Based On TLRs/MyD88 Signaling Pathway, The Mechanism Of Tongluo Zhitong Recipe On Synovial Inflammation Of Knee Osteoarthritis Was Investigated

Tongluozhitong Fang is the author’s experience in the treatment of Knee Osteoarthritis(KOA)for many years,and has carried out a series of clinical and basic research.Clinical trials found that Tongluozhitong KOA external treatment can effectively relieve the pain and improve knee function;animal experiment,Tongluozhitong treatment can significantly inhibit the formation of rabbit KOA synovial cell inflammatory factor;the principal drug effective components of Guizhi cinnamaldehyde in human KOA synovial cells,DAMPs/Toll like receptors(TLRs)NF-κB pathway mediated inflammation plays an important role in OA synovitis,many of the genes on the NF-κB pathway are involved in OA synovial inflammation.But the specific mechanism of the party is not yet clear.Therefore,this project intends to use Tongluozhitong as drug intervention,through the human body cell experiment,observe the application of real-time quantitative PCR and Western blotting using animal experiments(Western blot)technology to detect TLRs/MyD88 signaling pathway components(TLR2,TLR4,MyD88)changes,and enzyme-linked immunosorbent assay(ELISA the detection of inflammatory cytokines)pathway of interleukin-1(1L-1)tumor necrosis factor-α(TNF-α),matrix metallo proteinase-13(MMP-13)and the concentration of Tongluozhitong,molecular mechanism and target treatment of KOA.Section 1 clinical experimentThe correlation between the inflammatory factors associated with the synovial TLRs/MyD88 signaling pathway and the progression of knee osteoarthritis in patients with KOAObjective:to detect the concentration of inflammatory factors MMP-13 and IL-6 downstream of TLRs/MyD88 signal pathway in human KOA synovium.The relationship between the two and the analysis of X-ray staging(Kellgren&Lawrence standard)of osteoarthritis of the knee was observed.To explore the internal relationship between inflammatory cytokines and the severity of KOA,and to find the biochemical indicators that reflect the severity of KOA.It provides some useful reference and help for the early clinical diagnosis and treatment of KOA.Materials and methods:from the Department of orthopedics in outpatient or hospitalized patients randomly selected or arthroscopic knee arthroplasty for the treatment of knee osteoarthritis in 60 patients,the Chinese Medical Association orthopedics branch set"diagnosis and treatment of osteoarthritis guide"(2007 Edition)standard,according to the X-ray grade(Kellgren&Lawrence grade)standard is divided into early,in later,3 groups,20 patients in each group as the experimental group and control group were randomly selected 10 cases of acute lower extremity trauma(excluding knee injury)patients,the knee joint fluid was detected by ELISA after treatment,the concentration of MMP-13,IL-6 and other inflammatory mediators.There was no statistical difference in the concentration of MMP-13 and IL-6 between the experimental group and the control group and the experimental group,and the correlation between the concentration and the severity of KOA was analyzed.Results:1.the concentration of IL-6 in the synovial fluid of the experimental group decreased with the progression of KOA.The concentration of IL-6 in the early and middle stages was higher than that in the control group.The concentration of IL-6 in late stage was lower than that in the control group,P<0.05,there was statistical difference.2.,the concentration of MMP-13 in the synovial fluid of the experimental group increased with the concentration and concentration of KOA.The concentration of MMP-13 in the joint fluid of the experimental group was higher than that of the control group MMP-13,P<0.05,there was a significant difference.3.the concentration of 1L-6 and MMP-13 in different groups of experimental groups was statistically different,P<0.05.Conclusion:the concentration of KOA and the severity of MMP-13 and IL-6 is closely related with the progress of the course,the lower concentration of IL-6,with the progress of the disease,the higher the concentration of MMP-13,suggesting that at the early stage of KOA without bony changes,observe the content of MMP-13 and IL-6 in synovial fluid can be used as the diagnosis and treatment of early stage KOA index.Section 2 Cell experimentThe effect of Tongluozhitong prescription on KOA synovitis through TLRs/MyD88 signal pathwayExperiment1 Effect of Tongluozhitong Fang on the proliferation and cycle of synovial fibroblasts of human knee osteoarthritisObjective:To