| Objectives 1 To evaluate the therapeutic effect of Shexiang-Wulong Pill(SWP)in a mouse model of collagen-induced arthritis(CIA)by comparing the changes of clinical score,histopathology,bone microstructure and serological indexes of CIA mice.2 To explore the possible mechanism of SWP in the treatment of rheumatoid arthritis(RA)by comparing the effects of SWP on the number of synovial microvessels and the expression of vascular endothelial cadherin(VE-cadherin)and ZO-1(Zonula occludens-1)in the knee joints of CIA mice.Methods 1 Forty-five DBA/1 mice were randomly divided into the control group(n=15)and model group(n=30).Model mice(n=30)except the control group,were intradermally injected with 0.1 ml CII emulsion(Bovine type II collagen acetic acid solution was mixed with an equal amount of Freund's complete adjuvant)at the base of the tail for the first immunization and the booster dose was given on day 21.2 After the booster CII dose,the mice were randomized into the SWP-treated and untreated model groups(n=15 each),and accordingly administered SWP or saline via the gastrointestinal route till day 43;the control group mice also received saline for the same duration.3 Therapeutic evaluation: The body weight of all the mice was recorded every 7 days from tthe first day;The severity of paw arthritis was evaluated every 3 days from day 21;The histological changes in knee joints of mice were tested by hematoxylin and eosin staining(H&E),Safranin-O staining and Immunohistochemical(IHC)staining with hypoxia probe according to the manufacturer's instructions;Micro-computed tomography(micro-CT)was used to assess microstructural changes in the bone;Serum levels of Tumor necrosis factor alpha(TNF-?),Interleukin-6(IL-6)and Interferon-?(IFN-?)were measured by Enzyme-linked immunosorbent assay(Elisa)kit to evaluate the level of inflammation in mice.4 Evaluation of vascular number and connexin: IHC staining and Western blot were used to measure the expression of CD34 in knee synovium of mice;IHC staining was used to evaluate the expression of VE-cadherin and ZO-1 in knee synovium of mice.Results 1 Compared with the control group,the body weight of mice in CIA model group decreased significantly.However,the body weight of mice in SWP-treated group was significantly higher than that in CIA model group(P < 0.05).2 The clinical arthritis score in the CIA model group were significantly higher than that of in the control group from day 21 onwards(P < 0.05);From day 33,the clinical score of arthritis in the CIA model group continued to rise,while that in SWP-treated group began to decline.The arthritis clinical score in SWP-treated group was significantly less than that of in the model group(P < 0.05).3 The results of H&E staining showed that compared with the control group,the arthritic mice had synoviocyte hyperplasia,abnormal shape,disordered arrangement,severe inflammation and pannus formation,and these symptoms were significantly reduced in the SWP-treated mice.4 The results of Safranin-O staining showed that the cartilage layer of CIA model mice was thinner,the surface was rough,the number of chondrocyte decreased,the matrix lost staining seriously,the tidal line was destroyed,the cartilage structure was destroyed seriously and the mean Mankin score was significantly higher than that of the control group(P < 0.05);SWP treatment significantly restored the cartilage and lowered the mean Mankin score in the model group(P < 0.05).5 Compared with the control group,the area of hypoxia in CIA model group was larger,the positive staining was deeper,and the average optical density in hypoxic area was significantly higher than that in control group(P < 0.05).After treatment with SWP,the area of hypoxia in knee synovium of CIA mice decreased significantly,the positive staining became shallow,and the average optical density in hypoxic area decreased significantly(P < 0.05).6 Three-dimensional micro-CT revealed an intact knee joint structure with a smooth bone surface in the control group,whereas that of the arthritic mice were severely eroded,with signs of osteoporosis.SWP treatment markedly reduced the extent of joint damage.Compared to the control mice,the BV/TV,Tb.Th,Tb.N and BMD were significantly reduced(P < 0.05),and Tb.Sp,SMI were significantly increased in the CIA mice(P < 0.05);These parameters were significantly improved after SWP treatment(P < 0.05).7 Compared to the control group,the serum levels of TNF-?,IL-6 and IFN-? were significantly increased in the arthritic mice(P < 0.05),and markedly decreased by SWP treatment compared to the CIA group(P < 0.05).8 The results of immunohistochemistry and Western Blot showed that the relative expression of CD34 in CIA model group was significantly higher than that in control group(P < 0.05),while the expression of CD34 decreased significantly after treatment with SWP(P < 0.05).9 Immunohistochemical results showed that the positive expression and the average optical density of VE-cadherin and ZO-1 in CIA model group was significantly lower than that in control group(P < 0.05),while the positive expression and the average optical density of VE-cadherin and ZO-1 could be increased by SWP treatment(P < 0.05).Conclusions 1 SWP can effectively alleviate joint swelling in CIA mice,reduce arthritis index,inhibit synovial tissue hyperplasia,reduce inflammatory cell infiltration,relieve synovial hypoxia.and delay bone destruction.2 SWP can reduce the number of synovial microvessels in knee joints,and the anti-angiogenesis mechanism of SWP may be related to up-regulate the expression of VE-cadherin and ZO-1.Figure 12;Table 2;Reference 139... |
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