| 1 Objectives To explore the change of signal transduction of PHDs/p VHL/HIF-α pathway in adjuvant arthritis(AA)rats and the therapeutic effect of geniposide(GE).To investigate the effect of GE on cell proliferation via PHD2/p VHL/HIF-1α signaling pathway and its specific mechanism in MH7 A induced by hypoxia.2 MethodsIn vivo: After successful establishment of AA rats,they were randomly divided into 5groups,which were given equal volume of normal saline,GE(30mg/kg,60mg/kg,120mg/kg)and methotrexate(0.5mg/kg)for 7 days.The degree of arthritis was evaluated from arthritis index,paw swelling degree and global assessment score;the pathological changes of synovium were examined by HE staining;the levels of b FGF,IL-6 and IL-10 in serum were detected by ELISA;the expression of proteins related to PHDs/p VHL/HIF-α pathway in synovium was detected by immunohistochemistry.In vitro: After successful establishment of hypoxia model in vitro,GE(5 μM,10 μM,20 μM)and HIF-α inhibitor 2ME2(5 μM)were used to intervene in vitro,and PHD2 inhibitor IOX4(100 n M)and protease inhibitor MG132(100 n M)were separately or combined with GE to further explore the specific mechanism of GE.The proliferation activity of MH7 A was detected by CCK-8,the levels of b FGF,IL-6 and IL-10 in the supernatant were detected by ELISA;the expression of proteins related to PHDs/p VHL/HIF-α pathway was detected by Western blot;the cell cycle was detected by fluorescence activated cell sorting(FACS)and cell proliferation was detected by Ed U.3 Results3.1 Effect of GE on PHDs/p VHL/HIF-α pathway in adjuvant arthritis rats and its therapeutic effect3.1.1 Effect of GE on arthritis symptoms in AA ratsCompared with normal rats,AA rats showed obvious arthritis symptoms.The paw swelling degree,arthritis index and global assessment score were significantly increased,and pathological examination showed abnormal ankle cartilage and synovial hyperplasia.After GE administration,the paw swelling degree,arthritis index and global assessment score were decreased in varying degrees,abnormal hyperplasia of ankle cartilage and synovial tissue in AA rats was significantly inhibited,the infiltration of inflammatory cells was alleviated,and the arthritis of AA rats was ameliorated.3.1.2 Effect of GE on proliferation related factors in serum of AA rats Compared with normal rats,the levels of proliferation related factors b FGF and IL-6 in serum of AA rats were significantly increased,while the level of IL-10 was significantly decreased.GE can significantly down-regulate the secretion of b FGF and IL-6,up-regulate the secretion of IL-10.3.1.3 Effect of GE on expression of PHDs/p VHL/HIF-α related proteins in synovium of AA ratsCompared with normal rats,the expression levels of key enzymes PHD2,HIF-1α and HIF-2α in PHDs/p VHL/HIF-α pathway in AA rats were significantly increased.After GE administration,the expression of PHD2,HIF-1α and HIF-2α decreased in varying degrees,which are consistent with the manifestation of arthritis.It is suggested that GE may play a role in the treatment of RA by intervening in the pathway of PHDs/p VHL/HIF-α,inhibiting the abnormal hyperplasia of synovial tissue,alleviating the symptoms of joint inflammation.3.2 Effect of GE on proliferation of MH7 A induced by hypoxia by interfering with PHDs/p VHL/HIF-α signal transduction3.2.1 Effect of GE on proliferation activity of MH7 A induced by hypoxia in vitro Compared with normoxic normal group,the proliferation activity of MH7 A in hypoxia model group was significantly increased.After GE intervention in vitro,the proliferation activity of MH7 A induced by hypoxia was significantly decreased.3.2.2 Effect of GE on the secretion of proliferation related factors in MH7 A induced by hypoxia in vitroCompared with normoxic normal group,the levels of proliferation related factors b FGF and IL-6 in hypoxia model group were significantly increased,while the level of IL-10 was significantly decreased.After GE intervention in vitro,the levels of proliferation related factors b FGF and IL-6 of MH7 A induced by hypoxia were significantly decreased,and the level of IL-10 was significantly increased,suggesting that GE can restore the dynamic balance of proliferation related factors.3.2.3 Effect of GE on the expression of proteins related to PHDs/p VHL/HIF-αsignaling pathway in MH7 A induced by hypoxiaCompared with normoxic normal group,the expression of PHD2 and HIF-1α in MH7 A of hypoxia model group was significantly increased,and the expression of proliferation related proteins Cyclin D1,PCNA and BNIP3 was significantly increased.After GE intervention in vitro,the expression of PHD2 in MH7 A of hypoxia model group was continuously increased,and the expression of HIF-1α,Cyclin D1,PCNA and BNIP3 was significantly decreased.It is suggested that GE can interfere with the signal transduction of PHD2/p VHL/HIF-1α by up regulating the level of PHD2,inhibit the activation of downstream signal pathway,and then inhibit cell proliferation.3.2.4 Effect of GE on cell proliferation of MH7 A induced by hypoxia FACS and Ed U assay showed that compared with normoxia group,the proportion of S phase cells and positive expression percentage of Ed U in MH7 A of hypoxia model group were significantly increased,and the cell proliferation was abnormal.After GE intervention in vitro,the proportion of S phase cells and positive expression percentage of Ed U in MH7 A induced by hypoxia decreased significantly,and the abnormal proliferation was inhibited,which was consistent with the result of proliferation activity.3.3 Mechanism of GE intervention on proliferation of MH7 A induced by hypoxia3.3.1 Effect of GE on proliferation activity of MH7 A stimulated by inhibitor Compared with hypoxia model group,single proteasome inhibitor MG132 and PHD2 inhibitor iox4 groups enhanced the proliferation activity of MH7A;after GE combined with each inhibitor,compared with single inhibitor,the proliferation activity of MH7 A decreased significantly.3.3.2 Effect of GE on protein expression of PHDs/p VHL/HIF-α signaling pathway in MH7 A stimulated by inhibitorCompared with the hypoxia model group,the level of PHD2 was significantly increased in MG132 group and decreased in IOX4 group.The expression of HIF-1α,Cyclin D1,PCNA and BNIP3 increased significantly in both groups.After GE combined with inhibitor respectively,the level of PHD2 were significantly increased,while the expressions of HIF-1α,Cyclin D1,PCNA and BNIP3 were significantly decreased.3.3.3 Effect of GE on proliferation of MH7 A stimulated by inhibitor Compared with the hypoxia model group,the proportion of S-phase cells and positive expression percentage of Ed U in MG132 and IOX4 groups increased significantly.After GE combined with inhibitor respectively,the proportion of S-phase cells and positive expression percentage of Ed U decreased significantly compared with the single inhibitor,which was consistent with the results of proliferation activity.Conclusions1.GE can inhibit synovial hyperplasia and alleviate arthritis in AA rats,which may be mediated by interfering with signal transduction of PHDs/p VHL/HIF-α.2.GE can accelerate the hydroxylation and degradation of HIF-1α by up-regulating the level of PHD2,interfere with the signal transduction of PHD2/p VHL/HIF-1α,and then inhibit cell proliferation.3.GE can reverse the promotion of MG132 and IOX4 on the abnormal proliferation of MH7 A induced by hypoxia.In addition to promoting the degradation of HIF-1 α,GE may be involved in regulating the expression of HIF-1α through PHD2 dependent non hydroxylation pathway,blocking the activation of HIF-1α downstream signaling pathway,thereby inhibiting cell proliferation,improving joint synovial hypoxia and playing a therapeutic role in RA. |
PDF Full Text Request