| ObjectiveObserve the inhibitional effect of Kunmu Extract on human fibroblast-like synoviocytes of rheumatoid arthritis and learn about the effect on cell cycle、 apoptosis and VEGF expression. Explore the mechanism of the effect of Kunmu Extract on cell cycle and apoptosis of RA-HFLS through measuring cyclin D1、 bcl-2、bax and caspase-3. Reveal the possible mechanism by observing the phosphosylation role of ERK、JNK、P38 proteins in mitogen-acivated protein kinase pathway.MethodsHuman fibroblast-like synoviocytes are treated by Kunmu Extract of chloroform、ethyl acetate、petroleum ether and butanol. Inhibitional rate of growth is tested with CCK-8 in 24 hours、48 hours and 72 hours. Cell cycle and apoptosis is also detected through flow cytometry in 24 hours. Cyclin D1、 VEGF、bcl-2、bax and caspase-3 is determined by qPCR and ERK、 pER、JNK、 pJNK、P38 and pP38 is detected by western blot, both treated by Kunmu Extract of ethyl acetate and chloroform for 24 hours.Results1. The inhibitional effect of Kunmu Extract on RA-HFLS(1) Kunmu Extract of ethyl acetate:Comparing with non-treated group, high dose of the extract can inhibit the cell proliferation in 24 hours (P<0.05), while MTX group and all doses of the extract can significantly prevent the proliferation in 48 hours (each P<0.01) and in 72 hours (each P<0.01). The proliferation of HFS-RA in low dose is equal to that in MTX group (P>0.05), while that in the medium and high dose is stronger in 48 hours (each P<0.01) than that in MTX group. Comparing with MTX group, the preve-ntional effct is weaker in low dose, while stronger in medium and high dose in 72 hours (each P<0.01).(2) Kunmu Extract of chloroform:Comparing with non-treated group, none of the extract and MTX group can inhibit the cell proliferation in 24 hours (each P>0.05).although the inhibitoinal rate in high dose is a little bit higher than the other groups. Comparing with non-treated group, all groups can signicantly inhibit the proliferation of RA-HFLS in 48 and 72 hours (each P<0.01). Comparing with MTX group, the inhibitional effect in low dose is equal in 48h (P>0.05), and weaker in 72h (P<0.01), while it’s stronger in medium and high dose both in 48h and 72h (each P<0.01)(3) Kunmu Extract of petroleum ether:Comparing with non-treated group, none of the extract and MTX group can inhibit the cell proliferation in 24 hours (each P>0.05). Comparing with non-treated group, all groups can signicantly inhibit the proliferation of RA-HFLS in 48 and 72 hours (each P<0.01). Comparing with MTX group, the inhibitional effect in low dose group is equal in 48h (P>0.05), and weaker in 72 hours (P<0.01), while it’s stronger in medium and high dose both in 48h and 72 hours (each P<0.01)(4) Kunmu Extract of butanol:Comparing with non-treated group, none of the extract and MTX group can inhibit the cell proliferation in 24 hours (each P>0.05). Comparing with non-treated group, all groups can signicantly inhibit the proliferation of RA-HFLS in 48 and 72 hours (each P<0.01). Comparing with MTX group, the inhibitional effect in low dose is equal in 48 hours, and is lower in 72 hours (each P>0.05), while it’s stronger in medium and high dose both in 48 hours and 72 hours (each P<0.01)(5) In the same concentration group, the longer the treated time is, the stronger the inhibitional effect is,and the higher the inhibitional effect is. In the same time, the higher the extract concentration is, the stronger the inhibitional effect is, and the higher the inhibitional effect is. The inhibitional effect of Kunming Extract on RA-HFLS is dependant on the time and concentration.2. the effect of Kunmu Extract on cell cycle of RA-HFLSG1 phase and PI in non-treated group is compared with every group. G1 phase and PI in low dose group doesn’t change (P>0.05), while G1 phase rises and PI comes down significantly in both medium and