investigate the effect of Tongluozhitong Fang on the proliferation and cycle of synovial fibroblasts of human knee osteoarthritis(KOA).Methods:the synovial tissue was removed from the patients undergoing total knee arthroplasty in our hospital.After I collagenase digestion,primary culture was performed,and 3～4 fibroblasts were taken to intervene with high,middle and low concentration Tongluozhitong Fang.The proliferation and cell cycle of KOA synovial cells were measured by four methazolium salt method(MTT)and flow cytometry(FCM),and the morphology of cells was observed under inverted microscope.Results:the difference was statistically significant(P<0,05)between the addition group and the addition group and the blank group.MTT assay with Tongluozhitong concentration decreased KOA synovial cell number in the light absorption at the wavelength of 490nm was greater in high school;concentration group the inhibition rate was positive,the inhibition rate increased with the increase of concentration,the inhibition rate of low concentration is negative,with decreasing the concentration of inhibition rate decreased.High,middle and low concentrations Tongluozhitong Fang can prolong the G2(P<0,05)phase in the cell cycle and shorten the S(P<0,05)stage,and inhibit the proliferation of KOA synovitis cells.Conclusion:high and middle concentration Tongluozhitong Fang can inhibit the proliferation of synovial cells,and the Tongluozhitong Fang of low concentration group has a proliferation effect on synoviocytes,which needs further study.Experiment 2 Effect of Tongluozhitong Fang on 1L-1β,TNF-α,MMP-13 and NO of synovitis cells of human knee osteoarthritisObjective:To observe the effect of Tongluozhitong Fang on the content of 1L-1β,TNF-α,MMP-13 and NO in the supernatant of synovitis of knee osteoarthritis.Methods:our hospital waste in synovial tissue of patients with total knee replacement surgery,were cultured from 3～4 fibroblasts by different concentration of Tongluozhitong Fang,specifically divided into:high concentration group(2.5mg/mL),concentration(0.25mg/mL):low concentration group(0.025 mg/mL),control group(Omg/mL).The changes in the concentration of IL-1β,TNF-α,MMP-13 and NO in the supernatant of cell culture were detected by ELISA.Results:high,medium and low concentration group was significantly lower than the control group in the supernatant of IL-1β,TNF-α,MMP-13 and NO content(P<0.05),and the decreased amplitude and Tongluozhitong was positively related to the concentration of the 1L-1β:r=0.39,P<0.05;TNF-a:r=0.41,P<0.05;MMP-13:r=0.36 NO:r=0.52,P<0.05,P<0.05).The high concentration group of IL-1β,TNF-α,MMP-13 and NO reached the peak of inhibition,and the difference was statistically significant compared with the middle and low concentration groups(P<0.05).Conclusion:Tongluozhitong Fang has inhibitory effect on 1L-1β,TNF-α,MMP-13 and NO of human knee osteoarthritis synovitis,and the inhibition is more obvious with increasing concentration.Experiment 3 The effect of Tongluozhitong Fang on 1L-1β,HA and NO in the joint fluid of human knee osteoarthritisObjective:To observe the effect of Tongluozhitong Fang on the levels of 1L-1β,Hyaluronic(HA)and nitric oxide(NO)in human knee osteoarthritis synovial fluid.Methods:arthroscopic surgery patients,intraoperative intra-articular effusion in extraction,after centrifugation,the supernatant,adding different concentrations of Tongluozhitong Fang,divided into:high concentration group:2.5mg/ml;concentration:0.25mg/ml;low concentration group:0.025 mg/ml;and the control group(phosphate buffered saline,PBS).Enzyme linked immunosorbent assay(ELISA)was used to observe the changes in the content of 1L-1β,HA and NO in each concentration group.Results:the concentrations of 1L-1β and NO in the high,middle and low concentration groups were significantly lower than those in the control group(P<0.05),and the concentrations of 1L-1β and NO were positively correlated with the concentrations of Tongluozhitong Fang(1L-1β:r=-0.29,P<0.05;NO:r=-0.51,P<0.05).The content of HA in synovial fluid of high,middle and low concentration group was significantly higher than that of control group(P<0.05),and the concentration of HA was positively correlated with the concentration of Tongluozhitong Fang(HA:r=0.59,P<0.05).Conclusion:Tongluozhitong Fang has inhibitory effect on 1L-1β and NO in synovial fluid.The higher the concentration is,the greater the inhibition degree.Tongluozhitong Fang has a promoting effect on HA in the joint fluid,and the higher the concentration is,the greater the degree of promotion is.Experiment 4 Effect of