| BACKGROUND:Rheumatoid arthritis is an immune disease that is clinically characterized as erosive and symmetrical polygonitis,which often causes disability.The etiology and pathogenesis of RA are not fully understood.Scholars generally believe that infection,autoimmune response,genetic and environmental factors are involved in RA.Commonly used in RA treatment strategies such as disease-relieving drugs and non-steroidal anti-inflammatory drugs have unsatisfactory therapeutic effects,the degree of symptom relief is not high,and the toxic and side effects caused by long-term use are more common.Traditional Chinese Medicine has the advantages of fewer side effects and no immunodeficiency.Therefore,research on the pathogenesis of RA and safe and effective Traditional Chinese Medicine will help patients with RA to delay the progress of bone destruction and improve the long-term prognosis of the disease.OBJECTIVE:To study the anti-inflammatory and anti-bone destruction effects of ZhiWang Decoction(ZWT)on collagen-induced arthritis rats(CIA)and the synergistic mechanism of ZWT with methotrexate in the treatment of RA,and to explore the molecular mechanism of RA.That will provide a scientific experimental basis for elucidating the pathogenesis of rheumatoid arthritis,and provide new ideas for clarifying the targets and pathways of ZWT and optimizing the clinical treatment plan for rheumatoid arthritis.METHODS:1.The effect of ZhiWang Decoction on improving inflammation and bone destruction in rats with collagen-induced arthritisThe experiment in this chapter adopted the CIA modeling,and was divided into normal group,the model group,the methotrexate group(1.5 mg·kg-1),and the ZWT group(20.4 g·kg-1).The anti-inflammatory effect of ZWT was observed through arthritis index and histopathological methods.Immunohistochemistry were used to detect the effect of ZWT on the expression of TNF-α and MMP-13 in the ankle joint of CIA.PCR and WB were used to detect the effect of ZWT on the expression of TNF-α,MMP-13 mRNA and protein in the CIA.The TRAP staining was used to detect the effect of ZWT on the proliferation of osteoclasts in the ankle joint of CIA rats,and observe the anti-bone destruction effect of ZWT.2.Research on the mechanism of ZhiWang Decoction for the treatment of collagen-induced arthritis rats bone destruction by methotrexateThe experiment in this chapter adopted the CIA modeling,and the groups were set into the normal group,the model group,the methotrexate group(1.5 mg·kg-1),the low-dose ZWT group combined with methotrexate group(40.8 g·kg-1),and the high-dose ZWT group combined with methotrexate group(102.0 g·kg-1).Observing the anti-inflammatory effect of ZWT through weight,plantar thickness,arthritis index,organ index.Immunohistochemistry,PCR and WB were used to detect the effects of combined drugs on the expression of RANKL and OPG in the ankle joint tissues of CIA rats.TRAP staining were used to detect the proliferation of osteoclasts in the ankle joints of CIA rats to explore the effect and possible mechanism of ZWT combined with methotrexate in the treatment of CIA rats.RESULTS:1 The effect of ZhiWang Decoction on improving inflammation and bone destruction in rats with collagen-induced arthritis1.After the model was made,the rat’s coat color was dull,the diet were reduced,the spirit was poor,the feet appeared red and swollen,the movement was inflexible,and the movement was difficult.The general condition of the rat had improved after the administration.2.The AI scores of rats in the ZWT group at the 5th,6th weeks were lower than that of the model group.3.In normal group,the joint space in the ankle joint was normal,the bone surface was smooth,the cartilage tide line was smooth and clear,and there was no bone destruction.The joint space in the ankle joint of the model group was enlarged,the bone surface was not smooth and irregular and the cartilage was not smooth.There was hyperplasia,severe bone destruction,a large number of fibroblast-like cell proliferation and inflammatory cell infiltration.In the ankle joints of rats in the methotrexate group,there were normal joint spaces,mild cartilage tissue thickening,regular bone surfaces,and bone destruction was not serious,less neutrophils and lymphocytes infiltration.In the ankle joints of rats in the ZWT group,the joint space had enlarged,there had irregular bone surface,mild cartilage hyperplasia,bone destruction,and there was lymph Cell and neutrophil infiltration.4.Immunohistochemical results:The expression of TNF-α and MMP-13 in the ankle synovium of CIA model rats was significantly increased.Compared with the model group,the expression of TNF-α and MMP-13 in the ankle synovium of the methotrexate group and ZWT group decreased,the effects of methotrexate was better than ZWT.5.PCR results:CIA rats showed high expression of TNF-α and MMP-13 mRNA in the ankle joint tissue.Compared with the model group,TNF-α mRNA in the methotrexate group and MMP-13 mRNA in the ZWT group has decreased significantly.6.WB results:CIA rats showed high expression of TNF-α and MMP-13 protein in the ankle joint tissue.Compared with the model group,TNF-α protein in the methotrexate group and MMP-13 protein in the ZWT group has decreased significantly.7.TRAP results:The number of osteoclasts in the ankle joint tissue of CIA model rats was significantly increased.Compared with the model group,the number of osteoclasts in the ankle joint of the methotrexate group decreased,but there was no statistics.The