| OBJECTIVEExplore the role of Qingluoyin and Wenluoyin in RA angiogenesis and compare the differences between them,reveal mechanism of the effect, provide the experimental basis for clinical application for the treatment of RA.METHODSSD rats were continuous taken Qingluoyin and Wenluoyin by intragastric administration for five times, one hour later, abdominal aortic blood serum were separated. Qingluoyin containing medicine serum (QX) and Wenluo yin containting medicated serum (WX) was obtained. There are five experiental groups:normal mouse serum group (Con), IL-1 beta induced normal mouse serum group (Mod), QX (5%,10%, and 20%) group, WX (5%,10%, and 20%) group.30% ethanol reflux extraction of Qingluoyin and Wenluoyin, made Qingluoyin ethanol extract (QL), Qingluoyihn ethanol extract (WL). Consisted of the following groups:the blank group (Con), IL-1 beta induction group (Mod), IL-1 beta induction+QL (2,4,8 mg/ml) group, IL-1 beta+WL (2,4,8,16,32 mg/ml) group. TP group is divided into three Concentrations 1,10,50 ng/ml.RA-HFLS and HUVEC are inbubated With different Concentrations of Qingluoyin and Wenluoyin. Cell proliferative activity are detected by the incubation of MTS, to observe the effect of Qingluoyin and Wenluoyin on the proliferative of RA-HFLS and HUVEC; the influence of Qingluoyin and Wenluoyin on the migration of RA-HFLS and HUVEC are observed through the Transwell inserts; the effect of Qingluoyin and Wenluoyin on HFLS and HUVEC cells with the extracellular matrix adhesion are observed by using the extracellular matrix FN, one of main component, enveloped 96 well board; the role of Qingluoyin and Wenluoyin on RA-HFLS and HUVEC lumen formation ability are observed through the matrix gel enveloped 96 well board. In vivo Qingluoyin and Wenluoyin extracts have the influence on the angiogenesis in mice matrigel plug; Meanwhile ELISA test show Qingluoyin and Wenluoyin influence the expression level of angiogenic relevant cell factor (VEGF, Ang1, Ang2, TNF alpha, IL 17,) and HUVEC angiogenesis related receptor proteins VEGFR2 and Tie2.RESULTS:1. Effect on RA-HFLS proliferation, migration, adhesion and angiogenesis related factors (TNF alpha, IL-17, VEGF, Angl, Ang2)1.1. Effect on the proliferation of IL1-beta induced RA-HFLS of Qingluoyin and WenluoyinCompared with the Control group, IL-1 beta can obviously improve cell proliferation activity; QX group has no significant effect on IL-1 beta induced RA-HFLS proliferation; with the increased of Concentration,inhibition strengthen gradually, WX20 can significantly inhibit proliferation of RA-FLS (P<0.05). QL in lower Concentration 2-8 can inhibit the proliferation and WL in high doses 16-32 group just can significantly inhibit RA-FLS proliferation (P<0.01 or P<0.05), and shows the dose-dependence; Meanwhile TP10,50 group also inhibit the proliferation RA-HFLS,ands show dose-dependent (P<0.01)1.2. Effect on the migration of RA-HFLS of Qingluoyin and WenluoyinCompared with the Control group, VEGF can obviously increase the number of RA-HFLS cell migrated; With the increased Concentration,QX, QX20 group can significantly inhibit its migration (P<0.05); With the increased Concentration, WX10,20 group significantly inhibit its migration (P<0.05 or P<0.01); Meanwhile QL can inhibit the migration of RA-HFLS,show dose-dependent (P<0.05 or P<0.01); WL group with Concentration of WL8-32 group significantly inhibit the migration, with the increased Concentration, TP group inhibit the migration of RA-HFLS,show dose-dependent.1.3. Effect on RA HFLS adhesion of Qingluoyin and Wenluoyin.Compared with the Control group RA-FLS have strong adhesion ability to FN (P< 0.01); Meanwhile IL-1 beta induced RA-HFLS can significantly boost adhesion to FN (P < 0.05). QX, WX did show dose-dependent on the ability of adhesion. QL4 QL8, WL16, WL32. group significantly reduce the ability of adhesion (P<0.05 or P<0.01). At the same time, TP10,50 group, can significantly reduce reduce the ability of adhesion(P< 0.01). 