| ObjectiveIt aims to clarify the intervention effect and mechanism of Zhuanggu Jianxi Decoction on synovitis of knee osteoarthritis(KOA)from the perspective of inflammatory regulation on human joint synovial cells and tissues and key node proteins in LXRs/NF-?B pathway,so as to further enrich its scientific connotation of removing arthralgia in simultaneous treatment of arthralgia and flaccidity.Methods1.Literature review Systematically analyse literature,including the physiology and pathology of synovial tissue of knee joint,the role of synovitis in the pathogenesis of KOA,the relationships between cartilage degeneration,inflammation factors,matrix metalloproteinases and KOA ralated synovitis,signal pathway regulation of KOA synovitis,etiology and pathogenesis of traditional Chinese medicine,syndrome differentiation and treatment of KOA synovitis,the progress of rehabilitation of traditional Chinese and western medicine,the understanding of the research group on KOA,the basis of previous research and the principle of Zhuanggu Jianxi Decoction.Comprehensively comb and analyze the connotation of pathogenesis of KOA synovitis and cartilage degeneration,KOA related synovial inflammation,and pathogenesis of coexistence of arthralgia and flaccidity,and the key points of prevention,treatment and rehabilitation of KOA synovitis from the literature point.2.Experimental research Two in vitro experimental models of synovial tissue of patients with KOA synovitis and human knee synovial macrophage stimulated by LPS were used.The research was divided into two parts.2.1 Research of Zhuanggu Jianxi medicated serum on human knee synovial macrophage stimulated by LPS(1)The preparation of Zhuanggu Jianxi medicated serum Twelve New Zealand white rabbits aged 3 months were randomly divided into the blank serum group and the medicated serum group(n=6,respectively).The medicated serum group was administrated intragastrically with 4.58 mL/kg/d of Zhuanggu Jianxi Decoction liquid.The blank serum group was administrated intragastrically with equal volume of normal saline,twice a day for 3 days.An hour after the last gavage,blood of rabbit from the abdominal aorta was collected after anesthesia.After centrifugation,serum was then collected,56? water bath for 30 min for inactivation,filtration and divided for later use.(2)Selection of optimal concentration and time of LPS stimulating synovial macrophage Synovial macrophage in good growth condition were randomly divided into 0 ?g/mL,0.5?g/mL,1.0 ?g/mL,2.0 ?g/mL LPS groups,added DMEM culture medium containing 0?g/mL,0.5 ?g/mL,1.0 ?g/mL,2.0 ?g/mL LPS,intervene for 8 h,12 h,18 h and 24 h,respectively.Then collect the supernatant,and detect the contents of IL-1? and TNF-?,take the concentration and time of the highest expressed group as the most suitable time for LPS induced inflammatory cells models.(3)Determination of the optimal concentration and time of medicated serum intervention in synovial macrophage inflammation model LPS suspension concentration and intervention time determined before were used for culturing human knee synovial macrophage which were in good growth condition.And then randomly divided cells into 0%,5%,10%and 20%of Zhuanggu Jianxi Decoction medicated serum groups,complete culture medium containing 0%,5%,10%and 20%of Zhuanggu Jianxi Decoction medicated serum was added respectively and cultured for 12 h,24 h,36 h and 48 h.When intervention completed,ELISA was used to detect the contents of IL-1? and TNF-? in supernatant.The intervention concentration and time when the contents were at the lowest level were seen as the best concentration and time for the follow-up intervention.(4)The intervention of medicated serum on synovial macrophage Synovial macrophage in good growth condition were randomly divided into normal group and LPS group.Normal group was intervened with complete culture medium containing 10%FBS,while the LPS group was intervened with LPS of certain concentration determined before for certain time and then randomly divided into model group,medicated serum group,LXR?blocker group and N-CoR blocker group.Synovial macrophage in each group were intervened with the blank serum of the best concentration determined before,the medicated serum of Zhuanggu Jianxi Decoction and the DMEM medium of the corresponding blocker.After intervention,synovial macrophage and supernatant of each group were collected.ELISA was used to detect the contents of IL-1? and TNF-? in the synovial macrophage supernatant.Western blot and RT-qPCR were used to detect expressions of LXR?,N-CoR,P50 and P65 proteins and mRNA.Results were statistically analyzed and compared.2.2 Research of Zhuanggu Jianxi medicated serum on synovial tissue of patients with KOA synovitis(1)The medicated serum of Zhuanggu Jianxi Decoction was prepared by the same method as 2.1.