ABSTRACT
ObjectiveTo explore the effect of Danggui Niantongtang （DGNTT） on cell apoptosis and autophagy in rheumatoid arthritis （RA） fibroblast-like synoviocytes （FLS）. MethodRA-FLS were isolated and cultured from the synovial tissue of RA patients. The cells were treated with 10% blank serum （blank control group）， 10% sera containing low， medium and high doses of DGNTT. Wound healing assa and cell invasion test were applied to observe the effect of RA-FLS invasion technique. The apoptosis and autophagy level of RA-FLS cells was detected by Hoechst33342 method and monodansylcadaverine （MDC） staining. The mRNA and protein expression levels of B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， microtubule-associated protein 1 light chain 3 （LC3）， autophagy key molecular yeast Atg6 homolog 1 （Beclin1） were detected by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）and Western blot. ResultCompared with the blank control group，each dose of serum could slow down the wound healing and significantly Reduce the number of RA-FLS cells invading the lower chamber（P<0.01），the mRNA and protein expression levels of Bcl-2，LC3，Beclin1 were significantly decreased（P<0.01）， and Bax were significantly increased（P<0.01）. Hoechst33342 results showed that low， medium and high doses DGNTT could promote RA-FLS cell apoptosis. After MDC staining，autophagosome in low， medium and high doses DGNTT decreased significantly（P<0.01）. ConclusionDanggui Niantongtang can effectively inhibit the proliferation of fibroblast-like synoviocytes. Its mechanism may be related to promote apoptosis and inhibit autophagy of fibroblast-like synoviocytes.
ABSTRACT
ObjectiveTo observe the preventive and control effects of Danggui Niantongtang against adjuvant arthritis differentiated into wind-damp-heat impediment in rats and its influences on the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3）， homolog of yeast Atg6 （Beclin1） and p62. MethodThe six-week-old male SD rats were randomly divided into the normal group， wind-damp-heat impediment model group， low-， medium-， and high-dose Danggui Niantongtang （5.67， 11.34， 22.68 g·kg-1） groups， and methotrexate （MTX， 1.35 mg·kg-1） group， with 10 rats in each group. A rat model of adjuvant arthritis was established by subcutaneous injection of inactivated Mycobacterium tuberculosis into the tail root， followed by exposure to the manual climatic box for 16 d for inducing the wind-damp-heat impediment. The drugs were administered intragastrically on the day of immunization for 28 d. The general conditions of rats were observed and the swelling degree of toes and arthritis index （AI） were detected. The pathological changes in the synovial tissues of the knee joints were observed by hematoxylin-eosin （HE） staining. The mRNA expression levels of LC3， Beclin1， and p62 in the synovial tissues were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， followed by the assay of their protein expression by Western blot and immunohistochemistry. ResultCompared with the normal group， the wind-damp-heat impediment model group exhibited significantly increased swelling degree of toes （P<0.01）， increased AI （P<0.01）， proliferated synovial cells （P<0.01）， up-regulated LC3 and Beclin1 protein and mRNA expression （P<0.01）， and down-regulated p62 protein and mRNA expression （P<0.01） after 16， 20， 24， 28-d medication. Compared with the wind-damp-heat impediment model group， each medication group displayed alleviated toe swelling and synovial hyperplasia to different degrees， decreased mRNA and protein expression levels of LC3 and Beclin1 （P<0.01）， and increased p62 mRNA and protein expression （P<0.05，P<0.01）， with the best outcomes observed in the medium-dose Danggui Niantongtang group. ConclusionDanggui Niantongtang effectively relieves adjuvant arthritis due to wind-damp-heat impediment in rats， which may be related to its regulation of the expression of autophagy-related proteins LC3， Beclin1， and p62 and the inhibition of autophagy.
ABSTRACT
Objective:To observe the effect of Danggui Niantongtang on the protein and mRNA expression of key regulatory factors of the extrinsic and intrinsic apoptotic pathway in synovial tissue of adjuvant arthritis （AA） rats， and to further explore the mechanism of Danggui Niantongtang in the prevention and treatment of rheumatoid arthritis. Method:The general condition of AA rats， including its body weight， were observed. The changes of toe volume were detected by toe volume meter. Histopathological changes of synovium of knee joint were observed by hematoxylin-eosin （HE） staining. The mRNA and protein expression levels of tumor necrosis factor receptor super family 6 （Fas）， Fas-associating protein with a novel death domain（FADD）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax） and cysteinyl aspartate specific proteinase Caspase-3 （Caspase-3） were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Result:Compared with the normal group， the toe volume of the model group increased significantly （<italic>P</italic><0.01）， with significantly proliferated synovial cells， significantly reduced mRNA and protein expression levels of Fas， FADD， Bax and Caspase-3 in synovial tissues（<italic>P</italic><0.05，<italic> P</italic><0.01）， and significantly increased Bcl-2 level （<italic>P</italic><0.01）. Compared with the model group， the swelling degree of toes in Danggui Niantongtang group and Tripterygium group was significantly alleviated （<italic>P</italic><0.01）， with significantly improved synovial hyperplasia， significantly increased mRNA and protein expression levels of Fas， FADD， Bax and Caspase-3 （<italic>P</italic><0.05， <italic>P</italic><0.01）， and significantly decreased expression levels of bcl-2 mRNA and protein （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:Danggui Niantongtang can effectively reduce joint swelling and abnormal proliferation of synovial tissue in AA rats. Its mechanism may be related to regulating the expression of Fas， FADD， Bax， Bcl-2 and Caspase-3， and promoting the apoptosis of synovial cells.