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Objective:To investigate the mechanism of Duanteng Yimu decoction （DTYM） in the inhibition of pannus formation in collagen-induced arthritis （CIA） mice. Method:Twenty-four SPF-grade DBA/1 male mice were randomly divided into the following four groups： a blank group （NC group）， a model group （CIA group）， a methotrexate group （MTX group）， and a DTYM group， with six mice in each group. The mice， except for those in the NC group， were modeled. From the second immunization， the medium， MTX （1 mg·kg<sup>-1</sup>）， and DTYM （15.4 g·kg<sup>-1</sup>） were administered at an equal volume by gavage for 35 days. Mice were observed for general condition and the arthritis index. The knee and ankle joints were scanned by microcomputed tomography （micro CT）. Hematoxylin-eosin （HE） and safranin O/fast green staining were performed to observe pathological changes. Immunohistochemistry was performed to detect the expression of platelet/endothelial cell adhesion molecule-1 （CD31）， vascular endothelial growth factor-<italic>α</italic> （VEGF-<italic>α</italic>）， vascular endothelial growth factor receptor 2 （VEGFR2）， and phosphorylated（p）-VEGFR2. Result:Compared with the NC group， the CIA group showed red and swollen ankle joints， increased arthritis index scores （<italic>P</italic><0.05， <italic>P</italic><0.01）， manifest injury in the knee and ankle joints， reduced cartilage thickness， elevated Micro CT bone destruction scores of knee and ankle joints （<italic>P</italic><0.01）， and up-regulated absorbance values of synovial CD31， VEGF-<italic>α</italic>， VEGFR2， and p-VEGFR2 （<italic>P</italic><0.01）. Compared with the CIA group， the DTYM group showed relieved ankle joint redness and swelling， reduced arthritis index scores of mice three weeks after administration （<italic>P</italic><0.05， <italic>P</italic><0.01）， intact joint surfaces of the knee and ankle joints， thickened cartilage， declining Micro CT bone destruction scores in both the knee and ankle joints （<italic>P</italic><0.05， <italic>P</italic><0.01）， and lowered absorbance values of CD31， VEGF-<italic>α</italic>， VEGFR2， and p-VEGFR2 in the synovium （<italic>P</italic><0.01）. Conclusion:DTYM can inhibit the pannus formation in CIA mice presumedly by regulating the VEGF pathway.
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Objective:To explore the effect of Duanteng Yimu decoction （DTYM） on the activation of the human umbilical vein endothelial cell （HUVEC） model and the effect on related activated proteins and vascular endothelial growth factor （VEGF） signaling pathway. Method:After DTYM （200， 400 g·mL<sup>-1</sup>） treatment of HUVEC induced by VEGF and tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， cell proliferation， migration， and tubulogenesis were detected by cell counting kit-8 （CCK-8） assay， 5-ethynyl-2'-deoxyuridine （EdU） assay， transwell migration assay， phalloidin staining， and matrix gel card method. The mRNA expression of adhesion factors， including E-selectin， intercellular adhesion molecule-1 （ICAM-1）， and vascular cell adhesion molecule-1 （VCAM-1） was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The expression of von Willebrand factor （VWF）， platelet-endothelial cell adhesion molecule-31 （CD31）， angiogenic factor cysteine-rich-61 （CYR61）， angiopoietin-1 （ANG-1）， VEGF， and VEGF receptor-2 （VEGFR2） was detected by Western blot. Immunofluorescence was used to determine CD31 expression. Result:Compared with the normal group， the model group showed potentiated proliferation， migration， and tubulogenesis of HUVEC （<italic>P</italic><0.05， <italic>P</italic><0.01）， elevated mRNA expression of E-selectin， ICAM-1， and VCAM-1 （<italic>P</italic><0.01）， up-regulated protein expression of VWF， CD31， ANG-1， CYR61， VEGF-<italic>α</italic>， and phospho （p）-VEGFR2 （<italic>P</italic><0.05，<italic> P</italic><0.01）， and increased CD31 immunofluorescence intensity （<italic>P</italic><0.01）. Compared with the model group， the DTYM groups displayed blunted proliferation， migration， and tubulogenesis of HUVEC （<italic>P</italic><0.05，<italic> P</italic><0.01）， decreased mRNA expression of E-selectin， ICAM-1， and VCAM-1 （<italic>P</italic><0.05， <italic>P</italic><0.01）， down-regulated protein expression of VWF， CD31， ANG-1， CYR61， VEGF-<italic>α</italic>， and p-VEGFR2 （<italic>P</italic><0.05， <italic>P</italic><0.01）， and weakened CD31 immunofluorescence intensity （<italic>P</italic><0.01）. Conclusion:DTYM inhibits HUVEC proliferation， migration， adhesion， and tubulogenesis， which is associated with the regulation of CD31， VWF， CYR61， and ANG-1 expression in HUVEC and the VEGF signaling pathway.