(2)The collection and culture of synovial tissue Collect synovial tissue from patients who were diagnosed as primary KOA synovitis and meet the inclusion criterias in in surgery of joint,second bone area,Fuzhou Hospital of Integrated Traditional Chinese and Western Medicine during the procedure of total knee arthroplasty.Synovial tissue of knee joint in non synovitis patients who underwent arthroscopic repair due to cruciate ligament or meniscus injury were obtained.Samples of synovial tissues were put into EP tubes containing precooled sterile D-Hank's solution.After disinfection with 75%alcohol,samples were immediately transferred to the super clean workbench.Carefully remove the visible adipose tissue around the synovium with ophthalmic scissors,cut it into small pieces of about 1?2 mm3 visible to the naked eye,rinsing with D-Hank's solution and precipitate naturally.Then transferred into sterile 6-well plates with equal quantity.After inversion for 1 h,slowly added the complete culture medium containing 10%FBS and cultured at 37?and 5%CO2.(3)Determination of optimal concentration and time of medicated serum intervention in synovial tissue of patients with KOA synovitis Synovial tissues were randomly divided into 0%,5%,10%and 20%of Zhuanggu Jianxi Decoction medicated serum groups,0%,5%,10%and 20%of Zhuanggu Jianxi Decoction medicated serum was added respectively and cultured for 3 days,5 days,7 days and 10 days,respectively.After that,ELISA was used to detect the contents of IL-1? and TNF-? in synovial tissue homogenate supernatant.Take the concentration and time when these indexes express at the lowest level as the most appropriate concentration and time for intervention followed.(4)Intervention of medicated serum on synovial tissue Take certain amount of synovial tissues pieces and randomly divided into 4 groups as model group,medicated serum group,LXRa blocker group and N-CoR blocker group,3 wells in each group and 5 tissue blocks in each well.Take normal synovial tissues as the normal group.Then,culture medium containing blank serum,medicated serum and corresponding blockers whose concentrations were determined before was added into synovial tissues to culture for certain time determined during the pre-study.After that,observe the migration of cells around synovial tissue blocks in each group under light microscope,then discard the culture medium,collect and randomly take 6 small synovial tissue blocks in each group,and fix,dehydrate and embed them with 4%paraformaldehyde.HE staining was used to observe the morphological changes of synovium,Immunofluorescence staining was used to observe the expression intensity of specific markers of Smooth Musle Actin(SMA)in fibroblasts,and IHC were used to detect the expressions of LXR?,N-CoR,P50 and P65 proteins.After weighing the remaining synovial tissue in each group,take 0.2 g synovial tissue respectively,put it into the glass homogenization tube,add 1.8 mL PBS buffer,grind it fully,collect the synovial tissue homogenization,centrifuge and take the supernatant for ELISA which was used to detect IL-1?,TNF-?,MMP-3 and MMP-13 inflammatory factors,and the results were statistically analyzed.ResultsLiterature review(1)It is generally believed that KOA synovitis is an important cause of joint swelling,pain,stiffness and limited flexion and extension activities.KOA synovitis and KOA cartilage degeneration coexist and affect each other.(2)Researches have confirmed that LXRs/NF-?B pathway is closely related to the occurrence and development of KOA synovitis.The abnormal expression of key node proteins in the pathway such as LXRa,N-CoR,P50 and P65 may be involved in the inflammation of synovium.(3)“Coexistance of arthralgia and flaccidity" is the main feature of dysfunction caused by KOA.Synovitis belongs to the category of“arthralgia syndrome" and KOA cartilage degeneration belongs to the category of "flaccidity syndrome".However,arthralgia and flaccidity can affect each other during the occurrence and development of KOA,so it is appropriate to treat arthralgia and flaccidity together and remove arthralgia and flaccidity in the prevent and rehabilitation of KOA synovitis,so as to reflext the point of overall treatment.