.25g·Ng^-1·G¹,Control group:0.2ml/10g NS.Nude mice are put to death after 3 weeks and 6 weeks.We use electronic balance to detect weight of bearing cancer athymic mouse and tumor mass,and contrast the influence on weight of nude mouse body in every medicine group,and calculate inhibition ratio of tumor mass in every medicine group,and count hepatic metastasis lesion at the sixth week.We use immunohistochemistry method to detect microvessel density(MVD),and count inhibition ratio of MVD in tumor mass.And RT-PCR methods are used to detect expression of COX-2mRNA,NF-κBmRNA,PPARγmRNA of every group,and western blot methods are used to dtect expression-change of COX-2 proteinum.RESULTS:Study in vitro:Every medicine can inhibit cell growth,and the effect has time dependence.Celecoxib group has the best effect,The second one is CRP SD(?) grRXS,DnGL(?) inhibiting-effect is stronger than Astragalus Mongholicus group and Zedoray Rhizome group obviously.Rosiglitazone group has.The inhibition ratio of every medicine is:24h:11.28%, 3.91%,8.27%,8.86%,10.04%;48h:28.07%,15.11%,17.08%,24.57%,26.39%;72h:45.78%, 24.9%,28.34%,32.07%,39.68%.The inhibition effect of each medicine to cell differentiation enzyme begin at 48h,stronger at 72h.Celecoxib group is the best one.Every medicine can depress the expression of VEGF obviously.Totally,Celecoxib group and Compatibility group are best,and have manifest advantage than other 3 groups(P＜0.05).Following time goes,each medicine can down regulate COX-2mRNA and NF-κBmRNA at different extent.Excepting Zedoray Rhizome,each medicine can promote expression of PPARγmRNA.The expression-change of COX-2 proteinum coincide with COX-2mRNA.Study in vivo:In all medicines groups,only weight of nude mouse in Compatibility group are heavier than other groups(P＜0.01).The mouse weight of remanent 5 groups have no obvious disparity.Each medicine can restrain tumor growth after 3 weeks.Excepting Rosiglitazone group(P＜0.05),other medicine groups(P＜0.01).Inhibition ratio at this time are:25.27%,10.45%, 13.00%,13.72%,27.44%.After 6 weeks,every medicine group has manifest disparity,contrasting with control group(P＜0.01).Inhibition ratio at this time are:37.73%,20.45%,33.64%, 26.40%,43.12%.The restrain action on tumor MVD is apparent.After 3 weeks,Celecoxib grRXS'V DnG CRP SD(?) grRXS'V DrH 3＜0.01,Rosiglitazone group and Astragalus Mongholicus group are P＜0.05,contrasting with control group.Inhibition ratio to MVD at this time are:41.94%,12.90%,12.90%,25.81%,38.71%.After 6 weeks,every medicine groups has apparent disparity(P＜0.01).Inhibition ratio to MVD at this time are:43.75%,18.75%, 27.08%,21.88%,50.00%.We find no hepatic metastasis lesions at the third week.At the sixth week,hepatic metastasis lesions of every medicine group are obviously less than control group. hepatic metastasis lesions of Compatibility group are least than each other groups.No conspicuous disparities are found in other 5 groups.Results of gel electrophoresis products of RT-PCR and proteinum strap gray scale demonstrated that Celecoxib and Compatibility can depress expression of COX-2 apparently(P＜0.01),and after 6 weeks,effect of Compatibility is stronger than Celecoxib(P＜0.05),and Rosiglitazone also can inhibit COX-2,but the result has no statistically significance.To PPARγexpression,after 3 weeks,Rosiglitazone can promote PPARγexpression significantly(P＜0.01),and,after 6 weeks,Rosiglitazone group and Compatibility group have statistically significance,contrasting with control group(Rosiglitazone group:P＜0.01,Compatibility group:P＜0.05).To NF-κB expression,Rosiglitazone group and Compatibility group can restrain notably,after 3 weeks:P＜0.05,and after 6 weeks:P＜0.01. Astragalus Mongholicus also has statistically significance at the 6^th week.CONCLUSION:1.The compatibility of Astragalus Mongholicus and Zedoray Rhizome has synergistic action on depression of tumor cells growth.2.COX-2 is one of the important targets, through which the Compatibility of Astragalus Mongholicus and Zedoray Rhizome produce a marked effect to gastric cancer,and the Compatibility has synergistic action on depression of COX-2.3.The Compatibility of Astragalus Mongholicus and Zedoray Rhizome may regulation the expression of COX-2 partly through PPARγ/NF-κB single pathway.4.The Compatibility of Astragalus Mongholicus and Zedoray Rhizome can effectively improve survival quality and depress hepatic metastasis of cancer cells.