Tongluozhitong Fang on TOll like receptor of synovitis cells of human knee osteoarthritisObjective:To observe the effect of Tongluozhitong Fang on the TOll like receptor of synovitis cells of human knee osteoarthritis.Methods:our hospital waste in synovial tissue of patients with total knee replacement surgery,were cultured from 3～4 fibroblasts by different concentration of Tongluozhitong Fang,specifically divided into:high concentration group(2.5mg/mL),concentration(0.25mg/mL):low concentration group(0.025 mg/mL),control group(Omg/mL).The expression of TLR4 mRNA in cells was detected by al-time PCR,and the expression of TLR2 and TLR4 in cells was detected by flow cytometry(FCM).Results:compared with the control group,the expression level of TLR4 mRNA in the knee osteoarthritis synovitis cells of the high and middle concentration group Tongluozhitong Fang was significantly lower than that of the control group(P<0.05).The inhibitory effect of Tongluozhitong Fang in low concentration group on TLR4 mRNA of synovitis was similar to that of the control group,and the difference was not statistically significant.The inhibitory effect of medium and high concentration groups was proportional to the concentration.High,medium and low concentration of Tongluozhitong Fang group the expression rate of TLR4 compared with the control group were significantly decreased(P<0.05),and with the concentration of TLR4,the expression rate decreased;high,medium and low concentration of Tongluozhitong Fang group the expression rate of TLR2 compared with the control group,the difference was not statistically significant(P>0.05).Conclusion:high,middle and low concentrations Tongluozhitong Fang can significantly inhibit the expression of TOll like receptor,suggesting that Tongluozhitong Fang can affect or inhibit synovial inflammation of human knee osteoarthritis through TOll like receptor(Toll like receptor,TLR)pathway.Section 3 Animal experimentThe effect of Tongluozhitong Fang on TLRs/MyD88 signaling pathway of knee osteoarthritis in different degreesExperiment 1 The effect of Tongluozhitong Fang on histomorphology and inherent immunity of knee osteoarthritis of different degreesObjective:To evaluate the effect of Tongluozhitong Fang on cartilage and synovial structure and inherent immunity of different degrees of knee osteoarthritis.Methods:using the classical Hulth method to establish the mild,moderate and severe knee OA rat animal model,method of transdermal Tongluozhitong using ultrasound intervention on rat KOA model,respectively,to determine the extent of lesions by observing the changes of synovium and cartilage morphology,and the cartilage s score for Mankin ".The changes of major protein TLR4,NF-κB and MyD88,which were involved in the destruction of cartilage and synovial structure,were observed by double immunofluorescence technique.Results:the degree of synovium and cartilage injury in the group of Tongluozhitong Fang group in the 2,4 and 8 weeks group was significantly lower than that of the control group.In the innate immunity,the expression of TLR4,NF-κB and MyD88 in the group of Tongluozhitong Fang was weaker than that of the control group.Conclusion:Tongluozhitong Fang has inhibitory effect on knee osteoarthritis of different degrees.It is proved that Tongluozhitong Fang can inhibit the occurrence and development of knee osteoarthritis by inhibiting the expression of related TLRs/MyD88 signaling pathway.Experiment 2 The effect of Tongluozhitong Fang on the expression of TLRs/MyD88 signal pathway related original parts in different degree knee osteoarthritisObjective:To study the effect of Tongluozhitong Fang on the expression of TLRs/MyD88 signal pathway related original parts in different degree of knee osteoarthritis.Methods:using the classical Hulth method to establish the mild,moderate and severe knee OA rat animal model,method of transdermal Tongluozhitong using ultrasound intervention on rat KOA model,Western blot,PCR technology application effect of Tongluozhitong clear expression trend of varying degrees of KOA,MyD88,NF-κB and TLR4 in synovium.Results:the expression of the three indexes in the Chinese medicine group was significantly lower than that of the non dry pregroup and the saline group,and the expression level was similar to that in the untreated group and the saline group.Conclusion:Tongluozhitong Fang has inhibitory effect on knee osteoarthritis of different degrees.It is proved that Tongluozhitong Fang can inhibit the occurrence and development of knee osteoarthritis by inhibiting the expression of TLR4,NF-κB and MyD88.