(4)Zhuanggu Jianxi Decoction has the effect of simultaneous treatment of arthralgia and flaccidity.Based on the observation and interpretation of the influence of "treating flaccidity"on articular cartilage degeneration in the early stage,it is necessary to further explore its mechanism on removing arthralgia from the perspective of synovitis so as to enrich its scientific connotation on combined treatment of arthralgia and flaccidity.2.Experimental research2.1 The influence of Zhuanggu Jianxi medicated serum on human knee synovial macrophage stimulated by LPS(1)Concentrations and intervention time of LPS stimulated human knee synovial macrophage By comparing the contents of IL-1? and TNF-? of synovial macrophages stimulated by different concentrations of LPS for different times,took research period and cost into consideration,it's showed that the contents of IL-1? and TNF-? in synovial macrophages stimulated by LPS at 1.0 ug/mL for 12 h and 18 h were the highest.It‘s suggested that IL-1? and TNF-? expressed the highest level under the condition of 1.0 ug/mL LPS stimulated for 12 hours,which was used as the condition of follow-up intervention.(2)Concentrations and intervention time of medicated serum on human knee synovial macrophage inflammation models.By comparing the contents of IL-1? and TNF-? of synovial macrophages inflammation models intervened by different concentrations of medicated serum for different times,took research period and the cost of preparing medicated serum into consideration,The results showed that the contents of IL-1? and TNF-? were the lowest at 24 h,36 h and 48 h after intervention with 10%drug-containing serum.It's suggested that IL-1? and TNF-? expressed the highest level under the condition of 10%medicated serum intervened for 24 hours,which was used as the condition of follow-up intervention.(3)The results of related indexes1)Changes of expression of LXR?,N-CoR,P50 and P65 proteins and mRNA of synovial macrophage in each group after intervention.The F values of LXR?,N-CoR,P50 and P65 protein expression between groups after intervention were 47.946,50.648,40.317 and 35.553 respectively,which were of significant difference.Compared with the normal group,the expression of LXR?,N-CoR in the model group decreased significantly,P50 and P65 proteins increased significantly(P<0.01).Compared with the model group,LXR? and N-CoR in the medicated serum group were significantly increased,and P50 and P65 were significantly decreased(P<0.01 or P<0.05).Compared with the model group,LXR? and N-CoR of the LXR? blocker group had no significant differences(P>0.05),while the expression of P50 and P65 decreased significantly(P<0.01 or P<0.05).The expression of LXR? of the N-CoR blocker group increased significantly(P<0.01),while the expression of N-CoR,P50 and P65 decreased significantly(P<0.01 or P<0.05).Compared with the LXR? blocker group,the expression of LXR? and N-CoR of the medicated serum group increased significantly(P<0.01),while the expression of P50 and P65 decreased significantly(P<0.01).Compared with N-CoR blocker group,the expression of LXR? and N-CoR of the medicated serum group increased significantly(P<0.01),while the expression of P50 and P65 decreased significantly(P<0.01 or P<0.05).2)Changes of expression of LXR?,N-CoR,P50 and P65 mRNA of synovial macrophage in each group after intervention The F values of LXR?,N-CoR,P50 and P65 protein expression between groups after intervention were 60.123,48.313,38.036,42.682,respectively,which were of significant difference.Compared with the normal group,the expression of LXR?,N-CoR in the model group decreased significantly,P50 and P65 increased significantly(P<0.01).Compared with the model group,LXR? and N-CoR in the medicated serum group were significantly increased,and P50 and P65 were significantly decreased(P<0.01 or P<0.05).Compared with the model group,LXR? and N-CoR of the LXR? blocker group had no significant differences(P>0.05),while the expression of P50 and P65 decreased significantly(P<0.01 or P<0.05).The expression of LXRa of the N-CoR blocker group increased significantly(P<0.01),the expression of N-CoR had no significant difference(P>0.05),P50 and P65 decreased significantly(P<0.01 or P<0.05).Compared with the LXR? blocker group,the expression of LXR? and N-CoR of the medicated serum group increased significantly(P<0.01),while the expression of P50 and P65 decreased significantly(P<0.01).Compared with the N-CoR blocker group,the expression of LXR? and N-CoR of the medicated serum group increased significantly(P<0.01),while the expression of P50 and P65 decreased