| ObjectiveObserve the effect of Qingre Huoxue Decoction on synovial angiogenesis of rats with collagen-induced arthritis (CIA), and in vitro regulation of Rheumatoid Arthritis fibroblast-like synoviocytes (RA-FLS) and human umbilical vein endothelial cells (HUVEC) proliferation, migration, adhesion and tube formation, and explore the mechanism of action related to PI3K/Akt pathway, to provide an experimental evidence of Qingre Huoxue Decoction in the clinical treatment of rheumatoid arthritis.MethodsThe healthy female SD rats were randomly divided into 6 groups after weighing, named as Normal group, model group, Qingre Huoxue Decoction 15g/kg,20g/kg, 25g/kg in treatment group, and the inhibitor LY294002 group (20mg/kg). Inject type II collagen emulsifier in the root of rat tail subcutaneously, then needed a booster shot on day 7, to establish the CIA model. Groups of rats with CIA were treated with Qingre Huoxue Decoction and LY294002 orally according to the group from day 10 (the 10th immune day), and the normal group and CIA model group were given the same volume of saline. Continuously administrated of 28 days, observe and record changes in body weight, joint symptoms and swelling of rats in each group. The serum of abdominal aorta blood was collected by centrifugation, and the serum levels of interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF) were tested by ELISA; the protein levels of PI3K/Akt signal pathway related proteins P13K, p-Akt, PTEN of ankle tissues were tested by Western Blot; the mRNA levels of placental growth factor (PLGF) and its receptors Fltl, Flkl of synovial tissue, and PTEN, the negative regulation gene of PI3K/Akt pathway were tested by Real-time PCR.Primary human RA-FLSs were isolated from synovial tissues of RA patients and cultured with the tissue explants adherent method, of which passage 3 to 6 cells were used in experiments, while HUVECs were cultivated for experiments. Both RA-FLSs induced by TNF-a and HUVECs induced by VEGF165 were cultured with three concentrations (20,100,500ng/mL) of Qingre Huoxue Decoction. Cell proliferative activity was assayed by MTS; the influence of cell migration was observed by wound healing assay; the effect of cells with the extracellular matrix adhesion was observed by using fibronectin (FN), one of the major components of the extracellular matrix protein; the ability of HUVEC tube formation was observed though Matrigel-coated plates; ELISA showed expression levels on IL-1β, VEGF. PLGF and its receptors Plt 1, Flkl of RA-FLS; the protein levels of PI3K/Akt pathway relative proteins were tested by Western Blot.Results1. Effects of Qingre Huoxue Decoction on symptoms, joint swelling and weight in rats with CIACompared with the normal group, the joint red swelling of CIA rats was more obvious, and the degrees of paw swelling were significantly increased (P<0.001).Compared with the CIA model group, Qingre Huoxue Decoction could reduce joint swelling by dose-dependently, meanwhile the middle and high dose (20, 25g/kg), as well as LY294002, could significantly reduce paw swelling (P<0.05).Compared with the normal group, CIA model group had slowly growth in bodyweight. Qingre Huoxue Decoction could dose-dependently promote growth in bodyweight of rats with CIA, while the bodyweight of high dose (25g/kg) group and LY294002 group were significantly increased from 21st day of immunization (P<0.05).2. Effects of Qingre Huoxue Decoction on the level of serum IL-1β, VEGF in rat with CIAAfter administration for 28 days, the level of IL-1β and VEGF in serum of CIA rats were significantly higher than the normal group (P<0.01). Compared with CIA model group, Qingre Huoxue Decoction could reduce the level of IL-1β and VEGF by dose-dependently, and high dose (25g/kg) had the significant effect (P<0.05), while the level IL-1β of LY294002 group decreased significantly (P<0.05).3. Effects of Qingre Huoxue Decoction on protein levels ofPI3K/Akt pathway in ankle jointsAfter administration for 28 days, compared with the normal group, the protein levels of PI3K and phospho-Akt in CIA model group increased significantly (P<0.001), and Qingre Huoxue Decoction could down-regulate the expression of PI3K and p-Akt dose-dependently (P<0.01.P<0.001). The protein level of PTEN in CIA model group was lower than normal group (P<0.001), while middle and high dose (20,25g/kg) of Qingre Huoxue Decoction could up-regulate the expression ofPTEN (P<0.05, P<0.001). LY294002 significantly inhibited the expression of PI3K and p-Akt, conversely, increased expression of PTEN (P<0.001).4. Effects of Qingre Huoxue Decoction on mRNA levels of PLGF, Flt1, Flk1 and PTEN in synovial tissues After administration for 28 days, the mRNA levels of PLGF, Flt1 and Flk1 in CIA model group were higher than normal group (P<0.01), and Qingre Huoxue Decoction at doses of 20g/kg and 25g/kg could significantly inhibit the expression (P<0.05, P<0.01). Compared with normal group, CIA rats had much higher mRNA level of PTEN than normal group (P<0.001), and Qingre Huoxue Decoction at dose of 25g/kg could significantly promote the