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Objective::Duanteng Yimu decoction(DTYMD)is effective in treatment of rheumatoid arthritis (RA) by relieving joint inflammation and down-regulating some inflammatory factors in a short period of time, but the mechanism is still unclear. We aimed to investigate upstream kinase of mitogen activated protein kinases(MAPK) and define the anti-inflammatory mechanism of DTYMD. Method::Fibroblasts-like synovial cells(FLSs) were divided into blank group, model group (IL-1β), high-dose DTYMD group (1 000 mg·L-1), medium-dose DTYMD group (800 mg·L-1), low-dose DTYMD group (600 mg·L-1) and armour ammonia butterfly(MTX) group (20 μmol·L-1). The protein and mRNA expressions of mitogen-activated protein kinase kinase kinase 2 (MEKK2) were analyzed by real-time fluorescence quantitative PCR(Real-time PCR). Totally 42 male DBA/1J mice were randomly divided into 6 groups, with 7 mice in each group, namely normal group, model group and MTX group (2 mg·kg-1), low-dose DTYMD group (6.25 mg·kg-1), medium-dose DTYMD group (12.5 mg·kg-1), and high-dose DTYMD group (25 mg·kg-1). Except for the normal group, the other five groups were included in collagen-induced arthritis(CIA) model by secondary immunoassay. After administration, the posterior limbs and ankle joints were stained with htoxylin-eosin(HE), and the pathological scores of the joints were evaluated. Result::Compared with the model group, DTYMD inhibited the activity of FLSs in a concentration-dependent manner (P<0.01). Compared with the blank control group, the cell proliferation rate of the model group increased (P<0.01). Compared with the model group, high and middle-dose DTYMD groups could inhibit protein and mRNA expressions of MEKK2 (P<0.01), but there was no significant difference in low-dose group. However, the expression of DTYMD protein in high/medium/low-dose groups was significantly higher than that in blank group (P<0.01), but there was no significant difference in MTX group. Compared with the model group, the expressions of matrix metalloprotease-1 (MMP-1), tumor necrosis factor-α(TNF-α) and interleukin(IL)-6 were negatively regulated in different DTYMD groups(P<0.01), and the expressions of MMP-1, IL-6, TNF-α in the model group were significantly higher than those in the blank group (P<0.05, P<0.01). In the animal experiment, compared with the model group, high/middle-dose DTYMD groups could decrease the degree of joint swelling in CIA mice (P<0.01), but there was no significant difference in the low dose group, and the joint swelling in the model group was significantly higher than that in the blank group (P<0.05). In HE staining of ankle joint of CIA mice, the pathological scores of high/small-dose DTYMD groups were significantly lower those of model group (P<0.05, P<0.01), and the pathological score of model group was higher than that of blank group (P<0.01). Conclusion::DTYMD might down-regulate MEKK2 to negatively regulate inflammatory cytokines IL-6, TNF-α and MMP-1, thereby alleviating the inflammatory response in rheumatoid arthritis.
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Objective To observe the effect of Duanteng Yimu decoction (DYD) on bone turnover markers of C-terminal telopeptide of type Ⅰ collagen (CTX-Ⅰ) and N-terminal propeptide of type Ⅰ procollagen ( PⅠNP) of adjuvant-induced arthritis (AA) rats. Methods Seventy-two SD rats were divided into 9 groups, and complete Freund's adjuvant was injected into the rats to establish adjuvant-induced arthritis rat model. After medication for 36 days, the pedal swelling, the serum levels of CTX-Ⅰand PⅠNP, and ankle X-ray photographic scores were observed. Results The pedal swelling degrees and ankle X-ray scores in medication groups were lower than those in the model group ( P0.05). The CTX-Ⅰlevel in low-dose DYD group was decreased (P<0.05). Conclusion Caulis Celastri has two-way effects on CTX-Ⅰlevel of female AA rats, and its side effects can be reduced by Radix Dipsaci and Herba Leonuri, but their antagonism effect will disappear with the increase of the dose of Caulis Celastri.