significantly(P<0.01).3)Changes of the contents of inflammatory factors in synovial macrophage supernatant The F values of IL-1? and TNF-? between groups after intervention were 41.294,35.240,respectively,which were of significant difference.Compared with normal group,the contents of IL-1? and TNF-? of model group increased significantly(P<0.01).Compared with the model group,the contents of IL-1? and TNF-? in medicated serum group,LXR? blocker group and N-CoR blocker group decreased significantly(P<0.01 or P<0.05).Compared with the LXR? blocker group,the contents of IL-1?and TNF-? of the medicated serum group decreased significantly(P<0.01 or P<0.05).Compared with the N-CoR blocker group,the contents of IL-1? and TNF-? of the medicated serum group decreased significantly(P<0.01 or P<0.05).2.2 The influence of Zhuanggu Jianxi medicated serum on synovial tissue of patients with KOA synovitis(1)The comparison of observation of HE staining on synovial tissues between normal group and model group In the normal group,there were many adipocytes in the synovial tissue,the synovial surface is smooth,the synovial lining cells were 1-3 layers,the cells are evenly distributed and orderly,there is no vascular proliferation,no or occasional lymphocytes.In the model group,there were few adipocytes in the synovial tissue,the synovial surface was uneven,the number of cell layers in the synovial lining layer proliferated significantly,up to 5-9 layers,the cells were arranged in disorder,and more monocyte macrophages and lymphocytes were observed.(2)The concentration and intervention time of medicated serum on synovial tissues By comparing the effects of different concentrations of Zhuanggu Jianxi Decoction medicated serum on the influence of IL-1? and TNF-? in synovium of patients with KOA synovitis at different times,the results showed that the contents of IL-1? and TNF-? were the lowest when the concentration of 10%drug-containing serum was 7 days and 10 days.It showed that under the condition of 10%medicated serum intervened for 7 days,the contents of IL-1? and TNF-? were the lowest and as a condition for follow-up intervention.(3)The migration of small peripheral cells of synovial tissue in each group before and after intervention under light microscope Before intervention,observed under light microscope,there was a small number of adherent cells proliferated at the edges of synovial membranes in each group,and most of the cells were round,elliptical or long fusiform,arranged in a single layer of concentric clusters.After 7 days of intervention,observed under the light microscope,there were a large number of adherent cells along the edges of the normal group and the medicated serum group,and most of the cells were round,oval or long fusiform,arranged in a single layer in a centripetal shape,paving stone-like.There were a large number of adherent cells along the edges of the model group and LXR? and N-CoR blocker groups,and most of the cells were fusiform or stellate,the cells in the central part were arranged in multi-layer centripetal aggregation while the cells in the surrounding part grew without obvious arrangement direction.(4)Related index observation results1)After intervention,the level of immunofluorescence expression of fibroblast specific marker SMA in synovitis group was significantly higher than that in normal group(P<0.01).Compared with the modell group,the ID values of drug-containing serum group,LXR?blocker group and N-CoR blocker group were significantly decreased(P<0.01).Compared with LXR? blocker group,ID value of in the medicated serum group was significantly decreased(P<0.01).Compared withN-CoR blocker group,ID value in the medicated serum group was significantly decreased(P<0.01).2)Changes of HE staining of synovial tissue in each group after intervention Compared with the normal group,the model group had pathological changes like synovial hyperplasia,disorder and inflammatory infiltration,and there was significant difference in morphological score(P<0.01).Compared with the model group,the pathological changes of medicated serum group and LXR?,N-CoR blocker groups reduced,morphological score decreased(P<0.01 or P<0.05).Compared with LXR? blocker group,the pathological changes and the morphological score of the medicated group decreased(P<0.05).Compared with N-CoR blocker group,the pathological changes and the morphological score of the medicated group decreased(P<0.05).3)Changes of the contents of IL-1? and TNF-? in synovial tissue homogenate supernatant after intervention After intervention,the