expression of PTEN (P<0.001). LY294002 could down-regulate the mRNA levels of PLGF, Flk1 and Flt1, but up-regulate the expression of PTEN (P<0.01, P<0.001).5. Effects of Qingre Huoxue Decoction on proliferation of RA-FLS and HUVECCompared with the control group, TNF-α obviously induced the proliferative activity of RA-FLS (P<0.05), and Qingre Huoxue Decoction could inhibit the proliferation, which at concentrations of 2.5μg/mL and 12.5μg/mL had significant differences (P<0.05). Compared with the control group, VEGF could observably promote proliferation of HUVEC (P<0.05), Qingre Huoxue Decoction could inhibit the proliferative activity, and at concentration of 12.5μg/mL the difference was significant (P<0.05).6. Effects of Qingre Huoxue Decoction on migration of RA-FLS and HUVECAfter scratched on RA-FLS, there were few cells growing in the wound area of the control group after 24h compared with that at Oh, meanwhile TNF-a obviously induced RA-FLS to migrate to the wound area (P<0.001). Compared with TNF-a group, Qingre Huoxue Decoction can significantly concentration-dependently inhibit the migration of RA-FLS (P<0.01,P<0.001). After scratched, VEGF-induced HUVEC obviously migrated to the wound area after 24h compared with that at Oh (P<0.001), and compared with VEGF group, Qingre Huoxue Decoction could inhibit migration of HUVEC to the scratch area in concentration-dependence (P<0.001).7. Effects of Qingre Huoxue Decoction on adhesion of RA-FLS and HUVECCompared with 1% BSA negative group, RA-FLS had a significant capability of adhering to FN (P<0.001), moreover TNF-a induced the capability of adhesion increasingly enhanced (P<0.001).Qingre Huoxue Decoction could concentration-dependently inhibit the adhesion (P<0.001). HUVEC had a stronger capability of adhering to FN than 1% BSA negative group (P<0.001). VEGF could boost adhesion to FN (P<0.001), but Qingre Huoxue Decoction inhibited adhesion of VEGF-induced HUVEC significantly (P<0.001).8. Effects of Qingre Huoxue Decoction on tube formation of HUVECCompared with the control group, VEGF obviously induced the ability of tube formation of HUVEC in Matrigel (P<0.001). Qingre Huoxue Decoction could significantly inhibit tube formation of HUVEC in a concentration-dependent manner (P<0.05,P<0.001).9. Effects of Qingre Huoxue Decoction on angiogenic relative factors of RA-FLS and HUVECCompared with the control group, TNF-a induced a significant increasing levels of IL-1β, VEGF, PLGF in culture supernatants of RA-FLS (P<0.01). Qingre Huoxue Decoction at concentration of 500ng/mL could significantly reduce the levels of IL-1β and PLGF (P<0.05), and at concentration of 100ng/mL and 500ng/mL could significantly reduce the level of VEGF (P<0.05,P<0.01). Similarly, VEGF-induced the levels of VEGFR1, VEGFR2 in HUVEC significantly increasing (P<0.01), but Qingre Huoxue Decoction at concentration of 100ng/mL could significantly reduce the level (P<0.05).10. Effects of Qingre Huoxue Decoction on expressions of PI3K/Akt pathway-related proteins in RA-FLS and HUVECCompared with the control group, TNF-a could markedly promote the expression of PI3K. and phosphorylation of Akt of RA-FLS (P<0.001), while Qingre Huoxue Decoction could concentration-dependently down-regulate the expression of PI3K and p-Akt (P<0.001), as well as LY294002 could. Meanwhile, VEGF induced the levels of PI3K, p-Akt much higher than the control group (P<0.001), and Qingre Huoxue Decoction could inhibit the expression at various degrees (P<0.001), also, LY294002 could significantly inhibit VEGF-induced expression of PI3K and p-Akt in HUVEC.Conclusions1. Qingre Huoxue Decoction in oral treatment could alleviate joint symptoms of red swelling; reduce degree of joint swelling in CIA rats. Moreover Qingre Huoxue Decoction could dose-dependently reduce the level of serum pro-inflammatory cytokine (IL-1β) and pro-angiogenic factor (VEGF), and down-regulate the mRNA levels of PLGF, Fltl and Flkl in synovial tissue. Thereby it showed that Qingre Huoxue Decoction could improve the clinical symptoms, reduce synovial inflammation and inhibit pannus formation of CIA rats to delay the progression of arthritis.2. Qingre Huoxue Decoction could obviously inhibit the proliferation, migration and adhesion of RA-FLS, and reduce the level of pro-inflammatory cytokines (IL-1β) and pro-angiogenic factor (VEGF, PLGF). Synergistically, the decoction could inhibit the proliferation, migration, adhesion, tube formation and expression of VEGFR1 and VEGFR2 of HUVEC. Both results show that Qingre Huoxue Decoction played a role of angiogenesis inhibition.3. Qingre Huoxue Decoction could dose-dependently down-regulate the expressions of PI3K and p-Akt in CIA rats, RA-FLS and HUVEC, while up-regulate the expression of PTEN, the negatively regulated gene, at both protein and mRNA levels. It is shown that Qingre Huoxue Decoction could regulate the proliferation, migration, adhesion and expression of angiogenic related factors though P13K/Akt pathway to inhibit the formation of new blood vessels in synovium. |
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