number of osteoclasts in the ankle joint of rats in the ZWT group decreased significantly.2 Research on the mechanism of ZhiWang Decoction for the treatment of collagen-induced arthritis rats bone destruction by methotrexate1.After the model was established,the rats’ diet was reduced,their spirits were poor,and the feet gradually became red and swollen,movement was inflexible.After administration,the general condition of the rats improved,especially the high-dose ZWT combined group.After the second immunization,the body weight of the rats in each group was significantly reduced.The body weight of the rats in the low-dose ZWT combined group increased on the 28th day compared with the model group.The body weight of the high-dose ZWT combined group was significantly higher than that of the model group on the 19th,22ed,25th and 28th day,but there was no difference in the methotrexate group.2.After modeling,the thymus index and spleen index of the rats in the model group increased significantly.ZWT can significantly reduce the organ index of rats.3.The toe thickness of CIA rats increased significantly.The toe thickness of the high-dose combined group was significantly lower than that of the model group on the 19th day.There was no significant difference between the methotrexate group and the model group.4.The AI score of the ankle joint of CIA rats increased significantly.Compared with the model group,the AI score of rats in the high-dose ZWT combined group was significantly lower on the 13 th day,and there was no significant difference between the methotrexate group and the model group.5.Doppler ultrasound examination showed that after the model was established,the thickness of the synovial membrane of the ankle joint of rats in each group increased significantly.Compared with the model group,the synovial thickness of the rats in the low-dose ZWT combined group and the high-dose ZWT combined group was reduced,and the synovial thickness of the rats in the high-dose ZWT combined group was lower than that in the methotrexate group.6.Three methods were used to assess arthritis in rats.Compared with the normal group,all three methods could reflect the severe condition of synovitis in the modeled rats.However,the thickness of the toes and the AI score of the ankle joint showed that there was no statistical difference in each administration group,but the ultrasound results showed that compared with the model group,the synovitis condition of rats in the low/high-dose ZWT combined group improved,and the synovitis of the high-dose ZWT combined group improved better than the methotrexate group.7.ELISA results:TNF-α,IL-6 and IL-Iβ levels in the serum of CIA rats were significantly increased.Compared with the model group,TNF-α,IL-6,and IL-Iβ levels in the serum of the methotrexate group and low/high-dose ZWT combined group were decreased,and the level of TNF-α and IL-Iβ in the high-dose ZWT combined group was significantly lower than that of the methotrexate group.8.TRAP results:The number of osteoclasts in the ankle joint of CIA rats increased significantly.Compared with the model group,the number of osteoclasts in the ankle joint of rats in the methotrexate group and low/high-dose ZWT combined group decreased.The number of osteoclasts in the ankle joint of rats in the high-dose ZWT combined group was Less than that of methotrexate group.9.Immunohistochemical results:The expression of RANKL in the ankle joint of CIA rats was significantly increased,and the expression of OPG was decreased.Compared with the model group,RANKL in the ankle joint of rats in the methotrexate group was decreased and OPG increased,but there was no statistical significance.The expression of RANKL in the ankle joint of rats in the low-dose ZWT combined group and the high-dose ZWT combined group significantly decreased,OPG expression significantly increased,and the ratio of RANKL/OPG decreased significantly.The effect of combined medication group is better than the methotrexate group.10.PCR results:the expression of RANKL mRNA in the ankle joint of CIA rats was significantly increased,and the expression of OPG mRNA was decreased.Compared with the model group,RANKL in the ankle joint of rats in the methotrexate group and low/high-dose ZWT combined group was decreased and OPG increased,The ratio of RANKL/OPG decreased significantly.The effect of combined medication group is better than the methotrexate group.11.WB results showed that the expression of RANKL protein in the ankle joint of CIA rats was significantly increased,and the expression of OPG protein was significantly decreased;Compared with the model group,RANKL in the ankle joint of rats in the methotrexate group and low/high-dose ZWT combined group was decreased and OPG increased,the RANKL/OPG ratio decreased significantly.The effect of combined medication group is better than the methotrexate group.CONCLUSION:1.ZWT showed significant anti-inflammatory effects in the CIA model,and its mechanism may be to inhibit the expression of TNF-α and MMP-13 mRNA and protein,and regulated the expression of TNF-α and MMP-13 in the joints,thereby improving the inflammation of experimental animals.2.ZWT has a significant anti-bone destruction effect in the CIA.model,and its mechanism may be to inhibit RANKL and up-regulate the expression of OPG mRNA and protein.3.Doppler high-frequency ultrasound has application value in the assessment of bone and joint diseases in small animals. |
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