1.4. Effect on RA-HFLS secreted angiogenic related factors (TNF alpha, IL-17, VEGF, Angl, Ang2) of Qingluoyin and WenluoyinCompared with the Control group, group inducted with IL1-beta can increase TNF alpha in RA-HFLS supernatant, QX did not. compared with IL1-beta induced group, WX20 significantly reduced TNF alpha level (P< 0.05). QL4,8, WL16,32 groups can significantly inhibit the secrete of TNF alpha (P< 0.05 or P<0.01), TP10,50 also inhibit the secrete of TNF alpha (P<0.05 or P<0.01).Compared with the Control group, group induced IL1-beta can significantly raise IL-17 level in RA-HFLS supernatant. QL4,8, WL16,32 group can significantly inhibit expression of IL-17 (P< 0.05 or P< 0.01), IL-17 level in TP groups are significantly reduced(P<0.05 or P<0.01)Compared with the Control group, group induced IL1-beta can increase VEGF in RA-HFLS supernatant. There is no significant differences on QX group and WX group. QL4, 8, WL32 group can significantly inhibit VEGF level in supernatant (P<0.05) TP group can inhibit VEGF level in supernatant, and show dose-dependent (P<0.05 or P<0.01).Compared with the Control group, group induced IL1-beta can increase Ang1 level in RA-HFLS supernatant, There is no significant differences on QX group and WX group. QL4,8, WL8,16,32 groups can significantly inhibit Angl level in supernatant (P < 0.05 or P< 0.01) and Ang1 level in TP group were significantly lower (P<0.05 or P< 0.01).Compared with the Control group, group induced IL1-beta can significantly increase Ang2 level in RA-HFLS supernatant, There is no significant differences on QX group and WX group. Meanwhile QL group had no significant effect on Ang2 RA-FLS secretion, but WL32 group can significantly inhibite the Ang2 express (P< 0.05). Along with the increase of Concentration TP50 group, Ang2 were significantly reduced (P< 0.05).2. Effect on proliferation, migration, adhesion, lumen formation of HUVEC and the expression of angiogenesis related receptor proteins VEGFR2 and Tie22.1. Effect on HUVEC proliferation of Qingluoyin and WenluoyinQX10 and QX20 group significantly inhibit proliferation of HUVEC; WX inhibit the proliferation,and show dose-dependent(P<0.01)QL2, QL4, WL16, WL32 all can significantly inhibit proliferation of HUVEC (P< 0.01), but compared with the same dosage group of WL, QL has strong inhibition effect. Meanwhile TP10,50 group significantly inhibit the proliferation of RA-HFLS(P< 0.01).2.2. Effect on migration of HUVEC of Qingluoyin and WenluoyinCompared with the Control group, VEGF can obviously increase quantity of HUVEC cells migrated; QX20, WX20 group can significantly inhibit its migration (P<0.05 or P< 0.01); QL group inhibit its migration,and show dose-dependent (P<0.05 or P<0.01); WL8～32 obviously inhibit the migration. TP group inhibit RA-HFLS migration,and show dose-dependent.2.3. Effect on adhesion of HUVEC of Qingluoyin and WenluoyinCompared with the group, HUVEC has stronger adhesion ability to FN (P<0.01); Meanwhile IL-1 beta induction can significantly increase HUVEC adhesion of FN (P< 0.05). QX, WX with increased Concentration, there were no significant influence on HUVEC adhesion ability. And QL can dose-dependently inhibit HUVEC adhesion (P< 0.05 or P<0.01), along with the increase of WL Concentration, WL16, WL32 can significantly reduce HUVEC adhesion ability (P<0.05 or P<0.01). And with the increase of Concentration of TP group, all can significantly reduce HUVEC adhesion ability (P<0.01).2.4. Effect on tube formation ability of Qingluoyin and WenluoyinCompared with the Control group, VEGF induced can significantly increase HUVEC formation in the lumen Matrigel, QL, WL, TP group, with the increase of Concentratio,can dose-dependently inhibit HUVEC formation the lumen. (P<0.01).2.5. Effect on the expression level of angiogenic related receptor proteins Tie2 and VEGFR2 on HUVEC of Qingluoyin and Wenluoyin(1)VEGFR2 levelCompared with the Control group, WX QX dose-dependently promote VEGFR2 expression of the HUVEC (P<0.01), meanwhile QL2 group significantly inhibit the expression of VEGFR2 (P<0.05), QL8 promote VEGFR2 expression. And WL2 can down regulation its expression, with Concentration increases, TP1,10 group were significantly lower VEGFR2 level (P<0.05), along with the increase of dose, in TP50 group VEGFR2 express are on the rise.(2) Tie2 levelCompared with the Control group, QX, WX dose-dependently promote VEGFR2 expression of HUVEC (P<0.01), QL, WL group, except WL2 group, with the increase of Concentration, Tie2 expression level all shows ascendant trend, [P< 0.05 or P< 0.01); with TP Concentration increases, down regulation transforms to up regulation3. Effect on angiogenesis in the mice matrigel plug of Qingluoyin and Wenluoyin. Compared with the Control group, through the observation, in VEGF induction groups, the amount of endothelial cell infiltrates in internal matrix glue, and a tube sample structure formation, explain that VEGF have promote angiogenesis role; QL8, WL32 TP50 group, compared to VEGF induction group, vascular endothelial cells infiltration are significantly less. |
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