F values of IL-1? and TNF-? between groups were 55.306,73.699,respectively,which were of significant difference.Compared with the normal group,the contents of IL-1? and TNF-? in the model group were significantly increased(P<0.01).Compared with the model group,the contents of IL-1? and TNF-?significantly reduced in the medicated serum group,LXR? and N-CoR blocker groups after intervention(P<0.05 or P<0.01).Compared with LXR? blocker group,the contents of IL-1? and TNF-? of the medicated serum group were significantly decreased(P<0.01).Compared with N-CoR blocker group,the contents of IL-1? and TNF-? of the medicated serum group were significantly decreased(P<0.01).4)Changes of the contents of MMP-3 and MMP-13 in synovial tissue homogenate supernatant after intervention After intervention,the F values of MMP-3 and MMP-13 between groups were 422.496,106.809,respectively,which were of significant difference.Compared with the normal group,the contents of MMP3 and MMP-13 in the model group were significantly increased(P<0.01).Compared with the model group,the contents of MMP3 and MMP-13 were significantly decreased in the medicated serum group,LXR? and N-CoR blocker groups after intervention(P<0.01).Compared with LXR? blocker group,the contents of MMP3 and MMP-13 in medicated serum group were significantly decreased(P<0.01).Compared with N-CoR blocker group,the contents of MMP3 and MMP-13 in medicated serum group were significantly decreased(P<0.01).5)Changes of the expression of LXR?,N-CoR,P50 and P65 proteins of synovial tissue after intervention After intervention,the F values of LXR?,N-CoR,P50 and P65 IOD levels between groups were 73.205,14.235,48.416,124.092,respectively,which were of significant difference.Compared with the normal group,the IOD levels of LXR? and N-CoR in the model group decreased significantly(P<0.01),P50 and P65 increased significantly(P<0.01).Compared with the model group,the IOD levels of LXR? and N-CoR in the medicated serum group increased significantly(P<0.05),P50 and P65 decreased significantly(P<0.01);The IOD levels of LXR? and N-CoR in LXR? blocker group had no significant diffference(P>0.05),while the IOD level of P50 and P65 decreased significantly(P<0.01).The IOD levels of LXR? in N-CoR blocker group increased significantly,the IOD level of N-CoR had no significant difference(P>0.05),while P65 of P65 decreased significantly(P<0.05 or P<0.01).Compared with LXR? blocker group,IOD levels of LXR? and N-CoR in the medicated serum group increased significantly(P<0.01),P50 and P65 decreased significantly(P<0.05).Compared with N-CoR blocker group,the IOD levels of LXR? and N-CoR in the medicated serum group increased significantly(P<0.01),P50 and P65 decreased significantly(P<0.05).Conclusions1.According to TCM theory,“Coexistance of arthralgia and flaccidity" is the feature of dysfunction caused by KOA.Dysfunction caused by synovial inflammation of knee and cartilage degeneration belong to the category of "arthralgia syndrome""flaccidity syndrome"and can interact with one another,therefore,combined treatment of arthralgia and flaccidity to remove arthralgia and flaccidity should be applied.2.Zhuanggu Jianxi Decoction can improve the inflammatory state of synovial macrophages,decrease the secretion of IL-1? and TNF-?.This effect is probably related to its regulation on LXRs/NF-?B pathway in synovial macrophages,presented as upregulate the expression of LXR? and N-CoR proteins and mRNAs to affect the expression of P50 and P65 proteins and mRNAs,and then decrease the expression of downstream IL-1? and TNF-?.3.Fibroblast like synovial cell proliferation,inflammatory factors and matrix metalloproteinases increased in synovial tissue of KOA synovitis in vitro.Zhuanggu Jianxi Decoction can inhibit the proliferation of fibroblast like synovium in synovium of KOA synovitis,reduce the secretion of inflammatory factors,reduce the content of matrix metalloproteinase and reduce synovium inflammation.The mechanism probably related to the regulation of key node proteins in LXRs/NF-?B pathway in synovial tissue macrophage and reduce NF-?B signaling pathway activity.4.The effect of removing arthralgia of Zhuanggu Jianxi Decoction is closely related to the regulation of key node proteins in LXRs/NF-?B pathway in synovial macrophage,reduce NF-?B signaling pathway activity,and reduce the expression of inflammatory factors like IL-1? and TNF-?,improve the proliferation of synovial fibroblasts,thus reducing the synovial inflammation. |
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