high dose goups after treated by Kunmu Extract of chloroform、ethyl acetate and petroleum ether(each P<0.01). G1 phase and PI in low dose group doesn’t change (P>0.05), while G1 phase rises and PI comes down significantly in low、medium and high dose goups after treated by Kunmu Extract of butanol (each P<0.01).3. the effect of Kunmu Extract on apoptosis of RA-FLSAfter treated in 24 hours, every group is compared with non-treated group, apoptosis rates rise evidently in MTX group (P<0.01); there’s no change in low dose group of chloroform and petroleum ether (each P>0.05); the rates go up in low dose group of ethyl acetate and butanol (P<0.05、P<0.01). The rates increase significantly in all medium and high dose of Kunmu Extract (each P<0.01).Every group is also compared with MTX group. The apoptosis rates are lower in all low dose group of the extract (each P<0.01). The rates are equal in medium dose groups of chloroform、ethyl acetate and butanol (each P>0.05), while the rates are lower in medium dose groups of petroleum ether (P<0.01) The rates are higher in high dose groups of all Kunmu Extract (each P<0.01).4. the effect of Kunmu Extract on VEGF、cyclin D1、bax、bcl-2 and caspase-3 of RA-HFLS(1)VEGF expression in Kunmu Extract of chloroform and ethyl acetate groups: qPCR reveals that comparing with non-TNF-α group, VEGF expression goes up in TNF-α group (P<0.05). Comparing with TNF-α group, the expression goes down in MTX group (P<0.05)、high dose group of ethyl acetate(P<0.01) and all groups of chloroform (each P<0.01); there’s no change in low and medium dose of group of ethyl acetate (each P>0.05). Comparing with MTX group, VEGF expression is higher in low dose of ethyl acetate (P<0.05), equal in medium dose of ethyl acetate and in low dose of chloroform (each P>0.05), while lower in high dose of ethyl acetate and in medium and high dose of chloroform (each P<0.01)(2) Cyclin D1 expression in Kunmu Extract of chloroform and ethyl acetate groups:qPCR reveals that comparing with non-TNF-α group, Cyclin D1 expression rises dramaticly in TNF-α group (P<0.01). Comparing with TNF-α group, cyclin D1 declines remarkably in all groups of ethyl acetate (each P<0.01) and in medium and high dose of chloroform (each P<0.01), while there’s no noble change in low dose group of chloroform extract (P>0.05) Comparing with MTX group, cyclin Dl expression is higher in low dose group of ethyl acetate (P<0.05) and chloroform (P<0.01); it’s equal in medium dose group of ethyl acetate and in medium and high dose group of chloroform (each P>0.05), while the expression decreases significantly in high dose group of ethyl acetate (P<0.01)(3)bcl-2 expression in Kunmu Extract of chloroform and ethyl acetate groups:qPCR reveals that comparing with non-TNF-α group, bcl-2 expression goes up remarkably in TNF-α group (P<0.01). Comparing with TNF-α group, bcl-2 expression goes down dramatically in MTX group% the medium and high dose groups of ethyl acetate extract and chloroform (each P<0.01); The expression doesn’t change in low dose groups of ethyl acetate and chloroform (each P>0.05). Comparing with MTX group, bcl-2 expression is higher in both low dose group of ethyl acetate and chloroform (each P<0.01), equal in both medium dose group of ethyl acetate and chloroform (each P>0.05) and declines in both low dose group of ethyl acetate and chloroform (each P<0.01)(4)bax expression in Kunmu Extract of chloroform and ethyl acetate groups: qPCR reveals that comparing with non-TNF-α group, bax expression goes down in TNF-α group(P<0.05). Comparing with TNF-α group, bax expression in every group goes up dramatically (each P<0.01). Comparing with MTX group, it’s equal in low dose group of ethyl acetate (P>0.05) and goes up in medium and high dose groups of chloroform and in all groups of chloroform (each P<0.01).