| Objective:To observe the effect of Toddalia asiatica Lam alcohol extracts on the TLR4/NF-?B signaling pathway in the synovium of SD rats induced by collagen.To explore the biological mechanism of Toddalia asiatica Lam alcohol extract in the treatment of RA.Methods:1.Ten of 70 SD rats were randomly selected as normal group,and the rest were used to establish CIA model.Each emulsion of 0.3ml was injected into the right posterior foot,tail and back of the rat,and the negative control group was injected with the same volume of saline at the same site.The above steps were repeated 7 days later.After 14 days of second immunization,it was observed that the arthritis index of toe swelling and immunization group increased significantly,and three normal model rats were randomly selected.The synovial tissue sections of knee joint were taken for pathological observation,and the synovium thickened by staining.The inflammatory cell infiltration and AI score?4 indicated that the model had been successfully constructed.After 14 days of secondary immunization,the CIA model was successfully established,and the AI was divided into model group,high,medium and low dose groups and positive control group?MTX group?.The efthree of Toddalia asiatica Lam alcohol extracts group:reference to 60kg adult clinical commonly used dose converted to equivalent standard rat dose.The low,medium and high levels were1.2,2.4 and 4.8 mg·kg-1,respectively,once a day for 35 days.Positive control group:MTX dose reference 60 kg adult clinical dose 510mg kg-1·d-11 into rat dose 0.001g kg-1·d-11 once a day for 35 days..Normal group and model group:the same volume of saline was applied once a day for 35 days.2.The pathological changes of synovial tissue in rats were observed by who staining.The fixed synovial tissue was placed in the paraffin embedding box,marked for time and group,dehydrated overnight with 95%ethanol at room temperature,and the paraffin slices of suitable thickness were selected and placed on the slide.The proliferation of synovial cells and blood vessels and the infiltration of lymphocytes in synovial layer and subsynovial layer were observed under optical microscope.3.The serum levels of TNF-?and IL-1?were measured by ELISA 4 weeks after administration.4.The protein expression of NF-?Bp65 and MyD88,IRAK-1,TLR4 in synovial tissue was detected by Western blot.5.The mRNA expression of NF-?Bp65 and MyD88,IRAK-1,TLR-4 in synovial tissue was detected by RT-PCR.Results:1.General observations:rats in the normal group were in good condition,with normal drinking water,stable body weight,white nail and free joint movement.In the model group,the fur color of rats was dull and haggard,accompanied by shedding,reduced activity and food intake,swollen and deformed ankle joints and foot metatarsal joints,broken and claudicated foot surfaces of some rats,increased urine volume,loose stools and reduced body mass growth.In each dose group,the degree of joint swelling was reduced,food intake was slightly increased,and weight was gradually increased.Among them,the high dose group of Toddalia asiatica Lam alcohol extract was superior to other drug treatment groups.2.The swelling degree of the joints:the degree of swelling of the rats in the model group at week 1 presented an increasing trend after modeling,with significant difference?P<0.05?compared with that in the normal group;From the third week of the experiment,the degree of joint swelling in the treated group was significantly lower than that in the model group?P<0.05?.At the fourth week of the experiment,the degree of joint swelling in the Toddalia asiatica Lam medium and high dose group was significantly lower than that in the model group?P<0.05?,and the treatment was significantly improved.3.Observation on the pathological morphology of the synovial tissue:Normal synovial tissue of the knee joint in the Normal group,cells in neat arrangement,no hyperemia and edema,no infiltration of lymphocytes,mononuclear cells and other inflammatory cells,no proliferation of synovial cells;In the model group,synovial tissue hyperemia and edema were observed,and a large number of synovial cells were proliferated and disordered,and fibrous tissue exuded.Synovial cell hyperplasia was significantly reduced and synovial tissue showed slight hyperemia and edema in the high-dose group of Toddalia asiatica Lam alcohol extract.Synovial cell hyperplasia and synovial tissue hyperemia and edema were slightly reduced in the medium dose group.Synovial cell hyperplasia and synovial tissue hyperemia and edema were slightly reduced in the low dose group.Synovial cells in the positive control group were arranged in a relatively regular pattern,with mild tissue hyperemia and edema,a small amount of inflammatory cell infiltration,and some control of hyperplasia.4.ELISA:compared with the model group,the levels of TNF-?and IL-1?in the serum of the treatment group were significantly decreased?P<0.05?;compared with the MTX group,the drop level of the high-dose alcohol extract group was significantly higher than that of the MTX group?P<0.05?.5.Western blot:the protein expressions of TLR4,MyD88,IRAK-1,NF-?Bp65 in synovium of the CIA rat model group were significantly increased compared with the normal group.Compared with the model group,the protein expressions of TLR4,MyD88,IRAK-1 and NF-?Bp65 were decreased in the MTX group and the Toddalia asiatica alcohol extract group.The inhibitory effect of TLR4,MyD88,IRAK-1 and NF-?Bp65 on the expression level in the high dose group was more obvious than MTX.6.RT-PCR:compared with the normal group,the mRNA expression of TLR4,MyD88,IRAK-1,NF-?Bp65 in the synovium of CIA rat model group was significantly higher than normal group.Compared with the model group,the mRNA expression of TLR4,MyD88,IRAK-1,NF-?Bp65 in the high dose group was lower than model group.Among them,the mRNA expression of TLR4,MyD88,NF-?Bp65 in high dose group was significantly lower than that in MTX group.Conclusion:?1?Toddalia asiatica Lam sticks of ethanol extract can significantly improve the arthritis of CIA rats,improve the pathological changes of joint synovial membrane,and is correlated with the dose.The effect of the high-dose group is the best,slightly better than the methotrexate group.?2?It may reduce the inflammatory amplification effect of TNF-?and IL-1?activating TLR4/NF-?B signaling pathway by inhibiting the expression of inflammatory cytokines TNF-?and IL-1?in serum.?3?The mechanism of the IRAK-1 alcohol extract treatment for RA may regulate the expression of relevant genes on the TLR4/NF-?B signaling pathway. |
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