Study On The Mechanism Of FJHQ Decoction In The Treatment Of Knee Osteoarthritis By Regulating Lymphatic Drainage Function

Object: Knee osteoarthritis(KOA)is a chronic degenerative joint disease,characterized by articular cartilage rupture,subchondral bone remodeling,and synovial inflammation.In China,the prevalence of KOA is high and harmful,and there is still a lack of effective therapeutic drugs.Previous studies have shown that the progression of osteoarthritis is closely related to lymphatic drainage function,and that lymphatic system plays an important role in the development of osteoarthritis.At present,Fangji Huangqi(FJHQ)decoction has been widely used in the treatment of KOA,which can effectively improve the symptoms of pain and swelling in KOA patients.Our previous studies on FJHQ decoction have shown that FJHQ decoction can improve the production of lymphatic vessels in zebrafish and reduce the joint swelling of TNF-transgenic mice.In this study,we investigated whether FJHQ decoction improved joint structure of OA or delayed the process of knee joint degeneration in OA mice by regulating lymphatic drainage function.Method:1?Study on the relationship between early knee osteoarthritis and lymphatic drain function DMM surgical modeling of the right knee of 10-week-old male C57BL/6 mice and control of the left knee were performed.Samples were collected at 1,2,3,4 and 5weeks after surgery,respectively.We analyzed the correlation between lymphatic drainage function and KOA by detecting lymphatic drainage function,ABOG staining and immunofluorescence staining.2?Experimental study of fangji huangqi decoction in the treatment of knee osteoarthritis by promoting lymphatic drainage10-week-old male C57BL/6 mice were used to induce KOA by injection of type VII collagenase into the right knee,or the left knee as the control.The mice were randomly divided into three groups,including a group of OA models treated with PBS,a group of OA mice treated with FJHQ decoction,and a positive control group of OA mice treatedwith Mobic,a common nonsteroidal anti-inflammatory drug for KOA.Each mouse was given 0.2ml solution every other day.All mice were tested for lymphatic drainage at week 4,week 6,week 8 and week 10,then sacrificed for test(N=10/group).Using near infrared indocyanine green(NIR-ICG)lymphatic imaging system,immunohistochemical staining(IHC),immunofluorescence histochemical double staining(IF),and whole slide digital microscopy(WSD),we investigate lymphatic draining function,the distribution of lymphatic vessels in knee joints and degeneration of the knee.Type II collagen and NITEGE,accumulation of the aggrecan degradation product,were used for IHC to evaluate the degree of knee degeneration.The presence and alteration of lymphatic vessels were observed and analyzed using double immunofluorescence staining and a whole-slide digital imaging system.3.Effect of fangji huangqi decoction freeze-dried powder on lymphatic smooth muscle cells A primary mesenteric lymphatic smooth muscle cell culture system was established.Fangji huangqi decoction freeze-dried powder was prepared.Nitric oxide donor drug DETA NONoate was used to treat primary lymphatic smooth muscle cells.Results:1.The development of early knee osteoarthritis is related to the change of lymphatic drain function The lymphatic reflux function of the knee joint was detected 1-5 weeks after the operation,and it was proved that the lymphatic reflux function was gradually improved with the change of KOA.Analysis with ABOG staining showed progressive joint destruction and immunofluorescence lymphatic staining showed increased lymphatic involvement with KOA.2.Fangji huangqi decoction improves lymphatic drain function and delays the progression of early knee osteoarthritis We found that lymphatic draining function was significantly reduced in PBS group compared with the sham group.After treatment with FJHQ decoction,the lymphatic draining function improved at 4 wps and 6 wps.The results of analysis indicated a strong correlation between lymphatic draining function(ICG clearance)and tissue damage degree(OARSI score)in knee joints.By ABOG staining of the paraffin sections,FJHQ-treated group exhibited less cartilage destruction and lower OARSI scores.Moreover,the result of immunohistochemical staining(IHC)shows that FJHQ decoction increased the content of type II collagen in knee joints of OA mice at 4 wpsand 6 wps.By the immunofluorescence double staining of podoplanin and smooth muscle actin on the paraffin sections,the capillaries and mature lymphatics in the FJHQ group increased at 4 wps.3.Fangji huangqi decoction promoted the expression of the gene of lymphatic smooth muscle contraction function Under the intervention of nitric oxide donor drugs,lymphatic smooth muscle cells were pretreated with fangji huangqi decoction,and the expression of genes related to systolic function was increased.Conclusion:The lymphatic drain function of the early KOA model decreased significantly compared with the control group.Fangji huangqi decoction can improve the function of early KOA lymphatic drain and promote the increase of early KOA capillary lymphatics and mature lymphatics.Fangji huangqi decoction can delay the loss of type II collagen and the early KOA degeneration process.Fangji huangqi decoction can promote the expression of the contraction function gene of lymphatic smooth muscle cells.