(5) ratio between bcl-2 and bax in Kunmu Extract of chloroform and ethyl acetate groups:Comparing with non-TNF-α group, ratio goes up in TNF-α group (P<0.05). Comparing with TNF-α group, the ratio in every group declines dramatically (each P<0.01). Comparing with MTX group, the ratio is higher in low dose group of ethyl acetate; there is no change in medium dose group of ethyl acetate and in low and medium dose groups of chloroform (each P>0.05), while it goes down remarkably in high dose group of ethyl acetate and chloroform (each P<0.01)(6) caspase-3 expression in Kunmu Extract of chloroform and ethyl acetate groups:qPCR reveals that comparing with non-TNF-α group, caspase-3 expre-ssion goes down in TNF-α group (P<0.05). Comparing with TNF-α group, caspase-3 goes up in MTX group(P<0.05); there is no difference in low dose group of ethyl acetate and chloroform (P>0.05); it increases in medium and high dose group of ethyl actate (each P<0.01) and in medium and high groups of chloroform (P<0.05、P<0.01). Comparing with MTX group, there is no difference in medium and high dose group of ethyl acetate and in low and medium dose group of chloroform (each P>0.05); caspase-3 is lower in low dose group of ethyl acetate and is higher in high dose group of chloroform (P<0.05)5. the effect of Kunmu Extract on ERK、pERK、JNK、pJN、P38 and p-P38RA-FLS was treated by extract of ethyl ecetate and chloroform for 24 hours. Comparing with non-TNF-α group, the ratio between pERK and ERK goes up slightly in TNF-α group(P>0.05). Comparing with TNF-α group, as for ethyl acetate extract, the ratio doesn’t change in low dose group(P>0.05) and declines in medium and high dose groups (each P<0.01); as for chloroform extract, the ratio doesn’t change in low and high dose group (each P>0.05) and declines dramatically in medium dose group (P<0.01).Comparing with non-TNF-α group, the ratio between pJNK and JNK goes up in TNF-α group(P<0.05). Comparing with TNF-a group, the ratio comes down in low dose group of ethy ecetate and in low and medium dose group of chloroform (P<0.05、P<0.01、P<0.05), while the ratio’s higher in medium and high dose groups of ethyl acetate and in high dose group of chloroform (P<0.05、P<0.01、 P<0.05).Comparing with non-TNF-α group, the ratio between pP38 and P38 goes up in TNF-α group (P<0.05). Comparing with TNF-αgroup, the ratio decreases dramatically in all extract dose groups of ethyl acetate and chloroform (each F<0.01). Conclusion1. Kunmu Extract of chloroform、ethyl acetate、petroleum ether and butanol can suppress the proliferation of RA-HFLS. The suppression effect is dependant on the time and concentration. The longer the time is, the inhibitional rate is; The higher the concentration is, the inhibitional rate is. On the same concentration condition, the prohibital effect is stronger in chloroform、 ethyl acetate and petroleum ether than that in butanol.2. Kunmu Extract of chloroform and ethyl acetate may make G1 and S phase arrest happen through bringing down the expression of Cyclin D1, thus suppress-ing the proliferation of RA-HFLS.3. The apoptosis of RA-HFLS is triggered by Kunmu Extract of chloroform、 ethyl acetate、petroleum ether and butanol, which is dependent on concentration. The higher the concentration is, the higher the apoptosis rate is.4. The apoptosis triggered by Kunmu Extract of chloroform and ethyl acetate is regulated by a lot of factors. The mechanism may be bringing down bcl-2, raising bax and caspase-3 and bringing down the ratio between bcl-2 and bax.5. The time of phospholation that ERK、P38 and JNK is induced by TNF-α may be different. Kunmu Extract of chloroform and ethyl acetate may inhibit the phospholation of ERK、JNK and P38 in MAPK pathway. The prohibital effect on MAPK pathway is different in low、medium and high dose of chloroform and ethyl acetate extract. |
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